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PMC4512133

higher levels of cardiorespiratory fitness are associated with improved long - term cardiovascular outcomes among patients with type 2 diabetes mellitus ( t2 dm ) ( 1 ) . exercise training in patients with t2 dm has been shown to improve cardiorespiratory fitness , glycemic control , and other metabolic parameters ( 2,3 ) . however , the interrelationship between training - related changes in cardiorespiratory fitness and improvement in glycemic control and other metabolic parameters is not well understood . previous studies ( 4,5 ) have observed significant heterogeneity in the magnitude of change in measures of cardiorespiratory fitness in response to exercise training . recent analyses from the dose response to exercise training in women ( drew ) trial ( 6 ) showed that 30% of the study participants had no improvement in cardiorespiratory fitness after 6 months of supervised , moderate - intensity exercise training . it is unclear whether the metabolic benefits of exercise training are limited to those patients who improve their cardiorespiratory fitness . in this study , we examined the impact of exercise training on hemoglobin a1c ( hba1c ) levels and other metabolic parameters among fitness nonresponder and responder participants in the health benefits of aerobic and resistance training in individuals with type 2 diabetes ( hart - d ) study . we hypothesized that exercise training would be associated with significant improvements in glycemic control in fitness responders as well as nonresponders . the current study was performed as a secondary analysis of the hart - d trial . the full design and methodology of the hart - d study has been published previously ( 2 ) . briefly , the hart - d study was a 9-month randomized , controlled exercise - training trial comparing the effects of different modalities of exercise training on hba1c levels in sedentary participants with t2 dm . exclusion criteria for the study included a bmi > 48 kg / m , age < 30 or > 75 years , blood pressure 160/100 mmhg , fasting triglyceride levels 500 mg / dl , use of insulin pump , urine protein levels > 100 mg / dl , history of stroke , and advanced neuropathy or retinopathy or any serious medical condition that prevented adherence to the study protocol or the ability to exercise safely . the study protocol was reviewed and approved annually by the pennington biomedical research center institutional review board . the university of texas southwestern medical center institutional review board also approved the present substudy . the hart - d study recruited 262 participants who were randomized to one of four groups : 1 ) a nonexercise control group , 2 ) aerobic training only , 3 ) resistance training only , and 4 ) a combination of aerobic and resistance training . the nonexercise control group was offered weekly stretching and relaxation classes , and was asked to maintain their baseline activity levels during the 9-month study period . participants exercised 35 days / week at an intensity of 5080% of their maximum cardiorespiratory fitness for a total dose of 12 kcal / kg / week . the caloric dose was adjusted on a weekly basis based on the changes in body weight . american college of sports medicine equations were used to estimate caloric expenditure rates and , therefore , the time required per session ( 7 ) . participants exercised 3 days / week , with each session consisting of two sets of four upper - body exercises ( chest press , lateral pull - down , military press , and seated row ) , three sets of three lower - body exercises ( leg press , leg extension , and hamstring curl ) , and two sets of abdominal and back exercises . the prescribed weight was increased when the participant was able to complete 12 repetitions of a final set of each exercise on two consecutive sessions . participants had aerobic exercise training at a dose of 10 kcal / kg / body wt / week and two sessions of resistance training per week , with each session consisting of one set of each of the aforementioned nine exercises . the training regimen for the combination training group was consistent with federal physical activity guidelines and ensured equal time commitment among all exercise groups . among the hart - d study participants , cardiorespiratory fitness was measured as peak absolute oxygen uptake ( vo2peak , in liters per minute ) at baseline and at study completion using a treadmill test ( tmx425 ; trackmaster treadmills , newton , ks ) protocol , as previously described ( 2 ) . participants self - selected a walking pace at a level grade , and the grade increased by 2% every 2 min at a constant treadmill speed until they reached volitional exhaustion . breath - by - breath respiratory gases were measured using a trueone 2400 metabolic measurement cart ( parvo medics , salt lake city , ut ) . the same treadmill speed was used at the baseline and the end - of - study testing . baseline and post - training blood testing was performed after at least a 10-h fast . hba1c level was analyzed with a unicel dxc 600 pro ( beckman coulter , brea , ca ) . hba1c level was also assessed at the monthly visits with the diabetes educator using a finger - stick sample analyzed by an automated glycosylated hemoglobin analyzer ( dca 2000 ; bayer , dublin , ireland ) . weight was measured on a gse 450 electronic scale , and height was measured with a standard stadiometer . waist circumference ( wc ) was measured to the nearest 0.1 cm just above the iliac crest while the subject was at minimal expiration . body composition was measured by dual - energy x - ray absorptiometry with a qdr 4500/a whole - body scanner ( hologic , bedford , ma ) . diabetes medication type and dosage were assessed by detailed questionnaire with visual confirmation of prescription bottles . participants were categorized as having increased use of , decreased use of , or no change in use of diabetes medications based on baseline and follow - up medication dosages . for the present analysis , we have included all control participants and all exercise - training participants who had fitness test data available at baseline and follow - up , and had > 70% adherence to exercise training . cardiorespiratory fitness response was assessed as the change in measured vo2peak ( vo2peak , in liters per minute ) from baseline to follow - up . for the present analysis , a 5% increase in vo2peak from baseline to follow - up was defined as a clinically meaningful cardiorespiratory fitness response ( 8,9 ) , and the exercise - training participants were categorized as fitness nonresponders ( < 5% increase in vo2peak ) or fitness responders ( 5% increase in vo2peak ) . baseline clinical , demographic , and anthropometric characteristics were compared among the study groups ( control , fitness responders , and fitness nonresponders ) using the wilcoxon rank - sum trend test for all continuous variables and the trend test for categorical variables . the primary outcome for the present analyses was change in hba1c levels from baseline to completion of training . changes in hba1c levels , anthropometric measures , and exercise test parameters were assessed among the study groups using linear mixed - effect models for repeated measures over time . the models were adjusted for baseline values , age , sex , race / ethnicity , and duration of diabetes . for hba1c measurements , monthly data were available , whereas exercise test parameters and anthropometric measures were available only at baseline and trial completion . for participants with missing hba1c data at the 9-month follow - up , the change in hba1c level was calculated based on the difference in finger - stick measured hba1c levels at baseline and at the last recorded monthly visit . sensitivity analyses were performed among exercise - training participants after excluding resistance training only participants to compare the changes in metabolic parameters with exercise training between fitness responder and nonresponder groups . the association between continuous changes in fitness and change in hba1c levels from baseline to post - training was further characterized using multivariable adjusted linear regression analysis adjusted for age , sex , race / ethnicity , baseline hba1c level , diabetes duration , baseline fitness , baseline bmi , change in fitness , and change in the percentage of body fat . concomitant reductions in hypoglycemic medication use and reductions in hba1c levels were assessed as an additional secondary outcome using a composite dichotomous outcome variable . individuals who decreased diabetes medication or reduced hba1c level by 0.5% without increasing their use of medications multivariable adjusted logistic regression analysis models were constructed to determine the likelihood of achieving the composite secondary outcome in the study participants with exercise training after adjustment for age , sex , race / ethnicity , baseline hba1c level , diabetes duration , baseline fitness , baseline bmi , change in fitness , and change in the percentage of body fat . all statistical analyses were performed using sas for windows ( release 9.2 ; sas institute , inc . , cary , nc ) . the current study was performed as a secondary analysis of the hart - d trial . the full design and methodology of the hart - d study has been published previously ( 2 ) . briefly , the hart - d study was a 9-month randomized , controlled exercise - training trial comparing the effects of different modalities of exercise training on hba1c levels in sedentary participants with t2 dm . exclusion criteria for the study included a bmi > 48 kg / m , age < 30 or > 75 years , blood pressure 160/100 mmhg , fasting triglyceride levels 500 mg / dl , use of insulin pump , urine protein levels > 100 mg / dl , history of stroke , and advanced neuropathy or retinopathy or any serious medical condition that prevented adherence to the study protocol or the ability to exercise safely . the study protocol was reviewed and approved annually by the pennington biomedical research center institutional review board . the university of texas southwestern medical center institutional review board also approved the present substudy . the hart - d study recruited 262 participants who were randomized to one of four groups : 1 ) a nonexercise control group , 2 ) aerobic training only , 3 ) resistance training only , and 4 ) a combination of aerobic and resistance training . the nonexercise control group was offered weekly stretching and relaxation classes , and was asked to maintain their baseline activity levels during the 9-month study period . participants exercised 35 days / week at an intensity of 5080% of their maximum cardiorespiratory fitness for a total dose of 12 kcal / kg / week . the caloric dose was adjusted on a weekly basis based on the changes in body weight . american college of sports medicine equations were used to estimate caloric expenditure rates and , therefore , the time required per session ( 7 ) . participants exercised 3 days / week , with each session consisting of two sets of four upper - body exercises ( chest press , lateral pull - down , military press , and seated row ) , three sets of three lower - body exercises ( leg press , leg extension , and hamstring curl ) , and two sets of abdominal and back exercises . the prescribed weight was increased when the participant was able to complete 12 repetitions of a final set of each exercise on two consecutive sessions . participants had aerobic exercise training at a dose of 10 kcal / kg / body wt / week and two sessions of resistance training per week , with each session consisting of one set of each of the aforementioned nine exercises . the training regimen for the combination training group was consistent with federal physical activity guidelines and ensured equal time commitment among all exercise groups . participants exercised 35 days / week at an intensity of 5080% of their maximum cardiorespiratory fitness for a total dose of 12 kcal / kg / week . the caloric dose was adjusted on a weekly basis based on the changes in body weight . american college of sports medicine equations were used to estimate caloric expenditure rates and , therefore , the time required per session ( 7 ) . participants exercised 3 days / week , with each session consisting of two sets of four upper - body exercises ( chest press , lateral pull - down , military press , and seated row ) , three sets of three lower - body exercises ( leg press , leg extension , and hamstring curl ) , and two sets of abdominal and back exercises . the prescribed weight was increased when the participant was able to complete 12 repetitions of a final set of each exercise on two consecutive sessions . participants had aerobic exercise training at a dose of 10 kcal / kg / body wt / week and two sessions of resistance training per week , with each session consisting of one set of each of the aforementioned nine exercises . the training regimen for the combination training group was consistent with federal physical activity guidelines and ensured equal time commitment among all exercise groups . among the hart - d study participants , cardiorespiratory fitness was measured as peak absolute oxygen uptake ( vo2peak , in liters per minute ) at baseline and at study completion using a treadmill test ( tmx425 ; trackmaster treadmills , newton , ks ) protocol , as previously described ( 2 ) . participants self - selected a walking pace at a level grade , and the grade increased by 2% every 2 min at a constant treadmill speed until they reached volitional exhaustion . breath - by - breath respiratory gases were measured using a trueone 2400 metabolic measurement cart ( parvo medics , salt lake city , ut ) . the same treadmill speed was used at the baseline and the end - of - study testing . baseline and post - training blood testing was performed after at least a 10-h fast . hba1c level was analyzed with a unicel dxc 600 pro ( beckman coulter , brea , ca ) . hba1c level was also assessed at the monthly visits with the diabetes educator using a finger - stick sample analyzed by an automated glycosylated hemoglobin analyzer ( dca 2000 ; bayer , dublin , ireland ) . weight was measured on a gse 450 electronic scale , and height was measured with a standard stadiometer . waist circumference ( wc ) was measured to the nearest 0.1 cm just above the iliac crest while the subject was at minimal expiration . body composition was measured by dual - energy x - ray absorptiometry with a qdr 4500/a whole - body scanner ( hologic , bedford , ma ) . diabetes medication type and dosage were assessed by detailed questionnaire with visual confirmation of prescription bottles . participants were categorized as having increased use of , decreased use of , or no change in use of diabetes medications based on baseline and follow - up medication dosages . for the present analysis , we have included all control participants and all exercise - training participants who had fitness test data available at baseline and follow - up , and had > 70% adherence to exercise training . cardiorespiratory fitness response was assessed as the change in measured vo2peak ( vo2peak , in liters per minute ) from baseline to follow - up . for the present analysis , a 5% increase in vo2peak from baseline to follow - up was defined as a clinically meaningful cardiorespiratory fitness response ( 8,9 ) , and the exercise - training participants were categorized as fitness nonresponders ( < 5% increase in vo2peak ) or fitness responders ( 5% increase in vo2peak ) . baseline clinical , demographic , and anthropometric characteristics were compared among the study groups ( control , fitness responders , and fitness nonresponders ) using the wilcoxon rank - sum trend test for all continuous variables and the trend test for categorical variables . the primary outcome for the present analyses was change in hba1c levels from baseline to completion of training . changes in hba1c levels , anthropometric measures , and exercise test parameters were assessed among the study groups using linear mixed - effect models for repeated measures over time . the models were adjusted for baseline values , age , sex , race / ethnicity , and duration of diabetes . for hba1c measurements , monthly data were available , whereas exercise test parameters and anthropometric measures were available only at baseline and trial completion . for participants with missing hba1c data at the 9-month follow - up , the change in hba1c level was calculated based on the difference in finger - stick measured hba1c levels at baseline and at the last recorded monthly visit . sensitivity analyses were performed among exercise - training participants after excluding resistance training only participants to compare the changes in metabolic parameters with exercise training between fitness responder and nonresponder groups . the association between continuous changes in fitness and change in hba1c levels from baseline to post - training was further characterized using multivariable adjusted linear regression analysis adjusted for age , sex , race / ethnicity , baseline hba1c level , diabetes duration , baseline fitness , baseline bmi , change in fitness , and change in the percentage of body fat . concomitant reductions in hypoglycemic medication use and reductions in hba1c levels were assessed as an additional secondary outcome using a composite dichotomous outcome variable . individuals who decreased diabetes medication or reduced hba1c level by 0.5% without increasing their use of medications were defined as successfully achieving the hba1c - diabetes medication composite outcome . multivariable adjusted logistic regression analysis models were constructed to determine the likelihood of achieving the composite secondary outcome in the study participants with exercise training after adjustment for age , sex , race / ethnicity , baseline hba1c level , diabetes duration , baseline fitness , baseline bmi , change in fitness , and change in the percentage of body fat . all statistical analyses were performed using sas for windows ( release 9.2 ; sas institute , inc . , cary , nc ) . a total of 202 participants ( mean age 57.1 7.9 years , women 62.9% ) were included ( fig . 1 ) . all exercise - training participants had baseline and 9-month follow - up hba1c levels available . we observed a significant heterogeneity in vo2peak in response to comparable doses of exercise training , with 43% of exercise - training participants experiencing no improvement in fitness after exercise training . furthermore , only 36.6% of exercise - training participants had a clinically meaningful fitness response ( vo2peak 5% ) with exercise training . all control participants and exercise - training participants with available baseline and follow - up peak oxygen uptake ( in liters per minute ) data and > 70% adherence to exercise prescription were included in the current study . fitness responders were younger and had a greater proportion of combination training than fitness nonresponders . the proportion of fitness responders was similar in the aerobic training only ( 31.3% ) and resistance training only ( 33.9% ) groups . comparison of baseline characteristics of participants across the study groups defined by cardiorespiratory fitness response to exercise intervention data are presented as mean ( sd ) , except as noted . bp , blood pressure ; hr , heart rate ; na , not applicable ; rer , respiratory exchange ratio ; sbppeak , peak systolic blood pressure . * fitness responder vs. nonresponder . changes in selected exercise test parameters from baseline to the end of the trial among the fitness responders and nonresponders are compared in table 2 . fitness responders had a significantly greater increase in exercise time , peak exercise heart rate , peak exercise blood pressure , and maximum exercise heart rate reserve than fitness nonresponders . these findings validate the criteria for a clinically meaningful fitness response ( 5% increase in vo2peak ) that was used in the current study . change in total caloric intake from baseline to follow - up was not significantly different among the control , fitness responder , and fitness nonresponder groups ( median change in total daily caloric intake : control group 135.1 kcal , range 376.4 to 235.2 kcal ; fitness responder group 149.2 kcal , range 564.5 to 82.4 kcal ) ; fitness nonresponder group 134.4 kcal , range 330.1 to 107 kcal ; p = 0.44 ) . change in selected exercise test parameters from baseline to post training among exercise - training participants values are expressed as fitted mean ( 95% ci ) derived from linear mixed models that are adjusted for baseline value , age , sex , duration of diabetes , and race / ethnicity . predicted heart rate reserve = [ ( peak exercise heart rate resting heart rate)/(estimated maximum heart rate resting heart rate ) ] 100% where estimated maximum heart rate = 220 age ( years ) . figure 2 depicts the monthly mean hba1c levels across the control , fitness responder , and fitness nonresponder groups . in multivariable models adjusting for age , sex , race / ethnicity , diabetes duration , and baseline hba1c level , compared with the control group , mean hba1c levels measured over time were significantly lower for both the fitness responder group ( p = 0.01 ) and the fitness nonresponder group ( p = 0.003 ) . however , there was no significant difference between the fitness responder and fitness nonresponder groups for the trends in monthly mean hba1c levels ( p = 0.25 ) . the data are represented as fitted means derived from a linear mixed model that included the covariables age , sex , race / ethnicity , diabetes duration , and baseline hba1c level . the group effect was significant for comparison of trends in monthly mean hba1c levels between control vs. responder ( p = 0.014 ) , as well as control vs. nonresponder groups ( p = 0.0026 ) , but not for responder vs. nonresponder ( p = 0.254 ) . changes in vo2peak , hba1c levels , and anthropometric measures from baseline to the end of the trial within the study groups are shown in fig . 3 . while only fitness responders had a significant improvement in vo2peak , both fitness responders and nonresponders experienced significant reductions in hba1c level from baseline to final assessment ( fitness responders hba1c 0.26% [ 95% ci 0.5 to 0.01 ] , fitness nonresponders hba1c 0.26% [ 95% ci 0.45 to 0.08 ] ) . similarly , anthropometric measures such as wc and percentage of body fat improved significantly with exercise training among fitness responders ( wc 2.6 cm [ 95% ci 3.7 to 1.5 ] , body fat 1.07% [ 95% ci 1.5 to 0.62 ] ) as well as nonresponders ( wc 1.8 cm [ 95% ci 2.6 to 1.0 ] , body fat 0.75% [ 95% ci 1.09 to 0.41 ] ) . furthermore , the absolute change in hba1c levels and anthropometric measures with exercise training was not significantly different among fitness responders versus nonresponders . similar improvements in hba1c levels , wc , and percentage of body fat were observed among fitness responders and nonresponders from baseline to follow - up on sensitivity analyses after excluding resistance training only participants ( table 3 ) . comparisons of change in vo2peak , hba1c level , and anthropometric measures from baseline to trial completion among control , fitness responder , and fitness nonresponder groups . the values are expressed as fitted means , and all are adjusted for baseline value , age , sex , duration of diabetes , and race / ethnicity . * p < 0.05 for change from baseline to end of trial within a particular study group . changes in metabolic parameters from baseline to follow - up among fitness responders and nonresponders with and without resistance training participants values are expressed as fitted mean ( 95% ci ) derived from linear mixed models that are adjusted for baseline value , age , sex , duration of diabetes , and race / ethnicity . in adjusted linear regression analyses , exercise training , baseline hba1c level , and change in the percentage of body fat were identified to be significant predictors of change in hba1c level ( table 4 ) . change in fitness was not a significant determinant of change in hba1c level in adjusted analysis ( standardized -estimate = 0.13 , p = 0.69 ) . predictors of change in hba1c among the study participants model was adjusted for age , sex , race / ethnicity , baseline hba1c level , diabetes duration , lean body mass , baseline vo2peak , baseline bmi , vo2peak , medication change , exercise training , and change in percentage of body fat . n , no ; y , yes . the proportion of individuals who achieved the composite outcome of either decreasing glucose - lowering medication or reducing hba1c level by 0.5% without increasing the use of medications was numerically greater in the exercise - training groups compared with the control group ( 36% vs. 20.5% ; p = 0.08 ) . in multivariable adjusted logistic regression analysis , exercise training was associated with significantly greater odds of achieving the composite glycemic outcome ( odds ratio 3.0 [ 95% ci 1.049.2 ] ) . among exercise - training participants , the proportion of participants who achieved the diabetes medication - hba1c composite outcome was not significantly different between fitness nonresponders and responders ( 33% vs. 41% , p = 0.4 ) . first , a substantial proportion of participants with t2 dm have no significant improvement in cardiorespiratory fitness in response to exercise training . second , exercise training is associated with significant improvement in glycemic control among both fitness responders and fitness nonresponders . third , exercise training related improvement in glycemic control among fitness nonresponders is associated with a significant reduction in central obesity and percentage of body fat . taken together , these findings suggest that exercise training improves metabolic parameters in patients with t2 dm independent of changes in cardiorespiratory fitness . the prevalence of fitness nonresponse among t2 dm patients observed in the current study is greater than that previously reported among healthy adults ( 6 ) . this is particularly significant since the dose of exercise used in the current study was similar to that recommended by the national institutes of health ( 1012 kcal / kg / week ) ( 10 ) . the high fitness nonresponse rate observed among the exercise - training participants could be related to multiple factors . patients with diabetes have increased prevalence of chronotropic incompetence , left ventricular hypertrophy , and adverse remodeling , all of which are associated with a greater risk for fitness nonresponse ( 11,12 ) . also , previous studies ( 13,14 ) have shown that patients with diabetes have slowed muscle perfusion kinetics and slowed oxidative phosphorylation that could lead to a relatively blunted improvement in cardiorespiratory fitness with exercise training . our study findings have important clinical implications for exercise counseling provided to sedentary middle - aged adults with t2 dm . the current public health recommendations ( 10 ) suggest that such individuals should accumulate a minimum of 30 min of moderate - intensity physical activity on most days of a week with a goal to improve cardiorespiratory fitness . however , it may not be feasible to achieve this target in a significant proportion of patients with coexisting conditions such as obesity , older age , and cardiovascular disease ( 15 ) . the current study suggests that improvements in metabolic parameters are observed among exercise - training participants independent of their change in cardiorespiratory fitness . this highlights the importance of sustained exercise training even in individuals who do not demonstrate improvement in measures of cardiorespiratory fitness . furthermore , this study suggests that exercise - training programs should target and assess for improvement in metabolic parameters such as glycemic control , wc , and percentage of body fat among individuals with t2 dm . response to exercise training has been traditionally expressed in terms of improvement in cardiorespiratory fitness ( 4,16,17 ) . however , physiological adaptation to exercise training is a complex and heterogeneous process affecting multiple organ systems ( 18 ) . favorable effects of exercise on cardiac output and peripheral oxygen use mediate training - related improvement in cardiorespiratory fitness ( 19,20 ) . nonresponsiveness of cardiorespiratory fitness represents a failure of just one potential adaptation to exercise training . it is plausible that fitness - nonresponsive participants undergo favorable adaptations in other physiological pathways independent of fitness change . small longitudinal studies ( 21,22 ) from the 1980s reported a lack of association between the magnitude of improvement in vo2peak and aerobic performance among recreationally active participants who underwent short - term endurance training . more recently , vollaard et al . ( 23 ) showed significant adaptations associated with metabolic control and aerobic performance even among exercise - training participants who had minimal improvements in vo2peak after training ( low responders ) . the current study provides further evidence in support of this notion among participants with t2 dm , and shows that fitness nonresponders have significant improvement in metabolic parameters such as hba1c level and adiposity , changes that are not significantly different from those in fitness responders . thus , lack of improvement in one specific phenotype , specifically in vo2peak , does not negate other potential benefits of exercise and highlights the importance of assessing training response in a broader clinical context . recent studies ( 2426 ) have identified fitness improvement in response to exercise training as a significant predictor of improvement in glycemic control among participants with t2 dm . however , these studies used estimated mets , derived from the maximal speed and grade reached during a treadmill test , as a measure of fitness . the impact of changes in vo2peak , the gold standard measure of cardiorespiratory fitness , on metabolic outcomes in exercise - training trials is not well understood . while some studies ( 24,27,28 ) have identified an increase in vo2peak as a significant predictor of improvement in hba1c levels brennan et al . ( 27 ) evaluated the association between changes in vo2peak and insulin sensitivity among 60 participants with t2 dm who underwent aerobic training for 34 months , and reported that exercise - induced changes in vo2peak do not mediate changes in insulin sensitivity . in the current study , we have confirmed this lack of association between changes in vo2peak and improvement in hba1c in a much larger study population with a significantly longer duration of exercise training . in contrast , larose et al . ( 28 ) showed that change in vo2peak was a significant predictor of hba1c change in response to exercise training . this discrepancy between the study findings could be due to the different parameters used as a measure of fitness change in the two studies . while change in vo2peak ( in liters per minute ) was used as the measure of change in fitness in the current study , larose et al . ( 28 ) used the change in oxygen uptake scaled to body mass ( in liters per kilogram per minute ) as the measure of change in fitness . a significant reduction in body weight with exercise training was observed by larose et al . ( 28 ) , and , as the unit of indexation , influenced the estimated vo2peak independent of changes in absolute vo2 ( in liters per minute ) not scaled to weight . in that study , weight changes were significantly associated with improvement in vo2peak scaled to body mass as well as with hba1c , and could explain the observed association between changes in fitness ( oxygen uptake scaled to body mass ) and glycemic control . furthermore , there were significant differences in the analytical approach ( per group in larose et al . vs. combined in hart - d ) and dose of exercise training ( lower dose of training in the hart - d study ) used in the two studies that could also have contributed toward the discrepant study findings . the lack of association between change in cardiorespiratory fitness and improvement in glycemic control is likely due to differences in the underlying adaptive mechanisms that result from exercise training . improvement in cardiorespiratory fitness in response to exercise training is associated with central cardiovascular adaptations , physiological cardiac remodeling , and improvement in stroke volume ( 20,29,30 ) . in contrast , training - associated changes in glycemic control are more related to improvement in insulin sensitivity secondary to peripheral adaptations in the adipose and skeletal muscle tissue ( 31,32 ) . previous studies ( 26,33,34 ) have identified changes in measures of central adiposity as significant predictors of improvement in hba1c and glycemic control . similarly , the present observations suggest that reduction in the percentage of body fat is associated with significant improvement in hba1c , though whether it is causal or even contributory remains unclear . supporting a contributory role , it is notable that the magnitude of reduction in wc and the percentage of body fat with training was similar among fitness responders and nonresponders , which were associated in both groups with demonstrable improvements in glucose metrics . first , the participants of the hart - d study had relatively well - controlled diabetes with an average hba1c level of 7.6 . it is possible that among individuals with worse glycemic control , fitness responders may have a greater improvement in hba1c level with exercise training compared with nonresponders . second , we do not have a standard measure of insulin resistance that could have helped us to better understand the mechanism of improvement in glycemic control among fitness nonresponders . third , we used a cutoff of 5% change in vo2peak as a clinically meaningful fitness response based on previous studies and consensus in the literature . the threshold for meaningful improvement in fitness in these sedentary patients with t2 dm could be different . however , we observed similar results using a more lenient definition for fitness improvement ( change in vo2peak > 0 l / min , data not shown ) . furthermore , we did not observe any association between continuous change in fitness and change in hba1c level in linear regression analysis , suggesting that our observations are insensitive to the threshold of fitness response . finally , a food frequency questionnaire was used at baseline and follow - up to assess changes in diet , which limits our ability to identify changes in caloric intake and diet composition . in conclusion , exercise training is associated with significant improvement in glycemic control and measures of adiposity among participants with t2 dm independent of their change in cardiorespiratory fitness . further studies are needed to confirm the mechanisms by which training improves metabolic parameters among fitness nonresponders .

objectiveto evaluate the impact of exercise training ( et ) on metabolic parameters among participants with type 2 diabetes mellitus ( t2 dm ) who do not improve their cardiorespiratory fitness ( crf ) with training.research design and methodswe studied participants with t2 dm participating in the health benefits of aerobic and resistance training in individuals with type 2 diabetes ( hart - d ) trial who were randomized to a control group or one of three supervised et groups for 9 months . fitness response to et was defined as a change in measured peak absolute oxygen uptake ( vo2peak , in liters per minute ) from baseline to follow - up . et participants were classified based on vo2peak into fitness responders ( vo2peak 5% ) and nonresponders ( vo2peak < 5% ) , and changes in metabolic profiles were compared across control , fitness responder , and fitness nonresponder groups.resultsa total of 202 participants ( mean age 57.1 7.9 years , 63% women ) were included . among the exercise groups ( n = 161 ) , there was substantial heterogeneity in vo2peak 57% had some improvement in crf ( vo2peak > 0 ) , with only 36.6% having a 5% increase in vo2peak . both fitness responders and nonresponders ( respectively ) had significant improvements in hemoglobin a1c and measures of adiposity ( hba1c : 0.26% [ 95% ci 0.5 to 0.01 ] and 0.26% [ 0.45 to 0.08 ] ; waist circumference : 2.6 cm [ 3.7 to 1.5 ] and 1.8 cm [ 2.6 to 1.0 ] ; body fat : 1.07% [ 1.5 to 0.62 ] and 0.75% [ 1.09 to 0.41 ] ) . no significant differences were observed in the degree of change of these metabolic parameters between fitness responders and nonresponders . control group participants had no significant changes in any of these metabolic parameters.conclusionset is associated with significant improvements in metabolic parameters irrespective of improvement in cardiorespiratory fitness .

Introduction Research Design and Methods Study Design and Participants Exercise Intervention Aerobic Training Resistance Training Combination Training Exercise Testing Outcome Assessment Statistical Analysis Results Conclusions

PMC4663351

many human diseases , such as type ii diabetes mellitus , alzheimer 's disease , parkinson 's disease , and huntingdon 's disease , are associated with protein aggregation and amyloid formation [ 14 ] . in type ii diabetes mellitus , the cytotoxicity is most likely related to membrane damage , which leads to attrition of insulin - producing -cells [ 57 ] . the primary component of islet amyloid and actual fibril - forming molecule is human islet amyloid polypeptide ( hiapp or amylin ) , a 37-residue peptide which is synthesized in pancreatic islet -cells and cosecreted with insulin . the normal physiological role of hiapp is still unclear , but it is believed to have correlations with gastric emptying , suppression of food intake , and glucose homeostasis [ 810 ] . like other amyloidogenic peptides , it is believed that hiapp forms amyloid deposits via a nucleation - dependent aggregation pathway characterized by a lag phase associated with the formation of a nucleus . the early intermediates were reported to play important nucleating roles in hiapp fibrillation and nmr experimental studies have shown that these intermediates are large in size [ 1216 ] . increasing evidence suggests that the prefibrillar intermediates , such as oligomers and protofibrils , are the primary toxic species to trigger pathological processes [ 1214 ] , while mature amyloid fibrils themselves exert only a minimal cytotoxic effect on pancreatic -cells [ 1719 ] . the intermediate oligomers as well as the fibrillization process can disrupt membrane integrity and thereby cause toxicity [ 2022 ] . experimental studies reported that monomeric hiapp exhibits predominantly a random coil conformation in aqueous solution , and residues 8~19 of the peptide transiently adopt an -helical structure [ 2325 ] . in the presence of lipid membranes , hiapp initially binds to the membrane in a helical state electron paramagnetic resonance ( epr ) spectroscopy study showed that the -helical region of hiapp at neutral ph spans residues 9~22 and is oriented parallel to the surface of large unilamellar vesicles containing negatively charged lipids . an earlier nuclear magnetic resonance ( nmr ) study demonstrated that residues 7~17 and 21~28 adopt helical structure in sodium dodecyl sulfate ( sds ) detergent micelles . as the concentration of membrane - bound peptides rises , hiapps cooperatively convert from -helical intermediates to -sheet aggregates [ 24 , 2931 ] . it was reported that the n - terminal 1~19 fragment of hiapp is primarily responsible for membrane interaction , while the amyloidogenic 20~29 fragment is mainly responsible for fibrillar aggregates [ 26 , 32 , 33 ] . these hiapp aggregates may reconstitute membranes and form amyloid ion channels , which would mediate ion transport and destabilize the cell ionic homeostasis [ 5 , 3436 ] . nonselective ion channel activity of polymorphic hiapp double channels was reported recently by experimental and md simulation studies [ 37 , 38 ] . in addition , previous experiments demonstrated that the toxic hiapp and its variants primarily interact with the curved regions of the membrane , and lipids of phosphatidylethanolamine ( pe ) type exhibit intrinsic curvature strain , indicating that membrane curvature may play very important roles in the polymerization of hiapp . pore - like structures and channel activities are also reported in the studies of cytotoxicity induced by a and prp [ 35 , 41 ] . lipid composition was suggested to be one of the major factors that influence hiapp aggregation , and the presence of membranes that contain negatively charged lipids , such as phosphatidylglycerol ( pg ) or phosphatidylserine ( ps ) , can significantly accelerate the aggregation process [ 13 , 27 , 29 , 42 , 43 ] . however , the effect of lipid composition on the structures and orientations of hiapp on membrane surface at atomic level are not well understood . on the computational side , several studies have investigated the structures of monomeric / oligomeric species of full - length and the fragments of hiapp in aqueous solution [ 4451 ] and in membrane environment [ 5255 ] . our previous study demonstrated that monomeric hiapp has a preferential orientation on anionic palmitoyloleoyl - phosphatidylglycerol ( popg ) bilayers . as a first step to understand how lipid composition modulates the aggregation of full - length hiapp , in this study , we investigate the binding orientation and membrane interaction of hiapp at zwitterionic palmitoyloleoyl - phosphatidylcholine ( popc ) bilayer by conducting multiple atomistic md simulations and then compare the results with those obtained at anionic popg bilayers . through the comparison of binding behaviors and lipid interactions of monomeric hiapp at popc and popg bilayers , we try to understand at atomic level the membrane - modulated hiapp aggregation in the membrane environment . the amino acid sequence of hiapp is kcntatcatqrlanflvhssnnfga ilsstnvgsnty , with the cys2 and cys7 forming a disulfide bond that constrains the first four residues in a disordered hairpin loop . to mimic experimental conditions , the n - terminus was charged and the c - terminus was amidated . at neural ph , we constructed the zwitterionic membrane using popc lipids because phosphatidylcholine ( pc ) is the most abundant phospholipids in pancreatic islets . this model membrane consists of 2 64 popc lipids and the initial atomic coordinates were obtained from a previous computational study of a neat popc lipid bilayer by tieleman and bentz . na and cl ions were added to neutralize the system and provide an additional 0.1 m salt concentration . numerous experimental studies reported that hiapp adopt predominantly -helical structure when initially bound to the membrane [ 21 , 2327 ] . consistently , a recent spectroscopic study reported that hiapp transiently sample an -helical structure in solution that becomes fully stabilized when bound to the surface of a membrane containing negatively charged lipids . as the time scale for protein folding at water / membrane interface is on the order of milliseconds to seconds , it is still out of reach to sample conformational transition from a random coil to a helical structure at physiological temperature . therefore , we took one of the nmr - derived conformations ( pdb i d : 2kb8 ) solved in sds micelles as the starting point of our md simulations , as done in previous studies [ 54 , 6163 ] . it consists of a helix running from residue 5 to 28 and disordered structures for the n - terminal residues 14 and the c - terminal residues 2937 . the choice of an -helical conformation as a starting structure in our study is an md strategy to speed up the simulation outcome as the time for coil - to - helix transition at water / membrane interface is on the order of milliseconds to seconds . although the interfacial folding of shorter peptides could be probed by replica - exchange molecular dynamic ( remd ) simulations as we did for hiapp(119 ) in our recent study , it would be too expensive for a peptide of 37 residue with current computational resources . the helical region of hiapp monomer was initially orientated parallel to the membrane surface with a minimum distance 1.4 nm between the peptide and the popc bilayer . we chose four different starting orientations of hiapp with respect to popc bilayer surface ( see figure 1 ) so that the peptide was allowed to adjust itself before adsorption to the bilayer surface . in the initial state of s(0 ) , the side chain of residue k1 points toward the membrane surface . the initial state s(90 ) , s(180 ) , and s(270 ) were generated by rotating the hiapp peptide in s(0 ) by 90 , 180 , and 270 around the axis of the helix , respectively . all md simulations have been performed in the isothermal - isobaric ( npt ) ensemble using the gromacs 3.3.3 software package . currently , several force fields are available for protein - lipid system , such as gromos87/berger , opls - aa / berger , amber99sb - ildn / slipids , gromos54a7 , and charmm36 [ 6773 ] . the gromos force field and berger force field have been widely used for proteins and lipids , respectively . berger force field borrowed the standard parameters of the gromos force field for bonds , valence angles , improper dihedrals , and the dihedral angles in the headgroup region of lipids . thus , the combination of gromos force field with berger force field is appropriate for peptide - membrane system . the lipid is described with the berger force field , and the peptide is described with gromos87 force field . the popc parameters used in this study have the correction on the double bond suggested by bachar et al . . peptide bonds are constrained by the lincs algorithm and water geometries are constrained by settle . the pressure is maintained at 1 bar using a semi - isotropic scheme in which the lateral and perpendicular pressures are coupled separately with a coupling constant of 1.0 ps and a compressibility of 4.5 10 bar . the temperature of the system is maintained at 310 k with a coupling constant of 0.1 ps , above the gel - liquid crystal phase transition temperature ~270 k of popc and popg lipid bilayers [ 78 , 79 ] . long - range electrostatic interaction is calculated using the particle mesh ewald ( pme ) method with a real space cutoff of 1.2 nm , as recommended for membrane simulations , especially for those involving charged lipids . three independent 120 ns md runs were carried out for each system starting from the four initial states , using different initial velocity distributions . we analyze the md trajectories using our in - house - developed codes and the gromacs facilities . the z - position of each amino acid residue is described by the z - component of the main chain or side chain centroid with respect to the average z - position of the phosphorus atoms . a residue is considered to be the one closest to the bilayer surface if the z - position of its centroid is the smallest among all the residues . the number of hydrogen bonds ( h - bonds ) is calculated using gromacs tool g_hbond . a h - bond is considered to be formed if the distance between n ( h ) and o is less than 0.35 ( 0.25 ) nm and the angle of n h o is greater than 150. this geometrical criterion for hydrogen - bond formation is widely used in many previous studies [ 55 , 63 , 8287 ] . the interaction energy u between peptide and lipid is computed using the gromacs tools g_ener and mdrun - rerun ( using the formula uinter = u(peptide + lipid ) u(peptide ) u(lipid ) ) . to examine the effect of hiapp on the ordering of bilayer surface , we calculate the thickness of lipid bilayer and the order parameter scd of the lipid acyl chain ( sn-1 ) . the thickness of lipid bilayer is estimated by the average of the phosphorus - to - phosphorus distance . trajectory data of hiapp monomer at popg bilayer membrane are obtained from our previous study . the initial relative orientations of the peptide with respect to the membrane surface are the same for popc and popg lipid bilayers . we have calculated the z - position of the centroid of each residue with respect to the popc bilayer surface and present the closest residue index as a function of time in figure 2 . we observe that in 11 out of 12 md runs , the c - terminal residues are observed to adsorb to the popc bilayer surface prior to the n - terminal residues , namely , the adsorption of hiapp monomer is initiated from the c - terminal residues . this membrane adsorption behavior of hiapp may be attributed to the dipole - dipole interaction between the polar residues ( such as ser34 , asn35 , and thr36 ) and the zwitterionic popc lipids . in our previous study , we observed that the positively charged residues k1 and r11 in the n - terminal region have a preference to adsorb to the anionic popg lipid bilayer . previous experimental studies reported that the n - terminal residues are involved in the membrane entry of hiapp peptide , and result in membrane damage at high peptide concentration [ 19 , 89 ] . our results show that the adsorption process of hiapp monomer to popc and popg bilayer membranes is distinct , which may lead to different binding behaviors and may influence the aggregation of membrane - bound hiapps . to give the detailed adsorption process , we show in figure 3 the snapshots at different time points and the time evolution of the contact number and hydrogen bond number between residue 1~19/20~37 and popc headgroups in a representative md run started from the initial state s(0 ) . it can be seen from figure 3 that in the initial state , the hiapp monomer is placed in water parallel to the popc bilayer with the side chain of residue k1 pointing toward membrane surface . the contact numbers between the c - terminal residues 20~37 and popc lipids increase with simulation time . at t = 12 ns , the c - terminal residues 20~37 adsorb to the membrane surface prior to the n - terminal residues . then , it takes tens of nanoseconds for residues 20~37 to adjust their side chains . at t = 50 ns , hiapp monomer is mostly adsorbed to membrane surface and stays on the bilayer surface in the remaining 70 ns of md simulation . the larger contact number of c - terminal residues 20~37 with popc lipids with respect to the n - terminal residues 1~19 indicates that the c - terminal residues 20~37 interact with the membrane more strongly than the n - terminal residues 1~19 . as seen from figure 3(b ) , the adsorption process is accompanied by the formation of h - bonds between hiapp and the headgroups of popc lipids . figure 3(c ) gives the time evolution of the z - position of the positively charged residues ( k1 and r11 ) and their interaction energy with popc bilayer within the first 50 ns of md simulation . it is observed that k1 and r11 approach to the membrane surface at ~50 ns ( solid line in figure 3(c ) ) , while the c - terminal residues reach to the bilayer surface within 15 ns ( see figure 2 ) . in addition , the interaction energy between the popc bilayer and residue k1/r11 is positive during the first 40/30 ns of simulation , reflecting the existence of repulsive interaction between the positively charged residues and the popc lipids in the beginning of the simulations . it is known that a popc lipid molecule is composed of a positively charged choline , a negatively charged phosphate group and hydrophobic fatty acids . although the popc lipid has no net charge , the positively charged choline is located closer to the membrane - water interface than the negatively charged phosphate group ( see section 3.3 for more details about the location of choline and phosphate groups ) , which leads to net repulsive interactions during the adsorption process . this net repulsive interaction disfavors the n - terminal residues to adsorb first to the membrane surface , which explains the observed c - terminal - initiated adsorption behavior ( see figure 2 ) . interestingly , both insertion and some helical folding were observed in our recent remd study on hiapp(119 ) peptide . based on the results of our remd study , we deduce that the next step of hiapp - bilayer interaction might proceeds through insertion of partially ordered structures followed by helical folding within the interface [ 90 , 91 ] . however , the exact mechanism remains clearly to be determined . to investigate the peptide orientation at the membrane - water interface , we plot in figure 4 the z - positions of c-atom and side chain centroid of each residue . as experimental results have shown that the membrane - bound hiapp monomer adopts an -helix spanning residues 8~19 [ 2325 ] , we classify the membrane binding orientation of hiapp into four different of orientations ( labeled as fa , fb , fc , and fd ) according to z - positions of the residues in the core helix region ( residues 8~19 ) . our recent md study showed that the side chains of residues r11 , f15 , and s19 insert more deeply into the anionic popg bilayer than their neighboring residues . this binding resembles the binding orientation fd of hiapp at the zwitterionic popc bilayer ( figure 4 ) . however , four different membrane binding orientations are observed for hiapp at zwitterionic popc bilayers , with almost equal probability . we give in table 1 the initial states and the final hiapp orientations in each md run . as seen from table 1 , hiapp with the same initial orientation can lead to different final binding orientations , and those with different initial orientations can lead to the same final binding orientation . these results suggest that hiapp monomer adopts multiple binding orientations at popc membrane surface independent of its initial orientation . to identify the important interactions that stabilize each binding orientation of hiapp at popc bilayer , we plot in figure 5 the interaction energy between a peptide and a lipid bilayer ( per lipid ) . the interaction energy is decomposed into electrostatic and van der waals ( vdw ) components . as seen from figure 5 that hiapps with four binding orientations have nearly the same vdw interaction energy with lipids , and the electrostatic interaction energy is also similar . however , the electrostatic interaction is much stronger than vdw interaction , indicating that the former plays a dominant role in stabilizing the binding of hiapp monomer to the popc lipid bilayer although the net charge of a popc lipid is zero . different from our result , a recent md study by zhao et al . reported that the electrostatic and vdw interaction energy between hiapp ion - channel and a dopc bilayer are quite similar . it is of particular interest to note that for the four different binding orientations , the peptide - lipid electrostatic interaction energy overlaps with each other ( see the error bar of the average value ) . the small differences in total peptide - lipid interaction energy ( the same vdw + similar electrostatic interaction energy ) allow multiple binding orientations of hiapp at the popc lipid bilayer ; that is to say , hiapp has no preferred binding orientation to the membrane surface . in order to compare the binding orientations of hiapp at popc bilayer with those at popg bilayers , we plot in figure 6 the z - position of each amino acid residue of hiapp relative to the average z - position ( z = 0 ) of phosphorus atoms , averaged over the last 20 ns of twelve independent 120 ns md runs . it is seen that , at the popc membrane surface , the z - position of each residues is very close to the position of lipid phosphorus atoms ( green dotted line ) . the similar z - position of all residues reflects the uncertainty of peptide orientations . at the popg bilayer surface , the positively charged residues k1 and r11 anchor to the bilayer surface by electrostatic interactions , and the z - position gradually increases from the n - terminal to c - terminal residues . the z - position of residues in figure 6 shows that the -helix region ( residues 5~19 ) of hiapp is parallel to the popg bilayer surface , with residues r11 , f15 , and s19 pointing to the lipid bilayer while the hydrophobic residues l12 , a13 , l16 , and v17 exposed to the water , revealing a preferred binding orientation at anionic popg bilayers . previous experimental studies reported that the n - terminal residues of hiapp are mainly responsible for membrane insertion and the c - terminal residues for fibrillar aggregation [ 26 , 32 ] . figure 6 shows that the whole hiapp peptide binds tightly to the lipid headgroups of popc , with the c - terminal residues buried below the phosphorus atoms , thus restraining the flexibility of the c - terminal residues . when hiapp monomer binds to the popg membrane , the c - terminal residues 20~37 are immersed in the water solution . this allows the amyloidogenic 20~29 region to have more freedom than the residues inside lipid bilayer , which facilitate peptide - peptide interaction . on the other hand , the helix - helix association of hiapp is believed to proceed before the -sheet formation of the disordered c - terminal region [ 29 , 92 ] . our md simulations show that hiapp monomer exposes the hydrophobic face of the amphipathic helical region to the solvent when binding to popg lipid bilayer surface . the preferential binding orientation of hiapp may be attributed to the strong electrostatic interaction between the n - terminal positively charged residues k1 and r11 and the negatively charged popg lipid headgroups . when hiapp monomers interact with popc lipid bilayers , their multiple binding orientations would reduce the solvent exposure probability of hydrophobic residues , thus disfavoring the peptide - peptide association . these results are consistent with experimental reports that negatively charged membranes can promote hiapp aggregation [ 29 , 43 ] . through detailed structural analysis , we find that although the chemical components of popc and popg lipids are similar , the locations of these components in the membrane are different . we plot in figure 7 the electron density of popc and popg along the membrane normal ( i.e. , z - axis ) , as done in a previous study of lipid bilayers . it shows that the popc ester , phosphate , and choline groups are located in turn from the membrane center ( z = 0 ) to the water solution , while the popg phosphate and glycerol groups are nearly at the same depth in the membrane with ester groups buried deeper . in addition , the peak value of each popc headgroup component is smaller than that of popg , and the average area per lipid of popc membrane is higher than that of popg ( 61.6 0.7 versus 54.6 0.6 ) , consistent with previous computational and experimental studies [ 82 , 93 , 94 ] . overall , the headgroup region of the popc bilayer is less compact than that of the popg bilayer , which is helpful for the insertion of hiapp monomer into popc membrane . the symmetric distributions of popc / popg lipid atoms in the upper and lower leaflets of the bilayer reveal that hiapp monomer does not cause membrane disruption , in agreement with experimental observations [ 29 , 95 ] . we also calculate the number of hydrogen bonds formed between hiapp monomer and different groups of the popc and popg membrane . figure 7(c ) shows that hiapp monomer forms hydrogen bonds most with the phosphate groups and less with the ester groups and least with the glycerol groups . the formation of hydrogen bonds between the popc ester groups and residues n31 , s34 , n35 , and y37 of hiapp ( see figure 7(c ) ) allow hiapp to interact with the hydrophobic lipid tails , which may result in the c - terminal residues insertion deep into popc bilayer . it is noted that the popc choline groups can not form hydrogen bonds with hiapp monomer , while the popg glycerol groups can . the formation of h - bonds between hiapp and the glycerol groups , together with the formation of h - bonds between hiapp and the phosphate groups , would stabilize the specific binding orientation of the peptide at popg bilayer . on the other hand , the formation of these h - bonds would constrain hiapp monomer at the popg membrane surface and thus hinders the peptide inserting into the bilayer , which might be helpful for the peptide - peptide association through the water - exposed hydrophobic residues . this result is resembling the results reported for an antimicrobial peptide msi-78 by nmr and fluorescence experiments where the peptide insertion was measured with the variation of the pc : pg ratio , showing that the peptide inserts more deeply in zwitterionic lipid bilayers than that in anionic lipid bilayers . the toxicity of hiapp and membrane disruption are suggested to be associated with hiapp - membrane interactions [ 29 , 95 ] . to examine the effect of membrane - bound hiapp monomer on the popc membrane , we calculate the lipid tail order parameter scd of acyl chain 1 ( sn-1 ) and the local membrane thickness ( see figure 8) . the scd value is calculated by the formula scd = 0.53cos 1 , where represents the angle of the c h bond vector ( in the simulation ) or the c d bond vector ( in the experiment ) with the bilayer normal . as seen from figure 8 , the averaged scd value is within the error bar of the neat popc lipid bilayer , implying that lipid interaction of hiapp monomer does not disturb the membrane integrity . the calculated local thickness of lipid bilayers in figure 8(b ) using different cutoff demonstrates that hiapp - lipid interaction influences the local thickness of popc bilayer . the influence can be neglected when the cutoff is larger than 3 nm . these results suggest that the binding of hiapp monomer at the popc membrane surface has negligible disturbance on the integrity of the popc bilayer , which provides atomic - level evidence that membrane - bound hiapp monomer does not cause membrane disruption [ 29 , 95 ] . however , it is expected that when the concentration of membrane - bound peptide reaches a critical value , the hiapp - lipid interaction may cause membrane disruption . in this study , we have investigated the binding orientation and lipid interaction of hiapp monomer at the zwitterionic popc bilayer by carrying out multiple md simulations . we have also examined the influence of lipid composition on lipid binding by comparing results of hiapp at anionic popg bilayer . we have found that hiapp monomer adopts multiple orientations at popc bilayers while it has a preferential orientation at popg bilayers . the specific binding orientation of hiapp at popg bilayer allows the hydrophobic residues exposed to water , thus facilitating peptide - peptide association by hydrophobic interactions . our results also show that the hiapp monomer forms more hydrogen bonds with the headgroups of popg lipids , which constrains hiapp monomer to the popg bilayer surfaces , while the formation of less h - bonds allows hiapp ( especially the amyloidogenic c - terminal residues ) inserts deep into popc bilayer , thus reducing the probability of peptide - peptide interaction via solvent - exposed hydrophobic residues . our studies provide atomic - level information of the binding behavior of hiapp and the effect of lipid composition on hiapp - membrane interactions , which may improve our understanding of membrane - mediated hiapp aggregation .

increasing evidence suggests that the interaction of human islet amyloid polypeptide ( hiapp ) with lipids may facilitate hiapp aggregation and cause the death of pancreatic islet -cells . however , the detailed hiapp - membrane interactions and the influences of lipid compositions are unclear . in this study , as a first step to understand the mechanism of membrane - mediated hiapp aggregation , we investigate the binding behaviors of hiapp monomer at zwitterionic palmitoyloleoyl - phosphatidylcholine ( popc ) bilayer by performing atomistic molecular dynamics simulations . the results are compared with those of hiapp at anionic palmitoyloleoyl - phosphatidylglycerol ( popg ) bilayers . we find that the adsorption of hiapp to popc bilayer is mainly initiated from the c - terminal region and the peptide adopts a helical structure with multiple binding orientations , while the adsorption to popg bilayer is mostly initiated from the n - terminal region and hiapp displays one preferential binding orientation , with its hydrophobic residues exposed to water . hiapp monomer inserts into popc lipid bilayers more readily than into popg bilayers . peptide - lipid interaction analyses show that the different binding features of hiapp at popc and popg bilayers are attributed to different magnitudes of electrostatic and hydrogen - bonding interactions with lipids . this study provides mechanistic insights into the different interaction behaviors of hiapp with zwitterionic and anionic lipid bilayers .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

PMC3259319

an increasing number of patients have breast implants for cosmetic augmentation of the breast , reconstruction after mastectomy or correction of congenital malformations . implant rupture can have various causes , but most ruptures have no obvious traumatic origin and sometimes occur in asymptomatic patients . the incidence of rupture increases with implant age ; the average incidence is approximately 2 implant ruptures per 100 implant - years , with an estimated probability of being intact after 5 and 10 years of implantation of 98% and 8385% , respectively [ 36 ] . clinical diagnosis is difficult , being based solely on nonspecific findings such as palpable nodules , asymmetry or tenderness . free silicone from ruptured implants has in rare cases spread to distant body regions , giving rise to symptoms . if implant rupture is accompanied by loss of the shape of the breast , the diagnosis of breast implant rupture at physical examination is feasible . however , clinical evaluation may fail to detect breast implant rupture that occurs over time without loss of breast volume and misshapenness . breast pain on the clinical examination of implants is a strong predictor of rupture , but the absence of pain does not exclude rupture . according to tark et al . , the most common symptom in breast implant rupture is contour deformity ( 44% ) , followed by displacements ( 20% ) , mass formations ( 17% ) , pain ( 13% ) and inflammation ( 3% ) . however , physical examination fails to diagnose implant rupture in more than 50% of cases . magnetic resonance imaging ( mri ) , mammography , ultrasonography ( us ) and exceptionally computed tomography ( ct ) have all been used to diagnose silicone breast implant rupture . each technique has specific strengths and weaknesses that may make a particular technique the study of choice for an individual patient . familiarity with both the typical and atypical findings for implants is essential to enable abnormalities to be detected . many factors can influence which imaging technique should be used to evaluate the integrity of silicone breast implants in a particular patient . these factors include the cost of the examination , the availability of an imaging technique , the expertise of the radiologist performing and interpreting the study , and potential contraindications or limitations of a patient that would prevent the use of a specific imaging technique . furthermore , knowing which implant the patient has can help determine the type of imaging findings to expect in case of rupture . each type of silicone gel - filled implant has slightly different imaging findings for implant failure related to the manufacturing process and viscosity of the silicone gel . breast reconstruction may involve the insertion of various types of implant or the modelling of autologous myocutaneous flaps . according to the literature , breast implants can be categorised into five implant generations reflecting product development over time . the recent generations of silicone gel implants have a cohesive viscous silicone gel . as a result of this feature , these implants will rarely have a totally collapsed implant shell , differing from the older generations . the third and fourth implant generations offered models of breast implants with textured or uniformly smooth surfaces ( fig . 1 ) , and it seems that capsular contracture rarely occurs in women with recent generations of breast implants . all implants in this article are silicone breast implants including single - lumen implants ( the majority ) and less commonly double - lumen implants ( fig . 5 ) . single - lumen implants have a single lumen of silicone gel delimited by a multilayer shell , while double - lumen implants have a fixed amount of saline and silicone within them , or a fixed outer lumen usually filled with silicone and an inner lumen that can be expanded , as necessary , with saline ( inverse double - lumen ) .fig . ( d ) different implant types . a and b breast tissue expander with metallic component visualised by computed tomography scan . silicone gel implants with textured surface ( c ) and with smooth surface ( d ) finally , another surgical breast reconstruction technique uses autologous myocutaneous flaps . flaps are most commonly either transverse rectus abdominis musculocutaneous ( tram ) flaps or latissimus dorsi flaps , and are used to reform the breast either alone or in conjunction with an implant . another alternative more rarely encountered in clinical practice is direct silicone gel injection in the breast . breast implants may be placed in a subglandular ( anterior to the pectoralis major muscle ) or subpectoral ( posterior to the pectoralis major muscle ) location ( fig . ( left ) submammary implant located in front of the pectoralis major muscle and ( right ) submuscular implant located behind the pectoralis major muscle , visualised by mammography localisation of breast implants . ( left ) submammary implant located in front of the pectoralis major muscle and ( right ) submuscular implant located behind the pectoralis major muscle , visualised by mammography before implant insertion , especially in oncoplastic breast reconstruction , a tissue expander ( fig . 1 ) is usually placed in the mastectomy site to stretch the remaining skin in preparation for the placement of a permanent implant later . the expander is like an inflatable breast implant that is inserted into a pocket under the skin and muscle of the chest . the expander is usually placed in its collapsed form at the time of mastectomy and then , after surgery , fluid is introduced into the tissue expander to slowly inflate it . it is important to realise that , as their function is to expand , they might not be fully inflated and thus may appear to have multiple folds or wrinkles on the surface . depending on the type of expander , the fluid is either introduced directly into the expander ( magnetic marker ) or is injected into a distant port . this process continues for several weeks until the tissue expander is filled to an optimal volume , when a permanent breast implant can be inserted . notably , some kind of breast tissue expanders should be considered a contraindication to mri because of the magnetic marker of the filling valve : expander manufacturers list possible consequences such as overheating , possible expander displacement , and possible reduction of magnetisation of the marker . after placement , a thin fibrous capsule ( scar tissue ) normally forms around the prosthesis . this occurs around all silicone implants to some degree ; however , pronounced fibrous capsule formation causes discomfort and alters the shape of the breast . changes owing to marked fibrous capsule contraction often can not be appreciated on imaging , and clinical examination is the best way to diagnose this condition . magnetic resonance imaging ( mri ) its sensitivity for rupture is between 80% and 90% , and its specificity is between 90% and 97% [ 3 , 516 ] ( fig . right implant with extracapsular rupture exhibiting the typical linguine sign at the posterior margin of the implant . intact left silicone implant magnetic resonance imaging scan of a woman with bilateral breast silicone implants . right implant with extracapsular rupture exhibiting the typical linguine sign at the posterior margin of the implant . intact left silicone implant mri may be used to exclude a ruptured prosthesis , and it may aid explantation surgery as it documents the presence and extent of silicone leakage better than other imaging techniques . despite this , conventional breast imaging techniques are the methods of choice when breast implant failure is suspected because well - defined and discernible sonographic features have been established for ruptured implants , as we shall show throughout this article . moreover , mri is more expensive , and many women have contraindications ( cardiac pacemakers , aneurysm clips , metallic foreign bodies and claustrophobia ) to mri . in this article , we describe the preeminent role of mri in detecting implant failures , illustrating the spectrum of appearances of normal silicone gel implants and of implant ruptures . furthermore , we summarise the advantages and limitations of other imaging techniques and describe the key findings in detection of silicone implant failure . magnetic resonance imaging of silicone breast implants , with its high sensitivity and specificity for detection of rupture , is an excellent technique to assess implant integrity . mri has proven accurate in locating free silicone and evaluating implant rupture [ 3 , 516 ] . a dedicated mri breast coil should be used to obtain high - resolution images , allowing detection of subtle signs of implant leakage or rupture . mri s usefulness derives from its ability to suppress or emphasise the signal from water , fat , or especially silicone . its high spatial and soft - tissue resolutions make it ideal for the characterisation of breast implants . figure 4 shows the mri sequences used at our institution for breast implant study . the use of contrast agents in mri studies for assessment of breast implant integrity is not recommended . however , when the priority is the detection of recurrence or residual tumors , contrast - enhanced mri of the breast is useful for characterising parenchymal lesions [ 11 , 13 ] . high - field - strength magnets of at least 1.5 t are preferred when imaging patients with silicone breast implants because of the ability to use magnetic resonance sequences that can more readily suppress or emphasise the signal from water , fat or silicone . turbo - spin - echo t2-weighted images , short - time inversion recovery silicone excited ( silicone hyperintense , water suppressed ) , and silicone - saturated ( water hyperintense , silicone suppressed ) are the most common and most important sequences in silicone breast implant assessment . a single - lumen silicone implant has an outer shell containing homogeneous high - signal - intensity viscous silicone on t2-weighted images . a double - lumen silicone implant typically has an inner lumen of high - signal - intensity silicone surrounded by a smaller outer lumen that contains saline and has different signal intensities , depending on the pulse sequence ( fig . 4our mri examination protocol includes a 1.5-t superconducting mr system ( philips mr systems gyroscan nt ) with a sense - body coil , with the following sequences . 5mri of a 61-year - old woman with bilateral implants : a single - lumen implant ( right breast ) and a double - lumen implant ( left breast ) . ( a ) axial silicone - suppression and ( b ) axial t2-weighted turbo spin echo sequences . the right implant has homogeneous signal intensity , representing a single lumen with silicone gel ( * ) . the left implant has an inner lumen ( open arrow ) of low - signal - intensity or high - signal - intensity silicone surrounded by a smaller outer lumen ( solid arrow ) that contains saline our mri examination protocol includes a 1.5-t superconducting mr system ( philips mr systems gyroscan nt ) with a sense - body coil , with the following sequences . we always include a post - contrast study to detect possible malignant lesions mri of a 61-year - old woman with bilateral implants : a single - lumen implant ( right breast ) and a double - lumen implant ( left breast ) . ( a ) axial silicone - suppression and ( b ) axial t2-weighted turbo spin echo sequences . the right implant has homogeneous signal intensity , representing a single lumen with silicone gel ( * ) . the left implant has an inner lumen ( open arrow ) of low - signal - intensity or high - signal - intensity silicone surrounded by a smaller outer lumen ( solid arrow ) that contains saline breast implant ruptures can be divided into two major categories : intracapsular implant rupture and extracapsular implant rupture , which is less common [ 1 , 310 , 12 , 1518 ] . up to half of all ruptures in women with augmented breasts occur within 12 years . intracapsular implant rupture is defined as rupture of the implant shell with silicone leakage that does not macroscopically extend beyond the fibrous capsule . the most reliable mri criterion for intracapsular rupture is the presence of multiple curvilinear low - signal - intensity lines within the high - signal - intensity silicone gel , the so - called linguine sign . these curvilinear lines represent the collapsed implant shell floating within the silicone gel [ 5 , 7 , 9 , 10 , 12 , 13 , 16 ] ( fig . 6 ) . the linguine sign will not be present in an uncollapsed rupture . in an uncollapsed rupture , mri shows free silicone outside the implant shell but still contained by the fibrous capsule . focal silicone invagination between the inner shell and fibrous capsule are common , resulting in the teardrop sign and the key - hole sign [ 5 , 7 , 9 ] . an uncollapsed silicone implant rupture is defined as a tear of the silicone implant shell and is considered an intracapsular rupture ( fig . 7 ) . only these more subtle findings will be present in up to 52% of ruptured implants .fig . ( a ) axial t2-weighted turbo spin - echo and ( b ) axial silicone - excited sequence . the study shows a hypointense subcapsular line at the anterior margin of the implant ( solid arrow ) ; the teardrop sign and key - hole sign are also present ( open arrows ) . focal change in signal at the anterior margin of the implant ( white open arrow ) can also be observed magnetic resonance imaging scan of a woman with bilateral ruptured implants . typical linguine sign within implants representing collapsed implant shell mri of a woman with intracapsular rupture of a single - lumen silicone implant . ( a ) axial t2-weighted turbo spin - echo and ( b ) axial silicone - excited sequence . the study shows a hypointense subcapsular line at the anterior margin of the implant ( solid arrow ) ; the teardrop sign and key - hole sign are also present ( open arrows ) . focal change in signal at the anterior margin of the implant ( white open arrow ) can also be observed extracapsular silicone implant rupture is defined as rupture of both the implant shell and the fibrous capsule , with macroscopic silicone leakage that extends beyond the fibrous capsule into surrounding tissues . focal areas of high signal intensity in sequences that enhance only the silicone represent free silicone [ 5 , 9 , 12 , 16 ] ( fig . 8mri of a woman with an extracapsular rupture of a single - lumen silicone implant . a and b sagittal silicone - excited sequences demonstrate the presence of free silicone gel around the implant ( white arrows ) . ( c ) axial silicone - excited sequence shows free silicone gel located in the internal mammary chain ( black arrow ) mri of a woman with an extracapsular rupture of a single - lumen silicone implant . a and b sagittal silicone - excited sequences demonstrate the presence of free silicone gel around the implant ( white arrows ) . ( c ) axial silicone - excited sequence shows free silicone gel located in the internal mammary chain ( black arrow ) unlike rupture , gel bleed is microscopic silicone leakage through an intact implant shell . this phenomenon is closely related to the chemical affinity between the outer shell of the silicone elastomer and the gel contained therein . indeed , the gel , if in contact with the outer shell , can break the noncovalent molecular bonds between the polymer chains , causing swelling and weakening of the shell itself . once it separates from its shell , silicone can migrate , even reaching the upper limbs , the liver , the inguinal lymph nodes , the synovium , the skin , and the pleura fluid . for this reason , the presence of silicone gel in regional lymph nodes can be due to gel bleed and not be always indicative of a present implant rupture . with the introduction of new cohesive gel implants , most normal transudation of microscopic amounts of silicone gel can not be detected by mri . only when a gel bleed is extensive can silicone gel be detected outside the implant shell and form the inverted teardrop sign . an uncollapsed implant rupture can have a similar or identical magnetic resonance appearance to a large gel bleed . mri often shows radial folds or normal infoldings of the shell extending to the periphery of the implant . these folds may appear prominent and complex , but should not be confused with implant rupture or leak . this is one of the major pitfalls and causes of false positives on mri , mimicking the total collapse of the implant shell [ 9 , 10 , 13 , 16 ] . radial folds and periprosthetic fluid are considered normal variants of breast implants ( fig . this reactive fluid probably results from an inflammatory response and is not indicative of implant rupture.fig . 9axial t2-weighted turbo spin - echo image ( a ) and axial ct scan ( c ) of single - lumen implants show small amount of reactive fluid ( arrows ) . ( b ) axial t1-weighted turbo spin - echo image demonstrates normal radial folds of the membrane ( arrowhead ) . simple or complex folds are not in themselves indicative of rupture axial t2-weighted turbo spin - echo image ( a ) and axial ct scan ( c ) of single - lumen implants show small amount of reactive fluid ( arrows ) . ( b ) axial t1-weighted turbo spin - echo image demonstrates normal radial folds of the membrane ( arrowhead ) . simple or complex folds are not in themselves indicative of rupture radial folds are frequently observed in patients with capsular contracture . it is caused by excessive scar tissue around the implant that tightens and squeezes the implant . occasionally , the capsule tears , allowing part of the implant to herniate into adjacent parenchyma . the capsule sometimes calcifies , and rigid calcium deposits may be palpated immediately adjacent to the implant ( fig . 10chest plain film of a woman with capsule calcification ( arrowheads ) adjacent to the implant . many augmented patients develop capsular contracture chest plain film of a woman with capsule calcification ( arrowheads ) adjacent to the implant . many augmented patients develop capsular contracture rarely , intracapsular rupture will show multiple hyperintense foci on t2-weighted images or multiple hypointense foci on water - suppression images within the implant lumen ; these findings are referred to as the salad oil sign or droplet sign . without other mri evidence of implant rupture , like the identification of the collapsed implant shell or free silicone within the breast parenchyma ( with no previous history of implant rupture ) , water droplets or small amounts of air within a silicone implant are not reliable signs of implant rupture [ 9 , 10 , 16 ] . however , this sign should prompt the search for subtle signs of intracapsular rupture ( figs . the entire spectrum of mri findings for silicone breast implants is illustrated in figs . 11a sagittal silicone - excited mri sequence and ( b ) axial t2-weighted turbo spin - echo image of a 64-year - old woman with changes in the signal intensity of the silicone gel ( black arrows ) . the margins of the implant are slightly irregular and a small amount of fluid surrounds the prosthesis ( white arrow ) . 12mri of a 54-year - old woman with a ruptured breast implant confirmed at surgery . a axial silicone suppression . a moderate amount of water and probably serum is mixed in the silicone gel around the implant ( black asterisks ) . note also the punctuate changes in signal intensity droplets within the implant ( arrows ) and punctuate and hyperintense images due to calcifications in the implant periphery ( arrowheads)fig . a intact implant has an uninterrupted shell and fibrous capsule adjacent to the breast parenchyma . b periprosthetic fluid . presence of a small - to - moderate amount of reactive fluid surrounding the implant . lines extending from the surface of the implant and inwards in a rather perpendicular manner . the border of the implant is bulging more than usual ( called the rat - tail sign when very pronounced ) . the last two images represent silicone gel leakage through a small focal implant shell tearfig . the beginning and the end of the line can be followed to the surface of the implant . disruption of the shell and fibrous capsule will allow silicone to extravasate into surrounding breast tissue . c linguine sign . folded wavy multidirectional lines within the silicone gel , representing the collapsed implant shell . two parallel lines in close proximity forming a double - contoured subcapsular line within the silicone gel a sagittal silicone - excited mri sequence and ( b ) axial t2-weighted turbo spin - echo image of a 64-year - old woman with changes in the signal intensity of the silicone gel ( black arrows ) . the margins of the implant are slightly irregular and a small amount of fluid surrounds the prosthesis ( white arrow ) . mri of a 54-year - old woman with a ruptured breast implant confirmed at surgery . a axial silicone suppression . a moderate amount of water and probably serum is mixed in the silicone gel around the implant ( black asterisks ) . note also the punctuate changes in signal intensity droplets within the implant ( arrows ) and punctuate and hyperintense images due to calcifications in the implant periphery ( arrowheads ) variants of normal breast implants . a intact implant has an uninterrupted shell and fibrous capsule adjacent to the breast parenchyma . b periprosthetic fluid . presence of a small - to - moderate amount of reactive fluid surrounding the implant . c simple or complex radial folds . lines extending from the surface of the implant and inwards in a rather perpendicular manner . d calcification and thickening of the fibrous capsule findings of possible breast implant rupture . a deformity in contour . the border of the implant is bulging more than usual ( called the rat - tail sign when very pronounced ) . the last two images represent silicone gel leakage through a small focal implant shell tear definitive findings of breast implant rupture . a subcapsular lines . the beginning and the end of the line can be followed to the surface of the implant . disruption of the shell and fibrous capsule will allow silicone to extravasate into surrounding breast tissue . c linguine sign . folded wavy multidirectional lines within the silicone gel , representing the collapsed implant shell . two parallel lines in close proximity forming a double - contoured subcapsular line within the silicone gel although mri is the most accurate technique for evaluating implant integrity , it is important to know the mammographic , us and ct findings for implant rupture . mammography is of little value in the assessment of implant integrity , although it may be useful for the assessment of the surrounding breast tissue . silicone implants should be evaluated on a yearly basis to detect any change in configuration or identify any new free silicone within the breast parenchyma . mammographic features that are not specific to implant rupture include a measurable periprosthetic dense band , periprosthetic calcification , asymmetry of implant size or shape , and focal herniation of the implant . 16 ) is significantly associated with extracapsular rupture ( p 0.05 ) and inhomogeneous , reduced radiopacity seems to indicate rupture , without discriminating the type of rupture ( p 0.05 ) . specific mammographic evidence of implant rupture is extravasation of silicone outside the implant shell .fig . diffuse areas of increased density are visualised adjacent to the implant ( arrows ) oblique mammograms in a 29-year - old transsexual with subglandular implants and silicone injections . diffuse areas of increased density are visualised adjacent to the implant ( arrows ) because mammography can easily detect free silicone within the breast parenchyma , extracapsular silicone implant ruptures can be identified . unfortunately , silicone is dense and is not easily penetrated by the x - ray energies used for mammography ; therefore , intracapsular rupture can not be identified by mammography . the sensitivity of mammography for the rupture of silicone implants is variable but always low , ranging between 2530% [ 10 , 12 ] and 68% . the best mammograms are obtained when the breast is maximally compressed so the x - ray beam penetrates the thinnest possible layer of tissue . in 1988 , eklund et al . [ 13 , 15 ] introduced the displacement technique to facilitate mammography in augmented women ( fig . a thorough clinical history should be obtained before mammography to ensure that the patient has not had a previous implant rupture or has not had direct silicone injections.fig . patients with submammary implants have fewer visualised area compared to patients with submuscular implants . the displacement technique introduced by eklund is visualised with displacement ( below ) than with standard compression mammography ( above ) implant position is an important factor when studying the breasts . the displacement technique introduced by eklund facilitates mammography in women with implants . slightly more tissue assessments of the usefulness of ultrasonography ( us ) for detecting implant ruptures vary widely . typically , us breast implant examination involves evaluation of morphology , contour and contents , periimplant tissues and axillae . thus , the transversal - to - longitudinal ratio of the implants is calculated , the regularity of implant margins ( radial folds ) and the homogeneity of the implant lumen are checked , and signs of free silicone or granulomas in the breast or in the axillary lymph nodes are sought ( fig . a completely negative us examination strongly supports implant integrity , limiting the application of mammography or mri to cases suspicious at us [ 1 , 10].fig . breast gland ( black asterisk ) , pectoralis major muscle ( black arrow ) , and implant shell ( white arrow ) visualised as a thin and continuous echogenic line at the parenchymal tissue - implant interface , and silicone implant ( white asterisk ) . ( c ) a small fluid collection around the implant ( arrowhead ) and ( d ) a simple infolding of the shell silicone implant ( arrowhead ) a and b ultrasonography of a woman with an intact implant . breast gland ( black asterisk ) , pectoralis major muscle ( black arrow ) , and implant shell ( white arrow ) visualised as a thin and continuous echogenic line at the parenchymal tissue - implant interface , and silicone implant ( white asterisk ) . ( c ) a small fluid collection around the implant ( arrowhead ) and ( d ) a simple infolding of the shell silicone implant ( arrowhead ) reverberation artefacts are commonly encountered in the anterior aspect of the implant and should not be confused with abnormalities . one limitation of us is that marked attenuation of the ultrasound beam by silicone hinders the evaluation of the back wall of an implant and the tissue posterior to it . similarly , residual silicone granulomas from extracapsular rupture or from previous direct silicone injections compromise the evaluation of a new implant ( figs . 19 and 20).fig . 19(above ) a coronal maximum intensity projection from a silicone - excited sequence in a transsexual ( fig . 16 ) demonstrating multiple nodules with high signal throughout both breasts representing free silicone ( arrows ) . ( below ) it is extremely difficult to evaluate the silicone implants by ultrasonography because of attenuation of the ultrasound beam by the free injected silicone and granuloma formation in the subcutaneous tissuefig . 20a coronal silicone - excited sequence and ( b ) coronal contrast - enhanced fat - suppressed t1-weighted image of the previous patient show multiple nodules of free silicone ( siliconomas ) in the gluteal muscles ( above ) a coronal maximum intensity projection from a silicone - excited sequence in a transsexual ( fig . 16 ) demonstrating multiple nodules with high signal throughout both breasts representing free silicone ( arrows ) . ( below ) it is extremely difficult to evaluate the silicone implants by ultrasonography because of attenuation of the ultrasound beam by the free injected silicone and granuloma formation in the subcutaneous tissue a coronal silicone - excited sequence and ( b ) coronal contrast - enhanced fat - suppressed t1-weighted image of the previous patient show multiple nodules of free silicone ( siliconomas ) in the gluteal muscles radial folds present as echogenic lines that extend from the periphery to the interior of the implant . small amounts of free silicone mixed within the surrounding breast tissues give rise to the characteristic echogenic snowstorm ( statistically significant for extracapsular rupture , p 0.05 and the most sensitive and specific sign at us ) . this sign is the most reliable sign of extracapsular rupture and consists of a group of focal nodules with a generalised increase in echogenicity of the breast tissue and loss of normal parenchymal interfaces resulting from dispersion of the ultrasound beam . the nodules represent silicone granulomas , composed of free silicone and surrounding fibrous tissue reaction , lying outside the confines of the fibrous capsule . 21extracapsular silicone implant rupture in a 52-year - old woman with a history of breast cancer who presented with a palpable lesion in the supraclavicular right region . mammogram shows an irregular lump from the implant ( arrowhead ) and ultrasonography demonstrates the presence of a nodular lesion with typical inhomogeneity ( the snowstorm sign ) at the posterior margin , suspicious for a lymph node containing siliconefig . 22cytology of the node shows multinucleated foreign - body giant cells ( arrowhead ) with abundant birefringent particles inside and outside the cytoplasm , compatible with gel silicone ( arrows ) . axial t2-weighted turbo spin - echo mri study corroborated an extracapsular rupture of the implant extracapsular silicone implant rupture in a 52-year - old woman with a history of breast cancer who presented with a palpable lesion in the supraclavicular right region . mammogram shows an irregular lump from the implant ( arrowhead ) and ultrasonography demonstrates the presence of a nodular lesion with typical inhomogeneity ( the snowstorm sign ) at the posterior margin , suspicious for a lymph node containing silicone cytology of the node shows multinucleated foreign - body giant cells ( arrowhead ) with abundant birefringent particles inside and outside the cytoplasm , compatible with gel silicone ( arrows ) . axial t2-weighted turbo spin - echo mri study corroborated an extracapsular rupture of the implant us can also detect intracapsular rupture by identifying a series of horizontal echogenic straight or curvilinear lines , somewhat parallel , traversing the interior of the implant , commonly known as the stepladder sign ( statistically significant for intracapsular rupture , p 0.05 ) . this sign is analogous to the linguine sign seen on mri , and it is the most reliable sign of intracapsular rupture . it is important not to confuse the stepladder sign with normal prominent radial folds . at ct , an intact silicone implant is characterised by an oval shape and homogeneous grey density within a surrounding high - density ring . the ct findings of intracapsular silicone implant rupture are similar to the mri findings , and the linguine sign can also be seen at ct . because silicone and soft tissues have similar radiodensities , extracapsular silicone can be difficult to identify on ct . however , in most cases of extracapsular silicone implant ruptures , ct can detect the collapsed implant shell , so the ruptured implant is not usually missed.fig . a axial ct scan shows a severe deformity of the right implant surface representing a collapsed ruptured prosthesis ( arrow ) . b sagittal multiplanar reconstruction and ( c ) axial ct scan of the left implant show high - density curvilinear lines within the implant ( linguine sign , arrows)fig . ( a ) axial ct scan shows small high - density lines within the silicone gel in the right implant , suggestive of collapsed rupture ( arrow ) . ( b ) axial silicone - excited mri sequence confirmed intracapsular rupture , showing hypointense wavy lines at the posterior margin of the right implant ( linguine sign ) and subcapsular line at the anterior margin ( arrows ) . normal infoldings in the left implant ( arrowhead ) bilateral ruptured implants in a woman with primary lung cancer ( asterisk ) . a axial ct scan shows a severe deformity of the right implant surface representing a collapsed ruptured prosthesis ( arrow ) . b sagittal multiplanar reconstruction and ( c ) axial ct scan of the left implant show high - density curvilinear lines within the implant ( linguine sign , arrows ) unilateral implant rupture . ( a ) axial ct scan shows small high - density lines within the silicone gel in the right implant , suggestive of collapsed rupture ( arrow ) . ( b ) axial silicone - excited mri sequence confirmed intracapsular rupture , showing hypointense wavy lines at the posterior margin of the right implant ( linguine sign ) and subcapsular line at the anterior margin ( arrows ) . normal infoldings in the left implant ( arrowhead ) nevertheless , ct uses ionising radiation ; for this reason and its low sensitivity and specificity , ct should not be the study of choice , especially in young women . most ct images of ruptured breast implants are incidental findings in asymptomatic patients or in already known failed implants . all breast diseases can also occur in women with implants ; among these , the most important for its prognosis and treatment is breast cancer . the risk of breast cancer is not increased in augmented women [ 11 , 16 ] , and there is no significant difference in breast cancer survival rates between women with and those without implants [ 11 , 12 ] . however , because augmented women will sometimes develop breast cancer , there should be persistent precautions concerning cancer detection . implants might interfere with the earliest possible detection of a cancer by altering physical examination of the breast or impairing various imaging techniques ( figs . the sensitivity of mammography and us for cancer detection may be reduced in augmented woman . however , the presence of the implant does not seem to decrease the sensitivity of breast mri [ 11 , 14 ] . when evaluating these patients , physical and mammographic findings should be correlated . 25breast ultrasonography of a 39-year - old ( a ) and a 30-year - old ( b ) augmented women . in both studies there c ultrasonography of a 46-year - old augmented woman with a history of breast cancer . us shows a lesion suspicious of recurrent tumor ( arrow ) that was confirmed by histology . 26an oblique mammogram of a 57-year - old woman with a submammary implant ( a ) . a cluster of suspicious microcalcifications can be identified in the breast gland ( arrows ) . a magnified mammogram ( b ) confirms the presence of malignant microcalcifications ( arrows ) breast ultrasonography of a 39-year - old ( a ) and a 30-year - old ( b ) augmented women . in both studies there c ultrasonography of a 46-year - old augmented woman with a history of breast cancer . us shows a lesion suspicious of recurrent tumor ( arrow ) that was confirmed by histology . breast implant ( asterisk ) an oblique mammogram of a 57-year - old woman with a submammary implant ( a ) . a cluster of suspicious microcalcifications can be identified in the breast gland ( arrows ) . a magnified mammogram ( b ) confirms the presence of malignant microcalcifications ( arrows ) mri is clearly recommended in augmented woman with suspicion of a tumor . all suspicious lesions should be analysed by fine - needle aspiration or core needle biopsy with maximum caution to avoid damaging the implant ( fig . ( right ) real - time ultrasound is used to guide the needle tip ( arrow ) ( left ) metallic clip placed in the microcalcifications site after biopsy . ( right ) real - time ultrasound is used to guide the needle tip ( arrow ) the risk of implant rupture increases with implant age . radiologists need to be able to recognise the normal appearance of commonly used implants on various imaging techniques . the role of mri in the evaluation of breast implants resides in implant integrity evaluation and breast cancer detection ( especially in women with oncoplastic reconstructive surgery ) . intracapsular rupture , which is frequent but often clinically silent , can not be easily detected by mammography because of the radiopacity of the implant . the sensitivity of ultrasonography is greater if discontinuous echogenic lines within silicone gel ( stepladder sign ) are detected . periprosthetic hyperdensity on mammography and the snowstorm sign on ultrasonography are significantly associated with extracapsular rupture . mri is also the most accurate technique for differentiating intracapsular from extracapsular rupture and for assessing the extent of silicone leakage into the breast parenchyma and granuloma formation . mri can reliably reveal collapsed intracapsular rupture by detecting shell fragments floating inside the silicone gel implant ( linguine sign ) . an uncollapsed silicone implant rupture is seen as a sometimes subtle silicone invagination between the inner shell and fibrous capsule , known as the teardrop sign or the key - hole sign . other imaging signs , such as the salad oil sign or droplet sign , are nonspecific and not reliable without other mri evidence of implant rupture . it is essential to be aware of the spectrum of normal appearances to avoid false - positive findings ( prominent radial folds ) . moreover , the information provided by plastic surgeons is critical and often essential in the evaluation of breast implant integrity . the surgical technique of implantation , intraoperative complications that may have occurred , and the clinical monitoring of patients are very useful tools that the radiologist need to know about . in general , in symptomatic patients , after conventional imaging , non - contrast mri is recommended to confirm or exclude rupture . mri is not recommended for screening for implant rupture in asymptomatic patients . in patients with oncoplastic surgery

the number of women with breast implants is increasing . radiologists must be familiar with the normal and abnormal findings of common implants . implant rupture is a well - known complication after surgery and is the main cause of implant removal . although mammography and ultrasonography are the standard first steps in the diagnostic workup , magnetic resonance imaging ( mri ) is the most useful imaging modality for the characterisation of breast implants because of its high spatial resolution and contrast between implants and soft tissues and absence of ionising radiation . mri has the highest sensitivity and specificity for implant rupture , thanks to its sequences that can suppress or emphasise the signal from silicone . regardless of the technique used , the overall aim of imaging breast implants is to provide essential information about tissue and prosthesis integrity , detect implant abnormalities and detect breast diseases unrelated to implants , such as breast cancer .

Introduction Magnetic resonance imaging Mammography Ultrasonography Computed tomography Additional lesions found in breast implant study Conclusion

PMC5228587

estrogen receptor beta ( er ) was first discovered in the rat prostate ( kuiper et al . , 1996 ) . since then , there has been considerable interest in understanding its role in both breast and prostate cancer . despite a large body of literature , the function of er in these two cancers remains unclear ( haldosen et al . most authors agree that er has a predominantly antiproliferative , pro - apoptotic and tumor - suppressive role ( attia and ederveen , 2012 , bottner et al . , 2014 , chang and prins , 1999 , ellem and risbridger , 2007 , horvath et al . , 2001 , , 2014 , ruddy et al . , 2014 , zhu et al . , this is particularly in the context of castrate resistant prostate cancer ( crpc ) where it has been proposed as a driver of androgen receptor ( ar)-dependent gene transcription ( yang et al . , 2012 , yang et al . , 2015 ) , along with a potential role in mediating the transition from hormone - sensitive to crpc ( zellweger et al . , 2013 ) . in breast cancer bi - faceted role and should not simply be considered a tumor - suppressor ( jonsson et al . , 2014 ) . er has been reported to cross - talk with androgen receptor - positive breast cancer ( rizza et al . , 2014 ) and may be an important factor in er-negative breast cancer ( gruvberger - saal et al . , 2007 , smart et al . , 2013 ) . inconsistencies in the reported expression of er in breast and prostate cancers as determined by immunohistochemistry ( ihc ) have contributed to this uncertainty . in prostate , most data support the conclusion that er is highly expressed in benign epithelial cells , with expression declining in cancer development and inversely correlating with increasing gleason grade ( asgari and morakabati , 2011 , attia and ederveen , 2012 , dey et al . , 2014 , horvath et al . , 2001 , however , it has also been reported that er expression is high in bone and lymph node metastases ( bouchal et al . , 2011 , zhu et al . , 2004 ) and that high er expression correlates with poor clinical prognosis ( horvath et al . , 2001 , zellweger et al . , high er expression has been described both as a poor ( guo et al . , 2014a , guo et al . , 2014b ) and favorable ( esslimani - sahla et al . , 2002 , roger et al . , 2001 ) prognostic marker , with others finding no association between clinico - pathological parameters and er expression ( umekita et al . , 2006 ) . it is recognized that there is wide variability in the sensitivity and specificity of er antibodies , which may contribute to the uncertainties surrounding its molecular action and tissue expression ( choi et al . , 2001 , hartman et al . previous er antibody validation studies have been published ( carder et al . , 2005 , choi et al . , 2001 , 2002 , weitsman et al . , 2006 , wu et al . , 2012 ) , however some of them are limited by reliance on two key assumptions . firstly , that when assessing an antibody by western blotting in a cell line model , the factor of interest is expressed and secondly , when assessing an antibody 's specificity by ihc in tissue , the tissue expression of the factor has been well characterized . in the case of er , these assumptions are problematic , as its expression in commonly used cell line models and in tissues is not universally accepted ( al - bader et al . , 2011 , asgari and morakabati , 2011 , attia and ederveen , 2012 , bouchal et al . , 2011 , dey et al . , 2014 , gruvberger - saal et al . , 2007 , guo et al . , 2014a , guo et al . , 2014b , hieken et al . , 2015 , , 2001 , leav et al . , 2001 , nakajima et al . , 2011 , , 2007 , shaaban et al . , 2003 , skliris et al . , 2002 , umekita et al . , 2006 , , 2012 , zhu et al . , 2004 ) . in light of this , we sought to test and validate six commonly used , commercially available er antibodies and two non - commercially available er antibodies ( choi et al . , 2001 , wu et al . , 2012 ) in a systematic manner that addresses these assumptions . to achieve this , we employed a number of assays for antibody validation , including a novel proteomic - based pull down method called rapid immunopreciptation mass spectrometry of endogenous protein ( rime ) ( mohammed et al . , 2013 ) . we then applied successfully validated antibodies to cell line models of breast and prostate cancer commonly used for studies of er to assess them for er expression . er expression in the cell lines was validated by a non - antibody dependent , targeted proteomics method known as parallel reaction monitoring ( prm ) ( gallien et al . , 2012 ) . finally , benign and malignant prostate and breast tissues were stained with the validated er antibody to assess tissue expression of er by ihc . the cancer cell line mda - mb-231 with doxycycline - inducible er expression ( mda - mb-231-er ) ( reese et al . , 2014 ) was cultured in dulbeccos modified eagle medium with f12 supplement ( dmem / f12 ) with 10% heat - inactivated tetracycline - free fetal bovine serum ( fbs ) ( fisher - scientific ) , 2 mm l - glutamine , 50 u / ml penicillin , 50 g / ml streptomycin , 5 g / ml blasticidin s ( invivogen ) to select for the tetracycline repressor and 500 g / ml zeocin ( invitrogen ) to select for the er expression vector . to induce er expression in mda - mb 231-er cells , 15 cm plates were seeded with 5 10 cells and doxycycline added at either 0.1 g / ml ( for western blot , real - time polymerase chain reaction ( qrt - pcr ) and prm ) or 0.5 g / ml ( for rime ) for 24 h. the mcf-7 breast cancer cell line was cultured in dulbecco 's modified eagle medium ( dmem ) with 10% heat - inactivated fbs ( fisher - scientific ) , 2 mm l - glutamine , 50 u / ml penicillin and 50 g / ml streptomycin . the lncap prostate cancer cell line was cultured in rpmi 1640 with 10% heat - inactivated fbs ( fisher - scientific ) , 2 mm l - glutamine , 50 u / ml penicillin and 50 g / ml streptomycin . all cells were incubated at 37 c with 5% co2 and cultured to 8090% confluence . lncap and mcf-7 cell lines were obtained from atcc ( middlesex , uk ) and validated by str genotyping . mda - mb-231-er+ , mda - mb-231-er , mcf-7 and lncap cells were harvested for collection of mrna using the rneasy mini kit ( qiagen , california usa ) . on - column dnase digestion samples containing 250 ng random primers , 1 g rna , 1 l 10 mm dntp mix and water to a total volume of 13 l were heated to 65 c for 5 min , followed by 1 min incubation on ice . to each sample 4 l 5x first - strand buffer , 1 l 0.1 m dtt , 1 l rnaseout and 1 l superscript iii reverse transcriptase ( rt ) ( thermofisher scientific , leicestershire , uk ) were added and incubated at 25 c for 5 min then 50 c for 60 min followed by heating at 70 c for 15 min qrt - pcr primers for wild type er ( table 1 ) were designed based on published sequence of esr2 ( available from uscs genome browser at http://genome.ucsc.edu/ ) using the primer3 software package ( koressaar and remm , 2007 , untergasser et al . , 2012 ) available at http://bioinfo.ut.ee / primer3 - 0.4.0/primer3/. ubc primers ( sy121212648 ) were obtained from sigma - aldrich ( dorset , uk ) . each qrt - pcr reaction contained 7.5 l power sybr green pcr master mix ( applied biosystems , california usa ) , 0.5 l of 10 m primer mix , 2 l of a 1:5 dilution of cdna and nuclease - free water to a final volume of 15 l . reactions were performed with the stratagene mx3005p realtime machine in triplicate . hot - start taq polymerase was heat - activated at 95 c for 10 min followed by 40 cycles of 15 s at 95 c and 30 s at 60 c . fluorescence was read in each cycle and a melting curve constructed as the temperature was increased from 65 c to 95 c with continuous fluorescence readings . ubc was used as a control gene to normalize between the samples and relative expression determined using the delta - delta ct method ( livak and schmittgen , 2001 ) . mda - mb-231-er+ , mda - mb-231-er , mcf-7 and lncap cells were harvested for nuclear extract using the ne - per nuclear extraction kit ( thermo scientific pierce , rockford il usa ) according to the manufacturer 's instructions . extracted protein was quantified using the direct detect system ( merrick millipore , massachusetts usa ) . nuclear extracts were prepared with 4x protein sample loading buffer ( li - cor biosciences , usa ) , 10x nupage sample reducing agent ( thermofisher scientific , leicestershire , uk ) and water , and 15 g protein per lane loaded into bolt 412% bis - tris gels ( thermofisher scientific , leicestershire , uk ) . gels were run with mops running buffer for 30 min at 60 v followed by 30 min at 120 v. western transfer was performed using the iblot system ( invitrogen , paisley , uk ) according to the manufacturer 's instructions . odyssey blocking buffer ( li - cor biosciences , usa ) was added to membranes for one hour at room temperature . 1 ) were added at the following dilutions and incubated overnight at 4 c : novocastra - er - beta ( emr02-ncl - er - beta ) ( leica biosystems , newcastle , uk ) 1:100 , er1 ppg5/10 ( mai-81281 ) ( thermo scientific pierce , rockford il usa ) 1:100 , er-antibody h150 ( sc8974 ) ( santa cruz biotechnology , dallas tx , usa ) 1:200 , cwk - f12 , usa ) ( choi et al . , 2001 ) 1:200 , mc10 ( wu et al . , 2012 ) 1:300 , genetex er 70182 ( irvine , ca , usa ) 1:200 , er 06 - 629 ( merck millipore , watford , uk ) , 1:500 , abcam 288 [ 14c8 ] ( cambridge , uk ) 1:500 . the following were used as loading controls : rabbit anti - beta actin ( ab8227 ) ( abcam , cambridge , uk ) 1:5000 or mouse anti - beta actin [ ac-15](ab6276 ) 1:1000 according to the species of the er antibody . the membranes were washed three times with pbs/0.1% tween and incubated with secondary antibodies for one hour at room temperature : goat anti - mouse ( green ) 1:5000 with goat anti - rabbit ( red ) 1:20000 or goat anti - rabbit ( green ) 1:5000 with goat anti - mouse ( red ) 1:20000 according to the species of the er antibody . membranes were imaged using the li - cor odyssey fluorescent imaging system ( li - cor biosciences , usa ) . formalin - fixed , paraffin - embedded mda - mb-231-er and mda - mb-231-er+ cell pellets were generated , with 2 10 cells per pellet . er expression was induced with 0.5 g / ml doxycycline for 24 h. antigen retrieval was achieved by incubating in citrate - based retrieval solution for 20 min . sections were stained using cwk - f12 er antibody , diluted 1:250 in standard bond diluent using leica 's polymer refine kit ( catalogue no : ds9800 ) on the automated bond platform ( leica biosystems newcastle ltd , newcastle uk ) . images were captured using aperio software ( leica biosystems newcastle lt , newcastle uk ) . a prostate tissue microarray ( tma ) was created from a random selection of prostate cancers , including a range of different tumor grades , and benign prostatic tissue ( 10 cancer , 5 benign in total ) ( ethical approval : prompt study mrec/01/4/061 ) . the areas to be sampled from the formalin - fixed and paraffin embedded tissue blocks were marked on the corresponding haematoxylin and eosin stained paraffin sections . each block was assessed to ensure that there was an adequate amount of tissue for sampling , and cores of tissue punched from the selected area of the block using 5 mm skin biopsy punches . each core was re - embedded into a new recipient paraffin block and its position in the block recorded on a tma map . the breast tma was constructed using the chemicon advanced tissue arrayer ( merck millipore , germany ) according to the manufacturer 's instructions . this contained 30 benign samples , 56 grade i , 55 grade ii and 57 grade iii er alpha positive tumors . an additional tma was constructed from 10 invasive carcinomas and 10 non - malignant tissues for optimisation of antibody staining . to ensure adequate representation of the tissue , core size of 1 mm the study protocol for tissue collection was approved by the university of adelaide human research ethics committee ( # s h-2005 - 065 ) . for the prostate ihc , 3.5 m sections were cut and mounted onto charged slides , dried and sealed with paraffin . the cwk - f12 er antibody was further optimized to the clinical samples and diluted at 1:200 in diluent consisting of 1% donkey serum , 0.05% tween20 in 300 mm tbs to reduce background staining . antigen retrieval was achieved by incubating in tris edta for 20 min at 100 c . images were captured at 250 magnification using image pro - insight ( media cybernetics . , 4 m sections were cut and adhered to superfrost ultraplus slides ( thermo - fisher scientific # 1014356190 ) . endogenous peroxidase was quenched with 0.3% hydrogen peroxide ( ajax finchem # # 7722 - 84 - 1 ) . antigen retrieval was performed in 10 mm citric acid buffer ( ph 6.0 ) within a decloaking chamber ( biocare medical # dc2012 ) , for 5 min at 120 c . slides were blocked in 5% normal goat serum ( sigma - aldrich # g9023 ) in pbs for 30 min at room temperature . cwk - f12 antibody was added at a dilution of 1:100 and incubated overnight at 4 c . a second section of tma tissue that received buffer in the absence of primary antibody served as a negative control . secondary antibody ( biotinylated anti - mouse antibody ( dako # e0433 ) diluted in pbs with 5% normal goat serum was added and incubated for 60 min at room temperature . sections were washed twice in pbs followed by addition of hrp - conjugated streptavidin ( dako # p0397 ) . tissue was counterstained with haematoxylin and mounted under dpx mountant ( sigma # 06522 ) . rime experiments were conducted as previously described ( mohammed et al . , 2013 ) . briefly , mda - mb-231-er+ , mda - mb-231-er ( 2 10 cells per condition for antibody evaluation ) , lncap and mcf-7 cells ( 4 10 cells per condition for cell line characterization ) were grown in 15 cm plates to 90% confluency . cells were crosslinked with media containing 1% em grade formaldehyde ( tebu biosciences , peterborough uk ) for 8 min and the formaldehyde quenched with 0.1 m glycine . cells were washed , harvested and pelleted in cold pbs . to enrich the nuclear fraction the cell pellet was suspended in 10 ml of lysis buffer 1 ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 10% glycerol , 0.5% np-40 or igepal ca-630 , and 0.25% triton x-100 ) for 10 min at 4 c . cells were pelleted and resuspended in lysis buffer 2 ( 10 mm tris - hcl [ ph 8.0 ] , 200 mm nacl , 1 mm edta , and 0.5 mm egta ) for five minutes at 4 c . cells were pelleted and resuspended in 300 l of lysis buffer 3 ( 10 mm tris - hcl [ ph 8 ] , 100 mm nacl , 1 mm edta , 0.5 mm egta , 0.1% na - deoxycholate , and 0.5% n - lauroylsarcosine ) and sonicated ( diagenode bioruptor . diagenode , seraing belgium ) for 45 min 30 l of 10% triton - x was added and the sonicated lysate centrifuged at 17,000 g for 10 min to remove cell debris . the supernatant was incubated with 100 l of magnetic beads ( dynabeads , thermo fisher scientific , waltham ma usa ) pre - bound with antibody . for evaluation of the 8 er antibodies , immunoprecipitations ( ip ) were set up each for mda - mb-231-er and mda - mb-231-er+ cells using 10 g of antibody ( ncl - er - beta , genetex 70182 , millipore 06 - 629 , abcam 288 [ 14c8 ] , mc10 , cwk - f12 , sc8974 and ppg5/10 ) . for characterization of lncap and mcf-7 cells , in all cases , 10 g of e2f1-c20 ip was used as a positive control ( sc-193 , santa cruz biotechnology , dallas tx , usa ) and species - specific igg used to detect non - specific pull - down ( mouse sc2025 or rabbit sc2027 , santa cruz biotechnology , dallas tx , usa ) . beads were washed 10 times in 1 ml ripa buffer ( 50 mm hepes ph 7.6 , 1 mm edta , 0.7% na deoxycholate , 1% np-40 , 0.5 m licl ) and twice in 100 mm ammonium hydrogen carbonate ( ambic ) solution . dry , frozen beads were submitted for tryptic digestion of bead - bound protein , and peptides pulled down by ip identified by mass - spectrometry ( ltq velos - orbitrap ms , thermo fisher scientific , waltham ma usa ) . processed files were searched against the swissprot human database using the mascot search engine version 2.3.0 with a false discovery rate ( fdr ) of less than 1% . for each er antibody tested , the resulting list of purified peptides identified was filtered against the corresponding igg control to remove non - specific proteins pulled down . mean percentage er peptide coverage , and mean number of unique er peptides identified in biological duplicate experiments were calculated . nuclear pellets of mda - mb-231-er+ , mda - mb-231-er , lncap and mcf-7 cells were prepared using the panomics nuclear extraction kit ( affymetrix , ca usa ) as per the manufacturer 's provided instructions . nuclear pellets were lysed in 8 m urea , 0.1% sds in 50 mm teab by sonication twice , each for 5 min . after 50 mm of teab ( ph = 8) was added up to a total volume of 100 l . cysteines were reduced in 0.1 mm dtt for 1 h at room temperature and alkylated in 0.1 mm iaa for 30 min at room temperature in the dark . trypsin ( promega trypsin ( v5111 ) ) was added in a 1:100 trypsin : protein ratio for 1 h at room temperature . another batch of trypsin ( 1:100 ratio ) was added to have a final ratio of 1:50 for incubation overnight . samples were acidified to a final concentration of 1% formic acid ( fa ) and cleaned over c18 spin columns ( harvard apparatus c18 micro spincolumn ) . after elution from the columns samples were lyophilized in a speedvac and resolubilized in 0.1% fa , 5% acn to a final peptide concentration of 1 g/l . samples were subjected to liquid chromatography - electrospray ionization in an orbitrap nano - esi q - exactive mass spectrometer ( thermo scientific ) , coupled to a nanolc ( dionex ultimate 3000 uhplc ) . samples were trapped on a 100 m 2 cm , c18 , 5 m , 100 trapping column ( acclaim pepmap 100 ) in l - pickup injection mode at 4 l / min flow rate for 10 min . samples were loaded on a rapid separation liquid chromatography , 75 m 25 cm nanoviper c18 3 m 100 column ( acclaim , pepmap ) retrofitted to an easy - spray source with a flow rate of 300 nl / min ( buffer a , hplc h2o , 0.1% fa ; buffer b , 100% acn , 0.1% fa ; 60-min gradient ; 05 min : 5% buffer b , 545 min : 5 to > 56% buffer b , 45.150 min : 56% to > 95% buffer b , 50.160 min , 5% buffer b ) . peptides were transferred to the gaseous phase with positive ion electrospray ionization at 1.8 kv . precursors were targeted in a 2th selection window around the m / z of interest . precursors were fragmented in high - energy collisional dissociation mode with normalized collision energy dependent on the target peptide . the first mass analysis was performed at a 70,000 resolution , an automatic gain control target of 3 10 , and a maximum c - trap fill time of 200 ms ; ms / ms was performed at 35,000 resolution , an agc target of 5 10 , and a maximum c - trap fill time of 100 ms . differences in er mrna levels observed in mda - mb-231-er and mda - mb-231-er+ conditions were analyzed using unpaired t - tests . given the confusion in the er field and the concern associated with variable and potentially non - specific reagents , we sought to extensively validate commonly used er antibodies in a systematic manner that does not rely upon a priori assumptions regarding er expression in cell line models or in tissues . as a control , we employed a cell line system with doxycycline - inducible expression of the er protein , allowing us to assess antibodies in er negative and matched er positive conditions ( fig . one hundred - fold induction of er mrna in mda - mb-231-er cells treated with doxycycline 0.1 g / ml for 24 h ( p = 0.01 ) was confirmed by qrt - pcr ( fig . western blots of mda - mb-231-er+ and mda - mb-231-er cell lysates with 8 different er antibodies were performed ( fig . six commonly used antibodies in the literature were included ; ppg5/10 ( asgari and morakabati , 2011 , carder et al . , 2005 , ciucci et al . , 2014 , , 2003 , wimberly et al . , 2014 ) , ncl - er - beta ( ellem et al . , 2014 , hussain et al . , 2012 , mcpherson et al . , 2007 ; 2010 ; morais - santos et al . , 2015 , , 2007 , umekita et al . , 2006 , yang et al . , 2015 , zellweger et al . , 2013 ) , genetex 70182 ( celhay et al . 2015a , mak et al . , 2015b , ; nakajima et al . , 2011 ) , millipore 06 - 629 ( bouchal et al . , 2011 , chen et al . , 2009 , 2012 ) , abcam 288 [ 14c8 ] ( abd elmageed et al . , 2013 , carder et al . , 2005 , , 2012 , dey et al . , 2014 , setlur et al . , 2008 , shaaban et al . , 2003 , , 2010 , yang et al . , 2012 ) and santa cruz 8974 ( al - bader et al . , 2011 , foryst - ludwig et al . , 2008 , han et al . , 2015 , rossi et al . , 2011 , zhou et al . , 2012 ) antibodies . the ppg5/10 antibody detected a protein band of 77 kda with no difference between er+ or er conditions , suggesting it is recognizing a non - specific protein . similarly , the ncl - er - beta antibody detected a band of 59 kda , which is the correct size for er however , there was no difference between er+ or er conditions implying that this band was not er. the genetex 70182 antibody detected a band of 59 kda with differential signal between er+ and er conditions , and a non - specific band was present at around 65 kda . the millipore 06 - 629 antibody detected a band of 59 kda in both er+ and er conditions , however the band was stronger in the er+ condition , suggesting that the antibody could be cross - reacting with another protein of 59 kda in addition to detecting er. mc10 , cwk - f12 , abcam 288 [ 14c8 ] and sc8974 er antibodies all detected protein bands of 59 kda with differential signal between er+ and er conditions , confirming their specificity for er by western blotting . further confirmation of the specificity of cwk - f12 to er was demonstrated by ihc of mda - mb-231-er+ and mda - mb-231-er cell pellets ( fig . the 8 er antibodies were then assessed by an independent method called rime , which uses an antibody - based purification followed by mass spectrometry ( ms ) to identify enriched peptides . we conducted rime in mda - mb-231-er and mda - mb-231-er+ cells using all 8 antibodies . e2f1 antibody was included in parallel as a positive control since e2f1 is a ubiquitous protein ( fig . , no er peptides were purified by any of the er antibodies , confirming the er negative status of the uninduced mda - mb-231-er cell line ( fig . following er induction , rime revealed diverse coverage of the er protein by the different antibodies . the percent coverage of the er protein following purification with each of the er antibodies , and the location of the peptide fragments identified by ms are shown in fig . , we ranked all the proteins purified by the ip and identified by ms according to the number of unique peptides ( confirmed with a false discovery rate ( fdr ) of < 1% ) . we hypothesized that the higher the ranking of er , the greater the specificity of the antibody . hence , if er has the greatest number of unique peptides relative to all other proteins , it is ranked 1st . 3b ) , which is consistent with the lack of specificity identified from the western blot result ( fig . the millipore 06 - 629 antibody positively pulled down er in the test condition , although coverage and ranking were not as favorable as compared with some of the other antibodies . interestingly , lactb , a 60 kda protein was purified by millipore 06 - 629 in both er+ and er conditions ( data not shown ) , which may explain the 60 kda band identified from western blotting . whilst the ppg5/10 did not detect er by western blotting , by rime it detected er with 25% coverage , with er ranking 3rd in the list of identified peptides , suggesting differences in the specificity of this antibody from one experimental assay to another . ppg5/10 has been previously validated for ihc in a doxycycline - inducible u2os - er cell line , developed using the same plasmids as the mda - mb-231-er cell line ( wu et al . , 2012 ) . the abcam 288 [ 14c8 ] antibody is a very commonly used er antibody ( abd elmageed et al . , 2013 , colciago et al . , 2014 , , 2013 , dey et al . , 2012 , dey et al . , 2014 , , 2003 , vivar et al . , 2010 , yang et al . , 2012 ) , which performed well by western blotting , and also had the best antibody coverage by rime ( 31.9% ) . however er ranked 20th in the list of identified peptides when using abcam 288 [ 14c8 ] , suggesting that this antibody might also be purifying additional non - specific proteins . the mc10 antibody had the second - greatest coverage ( 28.2% ) with er ranking 1st in the list of identified peptides . in view of this finding , along with the positive western blot result ( fig . 1 ) , the mc10 antibody was carried forward into the rime experiments for the cell line characterization . the cwk - f12 antibody had 17.7% coverage , with er ranking 2nd in the list of purified peptides . as the cwk - f12 antibody produced very clean results by western blotting , ihc and ranked er second in the list of purified proteins , it was used for western blotting in the cell line characterization and directly compared against the non - specific ncl - er - beta antibody . the goal was to use independent validated er antibodies and additional independent methods to assess whether the most commonly studied breast and prostate cancer cell line models express er. given the wealth of publications assessing er in breast ( mcf-7 ) and prostate ( lncap ) cancer cell lines ( abd elmageed et al . , 2013 , al - bader et al . , 2011 , , 2014 , ellem et al . , 2014 , fuqua et al . , 1999 , , 2002b , lau et al . , 2000 , mak et al . , 2012 , yang et al . , 2015 , zhou et al . , 2012 ) , we sought to investigate the expression of er in these models , using the newly validated er antibodies . protein lysate and rna was collected from lncap and mcf-7 cells . using primers validated in the inducible mda - mb-231-er cell line , which binds to sequence common to wild type ( wt ) er and its isoforms ( fig . 1b ) , lncap and mcf-7 were shown to express no detectable levels of er mrna ( fig . er protein was undetectable by western blotting in these cells . by way of contrast , using the ncl - er - beta antibody on the same cell lysates , we detected a protein band of approximately 59 kda in all conditions tested , including the mda - mb-231-er cell line , confirming the non - specificity of this antibody to er ( fig . importantly , this demonstrates that the ncl - er - beta antibody is not detecting er in either lncap or mcf-7 cancer cell line models and is instead identifying a non - specific protein of similar molecular weight . furthermore , rime analysis of lncap and mcf-7 cells using the validated mc10 er antibody did not purify any er peptides by ms ( fig . this result was confirmed by an antibody - independent approach known as parallel reaction monitoring ( prm ) , which demonstrated that no er peptides were present in either of these cell lines ( fig . as such , our early passage lncap and mcf-7 cell line models are er negative and these cancer models should not be used for the analysis of this protein . importantly , whilst the lncap and mcf-7 cell - line models do not express er , application of the validated cwk - f12 er antibody to prostate and breast cancer tmas demonstrated variable er expression in differing cancer grades . in prostate tissue , previous reports have described an inverse correlation between er expression and increasing gleason grade of prostate cancer ( asgari and morakabati , 2011 , attia and ederveen , 2012 , dey et al . , 2014 , horvath et al . risbridger et al . , 2007 ) , whereas others have reported an association between increased er expression and higher gleason grade ( zellweger et al . , 2013 ) or increased expression of er in bone and lymph node metastases ( bouchal et al . , 2011 , zhu et al . , 2004 ) . in our prostate tma ( fig . 5a d ) we observed high expression of er in the basal epithelium of benign glands , with no expression in gleason grade 3 cancer . gleason grade 4 cancer showed weak nuclear staining of er and in areas of gleason grade 5 cancer , er nuclear expression was of moderate intensity . in breast tissue , previous studies have shown greatest er expression in benign tissue , with a gradual decrease in expression associated with increasing cancer grade ( guo et al . conversely , a non - statistically significant trend towards higher er expression in grade 3 tumors has also been reported ( myers et al . , 2004 ) . in our breast tma ( fig . 5f i ) , we observed greatest expression of er in benign epithelium , with a trend towards decreasing er expression associated with increasing cancer grade . one potential explanation for the inconsistencies in er tissue expression is the presence of er splice - variant isoforms , which are fully conserved in exons 16 , but have differing c - terminal domains ( leung et al . , 2006 ) . different antibodies that bind either to the conserved regions or only to the c - terminal domain of the full - length er protein may therefore give differing results ( supplementary fig . . this may particularly be the case in prostate cancer , where it has been reported that er isoform expression increases with the development of crpc ( dey et al . whilst this is likely to have an impact , our data suggest that some of the differing conclusions around er expression in primary tissues are a direct result of certain investigations utilizing non - specific reagents that lack specificity for er. despite a large body of published literature , the role of er in cancers of the prostate and breast is not clear . contradictions between ihc findings and antibody - dependent molecular biology methods have contributed to this uncertainty , particularly the lack of clear consensus regarding correlation between tissue expression of er and clinico - pathological parameters ( asgari and morakabati , 2011 , attia and ederveen , 2012 , bouchal et al . , 2011 , dey et al . , 2014 , esslimani - sahla et al . , 2004 , guo et al . , 2014a , guo et al . , 2014b , hieken et al . , 2015 , horvath et al . , 2001 , leav et al . , 2001 , our results have demonstrated marked variation in the ability of commonly used commercially available er antibodies to accurately detect er by western blotting and protein purification - ms based methods . most notably , ncl - er - beta , a commonly used antibody ( ellem et al . , 2014 , hussain et al . , 2012 , mcpherson et al . , 2007 ; 2010 ; this antibody consistently yields bands on western blots of the appropriate size for er ( 59 kda ) in all tested conditions ( figs . 1c and 3b ) , but we have confirmed that this protein band is not er through the use of the mda - mb-231-er inducible cell line system and the rime technique . as such , this non - specific 59 kda band is likely to be the source of much of the controversy and confusion surrounding the study and characterization of er. the ppg5/10 antibody targets the c - terminus of wt er , and as such may be useful for distinguishing wt er from expression of er isoforms . ppg5/10 identified er in the mda - mb-231-er cell line by rime , and has previously been shown to be er-specific by ihc in both an inducible cell line model ( wu et al . , 2012 ) and in breast tissue ( carder et al . , 2005 ) . however , in our study this antibody did not show specificity by western blot analysis ( fig . also assessed the abcam 288[14c8 ] antibody and found it to be er-specific for ihc in tissue ( carder et al . , 2005 ) . whilst our western blotting data support this assertion ( fig . 1c ) , our rime data suggest that this antibody also purifies additional , non - specific peptides , and as such should be used with caution for ip - based methods ( fig . , these findings reassert the importance of validating antibodies for individual experimental approaches , rather than assuming general applicability across methodological platforms ( baker , 2015 , bordeaux et al . , 2010 ) . rime was initially developed as a discovery tool to study the interacting proteomes of transcription factors in an unbiased manner ( mohammed et al . , 2013 ) . the advantage of using rime in antibody validation arises from being able to identify specific , named peptides purified by an antibody , rather than relying on the presence of a protein band of approximate size on a western blot . this is typified by the ncl - er - beta antibody , which gave bands on western blot in both er and er+ conditions and no er peptides identified by rime . taken together , these data confirm that this antibody is not specific to er. the non - commercially available er antibodies tested ( mc10 and cwk - f12 ) have been previously validated by other approaches ( choi et al . , 2001 , wu et al . , 2012 ) and our results add further confidence in their specificity using multiple independent assays . by comparing the peptide coverage of each antibody along with the er ranking ( as a surrogate of specificity ) rime facilitated an informed decision - making process in selecting which antibody to carry forward to the cell - line characterization . our multi - modal approach to cell - line characterization using both antibody - dependent ( western blotting and rime ) and antibody - independent ( qrt - pcr and prm ) approaches has shown that low - passage , genotyped lncap and mcf-7 cell lines do not express detectable er , despite numerous publications making conclusions about er biology using these cell line models ( abd elmageed et al . , 2013 , al - bader et al . , 2011 , , 2014 , ellem et al . , 2014 , fuqua et al . , 1999 , , 2002a , kim et al . , 2002b , lau et al . , 2000 , mak et al . , 2013 , weng et al . , 2013 , yang et al . whilst we acknowledge that immortalized cell lines may have variable expression of certain factors across passage numbers and laboratories ( masters , 2000 ) , our data suggest the need for caution in making this assumption with respect to er. reassuringly , we have confirmed expression of er in prostate and breast tissue using the validated cwk - f12 antibody . our ihc study is not intended to be an exhaustive analysis of er expression in prostate and breast tissue , and we acknowledge the limitations presented by our small sample size and lack of statistical correlation with clinico - pathological parameters . we have however , demonstrated that the cwk - f12 er antibody is validated for ihc and in principle can be used for larger scale assessment of er expression in tissue . epidemiological evidence suggests that estrogen and its receptors have important roles in the development and progression of prostate cancer . japanese men are known to have a very low incidence of prostate cancer ( ross et al . , 1992 ) , and it has been proposed that their traditional diet , which is high in er selective phytoestrogens may exert a protective role ( andres et al . , 2011 , attia and ederveen , 2012 , hori et al . , 2011 , 2000 , stettner et al . , 2007 , thelen et al . , 2007 , thelen et al . , 2005 , further evidence from studies of er knockout mice ( erko ) shows a clear phenotype and tumor - suppressive effect of er ( ricke et al . , 2008 ) . however , clinical trials of agents seemingly effective in vitro have demonstrated no clinical benefit of estrogen - selective agents in prostate cancer ( bergan et al . , 1999 , kim et al . there are numerous explanations as to why this might be , for example , expression of er in non - epithelial cell types ( gargett et al . , 2002 , 2010 ) modulating the tissue response to these agents , but in light of our findings we would suggest that use of poorly validated reagents and inadequately characterized cancer cell line models is an important contributing factor . in the presented study , detailed validation of commonly used er antibodies has demonstrated that some of these reagents either detect er in specific experimental conditions only or lack any specificity for er across multiple assays . er has been investigated in numerous cancers including prostate , breast , kidney ( yu et al . , 2013 ) , 2013 ) , endometrium ( han et al . , 2015 ) , ovary ( ciucci et al . , 2014 , 2008 ) bladder ( hsu et al . , 2013 ) and non - small cell lung cancer ( he et al . , 2015 , luo et al . , 2015 ) but in many cases , the findings are predicated on non - specific reagents . as such , a re - evaluation of er expression and biology is needed using reliable , specific reagents . our determination of er antibody specificity will contribute towards clarifying existing , conflicting data on the role of er in these diverse cancers and provide the necessary , validated tools with which to move forward our understanding of er biology . prostate tissue included in the tissue microarray was obtained under approval granted for the prompt study ( mrec/01/4/061 ) . breast tissue collection and assessment was approved by the university of adelaide human research ethics committee ( # s h-2005 - 065 ) . awn is supported by the medical research council ( mr / l00156x/1 ) and the urology foundation scholarship ( resch1302 ) . wdt is supported by the national health and medical research council of australia ( i d 1008349 and 1084416 ) , cancer australia / national breast cancer foundation ( i d 1043497 ) , national breast cancer foundation pilot study ( ps-15 - 041 ) and a prostate cancer research program transformative impact award from the us department of defense ( w81xwh-13 - 2 - 0093 ) .

estrogen receptor- ( er ) has been implicated in many cancers . in prostate and breast cancer its function is controversial , but genetic studies implicate a role in cancer progression . much of the confusion around er stems from antibodies that are inadequately validated , yet have become standard tools for deciphering its role . using an er-inducible cell system we assessed commonly utilized er antibodies and show that one of the most commonly used antibodies , ncl - er - beta , is non - specific for er. other antibodies have limited er specificity or are only specific in one experimental modality . er is commonly studied in mcf-7 ( breast ) and lncap ( prostate ) cancer cell lines , but we found no er expression in either , using validated antibodies and independent mass spectrometry - based approaches . our findings question conclusions made about er using the ncl - er - beta antibody , or lncap and mcf-7 cell lines . we describe robust reagents , which detect er across multiple experimental approaches and in clinical samples .

Introduction Materials and methods Results Discussion Ethics Financial support Conflicts of interest

PMC4393838

atrioventricular groove rupture has been recognized as the most fatal complication of mitral valve surgery . luckily , it is a very rare complication and is reported sporadically . even with preservation of the posterior leaflet , the frequency of left ventricular ( lv ) rupture is about 1% following mitral valve procedures with the mortality rates of over 50% . here the same surgeon experienced only these 2 cases in his 25-year - period of surgery , with both cases being treated successfully via the same technique . the study was approved by the hospital ethics committee of medicana international ankara hospital , and written informed consent was obtained from both patients . a 52-year - old female patient was admitted to the hospital with the complaints of dyspnea and fatigue . intraoperatively , cardiopulmonary bypass ( cpb ) was established following standard aorto - bicaval cannulation . a 31-mm ats mechanical prosthetic valve was positioned with interrupted 2/0 polyethylene terephthalate sutures , reinforced with teflon pledgets . a 56-year - old female patient was admitted to the hospital with the complaints of dyspnea , fatigue , and chest pain . echocardiographic examination revealed severe mitral stenosis with 1.2 cm of valve area and moderate to severe mitral insufficiency . the mitral valve leaflets were fibrotic , accompanied by severe annular calcification . the anterior and posterior leaflets were excised . a 31-mm st . jude medical mechanical valve was positioned with interrupted 2/0 polyethylene terephthalate sutures , reinforced with teflon pledgets . in both cases , cpb was successfully ceased without any inotropic support following extensive de - airing maneuvers . immediately after the cessation of cpb , excessive bleeding occurred from the posterior aspect of the heart . the valves were replaced with a 27-mm mechanical valve in both patients following the repair described in detail below . cpb was successfully weaned with the assistance of the intra - aortic balloon pump in order to decrease afterload . the patients were discharged on the 8 and 10 postoperative days with follow - up periods of 5.5 years and 10 months , respectively . following exposure of the left atrium , the mitral valve prosthesis is removed carefully to avoid further damage , and the interior rupture site is inspected . a fresh autologous pericardial patch is prepared for the internal support of the ruptured area . the pericardial patch is stabilized along the interior margin of the defect with interrupted 4/0 polypropylene sutures , reinforced with large teflon pledgets cut from the teflon strip on the ventricular side . deep myocardial sutures from the intact ventricle should be placed at adequate distance to the rupture site in order to provide enough strength to the repaired area since the lv is a high - pressure chamber . when the annulus is accessed on both sides , running polypropylene sutures are employed through the annulus beginning from each side . just before the last suture is tied , bioglue ( cryolife , usa ) is applied under the patch ( figure 1 ) . then a downsized mitral prosthesis is placed with interrupted 2/0 polyethylene terephthalate sutures at the original axis . pericardial patch is stabilized along the interior margin of the defect with interrupted 4/0 polypropylene sutures reinforced with large teflon pledgets . deep myocardial sutures from the intact ventricle should be placed at an adequate distance to the rupture site . when the annulus is reached on both sides , running polypropylene sutures are employed through the annulus beginning from each side . la , left atrium ; lv , left ventricle ; pm , papillary muscle ; pv , pulmonary vein a 52-year - old female patient was admitted to the hospital with the complaints of dyspnea and fatigue . intraoperatively , cardiopulmonary bypass ( cpb ) was established following standard aorto - bicaval cannulation . a 31-mm ats mechanical prosthetic valve was positioned with interrupted 2/0 polyethylene terephthalate sutures , reinforced with teflon pledgets . a 56-year - old female patient was admitted to the hospital with the complaints of dyspnea , fatigue , and chest pain . echocardiographic examination revealed severe mitral stenosis with 1.2 cm of valve area and moderate to severe mitral insufficiency . jude medical mechanical valve was positioned with interrupted 2/0 polyethylene terephthalate sutures , reinforced with teflon pledgets . in both cases , cpb was successfully ceased without any inotropic support following extensive de - airing maneuvers . immediately after the cessation of cpb , excessive bleeding occurred from the posterior aspect of the heart . the valves were replaced with a 27-mm mechanical valve in both patients following the repair described in detail below . cpb was successfully weaned with the assistance of the intra - aortic balloon pump in order to decrease afterload . the patients were discharged on the 8 and 10 postoperative days with follow - up periods of 5.5 years and 10 months , respectively . following exposure of the left atrium , the mitral valve prosthesis is removed carefully to avoid further damage , and the interior rupture site is inspected . a fresh autologous pericardial patch is prepared for the internal support of the ruptured area . the pericardial patch is stabilized along the interior margin of the defect with interrupted 4/0 polypropylene sutures , reinforced with large teflon pledgets cut from the teflon strip on the ventricular side . deep myocardial sutures from the intact ventricle should be placed at adequate distance to the rupture site in order to provide enough strength to the repaired area since the lv is a high - pressure chamber . when the annulus is accessed on both sides , running polypropylene sutures are employed through the annulus beginning from each side . just before the last suture is tied , bioglue ( cryolife , usa ) is applied under the patch ( figure 1 ) . then a downsized mitral prosthesis is placed with interrupted 2/0 polyethylene terephthalate sutures at the original axis . pericardial patch is stabilized along the interior margin of the defect with interrupted 4/0 polypropylene sutures reinforced with large teflon pledgets . deep myocardial sutures from the intact ventricle should be placed at an adequate distance to the rupture site . when the annulus is reached on both sides , running polypropylene sutures are employed through the annulus beginning from each side . just before the last suture is tied , la , left atrium ; lv , left ventricle ; pm , papillary muscle ; pv , pulmonary vein classified lv rupture following mitral valve replacement into two types based on the location of the epicardial tear . type 1 is defined as a defect along the posterior atrioventricular sulcus , whereas type 2 refers to the rupture of the lv posterior wall at the base of the papillary muscle . miller et al . proposed a third type defined as the rupture of the lv posterior wall between the base of the papillary muscle and the atrioventricular groove . the classification represents the location of the rupture , but it is not adequate for the preference of the repair technique . the primary goal is always survival , but postoperative intracardiac hemodynamics should be preserved for good cardiac status . the rapid expansion of the tear in most cases precludes definite distinction of the type and it is , therefore , mostly defined as the mixed type . excessive decalcification of the mitral valve annulus and use of an oversized prosthesis are usually causative for type 1 rupture . also , elevation of the lv is another contributing factor , which underscores the importance of the extensive de - airing of the lv . miller et al . suggested preservation of the posterior mitral apparatus in order to reduce the incidence of rupture . in both cases , the posterior valve leaflets were highly fibrotic and densely calcified and as such could not be preserved . the most effective repair technique for all types of lv rupture is utilizing an endocardially positioned patch , which is stabilized with interrupted prolene sutures reinforced with epicardial teflon pledgets , along the safe myocardial tissue ( intracardiac repair ) and downsizing the prosthetic valve size . extracardiac repair techniques and complete explantation of the heart , followed by repair and reimplantation , have also been described . reported the use of glue in the atrioventricular groove repair , but they used it in combination with a fibrin sheet in response to continued bleeding following repair only on the external surface . employed biological glue in a nearly similar way on the suture line and also filled the cavity in a few cases of destroyed annulus due to infection . zehr reported his experience with biological glue use in various cardiac procedures , and described its use along with the cellulose patch or the bovine pericardial patch in the repair of lv rupture . masroor et al . defined an alternative technique , named endoventricular pocket repair , with the use of teflon felt , bovine pericardium , and biological glue . briefly , our technique may be defined as intracardiac repair with a pericardial patch , positioned with 4/0 polypropylene sutures . along with the repair , we recommend the use of biological glue between the pericardial patch and the epicardial rupture area for perfect sealing . yamashita et al . measured the tensile strength of the pericardium and reported that the tensile strength of glutaraldehyde - treated pericardium was four times higher than that of non - calcified aortic valve leaflets . this finding is applicable to lv rupture repair because the aortic leaflets also face similar chamber dynamics . however , since glutaraldehyde fixation is time - consuming and the rupture is a very emergent state , a fresh pericardial patch is preferred . atrioventricular groove rupture following mitral valve replacement surgery is a devastating complication that requires prompt and urgent management . complete repair of the ruptured area , followed by implantation of a smaller prosthesis , is mandatory . biological glue use together with pericardial patch repair may increase the strength of the repaired area and ease the implantation of the valve .

abstractatrioventricular groove rupture is a rare , albeit mortal , complication following mitral valve surgery . avoidance is the best strategy but it can not fully prevent the occurrence of this complication . several repair techniques have been described with varying success rates ; however , the rarity of the complication precludes consensus about the safest technique.here we report two cases of posterior atrioventricular groove rupture . both cases were diagnosed immediately after the cessation of cardiopulmonary bypass . repair was performed successfully with a technique involving the use of biological glue . the postoperative course was uneventful for both of them . both cases are well with normally functioning mitral prostheses ; one with a follow - up time of 5.5 years and the other 10 months . we believe that the glue provides additional hemostasis and support to the repaired area .

Introduction Case #1 Case #2 Repair Technique Discussion Conclusion

PMC4932056

strength training involving repetitive eccentric exercises can cause muscle damage , resulting in muscle soreness , swelling and eventually reduction of muscle strength , especially when an athlete is unaccustomed to this type of muscle contraction1,2,3 . previous studies have found significant increases in self - reported pain after performing eccentric exercises involving the elbow flexor4 . a reduction in explosive force and increase of muscle soreness have also been reported after 12 sets of 10 eccentric squats in active physical education students5 . this muscle damage is usually defined as delayed - onset muscle soreness ( doms ) , and it appears between 8 to 20 hours after the exercise reaching its peak between 24 to 48 hours after the damage6 . in physiological terms , doms is characterized by a sensation of pain felt during movement or palpation of the affected muscle7 . inflammation , increased serum creatine kinase ( ck ) levels , muscle force loss , and decrease in performance have also been observed during this period8 , 9 . recently , whole - body vibration ( wbv ) has been proposed as a recovery treatment for reducing muscle soreness and muscle force loss after exercise10 , 11 . rhea et al.12 reported that applying wbv after strenuous resistance training and repeated sprints is effective at reducing the potential pain after a training session . similar results were found when wbv was applied before 6 sets of 10 maximal isokinetic eccentric contractions of the dominant - limb knee extensors13 . also , the application of vibrations at a frequency of 30 hz before 70%rm and 90%rm resistance exercises suppressed increases in the stress hormone epinephrine levels immediately after exercise14 . therefore , wbv appears to be effective regardless of the exposure time to vibration or moment ( i.e. before or after performing the eccentric exercise ) . wbv may increase blood flow and oxygen delivery to the muscles15 , 16 , and decrease the pain perception responses17 . additionally , wbv may avoid the disruption of muscle sarcomeres that occurs during eccentric exercises , and therefore prevent doms18 . however , the evidence regarding the use of wbv to reduce doms is controversial and limited . while some authors have found wbv to positively influence doms9 , 19 , others have concluded that the application of wbv after eccentric exercise does not alleviate the signs of muscle damage ( levels of serum ck and muscle soreness ) compared to other traditional ways of recovery ( i.e. stretching exercise or massage)8 . the large number of wbv training parameters ( vibration type , frequency , amplitude , rest interval between bouts and body position)20 together with limited existing research on the topic make it difficult to draw definitive conclusions on the effectiveness of wbv and the best recovery treatment to reduce doms signs . furthermore , the aforementioned studies implemented protocols with several vibration sessions after exercise , but to date no research has analyzed if a single session of wbv after eccentric exercise is enough to reduce muscle damage and stimulate the recovery process . therefore , the aim of this study was to examine whether or not a single whole - body vibration treatment after eccentric strength training can reduce muscle soreness and enhance muscle recovery . the experiment was approved by the committee of biomedical ethics of the university of extremadura ( spain ) on may 6th , 2014 , whose chairman was mr . fernando henao ( dossier number 31/2014 ) , and it was conducted in accordance with the principles of the declaration of helsinki . nine participants were excluded from the study because they did not want to provide blood samples ( n=4 ) , had some form of musculoskeletal problems ( n=2 ) or participated in a fitness program more than once per week ( n=3 ) . as a result , 20 participants were randomly assigned to the vibration group ( n=10 ) or the control group ( n=10 ) . the participants were asked to maintain their lifestyle , not to take any medication or nutritional supplements , and not to perform any exercise or sports activity during the period of the intervention . the participants attended the laboratory on 4 occasions and had to complete all sessions at the same time each day . five days before their eccentric training , all the participants performed a familiarization session involving the same kind of exercises to be performed in the subsequent training sessions . on the same day , peak quadriceps leg extension strength of 1 repetition maximum ( 1rm)21 was set and the workload for the eccentric exercise at 120% 1rm was calculated . during the first experimental visit , before starting the eccentric exercise session , blood samples were collected and stored for subsequent analysis . later , participants performed the eccentric strength training consisting of a 5-minute warm - up ( 30% of 1rm ) and 4 sets of 5 repetitions at 120% 1rm , with 4 min rest between sets , on a quadriceps leg extension ( selection med machine , technogym , spain ) . movement was performed with both limbs in a neutral position and along a range of motion of 90. subjects were asked to complete each repetition with maximal effort and a slow controlled movement ( 2 s ) during the eccentric phase . during the concentric phase of the movement , two assistants replaced the lift arm of the weight machine in the initial position using a mechanical jack , so participants did not make any effort during this phase . five minutes . after performing the eccentric training , the participants received 3 sets of 1 min whole body vibration ( 12 hz , 4 mm ) with 30 s of passive recovery between each set ( vibration group ) , or an identical protocol with the vibratory platform turned off ( control group ) . no information about the potential effects of the vibration treatment was given to the participants to minimize psychological effects . an oscillating vibratory platform ( galileo fitness , novotec medical , germany ) was used . all the participants were asked to stand on the platform barefoot in a static squat position with their knees bent 30 ( full extension=0 ) , hands on hips , and trunk straight . joint angle was measured with a goniometer ( 12 - 1001hr , baseline , usa ) . the vibration protocol was chosen based on previous research that showed that using similar frequencies leads to improvements in muscle recovery7 , 12 . assessments occurred immediately after the vibration , and at 24 h and 48 h post - exercise . body weight and height were measured in the first session using a portable measuring station ( seca 220 , germany ) . blood samples ( 5 ml ) were collected to assess serum ck and blood urea nitrogen ( bun ) concentrations . samples were drawn from the antecubital vein at each measurement time point : pre - exercise , post - exercise , and 24 h and 48 h post - exercise . before analysis serum concentrations were determined by a dry - chemistry automated multi - analyzer ( spotchem ez sp-4430 , menarini diagnostics , uk ) using standard menarini test strips . the level of muscle soreness was evaluated using a 100-mm visual analogue scale ( vas ) , with 0 at the left endpoint representing no pain and 100 at the right endpoint representing extreme pain . all of the participants were asked to point to their level of perceived soreness on the vas while they maintained a static squat position with their knees flexed at 30 for 5 s. muscle strength was assessed by recording pit through dominant - limb knee extensor testing on an isokinetic dynamometer ( system 3 , biodex medical systems , usa ) . participants performed two 5-s maximal isometric contractions at a knee joint angle of 45 ( 0 was considered as full extension ) , with a 1 min rest between each maximal effort . they were asked to generate maximal force as fast as possible when the signal was given . dynamometer calibration and protocol procedures were performed in accordance with the instructions provided by the manufacturer . the kolmogorov - smirnov test was conducted in order to verify the normality of the data distribution , and levene s test was used to assess the homogeneity of variance . comparisons between the intervention conditions ( vibration vs control ) over time for each variable were subjected to two - way repeated - measures anova . post hoc pairwise comparisons were performed using the bonferroni test to identify significant differences within groups from pre - exercise values . significance was accepted for p0.05 , a confidence level of 95% . means and standard deviations ( sd ) were used as descriptive statistics . the general characteristics of participants are shown in table 1table 1.general characteristics of the participantsvibration groupcontrol groupage ( years)24.2 0.523.4 1.4height ( cm)177.1 11.5175.6 5.3weight ( kg)75.2 11.573.2 5.9120% 1rm ( kg)123.0 19.2118.1 18.8values are mean sd .. there were no significant differences between the study groups . table 2table 2.markers of muscle damage , muscle soreness and muscle strengthvariablesprepost 24 h48 hserum ck ( u / l)control 100.5 4.7108.0 3.4300.5 26.1 * 285.2 19.7*vibration90.7 5.3102.5 5.6200.2 8.2*175.2 12.5*bun ( mg / dl)control14.4 3.214.9 3.716.8 3.715.4 2.8vibration16.3 2.817.73 2.319.6 2.016.8 2.8vas score ( mm)control6.6 11.536.6 20.568.3 15.1 * 65.2 13.2*vibration6.0 5.533.2 16.454.4 16.3 * 34.1 11.4pit ( nm)control235.3 53.7205.7 48.2187.3 49.0189.1 60.0vibration225.4 56.9201.2 62.1172.6 48.0177.2 bun : blood urea nitrogen ; vas : visual analog scale ; pit : peak isometric torque.*p0.05 , significant difference from pre - exercise . . shows the values of muscle damage , muscle soreness and muscle strength at the different measurement times . eccentric training elicited significant ( 0.05 ) increases in serum ck at 24 h and 48 h post - exercise in both groups , and vas scores were also significantly higher at 24 h ( both groups ) and 48 h ( control group only ) after the eccentric training . compared to the vibration group , the control group showed significant ( 0.05 ) increases in serum ck ( at 24 h and 48 h post - exercise ) and the vas score ( at 48 h post - exercise ) . bun : blood urea nitrogen ; vas : visual analog scale ; pit : peak isometric torque . * the participants baseline data was in the normal range for healthy people , showing that the subjects were not fatigued and had not done any strenuous physical activity prior to the experiment . therefore , the changes observed in the experiment resulted from the performance of the eccentric training . previous studies have reported that repetitive eccentric contractions can cause broadening or disruption of muscle sarcomeres18 . it is known that eccentric contractions may cause greater high - stress damage to recruited muscle fibers than concentric contractions2 . this muscle damage leads to inflammation , release of ck and an increased perception of muscle soreness for several days after the exercise13 , 22 . similar results were evident in our present research , which found there were increased concentrations of serum ck and high pain vas scores at 24 h and 48 h after eccentric exercise in both groups . analysis of the influence of wbv on the values of serum parameters related to muscle damage revealed that serum ck levels at 24 h and 48 h were lower in the vibration group than in the control group . given that serum ck activity is defined as an index of muscle damage23 , these results indicate that the vibration group sustained less muscle damage after eccentric training than the control group . it was previously demonstrated that muscle blood volume and lymphatic flow increase with vibration treatment and that the magnitude of this increase is positively associated with vibratory load15 . this rise in muscle perfusion may stimulate muscle recovery by improving oxygen delivery16 and removing pain substrates24 . to our knowledge , only two studies8 , 19 have investigated the effects of post - exercise vibration treatment on the reduction of serum ck concentrations after eccentric exercise in knee extensor muscles . caution is necessary when making comparisons because the intervention protocols and the type of vibration ( device , frequency and amplitude ) were different . protein catabolism produces urea , which may be a useful biomarker for monitoring fatigue and overtraining25 . however , fallon26 demonstrated that changes in serum urea are more useful for monitoring training status than for assessing short term fatigue , which may be the reason why there were no changes in bun after eccentric training in this study . vas scores of the vibration group at 48 h were lower than those reported by the control group , indicating less muscle soreness . this result is supported by previous research , which noted that post - exercise vibration alleviated doms and provided an analgesic effect . rhea et al.12 reported an attenuation of pain ranging from 2261% when untrained individuals received wbv . similar results were found by lau and nosaka22 for upper arm muscle soreness , with peak soreness being 1830% less for the vibration group than for the control group at 2 to 5 days after eccentric exercise . the benefits of vibration treatment on pain have been reported in both clinical and experimental settings for several decades27 . muscle vibration activates afferent inputs from sensory units in the muscle28 , and the subsequent afferent activity in myelinated sensory axons may inhibit nociceptive messages , possibly at spinal segmental levels , modifying the pain sensation and elevating the pain threshold29 , 30 . several studies have reported muscle strength loss following repetitive maximal eccentric contractions8 , 9 . however , no significant decrease in pit values was observed in our present study . one possible explanation for these conflicting findings could be the type of eccentric training performed . the training performed in the present study consisted of few repetitions of very high intensity , whereas the aforementioned studies used workouts with a high volume ( 100 and 300 repetitions , respectively ) , so it is possible that muscle involvement and fatigue were different in these trials . these findings are similar to those obtained by other research that concluded that standing on a vibrating platform9 or sitting on a vibrating cushion8 after a session of eccentric exercise did not improve the recovery of the knee extensor s pit . a limitation of our current study might be the choice of just one angle to evaluate muscle strength because pit values could vary at different angles . moreover , other serum markers such as lactate dehydrogenase , myoglobin , troponin or aspartate aminotransferase were not analyzed , and their analysis would have provided more information about muscle damage . the findings of the present study show that a single post - exercise wbv following eccentric training alleviated muscle damage but did not attenuate muscle strength loss . therefore , recreational athletes should know that their performance in training or competition could still be decreased at 48 hours after completing an intense eccentric training , regardless of being subjected to a post - exercise vibration treatment that attenuates doms . further studies to establish the appropriate duration , frequency and amplitude of vibration are needed to investigate the effectiveness of a single post - exercise wbv in the improvement of muscle recovery after eccentric training .

[ purpose ] the aim of this study was to investigate whether or not a single whole - body vibration treatment after eccentric exercise can reduce muscle soreness and enhance muscle recovery . [ subjects and methods ] twenty untrained participants were randomly assigned to two groups : a vibration group ( n=10 ) and control group ( n=10 ) . participants performed eccentric quadriceps training of 4 sets of 5 repetitions at 120% 1rm , with 4 min rest between sets . after that , the vibration group received 3 sets of 1 min whole body vibration ( 12 hz , 4 mm ) with 30 s of passive recovery between sets . serum creatine kinase , blood urea nitrogen , muscle soreness ( visual analog scale ) and muscle strength ( peak isometric torque ) were assessed . [ results ] creatine kinase was lower in the vibration group than in the control group at 24 h ( 200.2 8.2 vs. 300.5 26.1 u / l ) and at 48 h ( 175.2 12.5 vs. 285.2 19.7 u / l ) post - exercise . muscle soreness decreased in vibration group compared to control group at 48 h post - exercise ( 34.1 11.4 vs. 65.2 13.2 mm ) . [ conclusion ] single whole - body vibration treatment after eccentric exercise reduced delayed onset muscle soreness but it did not affect muscle strength recovery .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2982787

against this background , the recently - launched consortium for advanced research training in africa ( carta ) offers an opportune framework for building sustainable health research capacity in africa . the consortium brings together nine academic and four research institutions from west , east , central , southern africa , and select northern universities and training institutes . carta 's program of activities comprises two primary , interrelated , and mutually - reinforcing objectives : to strengthen research infrastructure and capacity at african universities , and to support doctoral training through the creation of a collaborative doctoral training program in population and public health . carta recognizes that health and research capacity - building are prominent , interlinked features of development , broadly defined . the risk factors for chronic diseases , such as diet , physical inactivity , smoking , sexual behavior , and environmental conditions associated with poverty are common worldwide , reflecting changing lifestyles linked to industrialization , urbanization , and the economic and political forces of globalization . context - specific , multidisciplinary research , and strong local research and training institutions are needed to strengthen health systems and ensure their responsiveness to social determinants of health . against this backdrop , carta 's overarching goal is to build a vibrant african academy able to lead world - class multidisciplinary research that impacts positively on public and population health . carta seeks to make a difference by building local research capacity to understand the determinants of population health and effectively intervene to improve health outcomes and health systems . to achieve its mission , carta aims to : foster the development of multidisciplinary research hubs at african universities , facilitate high - quality research on policy - relevant priority issues , create networks of locally trained internationally recognized scholars , and enhance the capacity of african universities to lead globally competitive research and training programs . high - quality doctoral training will address the present lack of research expertise in disciplines relevant to health . linking population and public health training with ongoing research will increase the number and quality of professionals able to design , implement , and evaluate public health policies and programs . policy - relevant research is needed to develop health interventions and measure their impact , address social determinants of health , understand failing health systems , and make sense of the inability of many health technologies to deliver their expected impacts in africa . carta is neither disease - focused nor discipline - specific , but brings together diverse methodological and disciplinary approaches to context - specific health and development issues facing africa . this resonates well with the current emphasis on multidisciplinary research as the key to addressing africa 's health and development needs . the consortium brings together ( a ) academic and research institutions with extant and longstanding relationships and memoranda of understanding and ( b ) a variety of related disciplines essential to enhancing health , well - being , and livelihoods in africa . these allow carta to build multidisciplinary research capacity in public and population health . currently funded by the wellcome trust ( under the african institutions initiative ) , the carnegie corporation of new york , the bill and melinda gates foundation , the ford foundation , and the department for international development ( under the delphe programme ) , carta has raised 60% of the us$20 million it needs to fully implement its program of activities over the next 5 years . carta is led jointly by the african population and health research center ( aphrc ) , kenya , and the school of public health , university of the witwatersrand ( wits ) , south africa ; these institutions have already collaborated successfully for the past 5 years . the aphrc provides a productive research environment , with a multidisciplinary team of well - qualified researchers from across sub - saharan africa . wits provides the institutional capacity of an internationally - rated , research - led , postgraduate - oriented african university . both institutions are internationally - recognized as leaders in research and training , have championed the cause of higher education in africa , have facilitated several partnerships and initiatives that strengthen collaborations among different universities and research organizations , and have strong systems of accountability . the african - led nature of carta and its focus on sustainable , measurable changes in research capacity and output are in tandem with the current emphasis on homegrown and intellectually - sound solutions to the problem of sustainable capacity for research for health and development in africa . carta comprises a balanced mix of institutions : established african universities with a history of producing phd graduates in public and/or population health ; promising african universities that aspire to develop multidisciplinary phd programs and research agendas and that are committed to building their institutional capacity ; established , well - managed african research institutions with longstanding research programs ; and northern partners that have longstanding research and/or training collaborations with african counterparts , a demonstrable record in equitable relationships , and a commitment to africa . they bring experience in establishing and running successful phd programs , ability to mentor both students and phd supervisors , and skills in building institutional research and training capacity . university faculty and administrative staff play a key role in graduate training and research management . yet , many of them lack opportunities to learn from best practices around the world and often work in isolation from each other . carta aims to strengthen human resources and university - wide systems critical to the success and sustainability of its program . carta will hold workshops targeted toward : ( a ) librarians , ( b ) graduate program coordinators including deans , ( c ) bursars , and ( d ) registrars . each group will attend one workshop ; thus , we envisage four workshops over the first 4 years . broad topical areas for these workshops include : information retrieval including relevant multidisciplinary literature searching , management of electronic resources , and e-publishing.higher education management training including efficient enrollment , monitoring and reporting procedures , identification of grant and fellowship opportunities for students and staff , generic professional skills training , and counseling and support services.effective student supervision , grant - writing , research management and administration , development and management of research partnerships , effective leadership , support for publication and current debates in social and health sciences.development of institutional policies and processes to support research including the establishment of institutional review boards where they do not exist , research and publication policies , and university - wide guidelines on management of externally funded research grants.strategic financial planning ; interpretation of financial information ; conducting reviews and evaluation for cost - reduction opportunities ; preparation and reconciliation of accounts ; budgetary controls , forecasting , and monitoring of cash flow ; etc.to promote the capacity of university academic and research faculty to train , supervise , and mentor graduate students , win research grants , conduct high - quality research , and manage collaborative research projects , carta will hold workshops to ensure supervisors and mentors have a clear understanding of , and commitment to , the goals of the carta program , as well as benefit from a collegial presentation of best practices in doctoral supervision and mentoring . these workshops will be developed and facilitated by local and international experts in doctoral supervision and mentoring . the joint development of joint advanced seminars ( jas ) modules will improve institutional capacity through local staff working with experts to develop the curriculum and deliver the courses . information retrieval including relevant multidisciplinary literature searching , management of electronic resources , and e - publishing . higher education management training including efficient enrollment , monitoring and reporting procedures , identification of grant and fellowship opportunities for students and staff , generic professional skills training , and counseling and support services . effective student supervision , grant - writing , research management and administration , development and management of research partnerships , effective leadership , support for publication and current debates in social and health sciences . development of institutional policies and processes to support research including the establishment of institutional review boards where they do not exist , research and publication policies , and university - wide guidelines on management of externally funded research grants . strategic financial planning ; interpretation of financial information ; conducting reviews and evaluation for cost - reduction opportunities ; preparation and reconciliation of accounts ; budgetary controls , forecasting , and monitoring of cash flow ; etc . to keep pace , african research and training institutions urgently require information , communication , and computing infrastructure ; supplementary sources of electric power supply ; and physical facilities , especially for hosting graduate seminars . strong doctoral programs are generally characterized by experienced faculty actively engaged in funded research , adequately motivated students and a stimulating institutional environment . a key element of the carta model is the selection of cohorts of candidates from various disciplines relevant to population and public health . each cohort will be fully funded through carta . to build capacity concurrently within affiliated african universities , carta will initially recruit , on a competitive basis , fellows who are junior faculty at these institutions . carta is committed to ensuring gender and regional equity in the selection of its fellows . fellows are expected to be substantially relieved of regular teaching and administrative responsibilities for the duration of their candidature . this will ensure that fellows progress through the program and thus complete their training in good time . all carta fellows are expected to obtain a phd or equivalent degree at the end of the 4-year fellowship . central to the training program of fellows will be a series of residential jas designed to enhance skills and knowledge ; guide and propel the fellows through the research process ; and provide a foundation for building networks of researchers , peers , and mentors . fellows will receive training on how to fully harness bibliographic resources and databases , retrieve web - based information , and benefit from list - serves and web - based lectures . residential training offers an advantage for the program in that students will be able to focus fully on specific program tasks , learn collaboratively , interact with local and international facilitators , and develop and consolidate professional networks . the jas will be offered once - annually for 4 years to each cohort , and will build skills and conceptual depth from year to year . specific topics to be covered will minimally include the following : jas-1 will build critical thinking , technical skills , and other core research competencies , and introduce students to the essential concepts and seminal articles of the disciplines brought together under carta.jas-2 focuses on data management and analysis . fellows will learn to use software packages for qualitative and quantitative data management and analyses . practice sessions will use real research data and current software packages for hands - on training.jas-3 will focus on data presentation , the doctoral dissertation , and scientific writing and communication skills to facilitate results dissemination and policy engagement.jas-4 will address professional development including skills necessary to raise and manage research funds , grant - writing and research management , development of courses , management of large class sizes , and supervision of graduate students . jas-4 will also be designed to serve as an opportunity for senior fellows to practice mentoring junior fellows through the discussion and laboratory sessions , software training , and general information sharing.jas activities will expose fellows to fundamental research concepts and facilitate their acquisition of necessary research skills . bringing fellows together at each jas will create a network of the next generation of african researchers , who are able to develop and lead research projects that expand the frontiers of knowledge and contribute to well - being . since they will generally come from related but different disciplinary backgrounds , fellows will develop , over the four years , a common research language and a strong understanding of their interrelatedness , and thus , engage more readily in collaborative and multidisciplinary research . jas-1 will build critical thinking , technical skills , and other core research competencies , and introduce students to the essential concepts and seminal articles of the disciplines brought together under carta . jas-2 fellows will learn to use software packages for qualitative and quantitative data management and analyses . practice sessions will use real research data and current software packages for hands - on training . jas-3 will focus on data presentation , the doctoral dissertation , and scientific writing and communication skills to facilitate results dissemination and policy engagement . jas-4 will address professional development including skills necessary to raise and manage research funds , grant - writing and research management , development of courses , management of large class sizes , and supervision of graduate students . jas-4 will also be designed to serve as an opportunity for senior fellows to practice mentoring junior fellows through the discussion and laboratory sessions , software training , and general information sharing . during each jas , different training modes will be used to facilitate student learning including formal lectures , open discussions , group work , assigned readings , tests , and examinations . fellows will also be affiliated with external supervisors who will support and mentor local supervisors where necessary . while we will apply rigorous criteria for the selection of our fellows to ensure their success , any fellows not performing well will be given further independent mentoring . following each jas , fellows will return to their respective universities to continue with their doctoral work . during the period between the jas series ( inter - jas period ) , we will implement a set of activities to keep fellows actively engaged and in continual communication with peers and mentors . we have established a carta website that will house reading materials , links to relevant online resources and tools , and other documents and information that fellows need to stay engaged with facilitators and meet clearly defined objectives and outputs . also during the inter - jas period , fellows will attend regional or international scientific conferences ; use carta materials and approaches to run training sessions for other students at their local institutions ; and may spend up to a semester as an intern at one of our northern partner institutions . deploying their skills in their research projects during the inter - jas period will equip fellows with experience needed to advance appropriately to the next jas and also transfer some of the key skills they have acquired to peers at their home institutions . further , as part of carta 's effort to strengthen the teaching and other skills of african university faculty , local supervisors will also attend the jas . the first cohort of carta fellows has been selected while the first jas will take place in march 2011 . university faculty and administrative staff play a key role in graduate training and research management . yet , many of them lack opportunities to learn from best practices around the world and often work in isolation from each other . carta aims to strengthen human resources and university - wide systems critical to the success and sustainability of its program . carta will hold workshops targeted toward : ( a ) librarians , ( b ) graduate program coordinators including deans , ( c ) bursars , and ( d ) registrars . each group will attend one workshop ; thus , we envisage four workshops over the first 4 years . broad topical areas for these workshops include : information retrieval including relevant multidisciplinary literature searching , management of electronic resources , and e-publishing.higher education management training including efficient enrollment , monitoring and reporting procedures , identification of grant and fellowship opportunities for students and staff , generic professional skills training , and counseling and support services.effective student supervision , grant - writing , research management and administration , development and management of research partnerships , effective leadership , support for publication and current debates in social and health sciences.development of institutional policies and processes to support research including the establishment of institutional review boards where they do not exist , research and publication policies , and university - wide guidelines on management of externally funded research grants.strategic financial planning ; interpretation of financial information ; conducting reviews and evaluation for cost - reduction opportunities ; preparation and reconciliation of accounts ; budgetary controls , forecasting , and monitoring of cash flow ; etc.to promote the capacity of university academic and research faculty to train , supervise , and mentor graduate students , win research grants , conduct high - quality research , and manage collaborative research projects , carta will hold workshops to ensure supervisors and mentors have a clear understanding of , and commitment to , the goals of the carta program , as well as benefit from a collegial presentation of best practices in doctoral supervision and mentoring . these workshops will be developed and facilitated by local and international experts in doctoral supervision and mentoring . the joint development of joint advanced seminars ( jas ) modules will improve institutional capacity through local staff working with experts to develop the curriculum and deliver the courses . information retrieval including relevant multidisciplinary literature searching , management of electronic resources , and e - publishing . higher education management training including efficient enrollment , monitoring and reporting procedures , identification of grant and fellowship opportunities for students and staff , generic professional skills training , and counseling and support services . effective student supervision , grant - writing , research management and administration , development and management of research partnerships , effective leadership , support for publication and current debates in social and health sciences . development of institutional policies and processes to support research including the establishment of institutional review boards where they do not exist , research and publication policies , and university - wide guidelines on management of externally funded research grants . strategic financial planning ; interpretation of financial information ; conducting reviews and evaluation for cost - reduction opportunities ; preparation and reconciliation of accounts ; budgetary controls , forecasting , and monitoring of cash flow ; etc . to keep pace , african research and training institutions urgently require information , communication , and computing infrastructure ; supplementary sources of electric power supply ; and physical facilities , especially for hosting graduate seminars . strong doctoral programs are generally characterized by experienced faculty actively engaged in funded research , adequately motivated students and a stimulating institutional environment . a key element of the carta model is the selection of cohorts of candidates from various disciplines relevant to population and public health . each cohort will be fully funded through carta . to build capacity concurrently within affiliated african universities , carta will initially recruit , on a competitive basis , fellows who are junior faculty at these institutions . carta is committed to ensuring gender and regional equity in the selection of its fellows . fellows are expected to be substantially relieved of regular teaching and administrative responsibilities for the duration of their candidature . this will ensure that fellows progress through the program and thus complete their training in good time . all carta fellows are expected to obtain a phd or equivalent degree at the end of the 4-year fellowship . central to the training program of fellows will be a series of residential jas designed to enhance skills and knowledge ; guide and propel the fellows through the research process ; and provide a foundation for building networks of researchers , peers , and mentors . fellows will receive training on how to fully harness bibliographic resources and databases , retrieve web - based information , and benefit from list - serves and web - based lectures . residential training offers an advantage for the program in that students will be able to focus fully on specific program tasks , learn collaboratively , interact with local and international facilitators , and develop and consolidate professional networks . the jas will be offered once - annually for 4 years to each cohort , and will build skills and conceptual depth from year to year . each jas will run for 35 weeks . specific topics to be covered will minimally include the following : jas-1 will build critical thinking , technical skills , and other core research competencies , and introduce students to the essential concepts and seminal articles of the disciplines brought together under carta.jas-2 focuses on data management and analysis . fellows will learn to use software packages for qualitative and quantitative data management and analyses . practice sessions will use real research data and current software packages for hands - on training.jas-3 will focus on data presentation , the doctoral dissertation , and scientific writing and communication skills to facilitate results dissemination and policy engagement.jas-4 will address professional development including skills necessary to raise and manage research funds , grant - writing and research management , development of courses , management of large class sizes , and supervision of graduate students . jas-4 will also be designed to serve as an opportunity for senior fellows to practice mentoring junior fellows through the discussion and laboratory sessions , software training , and general information sharing.jas activities will expose fellows to fundamental research concepts and facilitate their acquisition of necessary research skills . bringing fellows together at each jas will create a network of the next generation of african researchers , who are able to develop and lead research projects that expand the frontiers of knowledge and contribute to well - being . since they will generally come from related but different disciplinary backgrounds , fellows will develop , over the four years , a common research language and a strong understanding of their interrelatedness , and thus , engage more readily in collaborative and multidisciplinary research . jas-1 will build critical thinking , technical skills , and other core research competencies , and introduce students to the essential concepts and seminal articles of the disciplines brought together under carta . fellows will learn to use software packages for qualitative and quantitative data management and analyses . practice sessions will use real research data and current software packages for hands - on training . jas-3 will focus on data presentation , the doctoral dissertation , and scientific writing and communication skills to facilitate results dissemination and policy engagement . jas-4 will address professional development including skills necessary to raise and manage research funds , grant - writing and research management , development of courses , management of large class sizes , and supervision of graduate students . jas-4 will also be designed to serve as an opportunity for senior fellows to practice mentoring junior fellows through the discussion and laboratory sessions , software training , and general information sharing . during each jas , different training modes will be used to facilitate student learning including formal lectures , open discussions , group work , assigned readings , tests , and examinations . fellows will also be affiliated with external supervisors who will support and mentor local supervisors where necessary . while we will apply rigorous criteria for the selection of our fellows to ensure their success , any fellows not performing well will be given further independent mentoring . following each jas , fellows will return to their respective universities to continue with their doctoral work . during the period between the jas series ( inter - jas period ) , we will implement a set of activities to keep fellows actively engaged and in continual communication with peers and mentors . we have established a carta website that will house reading materials , links to relevant online resources and tools , and other documents and information that fellows need to stay engaged with facilitators and meet clearly defined objectives and outputs . also during the inter - jas period , fellows will attend regional or international scientific conferences ; use carta materials and approaches to run training sessions for other students at their local institutions ; and may spend up to a semester as an intern at one of our northern partner institutions . deploying their skills in their research projects during the inter - jas period will equip fellows with experience needed to advance appropriately to the next jas and also transfer some of the key skills they have acquired to peers at their home institutions . further , as part of carta 's effort to strengthen the teaching and other skills of african university faculty , local supervisors will also attend the jas . the first cohort of carta fellows has been selected while the first jas will take place in march 2011 . underlying the concept of carta is the recognition that individual african universities and countries lack the requisite human and financial resources and infrastructural capacity to deal single - handedly with the challenges facing phd training . carta makes a difference by offering a well thought through approach for rebuilding and strengthening the capacity of african universities to produce locally trained and skilled researchers and scholars . the initiative 's focus on the local production of networked and high - skilled researchers committed to working in sub - saharan africa , and on the concomitant increase in local research and training capacity of african universities and research institutes addresses the inability of existing programs to create a critical mass of well - trained and networked researchers across the continent . our focus on university - wide systems offers a potential solution for ensuring the sustainability of the carta intervention within each participating institution and to provide a basis for individuals to pursue a research and academic career in africa . the jas curriculum and the carta approach to phd training will be available to each participating university to incorporate as they see fit for phd students who are not part of the carta program . the carta approach also systematizes engagement with policymakers that augurs well for the relevance and use - value of the research undertaken . carta also addresses the increasing neglect of african university administrators and faculty in extant initiatives on the continent . the initiative 's goal of strengthening human resources and university - wide systems critical to the success and sustainability of research productivity in public and population health will rejuvenate institutional teaching , research and administrative systems , and improve the skills of faculty and administrative managers in graduate - level teaching and successful higher degree supervision , mentoring , grant application skills , research management , and the use of ict resources for knowledge management , dissemination , and information retrieval . in all , carta addresses important areas of local need and should produce well - prepared scientists and enriched institutional environments that together will strengthen doctoral training and research at participating institutions and nurture and sustain african researchers in the region . while the focus is on public and population health , success at this phase is expected to motivate expansions of the carta model to other disciplines . all authors , except undieh , are co - applicants on a grant funded by the wellcome trust to support the carta program . the consortium for advanced research training in africa has been funded by the wellcome trust ( uk ) ( grant : 087547/z/08/z ) , the department for international development under the development partnerships in higher education ( delphe ) , the carnegie corporation of new york ( grant : b 8606 ) , the ford foundation ( grant : 1100 - 0399 ) , and the bill and melinda gates foundation ( grant : 51228 ) . writing time for authors from the african population and health research center is supported by the william and flora hewlett foundation ( grant : 2009 - 4051 ) and the rockefeller foundation ( grant : 2009 scg 302 ) .

backgroundglobally , sub - saharan africa bears the greatest burden of disease . strengthened research capacity to understand the social determinants of health among different african populations is key to addressing the drivers of poor health and developing interventions to improve health outcomes and health systems in the region . yet , the continent clearly lacks centers of research excellence that can generate a strong evidence base to address the region 's socio - economic and health problems.objective and program overviewwe describe the recently launched consortium for advanced research training in africa ( carta ) , which brings together a network of nine academic and four research institutions from west , east , central , and southern africa , and select northern universities and training institutes . carta 's program of activities comprises two primary , interrelated , and mutually reinforcing objectives : to strengthen research infrastructure and capacity at african universities ; and to support doctoral training through the creation of a collaborative doctoral training program in population and public health . the ultimate goal of carta is to build local research capacity to understand the determinants of population health and effectively intervene to improve health outcomes and health systems.conclusionscarta's focus on the local production of networked and high - skilled researchers committed to working in sub - saharan africa , and on the concomitant increase in local research and training capacity of african universities and research institutes addresses the inability of existing programs to create a critical mass of well - trained and networked researchers across the continent . the initiative 's goal of strengthening human resources and university - wide systems critical to the success and sustainability of research productivity in public and population health will rejuvenate institutional teaching , research , and administrative systems .

The Consortium for Advanced Research Training in Africa (CARTA) The CARTA partnership CARTA's program of activities Strengthening university research capacity, infrastructure, and research agendas Model regional collaborative doctoral training program Conclusion: the difference CARTA will make Conflict of interest and funding

PMC4599476

this study was performed in accordance with the preferred reporting items for systematic reviews and meta - analyses ( prisma ) statement.36 ethics approval was not necessary for this study because only deidentified pooled data from individual studies were analyzed . a systematic literature search was conducted december 26 , 2014 , by searching medline via web of science , embase and journals@ovid full text via ovidsp , and the cochrane library database for trials . the search strategy in the current study was similar to those used in previous studies.37,38 rcts were eligible for inclusion if they met the following criteria : antiplatelet monotherapy versus monotherapy or dual versus monotherapy was tested in adult patients ( aged 18 years ) with ischemic stroke or tia and had a treatment duration of at least 1 year . because network meta - analysis requires a reasonably homogeneous sample,39,40 we did not include those rcts assessing antiplatelet therapy ( mostly aspirin ) versus placebo because such studies had a wide range of daily doses ( aspirin , from 75 to 1500 mg).41,42 another reason is that the evaluation of antiplatelet therapy versus placebo becomes less important . the abstracts of suitable titles were obtained and reviewed for suitability for full - text retrieval . ) using a predetermined data collection template . to resolve discrepancies about inclusion of studies and interpretation of data , a third investigator ( w.x . ) was consulted , and consensus was reached by discussion . the primary efficacy outcome was stroke recurrence , including ischemic , hemorrhagic , and unknown stroke , and fatal and nonfatal stroke . the secondary efficacy outcome was the composite outcome of vascular events and all - cause or vascular mortality . , b.z . , and x.s . ) who used the cochrane collaboration s risk - of - bias method.43 figure s1 shows the risk of bias of the included trials . network meta - analysis combines direct and indirect evidence for all relative treatment effects and provides estimates with maximum statistical power.4447 we fit the models within a bayesian framework using winbugs software ( version 1.4.3).48 the models , the winbugs codes , and r routines used in this study were open and could be found online.49 convergence was assessed by running 3 markov chains , and all results pertain to 100 000 markov chain monte carlo cycles after a 10 000-simulation burn - in phase . relative effect sizes were calculated as odds ratios ( ors ) with corresponding 95% credible intervals . we assessed the fitness of our model using the deviance information criterion , a measure of model fitness that penalizes model complexity . if the tradeoff between model fitness and complexity favored the model with assumed consistency , this model was preferred ( smaller deviance information criterion values correspond to more preferable values).50 as shown in table s3 , the assumption of consistency was supported for each outcome by a better tradeoff between model fitness and complexity ( a smaller deviance information criterion value ) when consistency was assumed rather than when it was not . we used the surface under the cumulative ranking curve , or sucra , probabilities to rank the antiplatelet therapies:47,51 sucra is a proportion expressed as the percentage of efficacy of an intervention on the outcome that would be ranked first without uncertainty , which equals 100% when the treatment is certain to be the best and 0% when it is certain to be the worst.47 the network results were assessed for consistency by comparing them with the results of pairwise meta - analyses . furthermore , we estimated inconsistency as the difference between direct and indirect estimates ( called the inconsistency factor ) and the corresponding 95% confidence interval ( ci ) for inconsistency factor in each closed loop by using the r code ifplot.fun , which could be found online.52 inconsistent loops are those that present inconsistency factors with 95% cis incompatible with zero . pairwise meta - analyses were performed by using stata ( version 11 ; stata corp ) within a random - effects framework that takes study heterogeneity into account to generate the pooled or and 95% ci . the percentage of variability across studies attributable to heterogeneity beyond chance was estimated using the i statistic . we did sensitivity analyses on the primary efficacy outcome to explore whether the results of the present network meta - analysis were sensitive to certain restrictions on the data included . those planned in advance were restricted to double - blind trials ( n=18 ) and true randomization and allocation - concealed trials ( n=16 ) . a systematic literature search was conducted december 26 , 2014 , by searching medline via web of science , embase and journals@ovid full text via ovidsp , and the cochrane library database for trials . rcts were eligible for inclusion if they met the following criteria : antiplatelet monotherapy versus monotherapy or dual versus monotherapy was tested in adult patients ( aged 18 years ) with ischemic stroke or tia and had a treatment duration of at least 1 year . because network meta - analysis requires a reasonably homogeneous sample,39,40 we did not include those rcts assessing antiplatelet therapy ( mostly aspirin ) versus placebo because such studies had a wide range of daily doses ( aspirin , from 75 to 1500 mg).41,42 the abstracts of suitable titles were obtained and reviewed for suitability for full - text retrieval . data were independently extracted and assessed by 2 authors ( f.z . and b.z . ) using a predetermined data collection template . to resolve discrepancies about inclusion of studies and interpretation of data , a third investigator ( w.x . ) the primary efficacy outcome was stroke recurrence , including ischemic , hemorrhagic , and unknown stroke , and fatal and nonfatal stroke . the secondary efficacy outcome was the composite outcome of vascular events and all - cause or vascular mortality . ) who used the cochrane collaboration s risk - of - bias method.43 figure s1 shows the risk of bias of the included trials . network meta - analysis combines direct and indirect evidence for all relative treatment effects and provides estimates with maximum statistical power.4447 we fit the models within a bayesian framework using winbugs software ( version 1.4.3).48 the models , the winbugs codes , and r routines used in this study were open and could be found online.49 convergence was assessed by running 3 markov chains , and all results pertain to 100 000 markov chain monte carlo cycles after a 10 000-simulation burn - in phase . relative effect sizes were calculated as odds ratios ( ors ) with corresponding 95% credible intervals . we assessed the fitness of our model using the deviance information criterion , a measure of model fitness that penalizes model complexity . if the tradeoff between model fitness and complexity favored the model with assumed consistency , this model was preferred ( smaller deviance information criterion values correspond to more preferable values).50 as shown in table s3 , the assumption of consistency was supported for each outcome by a better tradeoff between model fitness and complexity ( a smaller deviance information criterion value ) when consistency was assumed rather than when it was not . we used the surface under the cumulative ranking curve , or sucra , probabilities to rank the antiplatelet therapies:47,51 sucra is a proportion expressed as the percentage of efficacy of an intervention on the outcome that would be ranked first without uncertainty , which equals 100% when the treatment is certain to be the best and 0% when it is certain to be the worst.47 the network results were assessed for consistency by comparing them with the results of pairwise meta - analyses . furthermore , we estimated inconsistency as the difference between direct and indirect estimates ( called the inconsistency factor ) and the corresponding 95% confidence interval ( ci ) for inconsistency factor in each closed loop by using the r code ifplot.fun , which could be found online.52 inconsistent loops are those that present inconsistency factors with 95% cis incompatible with zero . pairwise meta - analyses were performed by using stata ( version 11 ; stata corp ) within a random - effects framework that takes study heterogeneity into account to generate the pooled or and 95% ci . the percentage of variability across studies attributable to heterogeneity beyond chance was estimated using the i statistic . we did sensitivity analyses on the primary efficacy outcome to explore whether the results of the present network meta - analysis were sensitive to certain restrictions on the data included . those planned in advance were restricted to double - blind trials ( n=18 ) and true randomization and allocation - concealed trials ( n=16 ) . fifty - five articles were reviewed by full text for details , and 31 of those were excluded . finally , a total of 24 rcts with 85 667 patients were included in the present network meta - analysis.528 tables 1 and 2 summarize the characteristics of the 24 included trials . the following antiplatelet therapies were tested in the trials : cilostazol versus aspirin ( 3 trials with 3459 patients),9,11,15 clopidogrel versus aspirin ( 1 trial with 6431 patients),8 dipyridamole versus aspirin ( 1 trial with 3303 patients),13 sarpogrelate versus aspirin ( 1 trial with 1499 patients),23 terutroban versus aspirin ( 1 trial with 19 100 patients),21 ticlopidine versus aspirin ( 4 trials with 5488 patients),5,16,18,27 ticlopidine versus clopidogrel ( 1 trial with 1151 patients),14 triflusal versus aspirin ( 2 trials with 2536 patients),25,26 aspirin plus clopidogrel versus aspirin ( 2 trials with 7340 patients),10,24 aspirin plus clopidogrel versus clopidogrel ( 1 trial with 7599 patients),19 aspirin plus dipyridamole versus aspirin ( 5 trials with 8622 patients),6,7,12,13,17 aspirin plus dipyridamole versus clopidogrel ( 1 trial with 20 332 patients),22 aspirin plus dipyridamole versus dipyridamole ( 2 trials with 3490 patients),13,20 and aspirin plus ticlopidine versus ticlopidine ( 1 trial with 270 patients).28 study selection flow diagram adapted from the preferred reporting items for systematic reviews and meta - analyses statement . baseline characteristics of included trials is indicates ischemic stroke ; ns , not specified ; ri , retinal ischemia ; tia , transient ischemic attack . antiplatelet treatments and outcomes of included trials asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . the network meta - analysis results for stroke recurrence and intracranial hemorrhage are reported in table 3 . cilostazol significantly reduced stroke recurrence compared with aspirin ( or 0.66 , 95% credible interval 0.44 to 0.92 ) and dipyridamole ( or 0.57 , 95% credible interval 0.34 to 0.95 ) , respectively . intracranial hemorrhage was also significantly reduced by cilostazol compared with aspirin , clopidogrel , terutroban , ticlopidine , aspirin plus clopidogrel , and aspirin plus dipyridamole . the size of the node corresponds to the total sample size of the treatment from all included trials . directly comparable treatments are linked with a line , the thickness of which corresponds to the total sample size for assessing the comparison . results for stroke recurrence ( upper diagonal part ) and intracranial hemorrhage ( lower diagonal part ) from network meta - analyses each cell gives an odds ratio ( or ) and 95% credible interval . in the upper diagonal part , the or compares the column condition with the row condition , and in the lower diagonal part , this or compares the row condition with the column condition . asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . similarly , cilostazol significantly reduced the composite outcome compared with aspirin ( or 0.68 , 95% credible interval 0.48 to 0.93 ) and dipyridamole ( or 0.59 , 95% credible interval 0.39 to 0.95 ) and reduced major bleeding compared with aspirin , clopidogrel , terutroban , aspirin plus clopidogrel , and aspirin plus dipyridamole ( table 4 ) . in addition , dipyridamole , ticlopidine , and triflusal caused significantly less major bleeding than aspirin ; terutroban caused significantly more major bleeding than dipyridamole and triflusal ; aspirin plus clopidogrel caused significantly more major bleeding than aspirin , clopidogrel , dipyridamole , ticlopidine , triflusal , and aspirin plus dipyridamole ; and aspirin plus dipyridamole caused significantly more major bleeding than dipyridamole and triflusal . results for composite outcome ( upper diagonal part ) and major bleeding ( lower diagonal part ) , from network meta - analyses each cell gives an odds ratio ( or ) and 95% credible interval . in the upper diagonal part , the or compares the column condition with the row condition , and in the lower diagonal part , this or compares the row condition with the column condition . asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . table 5 shows the mean values of sucra probabilities that provided the hierarchies for the efficacy and safety of the 11 antiplatelet therapies . in particular , cilostazol displayed the best risk benefit profile , with sucra probabilities of 0.9343 , 0.9252 , 0.9718 , and 0.8850 for reducing stroke recurrence , composite outcome , intracranial hemorrhage , and major bleeding , respectively . the sucra probabilities of antiplatelet therapies on efficacy and safety outcomes sucra indicates surface under the cumulative ranking curve . ranking sucra probabilities in order as the best treatment , the second best , the third best , and so on , among the antiplatelet therapies . no inconsistent loop was identified in the analyses of inconsistency factor ( figure s6 ) . we examined pairwise comparisons of all interventions with available head - to - head data . the results are presented in figures 3 through 6 . in general , the results obtained from pairwise meta - analysis closely matched those of the network meta - analysis . stroke recurrence , composite efficacy outcome , intracranial hemorrhage , and major bleeding were all significantly reduced by cilostazol versus aspirin . among the 22 pairwise meta - analyses , each of which included at least 2 trials ( figures 3 through 6 ) , one of the 2 pairwise meta - analyses compared ticlopidine with aspirin for preventing stroke recurrence ( including 4 trials , i=69.9 , p=0.019 ) ( figure 3 ) , and the other compared ticlopidine with aspirin for composite outcome ( including 4 trials , i=73.1 , p=0.011 ) ( figure 4 ) . there was no evidence of heterogeneity across trials in the remaining 20 pairwise meta - analyses . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . the pooled risk estimates did not change substantially in the sensitivity analyses on the primary efficacy outcome from both network meta - analyses and pairwise meta - analyses . tables s4 and s5 and figures s7 and s8 show the full results of the sensitivity analysis . fifty - five articles were reviewed by full text for details , and 31 of those were excluded . finally , a total of 24 rcts with 85 667 patients were included in the present network meta - analysis.528 tables 1 and 2 summarize the characteristics of the 24 included trials . the following antiplatelet therapies were tested in the trials : cilostazol versus aspirin ( 3 trials with 3459 patients),9,11,15 clopidogrel versus aspirin ( 1 trial with 6431 patients),8 dipyridamole versus aspirin ( 1 trial with 3303 patients),13 sarpogrelate versus aspirin ( 1 trial with 1499 patients),23 terutroban versus aspirin ( 1 trial with 19 100 patients),21 ticlopidine versus aspirin ( 4 trials with 5488 patients),5,16,18,27 ticlopidine versus clopidogrel ( 1 trial with 1151 patients),14 triflusal versus aspirin ( 2 trials with 2536 patients),25,26 aspirin plus clopidogrel versus aspirin ( 2 trials with 7340 patients),10,24 aspirin plus clopidogrel versus clopidogrel ( 1 trial with 7599 patients),19 aspirin plus dipyridamole versus aspirin ( 5 trials with 8622 patients),6,7,12,13,17 aspirin plus dipyridamole versus clopidogrel ( 1 trial with 20 332 patients),22 aspirin plus dipyridamole versus dipyridamole ( 2 trials with 3490 patients),13,20 and aspirin plus ticlopidine versus ticlopidine ( 1 trial with 270 patients).28 study selection flow diagram adapted from the preferred reporting items for systematic reviews and meta - analyses statement . baseline characteristics of included trials is indicates ischemic stroke ; ns , not specified ; ri , retinal ischemia ; tia , transient ischemic attack . antiplatelet treatments and outcomes of included trials asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . the network meta - analysis results for stroke recurrence and intracranial hemorrhage are reported in table 3 . cilostazol significantly reduced stroke recurrence compared with aspirin ( or 0.66 , 95% credible interval 0.44 to 0.92 ) and dipyridamole ( or 0.57 , 95% credible interval 0.34 to 0.95 ) , respectively . intracranial hemorrhage was also significantly reduced by cilostazol compared with aspirin , clopidogrel , terutroban , ticlopidine , aspirin plus clopidogrel , and aspirin plus dipyridamole . the size of the node corresponds to the total sample size of the treatment from all included trials . directly comparable treatments are linked with a line , the thickness of which corresponds to the total sample size for assessing the comparison . results for stroke recurrence ( upper diagonal part ) and intracranial hemorrhage ( lower diagonal part ) from network meta - analyses each cell gives an odds ratio ( or ) and 95% credible interval . in the upper diagonal part , the or compares the column condition with the row condition , and in the lower diagonal part , this or compares the row condition with the column condition . asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . similarly , cilostazol significantly reduced the composite outcome compared with aspirin ( or 0.68 , 95% credible interval 0.48 to 0.93 ) and dipyridamole ( or 0.59 , 95% credible interval 0.39 to 0.95 ) and reduced major bleeding compared with aspirin , clopidogrel , terutroban , aspirin plus clopidogrel , and aspirin plus dipyridamole ( table 4 ) . in addition , dipyridamole , ticlopidine , and triflusal caused significantly less major bleeding than aspirin ; terutroban caused significantly more major bleeding than dipyridamole and triflusal ; aspirin plus clopidogrel caused significantly more major bleeding than aspirin , clopidogrel , dipyridamole , ticlopidine , triflusal , and aspirin plus dipyridamole ; and aspirin plus dipyridamole caused significantly more major bleeding than dipyridamole and triflusal . results for composite outcome ( upper diagonal part ) and major bleeding ( lower diagonal part ) , from network meta - analyses each cell gives an odds ratio ( or ) and 95% credible interval . in the upper diagonal part , the or compares the column condition with the row condition , and in the lower diagonal part , this or compares the row condition with the column condition . asp indicates aspirin ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; ns , not specified ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . table 5 shows the mean values of sucra probabilities that provided the hierarchies for the efficacy and safety of the 11 antiplatelet therapies . in particular , cilostazol displayed the best risk benefit profile , with sucra probabilities of 0.9343 , 0.9252 , 0.9718 , and 0.8850 for reducing stroke recurrence , composite outcome , intracranial hemorrhage , and major bleeding , respectively . the sucra probabilities of antiplatelet therapies on efficacy and safety outcomes sucra indicates surface under the cumulative ranking curve . ranking sucra probabilities in order as the best treatment , the second best , the third best , and so on , among the antiplatelet therapies . no inconsistent loop was identified in the analyses of inconsistency factor ( figure s6 ) . we examined pairwise comparisons of all interventions with available head - to - head data . the results are presented in figures 3 through 6 . in general , the results obtained from pairwise meta - analysis closely matched those of the network meta - analysis . stroke recurrence , composite efficacy outcome , intracranial hemorrhage , and major bleeding were all significantly reduced by cilostazol versus aspirin . among the 22 pairwise meta - analyses , each of which included at least 2 trials ( figures 3 through 6 ) , one of the 2 pairwise meta - analyses compared ticlopidine with aspirin for preventing stroke recurrence ( including 4 trials , i=69.9 , p=0.019 ) ( figure 3 ) , and the other compared ticlopidine with aspirin for composite outcome ( including 4 trials , i=73.1 , p=0.011 ) ( figure 4 ) . there was no evidence of heterogeneity across trials in the remaining 20 pairwise meta - analyses . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . squares represent point estimates for effect size expressed as an odds ratio , with the size proportional to the inverse variance of the estimate . asp indicates aspirin ; ci , confidence interval ; cilo , cilostazol ; clop , clopidogrel ; dip , dipyridamole ; or , odds ratio ; sarp , sarpogrelate ; teru , terutroban ; tic , ticlopidine ; trif , triflusal . the pooled risk estimates did not change substantially in the sensitivity analyses on the primary efficacy outcome from both network meta - analyses and pairwise meta - analyses . tables s4 and s5 and figures s7 and s8 show the full results of the sensitivity analysis . our network meta - analysis provided evidence - based hierarchies for the efficacy and safety of long - term antiplatelet mono- and dual therapies among patients with ischemic stroke or tia . it overcame the major limitation of conventional pairwise meta - analyses by combining direct and indirect evidence of relative treatments in the analysis . results from this study indicated that when compared with antiplatelet monotherapy , dual therapy was not associated with a reduction in stroke recurrence and composite outcome but rather with a significant increase in the risk of major bleeding , especially aspirin plus clopidogrel . in addition , our results showed that cilostazol displayed the best risk benefit profile among the 11 antiplatelet treatments . the effects of dual therapy in short- and long - term prevention of recurrent stroke might be different . a recent meta - analysis that combined the results from 14 rcts reported that dual therapy was more effective than monotherapy in reducing the risk of early recurrent stroke in patients with an index stroke in the previous 3 days.37 the latest aha / asa guidelines also recommend that the combination of aspirin and clopidogrel might be considered for initiation within 24 hours of a minor ischemic stroke or tia.4 for long - term secondary prevention , however , the combination of aspirin and clopidogrel is not recommended by the aha / asa guidelines for routine long - term secondary prevention of stroke due to high risk of bleeding,4 which is consistent with our results . moreover , a recent pairwise meta - analysis based on 7 rcts that involved 39 574 patients with ischemic stroke or tia reported that antiplatelet dual therapy lasting > 1 year is not associated with a greater reduction in overall recurrent stroke risk than monotherapy , and that finding also supported our results.29 as far as we know , this network meta - analysis is the first to evaluate the efficacy and safety of long - term antiplatelet therapies after ischemic stroke or tia and provides the most robust evidence in support of long - term monotherapy as a better choice than long - term dual therapy . the present study indicated that cilostazol had the best risk benefit profile among 11 antiplatelet therapies and supported cilostazol as a possible therapeutic option to recommend for secondary prevention of stroke . in the casisp trial , which included 720 chinese patients with ischemic stroke within the previous 1 to 6 months , cilostazol reduced the rate of recurrent stroke compared with aspirin ( hazard ratio 0.62 , 95% ci 0.30 to 1.26 ) , although the benefit was not significant.9 the rate of any hemorrhagic event was also lower in the cilostazol group than in the aspirin group.9 the csps 2 study in 2757 japanese patients is another trial conducted in an east asian population to compare the efficacy and safety of cilostazol and aspirin in patients with ischemic stroke.11 this trial found that cilostazol significantly reduced the recurrence rate of stroke compared with aspirin ( hazard ratio 0.74 , 95% ci 0.56 to 0.98 ) and that major bleeding events occurred in fewer patients on cilostazol than on aspirin ( hazard ratio 0.46 , 95% ci 0.30 to 0.71).11 on the basis of this evidence , cilostazol has been approved by the china food and drug administration for treatment of noncardioembolic ischemic stroke ( license number h10960014 ) , and the latest chinese guidelines for secondary prevention of stroke recommends cilostazol ( 100 mg bid ) as an alternative to aspirin.53 similarly , cilostazol is used in japan for secondary prevention of stroke and is included in the japanese guidelines for the treatment of ischemic stroke.54 cilostazol is not licensed in the united states for ischemic stroke or tia treatment because the efficacy and safety of cilostazol have not been tested in non east asian patients . generalizing the effect of cilostazol to other groups can be challenging because the risk of both ischemic and hemorrhagic stroke is higher in the east asian population compared with other populations . further trials in non east asian patients are needed to confirm whether cilostazol is effective and safe as a monotherapy for long - term secondary prevention after ischemic stroke or tia . in addition , cost - effectiveness studies are also required to explore whether long - term use of cilostazol is cost - effective compared with other monotherapies . two previous network meta - analyses have been conducted to compare the effect of antiplatelet therapies after ischemic stroke or tia34,35 ; however , neither provided hierarchies for the efficacy and safety of antiplatelet therapies . in one of the studies,34 thijs et al found that the combination of aspirin and dipyridamole was more effective than aspirin , ticlopidine , and clopidogrel in the prevention of serious vascular events ; this finding was not consistent with our analysis . we consider the main reason to be that 13 of the 24 trials identified by thijs et al did not meet the inclusion criteria for our study because of placebo control or treatment duration < 1 year . in addition , the network meta - analysis by thijs et al excluded trials assessing triflusal , cilostazol , and sarpogrelate and did not report safety data.34 in the other study,35 malloy et al reported that more overall hemorrhagic events seemed to occur with the combination of aspirin and clopidogrel than with other treatments , and that finding supported our results . nevertheless , they found that aspirin plus dipyridamole was more protective than aspirin alone , which was not consistent with our results . similarly , we consider the main reason to be that 9 of the 24 trials identified by malloy et al did not meet the inclusion criteria for our study . the main strength of our study is the inclusion of 24 rcts with 85 667 patients , thus it is the largest evaluation of long - term antiplatelet therapies for stroke recurrence to date . furthermore , the network meta - analysis based on a bayesian model makes indirect comparison among multiple treatments available , especially when there are few trials for direct comparison between different antiplatelet therapies . consequently , this study can provide evidence - based hierarchies for the long - term efficacy and safety of all available antiplatelet therapies among patients with ischemic stroke or tia . first , the full - text articles reviewed were limited to english- and chinese - language studies , and that can introduce selection bias . a relevant article in french identified from the literature was not included in this study.55 nonetheless , we believe that the possibility of selection bias is reduced by the relatively large number of studies available in english and chinese . in addition , previous studies demonstrated that excluding studies published in languages other than english generally has little effect on summary effect estimates.56,57 second , not all included trials reported the results of intracranial hemorrhage or major bleeding , thus some comparisons between antiplatelet therapies for safety outcomes were not available . third , all comparisons involving aspirin plus ticlopidine are tenuous , given that only 1 small trial was included in this study , and that may affect the stability of relevant results . finally , most pairs for comparison included only 1 trial , and cilostazol versus aspirin has not been tested in non east asian patients , which might undermine the strength of our results to affect clinical practice . in conclusion , based on this network meta - analysis , we suggested that long - term monotherapy was a better choice than long - term dual therapy and that cilostazol had the best risk benefit profile for long - term secondary prevention after stroke or tia . more high - quality trials in non east asian patients are needed to determine whether long - term use of cilostazol is the best option for the prevention of recurrent stroke . this study was funded by the national natural science foundation of china ( contract no . the funder provider had no role in study design , data collection , and analysis , decision to publish , or preparation of the manuscript . sensitivity analysis on the primary efficacy outcome restricted to 18 double - blind trials , results from a network meta - analysis table s5 . sensitivity analysis on the primary efficacy outcome restricted to 16 true randomization and allocation concealed trials , results from a network meta - analysis figure s1 . cumulative probability and probability of surface under the cumulative ranking curve ( sucra ) for stroke recurrence . figure s3 . cumulative probability and probability of surface under the cumulative ranking curve ( sucra ) for the composite outcome . figure s4 . cumulative probability and probability of surface under the cumulative ranking curve ( sucra ) for intracranial hemorrhage . figure s5 . cumulative probability and probability of surface under the cumulative ranking curve ( sucra ) for major bleeding . sensitivity analysis on the primary efficacy outcome restricted to 18 double - blind trials , results from pairwise meta - analyses . sensitivity analysis on the primary efficacy outcome restricted to 16 true randomization and allocation - concealed trials , results from pairwise meta - analyses .

backgroundthe latest guidelines do not make clear recommendations on the selection of antiplatelet therapies for long - term secondary prevention of stroke . we aimed to integrate the available evidence to create hierarchies of the comparative efficacy and safety of long - term antiplatelet therapies after ischemic stroke or transient ischemic attack.methods and resultswe performed a network meta - analysis of randomized controlled trials to compare 11 antiplatelet therapies in patients with ischemic stroke or transient ischemic attack . in december 2014 , we searched medline , embase , and the cochrane library database for trials . the search identified 24 randomized controlled trials including a total of 85 667 patients with antiplatelet treatments for at least 1 year . cilostazol significantly reduced stroke recurrence in comparison with aspirin ( odds ratio 0.66 , 95% credible interval 0.44 to 0.92 ) and dipyridamole ( odds ratio 0.57 , 95% credible interval 0.34 to 0.95 ) , respectively . cilostazol also significantly reduced intracranial hemorrhage compared with aspirin , clopidogrel , terutroban , ticlopidine , aspirin plus clopidogrel , and aspirin plus dipyridamole . aspirin plus clopidogrel could not significantly reduce stroke recurrence compared with monotherapies but caused significantly more major bleeding than all monotherapies except terutroban . the pooled estimates did not change materially in the sensitivity analyses of the primary efficacy outcome.conclusionslong-term monotherapy was a better choice than long - term dual therapy , and cilostazol had the best risk benefit profile for long - term secondary prevention after stroke or transient ischemic attack . more randomized controlled trials in non east asian patients are needed to determine whether long - term use of cilostazol is the best option for the prevention of recurrent stroke .

Methods Data Sources and Search Strategy Study Selection Data Extraction and Quality Assessment Data Synthesis and Analysis Results Study Selection and Characteristics Network Meta-Analysis Pairwise Meta-Analysis Sensitivity Analysis Discussion Sources of Funding Disclosures Supporting Information

PMC4713810

patellofemoral pain ( pfp ) is a common musculoskeletal condition of the knee which is believed to occur because of lateral patellar malalignment1,2,3,4,5 . closed - chain exercises for quadriceps strengthening combined with flexibility exercises for the lower limb musculature have been widely used in pfp treatment6 . despite the clinical effectiveness of the leg press ( lp ) exercise , which along with lower - limb stretching has been shown to ease pain and promote functional ability7 , 8 , few studies have looked at the strengthening and stretching exercise s effect on patellar realignment8 . although a recent magnetic resonance imaging study identified increased patellofemoral joint contact area as a potential source of pain reduction8 , their results did not indicate any positive effects of strengthening and stretching exercise training on patellar alignment ( i.e. , patellar tilt angle ) . furthermore , there was no mention of the patellar displacement8 . the vastus medialis obliquus ( vmo ) muscle is an important medial stabilizer of the patella , and it resists the lateral pull of the vastus lateralis ( vl)9 . an in vitro biomechanical study showed that an insufficient vmo strength can reduce patellar lateral stability by 30%10 . using a regression model , another in vivo dynamic computed tomography ( ct ) study indicated the cross - sectional area of the vmo is predictive of patellar tilt11 . the role of the vmo is therefore considered important in the rehabilitation of patients with pfp , especially those with an extremely , laterally malaligned patella11 . resistance training of the quadriceps may possibly influence patellar alignment by altering the sensorimotor control of the vastus muscles or the cross - sectional area of the vmo12 , 13 . incorporating hip adduction into lp exercises is another potential way of achieving this7 , 14 . lp with hip adduction ( lpha ) within 45 of knee extension / flexion promoted a greater vmo - to - vl ratio as well as vmo hypertrophy7 , 14 . it was speculated by the authors that incorporating hip adduction is more likely to realign the patella7 , 14 . however , to the best of our knowledge , no studies have examined this issue . it remains unclear whether the degree of patellar alignment is mediated by a hip adduction exercise strategy ( i.e. , lpha ) , and if it is , to what extent . the importance of axial ct for examining the patellofemoral joint is well - established11 , 15,16,17,18 , and the ct gantry is spacious enough for the examination of dynamic movements11 . thus , a dynamic technique could be used to assess the role of the quadriceps in repositioning the patella . the purposes of this study were to investigate the effects of lp combined with lower - extremity stretching exercises on the patellar alignment in patients with pfp , and to examine the effect of lpha exercise on patellar alignment . the patellar alignment was assessed by using ct , with the quadriceps both relaxed and contracted to determine whether the exercise intervention changed both the static and dynamic patellar alignment . it is generally assumed that decreased pain is associated with changes in patellar alignment . to clarify the interrelationship between patellar alignment and clinical symptoms , pain severity it was our hypothesis that both exercises would reduce pain with substantial changes in patellar alignment , but of differing degrees . the inclusion criteria were : insidious onset of symptoms unrelated to traumatic accident ; the presence of pain for more than 1 month ; and experience of anterior or retropatellar knee pain after performing at least 2 of the following activities : prolonged sitting , stair climbing , squatting , running , kneeling , hopping / jumping , and deep knee flexing . in addition , participants had to exhibit at least 2 of the following positive signs of anterior knee pain during the initial physical examination : patellar crepitus ; pain following isometric quadriceps contraction against suprapatellar resistance with the knee in slight flexion ( clarke s sign ) ; pain following compression of the patella against the femoral condyle with the knee in full extension ( patellar grind test ) ; tenderness upon palpation of the posterior surface of the patella or surrounding structures ; and pain following resisted knee extension . patients were excluded if they had a diagnosis of other knee pathology , a history of patellar subluxation or knee surgery , central or peripheral neurological pathology , lower extremity malalignment ( i.e. , pronated foot ) , or severe knee pain ( visual analog scale : vas > 8) , or had received nonsteroidal anti - inflammatory drugs , injections , or physical therapy in the preceding 3 months . all subjects were enrolled after providing their written informed consent and the study procedure was approved by the research ethics committee of the national taiwan university hospital . using numbered opaque envelopes , 9 and 8 participants were randomly assigned to the lp and lpha intervention groups , respectively . of these , 6 and 5 patients , respectively , presented with bilateral symptoms . thus , a total of 15 and 13 pfp knees were studied in the lp and lpha groups . there were no significant differences in the basic demographic data between the groups ( table 1table 1.demographic data of the participantslplphagender ( male : female ) 2:73:5age ( years)40.3 10.538.3 11.3height ( cm)161.1 7.4163.9 7.8weight ( kg)59.1 10.555.5 8.7involved side ( bilateral : unilateral)6:35:3results are shown as mean standard deviation.lp : leg press exercise ; lpha : leg press with hip adduction exercise ) . lp : leg press exercise ; lpha : leg press with hip adduction exercise patients performed triweekly lp or lpha exercise three times a week , for a total of 8 weeks , according to the method of song et al7 . exercises were performed by using an en - dynamic track machine ( enraf - nonius b.v . , patients were unilaterally trained at 60% of 1 repetition maximum for 5 sets of 10 repetitions each . for training resistance advancement , the 1 repetition maximum was remeasured every 2 weeks , and the exercise intensity was adjusted accordingly . in addition , patients were taught to perform static stretching of their quadriceps , hamstring , calf , and iliotibial band muscles . table 2table 2.exercise regimens of lp and lpha exercisesexercise regimenlp exercise1 ) hot pack to quadriceps ( 15 minutes)2 ) lp from 45 of knee flexion to full extension 2-second concentric and eccentric contractions paced by using a metronome 60% of 1 rm for 5 sets of 10 repetitions left and right limbs alternatively trained in each exercise set 2-second break between each repetition and a 2-minute break between each set3 ) self - stretches of quadriceps , hamstrings , iliotibial bands and calf muscle groups ( 30 seconds 3 repetitions/ muscle group)4 ) cold pack to knee joints ( 10 minutes)lpha exercisesame as lp exercise with the resisted hip adduction addedlp : leg press exercise ; lpha : leg press with hip adduction exercise ; rm : repetition maximum summarizes the detailed exercise regimen . the only difference between the lp and lpha intervention was the resisted hip adduction . for lpha , patients forcefully pressed their leg with concurrent isometric hip adduction to resist 50 n resistance offered by the blue theraband ( the hygenic corporation , akron , oh , usa ) . in the lp group , the theraband was only wrapped around each patient s thigh , without resistance , to mimic the tactile received by the lpha group . during the intervention period , all participants were asked not to perform or receive any other exercise program or intervention . lp : leg press exercise ; lpha : leg press with hip adduction exercise ; rm : repetition maximum the pre- and post - training assessment of patellar alignment and pain were performed within 1 week before and after finishing 8 weeks of exercise intervention by the same physical therapist who was blinded to patient treatment . all participants underwent axial ct imaging by a pace general electric machine ( ge medical systems , milwaukee , wi , usa ) of the symptomatic knees , with their quadriceps muscle in relaxation and maximal voluntary isometric contraction for the assessment of patellar alignment . the ct images ( 5-mm slice thickness ) were obtained at full knee extension through the widest diameter of the patella , which facilitated an optimal view of the patellofemoral joint for measurement11 , 17 . imaging was performed with the subjects in a supine position , with their ankles restrained by using felt strips to prevent leg rotation . a rigid support and soft padding centricity radiology ra 600 image software ( version 6.1 ; ge medical systems ) was used to determine the patellar alignment ( i.e. , tilt and displacement ) . the mediolateral tilt of the patella was measured along the patellar tilt angle ( pta ) , which is the angle formed by the line joining the maximum width of the patella and the line joining the posterior femoral condyles3 , 19 . mediolateral patellar displacement was assessed by using the bisect offset index ( bso)3 , 19 . the bso was measured by drawing a line connecting the posterior femoral condyles and then projecting a perpendicular line anteriorly through the deepest point of the trochlear groove . when the trochlear groove was flattened , the perpendicular line was projected from the bisection of the posterior condyle line . this perpendicular line intersected the patellar width line , and the percentage of the patella lateral to the midline was calculated as the bso3 , 19 . larger values of pta and bso indicated more lateral tilt or displacement of the patella . the worst pain that subjects experienced in the week before assessment was evaluated by using a 10-cm vas , where 0 indicated no pain and 10 indicated extreme / maximal perceived pain20 . chicago , il , usa ) . for comparisons of age , body height , and weight , the independent t - test was performed . the gender and number of afflicted sides ( bilateral vs. unilateral ) were compared by using the test , with an level of 0.05 . three - way mixed analyses of variance ( anova ) were performed on both pta and bso measures with group ( lp or lpha ) as between - subject factor and assessment time point ( pre- or post - training ) and muscle state ( quadriceps relaxation or contraction ) as the within - subject factors . the vas pain score was compared between groups and assessment times by performing a 2-way mixed anova . pilot work was conducted to ascertain the reliability of patellar alignment measurement on the image . the intraclass correlation coefficient ( icc(3,1 ) ) values for between - day test - retest reliability of pta and bso measurements were 0.91 and 0.98 , respectively , indicating high reproducibility . the standard error of measurement ( sem ) was 1.2 for pta and 2% for bso . to calculate power and sample size , we used the concept of the smallest real difference ( srd = 1.96 2 sem , with sem = 1.2 from a pilot test of reliability ) for predetermining the smallest measurement change that can be interpreted as a real difference . using the srd between pre- and post - training of 3.4 for pta and assuming a standard deviation of 3 based on previously published data21 , at least 13 pfp knees per group would attain 80% power at the level of 0.05 . table 3table 3.comparison of pre- and post - training changes in patellar alignment and pain in the lp and lpha groupslp ( n = 15)lpha ( n = 13)pre - trainingpost - trainingpre - trainingpost - trainingpta ( )without contraction17.4 4.717.6 5.419.2 2.418.9 3.6with contraction 17.8 5.517.6 6.418.7 4.518.3 4.7bso ( % ) without contraction63.9 12.264.7 8.466.4 12.966.4 13.5with contraction 67.0 14.668.9 13.968.7 15.868.9 12.6vas pain score ( cm)4.65 2.182.25 2.364.55 1.902.65 lp : leg press exercise ; lpha : leg press with hip adduction exercise ; pta : patellar tilt angle ; bso : bisect offset index ; vas : visual analog scale summarizes the main outcomes of the patellar alignment measures and vas pain scores . both groups were similar with respect to the pta and bso at baseline , regardless of the quadriceps state ( relaxation or contraction ) . there were no significant interaction effects or main effects on the pta or bso ( table 4table 4.insignificant main and interaction effects from the 3-way analyses of variance ( group by time by muscle ) performed on the pta and bsosourceptabsosum of squaresdfmean squaresum of squaresdfmean squaretime0.81210.8120.00110.001time by group 1.01411.0140.00110.001error ( time)193.235267.4320.193260.007muscle0.86210.8620.02510.025muscle by group 3.66213.6620.00110.001error ( muscle)426.4782616.4030.251260.010time by muscle0.44510.4450.00010.000time by muscle by group0.17910.1790.00010.000error ( time by muscle)76.932262.9590.130260.005group37.767137.7670.00610.006error1,750.4542667.3251.208260.046pta : patellar tilt angle ; bso : bisect offset index ; df : degrees of freedomgroup refers to leg press exercise / leg press with hip adduction exercise ; time refers to pre-/post - training ; and muscle refers to quadriceps relaxation / contraction . ) , indicating there were no significant intra- or inter - group differences in patellar alignment following the exercise interventions with the quadriceps either relaxed or contracted . as for pain , no group - by - time interaction was identified with respect to the vas pain score ( f= 0.198 , p = 0.66 ) . statistically significant reductions in pain were found in both groups after the intervention ( f = 14.286 , p < 0.01 ) . the mean decrease of the vas pain score was 2.4 and 1.9 for the lp and lpha groups , respectively . the degree of pain reduction , however , was not significantly different between the groups ( f = 0.072 , p = 0.79 ) ( table 3 ) . lp : leg press exercise ; lpha : leg press with hip adduction exercise ; pta : patellar tilt angle ; bso : bisect offset index ; vas : visual analog scale pta : patellar tilt angle ; bso : bisect offset index ; df : degrees of freedom group refers to leg press exercise / leg press with hip adduction exercise ; time refers to pre-/post - training ; and muscle refers to quadriceps relaxation / contraction . quadriceps strengthening together with lower - extremity stretching exercises is one of the most common therapeutic approaches for clinically treating pfp clinically6 , 22 . consistent with a previous systematic review6 , our study provides further evidence of the effectiveness of therapeutic exercise in the reduction of pain . both the lp and lpha exercise interventions for 8 weeks were found to significantly reduce pfp , but this occurred in the absence of significant changes in patellar alignment . furthermore , hip adduction had no added effect on patellar realignment or pain relief . despite the fact that patellar malalignment is thought to contribute to pfp1,2,3,4,5 , our findings are in agreement with a recent report that weight - training exercise did not change the patellar tilt angle8 . imaging techniques are clinically helpful in the diagnosis of patellar malalignment associated with pfp3 , 19 . the pta and bso measured in this study similar in magnitude to the values reported by previous studies8 , 13 , 19 , 23 . using imaging techniques to explore the effect of exercise on patellar alignment , an early radiographic study had examined the effects of individualized and progressive therapeutic exercises on patellar alignment at 30 of knee flexion in patients with pfp24 . the findings revealed that patients who were pain - free after exercises demonstrated significant mean decreases in patellofemoral congruence angles , but not in the patellofemoral index ( an indicator of patellar tilt ) . it is noteworthy that the patients in that study exhibited a mean congruence angle of 8.03 before training , leaving high scope for improvement in patellar displacement . it is difficult to compare our findings with those of the cited studies mentioned , because the manner of exercise , imaging techniques , indices chosen for assessing patellar alignment , and knee flexion angle tested were different . however , it also appears that patients who have a certain patellar malalignment type may respond differently to exercise intervention . quadriceps contraction plays an important role in mediating the patellar alignment during full knee extension , when the patella is less stable15 , 25 . in the present study , this finding is similar to the conclusions of previous studies that found no difference in lateral patellar tilt or displacement between quadriceps states in extended knee positions11 , 15 , 17 . another study also indicated there was a significant and proportional relationship between the ct measurement of the lateral patellofemoral angle and lateral patellar shift during quadriceps contraction or relaxation at either 0 or 20 of knee flexion ( r = 0.930.96 , p < 0.01)16 . conversely , some investigators have reported that quadriceps contraction results in increased patellar lateralization or lateral tilt11 , 18 , 23 , 26 . hence , at the present time , there is no consensus about the effect of quadriceps contraction on patellar alignment26 . resistant hip adduction ( lpha ) was additionally performed to specifically target the vmo , because vmo dysfunction is manifest in patients with pfp27 . the effects of the 2 interventions were comparable , indicating that there is no additive beneficial effect of incorporating hip adduction into the lp exercise , at least with regarding to patellar alignment . adding isometric hip adduction to the lp exercise did not elicit more vmo hypertrophy than did lp alone7 , although it is possible that the simple lp exercise simultaneously activates the hip adductor magnus and longus muscles as well as the knee extensor28 . it should be noted that the hip adduction load was fixed ( i.e. , 50 n ) in the present study , and vmo activation may vary with different levels of hip adduction load29 . on the other hand , squat exercise with hip adduction offers an alternative method of strengthening the vmo29 , 30 . although no significant changes in patellar alignment were detected following both exercises , clinically significant pain reductions ( > 1.5 points on the 10-cm vas)20 were detected in both groups . this finding is in accordance with that from a patellar bracing study conducted by powers et al.19 , who reported large decreases in pain without obvious changes in patellar alignment . we speculate that the decrease in pain detected in our patient cohort was probably the result of increased quadriceps muscle strength and enhanced flexibility of the soft tissue surrounding the knee7 , 31 , 32 . it is likely that quadriceps strength and patellofemoral joint contact area were altered by the exercise intervention , leading to redistributing joint contact pressure , and that pain was decreased as a consequence8 . furthermore , it is known that pfp is a chronic pain condition in which central pain mechanisms may be important33 , 34 . hence , the repeated and rhythmic lp training and lower - extremity stretching ( as a form of proprioceptive input ) , the patients belief in treatment or therapist , and accompanying cognitive or illness behavior changes ( through psychophysiological mechanism ) may evoke neuroplastic changes in the central nervous system , and consequently , alter the pain34 . lateral patellar tilt and displacement were assessed only in the fully extended knee position , and patellar alignment may exhibit changes at different knee flexion angles . future studies should examine patellar alignment changes during dynamic tasks and during weight - bearing movements , because femoral motion may influence the patellofemoral kinematics4 . it would be of interest to ascertain whether patients with different types of malalignment ( e.g. , lateral displacement alone , lateral tilt alone , or a combination of both ) would show different responses in terms of patellar movement in response to therapeutic exercise training . such classifications may facilitate the clinical management of pfp in a more selective manner . furthermore , because hip external rotator and abductor muscle strengthening exercises are effective in pfp management35 , future studies should examine their biomechanical influences on patellar alignment . in conclusion , although lp and lpha exercises were both effective at reducing pfp , neither of them were beneficial for realignment of the patella . the results indicated that patellar realignment does not appear to mediate pain alleviation , since adding hip adduction to lp had no more beneficial effect on patellar realignment or pain reduction than lp exercise alone .

[ purpose ] the purpose of this study was to investigate the effect of leg press and leg press with hip adduction exercise training on patellar alignment and pain in patients with patellofemoral pain ( pfp ) . [ subjects and methods ] seventeen patients participated in this study . eight weeks of leg press or leg press with hip adduction training , including progressive lower - limb weight - training and stretching , was given . patellar alignment ( tilt and displacement ) and pain measurements were conducted before and after leg press or leg press with hip adduction training . patellar tilt angle and the bisect offset index were measured on axial computed tomography scans of the fully extended knee position with the quadriceps relaxed and contracted . pain was assessed by using a 10-cm visual analog scale . [ results ] no differences were found in patellar tilt and displacement with the quadriceps either relaxed or contracted after leg press and leg press with hip adduction . however , significant pain reduction was evident in both leg press and leg press with hip adduction . [ conclusion ] the results indicated that patellar realignment does not appear to mediate pain alleviation . furthermore , hip adduction in addition to leg press training had no additive beneficial effect on patellar realignment or pain reduction .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2323520

despite the general use and success of systemic therapies , there has always been an interest in maximizing drug availability in target tissues and minimizing adverse reactions . as a result , many simple , local , and highly effective drug treatments have been developed including drops for eyes and ears , puffers for the respiratory tract , sprays for the sinuses , creams for skin and mucosa , central nervous system shunts and other implants . local therapy , where appropriate , allows maximum bioavailability in target tissues while minimizing systemic interactions and allowing for the lowering of drug dosages needed for therapeutic effectiveness . interest in the development of new drugs is therefore balanced by the increasing emphasis placed on novel delivery systems ( 1,2 ) . such technologies have been subject to intense development over the past decade and now occupy a multibillion dollar per annum industry in the united states ( 1 ) . in particular , polymers capable of controlled pharmaceutical release are of significant interest and offer to expand the range of diseases amenable to more localized treatment . the most classic systems rely upon temperature , ph , magnetic fields , light , mechanical force , or ultrasound ( as in the presently described system ) to cause a change that facilitates drug release by diffusion ( 3,4,5,6 ) . such " smart polymers " often work well in vitro but demonstrate severe limitations when constrained to more physiological conditions . as a simple alternative , we propose a drug delivery vehicle based upon calcium carbonate powder suspended in particles of a calcium alginate hydrogel matrix . alginate hydrogels are soft and non - toxic polymers traditionally used as food thickeners , but have more recently found applications in both industrial and medical encapsulation technologies for controlled release ( 7 ) . calcium carbonate powder is insoluble , non - toxic , and known to be susceptible to ultrasound ( 8) . the combination of these two inexpensive materials produces a medically useful hydrogel matrix that can be disintegrated with high intensity sound waves . so far as the authors are aware , this is the first attempt to combine these materials for the purposes of creating an ultrasound activated drug delivery device . a co - axial airflow encapsulation nozzle was constructed as described by fiszman et al ( 7 ) . the authors varied the flow rates described below until macroscopic particles of desirable diameter were attained . it was thought that a 1 mm spherical diameter offered balance between a small enough object to theoretically inject and yet large enough to work with on a macroscopic scale . briefly , a 16 gauge needle , connected to a peristaltic pump via lab tubing , was passed through a inch " t " junction and secured in place , thus plugging one of the t 's three ports . to the horizontal inlet was attached an air hose and pump such that air could be forced around the 90 degree bend and over the needle tip . via a procedure modified from yang ( 9 ) , a 1% ( m / v ) algin solution was prepared by dissolving 1000.0 mg of sodium alginate ( sigma ) in 100.0cc of distilled water with intense agitation via vortex for 30min . preparations were made including 50 g , 75 g and 100 g of calcium carbonate ( sigma ) in the sodium alginate solution to create final 50% , 75% and 100% ( m / v ) mixtures . suspension was forced through the needle by a peristaltic pump at a rate of 8cc / min while air flowed around the tip at 4l / min , creating enough shear force to convert the liquid stream into fine droplets . these fell 10 cm into a 4% ( m / v ) cacl22h20 hardening solution below . four hours were allowed for full polymerization of the particles before they were spun out of solution by gentle centrifugation and re - suspended in normal saline after three washes . spheres produced by the above method measured approximately 1 mm in diameter and were of an opaque white color . three particles of each final calcium carbonate concentration were separated into each of three plastic centrifuge tubes and used for the sonication trials . the tubes were subjected to bursts of ultrasound at 85 watts , one second on and one second off , until fully disintegrated . similarly , an identical set of three tubes , three alginate spheres per tube , were agitated by vortex for a period of twenty minutes to gain a rough assessment of the product 's mechanical stability without the use of ultrasound disruption . using the same parameters of three particles per plastic tube and 85 watt ultrasound bursts of 1 second on / off , the time necessary to disintegrate the particles was found as a function of calcium carbonate concentration in the initial encapsulation mixture ( see table 1 ) . disintegration of the spheres was determined qualitatively by viewing the attained product under a microscope . similarly , three spheres of each of the three calcium carbonate concentrations were subjected to three trials of agitation by vortex . in each case , no signs of particle disintegration could be observed after thirty minutes ( see table 2 ) . it is clear that an increasing concentration of calcium carbonate leads to a decrease in the time necessary for particle disintegration . increasing the concentration of calcium carbonate both raises the amount of material in each sphere sensitive to ultrasound and reduces the relative concentration of alginate . we experimented with a range of values before running final trials at 50% , 75% and 100% calcium carbonate . using calcium carbonate concentrations of less than 50% drastically increased the time necessary for ultrasound disruption , while concentrations greater than 100% increased viscosity of the mixture and made extrusion through the needle very difficult . time - dependent diffusion of substances through an alginate matrix has been previously demonstrated , with diffusion times varying over minutes to hours depending upon the weight of the molecule in question and the initial concentration of sodium alginate used to make an encapsulation polymer ( 10 ) . while we may reasonably conjecture that the particles generated in this study would share diffusion characteristics with those published , our initial goal was to dry particles in order to create a hard external calcium carbonate diffusion barrier . drying particles in an incubator , however , generated a product that adhered too firmly to the drying surface to be removed without damage . . limitations of this study include the lack of a quantitative method to determine the rate of sphere disintegration as a function of the number of ultrasound bursts to which they were subjected . second , a method to quantify the amount of agitation delivered would be helpful so that the maximum mechanical disruption tolerated by spheres can be identified . finally , although the coaxial airflow technique described in this study is a simple and widely used system of generating even micro - scale hydrogel particles , we found the reproducibility of particle shape and size to be limited from batch to batch . there are techniques available which offer greater reproducibility of product and smaller sphere size , albeit at much greater cost ( 11 ) . we have demonstrated a process by which routinely available and non - toxic materials can be used to create particles of encapsulated calcium carbonate in a calcium alginate hydrogel . the resulting product is easily disrupted with ultrasound and yet retains structural integrity under agitation by vortex for an extended period . there are logical implications for these particles to be injected into a target tissue under image guidance and subsequently identified via fluoroscopy or ct scan . destruction of particles with therapeutic ultrasound and the controlled release of encapsulated drugs is a reasonable future experimental goal . our next set of experiments will involve a system to dry spheres while they are simultaneously agitated . it will thus be possible to quantify the diffusion characteristics and parameters of ultrasound disruption on dried particles . we are also working with microscopes that can photograph particles , the disruption products , as well as potentially measure the reduction of particle radius as a function of ultrasound delivery .

a calcium carbonate suspension , encapsulated within particles of calcium alginate hydrogel , is proposed as a drug delivery device susceptible to ultrasound disruption . spheres approximately 1 mm in diameter were prepared by the coaxial airflow method from mixtures of 1% sodium alginate ( m / v ) and each of 50% , 75% and 100% calcium carbonate ( m / v ) in distilled water . this product was subjected to cycles of 85 watt ultrasound in 1 second on / off bursts via a lab sonication system until fully disintegrated , a process requiring between 8 and 20 minutes depending upon initial calcium carbonate concentrations . the spheres subjected to vortex did not demonstrate any signs of mechanical degeneration after 30 minutes . before use as a model implant , further work is required to develop a method of drying the particles to make them impermeable to drug diffusion before the time of their disruption with ultrasound .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC2855448

cryptorchidism ( from the greek kryptos , meaning " hidden , " and orchis , meaning " testis " ) refers to the absence of a testis from the scrotum . isolated cryptorchidism is the most common congenital anomaly of the male genitalia , affecting almost 1% of full - term infants at the age of 1 year . during embryonic life , the testes form beside the mesonephric kidneys and descend via the inguinal canal to the scrotum . if this process is faulty , a cryptorchid testis may halt along the normal path of descent ( undescended or retractile testis ) , may travel off the normal path of decent ( ectopic testis ) , or may die or never develop ( absent testis ) . the history of the study of cryptorchidism and the first attempt to correct it began in the 18th century . like other areas of medicine , which is a combined action of art and science , the progress of techniques in orchiopexy has been supported by an improved understanding of cryptorchidism . in this context , this review aimed to describe the historical landmarks in the progress of orchiopexy in parallel with the growing knowledge of cryptorchidism that had spurred the technical advances in orchiopexy . the theoretical bases that justify orchiopexy in patients with cryptorchidism originated from some critical observations by two pioneers in the 18th century , baron albrecht von haller and john hunter . over the ensuing years , theories on the mechanism of descent , and study of the histological and physiological alterations in the cryptorchid testis came out , aiding the development of orchiopexy . baron albrecht von haller , who became the chairman in anatomy and surgery at gttenger university in the 1730s , described the abdominal position of the fetal testis in his famous work ' opuscula pathologica , ' published in 1755 . in the chapter regarding congenital hernia , he accurately indicated the presence of abdominal testis , though he did not know the exact timing of testicular descent . also , his explanation of the phenomenon responsible for the descent of the testis was wrong . however , it was important that his description attracted the interest of john hunter , who later made some great observations that still hold true today . john hunter is known as one of the fathers of modern surgery and anatomy . he was born on a small farm near glasgow , scotland , in 1728 , the youngest of 10 children . among his 10 siblings , he and his older brother william made a significant contribution to medicine . john hunter was a brilliant anatomist with unending curiosity , a variety of interests , and a colorful character . although william wanted his younger brother to be a classic gentleman , his character made him a house - surgeon and later a partner in the anatomy school . he was the leading expert in the study of comparative anatomy , infectious diseases , and gunshot wounds . john hunter began to study on the descent of fetal testis with an interest in baron von haller 's observations . in 1762 , hunter confirmed the abdominal position of the fetal testes as well as the neurovascular supply and the cremaster or musculus testis ( a more proper name , according to hunter ) . through postmortem dissection he did not agree with the current suggestion that the testis was forced into the scrotum by the compressive force of respiration , or pulled by the cremaster muscle . instead , he proposed the significance of gubernaculum as a helm or rudder for testicular descent . although the precise cause of failure to descend has not been clearly understood at this time , his association of cryptorchidism with faulty testis appears interesting , as he stated that : " when one or both testicles remain through life in the belly , i believe that they are exceedingly imperfect and probably incapable of performing their natural functions , and that this imperfection prevents the disposition for descent from taking place . " he believed in the necessity of treatment of undescended testis after a period of patient observation . he wrote " as this progress is very slow , especially when the testicle is creeping through the ring , a doubt often arises whether it is better entirely to prevent its passage or to assist it by exercise or other means ; and it would certainly be the best practice to assist it , if that could be done effectually and safely . " hunter 's accurate description of fetal testicular descent made great progress in the understanding of cryptorchidism . although testicular decent and maldescent are now being explored on the molecular level , most of our knowledge on cryptorchidism is still based on his observations . furthermore , even though many theories such as abdominal pressure , endocrine factors , cremasteric muscle contraction and gravity have been proposed to explain testicular descent , none is more important than the gubernaculum as hunter claimed more than 2 centuries ago . after the great discovery of hunter , the discussion of the nature of the gubernaculum , the role of the cremaster , and the process of descent continued . in 1866 thomas b. curling summarized what was known at that time regarding undescended testicles in his book , a practical treatise on the diseases of the testis . some of his observations hold true today , such as abnormal testicular function in undescended testis and proper evolutionary time limits to recovery of retained testicles . he also summarized the possible cause of the retained testicle : either the defective development of the cremasteric muscle , adhesions secondary to peritonitis , or a contracted external ring . although he reported some misconceptions in current understandings , his observations surely gave a theoretical basis on which later doctors attempted the first orchiopexy . the structure of the gubernaculum , or genitoinguinal ligament , was first described and named by hunter in 1762 . although most authors believe that it contributes significantly to testicular descent , there is little consensus on the mechanism involved . controversies around gubernaculum were related to its tail structures and its role in the development of ectopic testis . in 1840 , curling noted the gubernaculum to be a soft , solid protruding body that varied in shape and size at different stages of testicular descent . he also claimed that the gubernaculum terminated in three muscular processes , which he could trace before , during and after testicular descent . he listed these as ( i ) external - connected to poupart 's ligament ; ( ii ) middle - through the external ring to the bottom of the scrotum ; and ( iii ) internal - to the os pubis and the rectus muscle sheath . since the gubernaculum contains muscle that is likely to contract , this led lockwood to propose the ' traction theory ' of testicular descent . following the examination of eight fetuses ( 7 weeks ' gestation to full term ) , he noted the change of distal gubernaculum from a soft , jelly - like mass into a leash of fibers that spread out to several areas and pulled the testis into the scrotum . sometime later , his description of the fibers became known as the ' tails of lockwood ' and the presumed cause of testicular ectopia ( fig . although lockwood himself did not mention that abnormal development of the gubernacular tail could be the cause of testicular ectopia , some authors referred to lockwood 's work as an explanation for testicular ectopia [ 9 - 11 ] . in the early 20th century , the concept of gubernacular tails as a cause of testicular maldescent prevailed . coley found many similarities between curling 's and lockwood 's theories and tried to amalgamate both theories . sonneland could not find any corroborative evidence either embryological or anatomical for the existence of multiple processes . instead of multiple gubernacular tails , he claimed a singular gubernacular process and believed that the fibrous attachment of ectopic testis was just a result and not the cause of testicular maldescent . mcgregor also failed to find any subdivision of the gubernaculum and concluded that lockwood 's theory was unproved and proposed the ' third inguinal ring ' or congenital fascial packets or barriers as a cause of testicular ectopia . he described the gubernaculum as consisting only of mesenchyme ( not muscles and fibers , as previous believed ) , which formed a column in the abdomen at taching the testis to the inguinal region and then to the floor of the scrotum . his theory was that the gubernacular mesenchyme could be disrupted by surrounding fibrous tissues to cause undescended or maldescended testis . he proposed that ' a fibrous band is formed ( a " tail of lockwood " ) which anchors the gubernaculum and later the testicular apparatus to the surrounding tissues , and , by virtue of the processus vaginalis having being formed normally elsewhere , the descending testis is diverted from its course in the direction of the band . this then is the aetiology of an ectopic testis and also the formation of the tails of lockwood , which are a pathological feature rather than a normal component of the gubernaculum . ' according to his theory , the tail of lockwood indicated either a gubernacular process or an abnormal fibrous attachment , causing great confusion . by the mid 1980s most investigators completely rejected the theory of multiple gubernacular tails providing traction to cause testicular descent . in 1987 , heyns published a landmark paper describing testicular descent in 178 human fetuses , the largest number of fetuses examined at the time . what he found was no inclusion of muscle in the gubernaculum and singular rather than multiple processes of distal gubernaculum . at present , many studies have now shown conclusively that multiple gubernacular processes ( or tails ) do not exist and that the distal gubernaculum is unattached to surrounding tissues during inguinoscrotal migration in both rodents and humans . attachment to the scrotum ( or elsewhere ) occurs only after normal ( or abnormal ) testicular descent is complete . although some recent papers still referred to tails of lockwood as a cause of ectopic testis and even transverse testicular ectopia , it is certainly time to dispel the myth of the tails of lockwood as a cause for ectopic testis . before the periods when orchiopexy was widely accepted , an inguinal ascended testis was managed primarily with the use of truss or castration . it was said the surgery for correction of undescended testis was attempted by several german doctors such as j.f . however , the first recorded attempt was performed by james adams in the london hospital in 1871 on an outpatient . he proposed 3 reasons to operate on an undescended testis : poor scrotal development due to cryptorchidism , the risk of atrophy of the abdominal testis , and the likelihood of injury or pain associated with the abnormal location . he reported on one 11-week - old patient who was referred for an empty left scrotum , normal right testicle and an " oval swelling in the perineum , to the left of the middle line .. in front of the anus . " adams performed the orchiopexy with curling through a 1.5 inch incision over the external ring . the tunica vaginalis was uninjured and a catgut suture was used to affix the testicle into the scrotal pouch , after which the wound was closed . owing to the high prevalence of erysipelas in the hospital , the operation was performed on an outpatient basis and the child was sent home . nevertheless , the patient developed a wound infection on postoperative day 3 that progressed to fatal erysipelas . adams personally performed the autopsy and concluded that " death was caused by peritonitis commencing in the tunica vaginalis and extending upwards . " the importance of identifying and ligating a patent processus vaginalis during orchiopexy was not recognized at that time . in adams concluded that " no operation should be undertaken in early life " because of the high likelihood of wound infection and subsequent peritonitis , unless the patient is " destined to become an equestrian . " because most orchiopexies at that time were conducted for a perineal ectopic testis , which could have caused significant discomfort to the horseman , he seemed to suggest orchiopexy limited to this kind of occupation . thomas " tommy " annandale was born at newcastle - on - tyne on february 2 , 1838 . at the age of 15 years tommy began work as an apprentice to his father , and at age 18 he matriculated at the university of edinburgh , receiving a doctorate in medicine 4 years later in 1860 . for his surgical training , he remained in edinburgh under the tutelage of james syme . then he served as private assistant for 10 years until syme 's death in 1870 . at that time , he became acquainted with joseph lister , who had already been the senior assistant under syme . besides , he also got the idea of antiseptics by using a carbolic acid wound dressing from lister . the concept of an antiseptic wound dressing technique , first reported by lister , was revolutionary at that time , significantly reducing the risk of wound infection . in 1877 , he succeeded joseph lister as the regius professor ( chair ) of clinical surgery at the university of edinburgh . he occupied the surgical chair at edinburgh for 30 years , from 1877 to 1907 , maintaining an active surgical practice in general surgery , orthopedics , otolaryngology , urology , and other subspecialties . on june 1877 , annandale was referred a 3-year - old boy with pain in the perineum on walking and running . annandale described the care of this patient in ' the british medical journal ' in 1879 , detailing the first recorded successful orchiopexy . the patient had a right ectopic testis palpated on ipsilateral perineum . on july 5 , 1877 , he did orchiopexy after freeing the testicle and gubernaculum from its attachment . annandale credited curling , who had attempted to perform orchiopexy with adams , with the idea of anchoring the testis to the bottom of the scrotum , and his patient further benefited from the application of lister 's antiseptic technique as a dressing of carbolic acid ( phenyl alcohol ) was applied to the wound . unlike the cases of adams , the postoperative course was " satisfactory in every way , " possibly due to the proper use of antiseptic techniques as annandale has written " the whole of the operation was performed antiseptically . " unquestionably , in the right place and at the right time , annandale integrated the art and science of medicine . he recognized the importance of lister 's work and curling 's previous experiences , and fused their ideas into the first successful orchiopexy . following the successful orchiopexy by annandale , several authors refined the techniques , which helped to give birth to the current technique . max schller , in the 1881 annals of anatomy and surgery , wrote an extensive treatise on undescended testis that included a description of the malignant potential of cryptorchidism . in describing the surgical technique , he first advocated the division of the processus vaginalis to mobilize the spermatic cord in correction of the malpositioned testis . additionally , he stressed the full division of cremaster upon the testicle and obstruction of the inguinal canal to reduce the re - ascent of testis . in contrast to schller 's technique , in 1893 , leonard bidwell , assistant surgeon of the west london hospital , described a technique for inverting the testis to gain approximately an inch and a half of length and anchoring the testis to an external wire cage to provide continuous traction . arthur dean bevan was professor and head of the department of surgery at rush medical college in chicago and later president of the american medical association and the american surgical association . in 1899 , he brought the schller 's concepts , such as division of the processus vaginalis , to the united states for the first time and took another step further , emphasizing the tension - free mobilization of the testis to the scrotum by releasing the spermatic vessels to the retroperitoneum and possible resection of them to gain further length . he claimed that the need for spermatic vessel division would be lessened after increased study and experience . he also described sewing the deep layer of the superficial fascia to the aponeurosis of the external oblique to prevent retraction of the testis , using a purse - string technique . with these kinds of modifications , he reported his results in over 400 cases with an overall success rate of approximately 95% . his statement was further supported by eisendrath , who showed a 90% occurrence of seminiferous tubule atrophy by 2 years of cryptorchidism . this was followed by the landmark animal study by carl moore , who observed loss and recovery of testicular function after surgical cryptorchidism and orchiopexy , respectively . from bevan 's work , three main issues concerning the surgical treatment of the undescended testis became apparent : the requirement for mobilization of the cord , the questionable necessity of division of the spermatic vessels to gain additional cord length , and the debate between traction and tension - free repositioning of the testis within the scrotum . in the beginning of the 20th century , several concepts concerning cryptorchidism that are currently useful for understanding the disease were accepted , such as the prevalence of hernias , the possibility of torsion , the malignant potential of the " arrested " testis , and functional limitations of cryptorchid testis , both in terms of spermatogenesis and hormone production . these were well summarized by eccles in a lecture named " the anatomy , physiology , and pathology of the imperfectly descended testis " . they fixed the testis to another site , such as the fascia lata or contralateral testis , for the purpose of possible lengthening of the cord . torek in newyork and keetley in england independently reported the technique of fixation in the fascia lata in a similar period . the testis was recommended to be kept in situ for 3 to 6 months and then detached carefully and repositioned in the scrotum . torek also reported 64 cases of successful staged operation , which did not need to divide the spermatic vessels . the concepts of continuous traction were revisited by cabot and nesbit of michigan university again with the use of a rubber band and wire cage for approximately 12 days . although there is enthusiasm for one stage repair of orchiopexy , some difficult cases with short testicular cord will benefit by the judicious use of staged operations with traction of the testicular cord for the time being . robert j. prentiss of san diego county hospital added additional technical insight with his detailed depiction of the surgical anatomy of the spermatic vessels and the anatomic proof that relative lengthening of the spermatic cord could be achieved by division of the inferior epigastric vessels and medial displacement of the spermatic vessels . the current method of testicular fixation within the subdartos pouch was first described by schoemaker in 1932 but was popularized by john k. lattimer , at columbia university , in 1957 . he also worked out a way to implement gentle traction via an elastic band anchored in the vicinity of the patient 's knee for 10 days . with the inclusion of the subdartos pouch technique , the standard orchiopexy can be applied to almost all undescended testes with the exception of high undescended testes . therefore , attention has turned to the treatment of high undescended testes which were not adequately treated by standard orchiopexy . in 1979 , jones and bagley suggested a high inguinal incision as the open surgical alternative for high canalicular or intraabdominal testes . a transverse incision is made medial to the anterior superior iliac spine and carried down to the external oblique fascia just superior to the internal ring . this incision made it possible to approach the peritoneal cavity easier than through a standard inguinal incision . the point is the preservation of the spermatic vessels , high retroperitoneal mobilization of the spermatic vessels , and passage of the testis directly through the abdominal wall at the pubic tubercle ( prentiss maneuver ) . this procedure shares similar indications and surgical principles with laparoscopic orchiopexy and was the popular surgical approach before the advent of laparoscopic management of an intra - abdominal testis . in case of a high undescended testis , the testicular artery and veins often limit the distal mobility of these testes . as mentioned earlier , attempts to divide the testicular artery were made well before the 20th century . however , a high atrophy rate precluded wide application . in 1959 , fowler and stephens studied the vascular anatomy of the testis and devised a means to repair a high undescended testis and preserve its blood supply via collateral circulation . children with a long , looping vas that extends down the inguinal canal are the ideal candidates for this surgery , but less than one third of the children with intraabdominal testes were found to have this condition . originally , fowler and stephens orchiopexy was known as a staged technique but it was further modified into a 2-staged operation with a better success rate ( 77% vs. 67% ) . prior to 1976 , the non - palpable testis was only located by inguinal exploration . however cortesi et al first described the laparoscopy as a modality that could reveal the location of non - palpable testis . with increased experience therapeutic laparoscopy has the advantage of 1 ) high magnification and improved visualization 2 ) capability of extensive vascular dissection up to the origin of gonadal vessels , 3 ) minimal morbidity , and 4 ) the ability of creating a new internal ring medial to inferior epigastric vessels to achieve the straight vascular course to the scrotum . laparoscopic orchiopexy can be conducted as either one - stage orchiopexy with preservation of spermatic vessels or fowler and stephens orchiopexy . while current orchiopexy includes a variety of methods , all methods stem from the basic concepts of standard orchiopexy . more than 200 years ago , the discussion of cryptorchidism began and the surgical techniques and philosophies have continued to evolve . the current technique of standard orchiopexy is the end result of evolved concepts . a study of the history of surgical management of the undescended testis sheds light on the rationale behind the current management .

the history of treatment for cryptorchidism dates back more than 200 years . this review is intended to highlight some historical aspect that led us to our current surgical treatment of this condition . the medical and historical surgical literatures pertaining to cryptorchidism were reviewed . data sources were pubmed , embase , conference proceedings , and bibliographies . no language , date , or publication status restrictions were imposed . the study of cryptorchidism began with the anatomical descriptions of baron albrecht von haller and john hunter . attempts at surgical correction of the undescended testis began in the early 1800s , culminating in the first successful orchiopexy by thomas annandale in 1877 . max schller , arthur dean bevan and lattimer contributed to the establishment of current techniques for standard orchiopexy . later , laparoscopy , high inguinal incision ( jones ' approach ) and scrotal approach were added to the list of current orchiopexy .

INTRODUCTION THE ERA OF PIONEERS UNDERSTANDING AND CONTROVERSIES ABOUT GUBERNACULUM THE FIRST ATTEMPT AT SURGICAL CORRECTION OF CRYPTORCHIDISM ANNANDALE'S SUCCESS FOR 1 ESTABLISHMENT OF MODERN STANDARD ORCHIOPEXY: INTEGRATING MORE KNOWLEDGE INTO PRACTICE ESTABLISHMENT OF CURRENT ORCHIOPEXY CONCLUSIONS

PMC3857152

bacterial resistance to -lactams and -lactamase inhibitors is an ever - increasing problem that compromises their clinical utility . among gram - negative bacteria , of particular concern are enzymes able to target the expanded - spectrum -lactams , including the ampc enzymes ( class c cephalosporinases ) , the so - called extended - spectrum--lactamases ( esbl ; classes a and d ) , and the carbapenemases , which hydrolyze most -lactams , including the carbapenems ( classes a , b , and d).1 in order to restore their antibacterial activity against gram - negative pathogens , -lactams have been paired with inhibitors of -lactamases . those currently used in the clinical setting ( clavulanate , tazobactam , and sulbactam ) have a spectrum of inhibition essentially covering class a enzymes . all three marketed inhibitors contain a -lactam core and share a similar mechanism of inhibition . they react with serine enzymes to form a covalent acyl - enzyme intermediate ; opening of the four - member -lactam ring is followed by considerable molecular rearrangement before hydrolysis to regenerate the active enzyme.2 avibactam ( nxl104 ) is a non--lactam -lactamase inhibitor that displays a broad - spectrum inhibition profile , with potent inhibition of class a , class c , and some class d enzymes . the inhibitor is characterized by high carbamylation efficiency and slow decarbamylation , resulting in a long half - life of the inactive covalent adduct.3 in addition , the decarbamylation step results in regeneration of intact avibactam , and not hydrolysis.4 avibactam has little intrinsic antibacterial activity , but efficiently protects -lactams from hydrolysis in a variety of class a , class c , and some class d - producing strains , including esbl , klebsiella pneumoniae carbapenemase ( kpc ) , and oxa-48 producers.57 many strains of the enterobacteriaceae family , as well as some nonfermenters , such as pseudomonas aeruginosa and acinetobacter baumannii , encode a chromosomal ampc -lactamase , although the regulation of enzyme production is different among the various enzyme - producing strains . in escherichia coli , enzyme expression is usually constitutive and low - level , whereas in other species , such as enterobacter spp . , it can be transiently induced to higher levels by several -lactam compounds , with carbapenems and cephamycins generally having the highest induction potential.8 derepression can also occur by mutations favoring constitutive production of very high levels of -lactamase . the mechanism of induction is complex , via a system involving ampd , ampr , ampg , and intermediates in peptidoglycan recycling.9 induction of ampc does not necessarily correlate with a risk of clinical failure , particularly when the rate of bactericidality is high . however , the potential for ampc induction has to be carefully examined when considering administration of a -lactamase inhibitor , because it can antagonize the antibacterial activity of its partner -lactam.1012 indeed , the antibacterial activity of a given -lactam with limited stability to ampc is preserved , provided that its potential for ampc induction is low . in contrast , its activity would be compromised if combined with a -lactamase inhibitor that induces significant ampc production . the aim of this study was to investigate the ability of avibactam to induce ampc expression in e. cloacae strains in vitro . because -lactamase / avibactam complexes are known to have a long half - life , it was not possible to evaluate ampc induction by measuring directly the -lactamase activity produced by avibactam - treated cells . thus , it was measured by quantitation of cellular ampc messenger ribonucleic acid ( mrna ) . e. cloacae isolates used in this study were obtained from the novexel culture collection , originally collected from a variety of clinical or laboratory sources . minimal inhibitory concentration ( mic ) determinations were performed according to the clinical and laboratory standards institute broth - microdilution methods using cation - adjusted mueller hinton broth.13 mic values were measured for cefoxitin ( sigma - aldrich , st louis , mo , usa ) and ceftazidime ( novartis , basel , switzerland ) ; the latter was tested alone or in association with clavulanate ( us pharmacopeial convention , rockville , md , usa ) or avibactam , at a constant inhibitor concentration of 4 mg / l . bertani broth ( interchim , montlugon , france ) , then diluted to an optical density ( od600 nm ) value of 0.1 and incubated with shaking for 24 hours to reach midlog - growth phase . at this point , the test inducer ( cefoxitin , clavulanate , or avibactam ) was added at the appropriate concentration ( 8 , 16 , 32 , or 64 g / ml ) , whereas control cultures were grown in the absence of inducer . approximately 5 10 cells were sampled for rna extraction just before addition of inducer , and at timed intervals thereafter up to 6 hours . each induction experiment was performed at least three times for all three e. cloacae strains . total cellular rna was extracted with an rneasy rna protect mini kit ( qiagen , venlo , the netherlands ) , and residual deoxyribonucleic acid ( dna ) was eliminated by treatment with an rnase - free dnase set ( qiagen ) . analysis of rna integrity and total rna quantification was performed using the agilent 2100 rna bioanalyzer and the nano 6000 kit ( agilent technologies , santa clara , ca , usa ) . polymerase chain reaction ( pcr ) primers were designed with primer express software for ampc ( forward 5-tggcgtatcgggtcaatgt-3 ; probe 5-tcagggtctgggctgggagatgc-3 ; reverse 5-cctccacgggccagttg-3 ) and for rpls ( forward 5-caggtgacaccgtggaagtg- ; probe 5-aagtatgggttgttgaaggttccaa-3 ; reverse 5-cgaatgcctgcagacgttt-3 ) . the probe primers were modified by addition of 6-fam ( 6-carboxy - fluorescein ) at the 5 end and tamra ( 6-carboxy - tetramethyl - rhodamine ) at the 3 end . real - time pcr ( rt - pcr ) reactions were carried out in the abi prism 7000 sequence - detection system ( life technologies , carlsbad , ca , usa ) using a quantitect probe rt - pcr kit ( qiagen ) . individual reactions were set up in triplicate for either ampc or rpsl genes , according to the manufacturer s recommendations . briefly , complementary dna was syn - thesized from 0.5 ng of rna using moloney murine leuke - mia virus reverse transcriptase and 0.7 m of each primer ; reverse transcription was carried out at 50c for 30 minutes . pcr conditions were as follows : initial activation of dna polymerase at 95c for 15 minutes , and pcr for 40 cycles at 95c for 15 seconds , 60c for 60 seconds . absence of genomic dna contamination was verified for each rna preparation by running the assay in the absence of reverse transcriptase . data were analyzed using sequence detection 2.0 software ( life technologies ) . to correct for differences in the amount of starting material , the ribosomal e. cloacae rpls gene ( encoding ribosomal protein 19 ) was chosen as a housekeeping reference gene . values obtained were then normalized to that of ampc from e. cloacae strain p99 for measurement of basal expression , or to that of ampc in the test strain before induction . relative quantitation was carried out by using the 2 t method , as recommended by the manufacturer.14 normalized ampc expression in culture 2 relative to that in culture 1 was calculated as follows : crude bacterial extracts were prepared by vortexing bacterial cells with glass beads in 100 mm phosphate buffer ph 7 containing 0.1 mg / ml of bovine serum albumin and 2% v / v glycerol ( about 10 bacterial cells / ml ) . -lactamase activity was measured using a spectrophotometer at 485 nm for 15 minutes using 180 l of crude cell lysate at appropriate dilution , and 20 l of 1 mm nitrocefin ( oxoid sr112c ) . results were expressed as initial reaction rates ( a485 nm / minute ) per 10 cells or per milligram of protein . e. cloacae isolates used in this study were obtained from the novexel culture collection , originally collected from a variety of clinical or laboratory sources . minimal inhibitory concentration ( mic ) determinations were performed according to the clinical and laboratory standards institute broth - microdilution methods using cation - adjusted mueller hinton broth.13 mic values were measured for cefoxitin ( sigma - aldrich , st louis , mo , usa ) and ceftazidime ( novartis , basel , switzerland ) ; the latter was tested alone or in association with clavulanate ( us pharmacopeial convention , rockville , md , usa ) or avibactam , at a constant inhibitor concentration of 4 mg / l . bacterial strains were grown overnight at 37c in luria bertani broth ( interchim , montlugon , france ) , then diluted to an optical density ( od600 nm ) value of 0.1 and incubated with shaking for 24 hours to reach midlog - growth phase . at this point , the test inducer ( cefoxitin , clavulanate , or avibactam ) was added at the appropriate concentration ( 8 , 16 , 32 , or 64 g / ml ) , whereas control cultures were grown in the absence of inducer . approximately 5 10 cells were sampled for rna extraction just before addition of inducer , and at timed intervals thereafter up to 6 hours . each induction experiment was performed at least three times for all three e. cloacae strains . total cellular rna was extracted with an rneasy rna protect mini kit ( qiagen , venlo , the netherlands ) , and residual deoxyribonucleic acid ( dna ) was eliminated by treatment with an rnase - free dnase set ( qiagen ) . analysis of rna integrity and total rna quantification was performed using the agilent 2100 rna bioanalyzer and the nano 6000 kit ( agilent technologies , santa clara , ca , usa ) . polymerase chain reaction ( pcr ) primers were designed with primer express software for ampc ( forward 5-tggcgtatcgggtcaatgt-3 ; probe 5-tcagggtctgggctgggagatgc-3 ; reverse 5-cctccacgggccagttg-3 ) and for rpls ( forward 5-caggtgacaccgtggaagtg- ; probe 5-aagtatgggttgttgaaggttccaa-3 ; reverse 5-cgaatgcctgcagacgttt-3 ) . the probe primers were modified by addition of 6-fam ( 6-carboxy - fluorescein ) at the 5 end and tamra ( 6-carboxy - tetramethyl - rhodamine ) at the 3 end . real - time pcr ( rt - pcr ) reactions were carried out in the abi prism 7000 sequence - detection system ( life technologies , carlsbad , ca , usa ) using a quantitect probe rt - pcr kit ( qiagen ) . individual reactions were set up in triplicate for either ampc or rpsl genes , according to the manufacturer s recommendations . briefly , complementary dna was syn - thesized from 0.5 ng of rna using moloney murine leuke - mia virus reverse transcriptase and 0.7 m of each primer ; reverse transcription was carried out at 50c for 30 minutes . pcr conditions were as follows : initial activation of dna polymerase at 95c for 15 minutes , and pcr for 40 cycles at 95c for 15 seconds , 60c for 60 seconds . absence of genomic dna contamination was verified for each rna preparation by running the assay in the absence of reverse transcriptase . data were analyzed using sequence detection 2.0 software ( life technologies ) . to correct for differences in the amount of starting material , the ribosomal e. cloacae rpls gene ( encoding ribosomal protein 19 ) was chosen as a housekeeping reference gene . values obtained were then normalized to that of ampc from e. cloacae strain p99 for measurement of basal expression , or to that of ampc in the test strain before induction . relative quantitation was carried out by using the 2 t method , as recommended by the manufacturer.14 normalized ampc expression in culture 2 relative to that in culture 1 was calculated as follows : crude bacterial extracts were prepared by vortexing bacterial cells with glass beads in 100 mm phosphate buffer ph 7 containing 0.1 mg / ml of bovine serum albumin and 2% v / v glycerol ( about 10 bacterial cells / ml ) . -lactamase activity was measured using a spectrophotometer at 485 nm for 15 minutes using 180 l of crude cell lysate at appropriate dilution , and 20 l of 1 mm nitrocefin ( oxoid sr112c ) . results were expressed as initial reaction rates ( a485 nm / minute ) per 10 cells or per milligram of protein . both cefoxitin and ceftazidime are good ampc substrates ; cefoxitin is also a good ampc inducer , whereas ceftazidime has limited potential for induction . the differential activity of cefoxitin and ceftazidime can therefore be used to infer the presence of an inducible ampc gene , as strains without significant levels of ampc enzyme remain susceptible to cefoxitin.9,15 in order to select potentially inducible strains for this study , three e. cloacae strains ( 293la2 , 293ht107 , and 293uc1 ) were chosen on the basis of resistance to cefoxitin and susceptibility to ceftazidime . the e. cloacae p99 strain was included in this study as a reference , having a cefoxitin - resistant ( cefoxitin - r ) and ceftazidime - r phenotype ( stably derepressed high - level ampc producer ) . the mic values obtained for these four strains are shown in table 1 . as expected , owing to its spectrum of coverage limited to class a enzymes , clavulanate had no effect on the high ceftazidime mic value for the p99 strain ; in contrast , avibactam reduced ceftazidime mic to a susceptible level in this strain . induction of -lactamases is most frequently assessed by assaying -lactamase activity using spectrophotometric assays of nitrocefin hydrolysis , in the presence or absence of an inducer . however , this is not technically possible when testing the induction potential of a compound that can form highly stable complexes with ampc enzymes ( half - life around 7 days for avibactam / p99 ampc complex).5 therefore , induction was measured by quantitation of ampc transcripts using rt - pcr . however , -lactamase enzymatic activity was measured using nitrocefin in parallel with rt - pcr in the experiments that did not involve exposure to -lactamase inhibitors . basal expression of ampc mrna in those three e. cloacae strains was compared to that of derepressed ampc p99 and found to be 150- to 300-fold lower ( table 1 ) . concomitantly , basal -lactamase activity in crude cell extracts reported using the nitrocefin substrate was also higher ( 1,000- to 1,800-fold ) in p99 than in the three selected e. cloacae strains . basal levels of ampc transcripts and -lactamase activity were therefore fully consistent with the susceptibility / resistance phenotype of the strains . the inducibility of -lactamase activity was studied in the presence of 132 mg / l cefoxitin . the three e. cloacae strains tested showed similar induction profiles : ampc mrna peaked at 12 hours following induction , then slowly declined to reach basal levels at 46 hours . figure 1 shows the kinetics of one representative experiment with the strain 293ht107 treated with cefoxitin at 1 or 2 mg / l . increased mrna concentrations were detectable soon after incubation start ( as soon as 10 minutes ; data not shown ) and peaked after 12 hours of incubation . -lactamase activity was delayed slightly when compared to ampc mrna and continuously increased throughout the 4 hours of incubation with cefoxitin . when treated with 1632 mg / l of cefoxi - tin , the maximal ampc transcriptional level after 2 hours of culture was between 100 and 200 times the basal level of both 293ht107 ( figure 2 g ) and 293la2 ( figure 2d ) strains , and around 50 times the basal level of the 293uc1 strain ( figure 2a ) . the potential of avibactam and clavulanate for induction of ampc expression was evaluated on the three e. cloacae strains at 1664 mg / l . clavulanate had no significant effects on 293uc1 and 293la2 strains during the 6-hour incubation period ( figure 2b and e ) , whereas it was a moderate ampc inducer for 293ht107 with about a 40-fold increase of ampc mrna at 64 mg / l after 2 hours of incubation ( figure 2h ) . in contrast , avibactam had no detectable effect on ampc mrna levels in the three strains tested ( figure 2 , c , f , and i ) . at the concentrations used for induction studies , avibactam had no effect on the growth of the bacterial strains tested , as testified by the od values measured at each time point . enterobacter spp . are recognized to be among the most common nosocomial pathogens , with current resistance rates presenting a serious therapeutic dilemma . resistance through overexpression of ampc can occur in the vast majority of strains possessing a chromosomally encoded cephalosporinase , and ampc induction is recognized as a widespread resistance mechanism . in a study examining 200 clinical isolates of p. aeruginosa , enterobacter spp . , it was shown that 85% of the collected strains showed inducible ampc production , of which 11% were stably derepressed and only 3% were not induced by either cefoxitin or imipenem.16 approximately 12% of hospital strains of the european meropenem yearly susceptibility test information collection ( mystic ) program in the years 19972000 were due to potential ampc - producing strains of enterobacter spp . s. marcescens , in which 28% represented stably derepressed ampc - producing phenotypes.17 much of what is known about ampc regulation is from studies in e. coli , c. freundii , and e. cloacae ; the induction mechanism in response to exposure to certain -lactams is complex and closely linked to the peptidoglycan - recycling pathway.9 different effector proteins and regulation mechanisms have been recently evidenced for p. aeruginosa ampc induction , suggesting that the process is more complex in that species and distinct from the current paradigm established following studies of enterobacteriaceae species.18 -lactams differ in their inducing abilities , with carbapen - ems and cephamycins having the highest potential.8 the clavu - lanate -lactamase inhibitor is also an ampc inducer , and was shown in vitro to antagonize the antibacterial activity of various -lactams.10,11 in this context , the potential for induction of the new -lactamase inhibitor avibactam was evaluated . at sub - mic concentrations , cefoxitin induced a major dose - dependent synthesis of ampc in all three e. cloacae strains tested here , whereas clavulanate triggered synthesis of ampc mrna in two out of the three strains , at the highest concentration tested ( 64 mg / l ) . in contrast , in the same range of concentrations , avibactam had no effect on cellular ampc mrna concentration in any of the three e. cloacae strains during the 6-hour incubation period . from these initial findings , it is concluded that there is little likelihood of antagonism between -lactam antibiotics and the novel -lactamase inhibitor avibactam in enterobacter spp . questions remain about other bacterial species producing inducible chromosomal ampc enzymes , and will be the focus of future studies . in contrast with the inhibitors currently available ( clavulanate , tazobactam , and sulbactam ) , which all have relatively limited activity against the class c enzymes , avibactam is a potent inhibitor of ampc -lactamases.3,5 it is the first non--lactam -lactamase inhibitor to advance to clinical development , currently undergoing phase ii iii clinical trials in combination with ceftazidime and with cef - taroline ( http://www.clinicaltrials.gov ) . ceftaroline , like some third - generation cephalosporins , is a weak inducer of ampc enzymes at sub - mic concentrations , resulting in a propensity to select ampc - derepressed or ampc - hyperinducible mutants.19 pairing ceftaroline with avibactam should thus be an effective strategy to limit the risk of selection of mutants , and to restore ceftaroline activity against ampc - hyperproducing strains , as well as to other -lactamase producers .

induction of ampc -lactamase expression can often compromise antibiotic treatment and is triggered by several -lactams ( such as cefoxitin and imipenem ) and by the -lactamase inhibitor clavulanic acid . the novel -lactamase inhibitor avibactam ( nxl104 ) is a potent inhibitor of both class a and class c enzymes . the potential of avibactam for induction of ampc expression in enterobacter cloacae was investigated by ampc messenger ribonucleic acid quantitation . cefoxitin and clavulanic acid were confirmed as ampc inducers , whereas avibactam was found to exert no effect on ampc expression . thus , avibactam is unlikely to diminish the activity of any partner -lactam antibiotic against ampc - producing organisms .

Introduction Materials and methods Bacterial strains and susceptibility testing Induction experiments Reverse-transcription polymerase chain reaction -Lactamase activity assays Results Discussion

PMC5359996

louis wilson , the first pathologist of the mayo clinic group practice in rochester , minnesota , described a reproducible method for rapid histologic evaluation of surgical specimens . this methodology involves rapid freezing of fresh tissue on a specially designed microtome down to temperatures below those used in modern cryostat microtomes , allowing evaluation of a wider variety of tissue types while minimizing freezing artifacts . the frozen section technique allows for intraoperative diagnosis and evaluation of tumor margin status , leading to improved patient outcomes . in 2014 , approximately 20,000 frozen section intraoperative consultations were performed at mayo clinic in rochester ( i.e. , 7080 frozen section cases per weekday ) using this technique , with the interpretation of more than 150,000 histologic blocks and 100,000 frozen section slides . to enable rapid diagnoses with such high case volumes , pathologic , radiologic , surgical , and clinical information is gathered and synthesized for each case before the day of surgery by surgical pathology residents and fellows . on the day of surgery , the information is then disseminated to the frozen section laboratory team during a trainee - led morning report . morning report involves a synoptic discussion of the day 's patients and requires concise and accurate transmission of large volumes of case information to a diverse audience in a limited time frame . case information is then applied in real time by laboratory staff to guide gross dissection and microscopic diagnosis . before the work described herein , aspects of the case review process were carried out in an idiosyncratic and manual fashion . review of pending surgical pathology cases was a largely paper - based process that required extensive transcription of information from the electronic health record ( ehr ) . transmission of the gathered information then occurred through a fast - paced verbal morning report in which all participants transcribed relevant case information onto their own printed copies of the day 's surgical list . although this challenging task was quite functional and well handled by trainees and staff , it was somewhat laborious and bore an inherent risk for errors to occur in either collection , transmission , or recording of clinical information . to help address perceived inefficiencies in the list review process and to eliminate , where possible , the potential for errors in the communication of case information , a process improvement effort was undertaken . herein , we describe that improvement effort , the software application that resulted from it , and the use of that application to facilitate improved communication of case review information and enable real - time case tracking during surgical pathology consultation . a lean process improvement strategy was utilized to identify areas of waste in the surgical pathology list review process . a workflow process map [ figure 1 ] was developed to capture the essential inputs and outputs required to complete the list review process and then utilize the gathered information for surgical pathology diagnoses . specific areas for process improvement were identified by interviewing stakeholders ( residents , fellows , pathology assistants , and staff pathologists ) , with a focus on tasks perceived as inefficient or unproductive , and by independently observing and mapping the current processes . presurgical case preparation was identified as an area of significant potential inefficiency , with residents and fellows reporting that case review using the ehr involved several repetitive activities . abstraction of information from the ehr for pathological diagnosis can be a complex and time consuming ; in our practice , the average reported time to complete the list review and case annotation ( ~125 total cases with ~35 frozen section cases ) was 4 hours per person per day ( with single fellow and/or resident participating ) . significant time was consumed in shifting attention between a printed list of surgical patients ( used by residents and fellows to identify potential cases for review ) and the pathology , radiology , and clinical note sections within the ehr . once cases were identified , pertinent details then had to be abstracted from the ehr by manual transcription to either a printed surgical patient list or separate note sheet . in the absence of an established mechanism for collating the list review work product before morning report , residents and fellows independently reviewing the list ( typically accomplished at night and/or from home ) sometimes duplicated case review work . process flow diagram for ( a ) presurgical case review , ( b ) morning report , and ( c ) real - time case review . an accessory process for surgical listing information recipients who participate in specimen grossing is shown in tan . information flows between processes are designated by red arrows other areas identified for improvement by stakeholders were clarity and completeness of morning report ( during which information from the case review is disseminated to the frozen section laboratory team ) and situational case awareness during the intraoperative case review . interestingly , the physical size of the existing printed surgical list was identified as a potential logistical issue in case review . although only 3040 surgical cases in a day ( out of 75175 ) might require a frozen section consultation , the electronic listing system in place was only capable of printing a surgical list including all cases ( typically 3040 pages in length ) . stakeholders reported that a nontrivial amount of time was used to simply search through the printed list to locate a specific listing ( and associated case notes ) when tissue from a new case would arrive in the laboratory . after mapping the case review process in its entirety , three major subprocesses were identified [ figure 1 ] in the creation and application of the surgical list for frozen section pathology : ( 1 ) generation of the pathology notes for the surgical list ; ( 2 ) communication of those findings to the laboratory staff ; and ( 3 ) utilization of those notes to aid in the diagnostic process . furthermore , the utilization of pathology notes by other staff ( junior residents and pathology assistants ) to perform the gross dissection of frozen section specimens was identified as an important accessory process [ figure 1 ] . to increase efficiency and reduce the possibility for errors in the gathering , recording , communicating , and application of case information , it was determined that a software application should be developed . modification of an existing surgical and procedure scheduling ( sps ) software application was identified as the best approach for meeting the needs of the surgical pathology practice . the sps application is custom software developed in - house and primarily used by the mayo clinic department of surgery to create and manage the institutional surgical schedule . a listing in the application contains patient demographics , procedure type , procedure indication , diagnosis ( if available ) , medication orders , patient assessments , required equipment or supply notes , and pharmacy and anesthesia requests . the sps application was designed to accommodate modules with additional functionality from other departments , such as anesthesia or pharmacy , to facilitate total care of surgical patients . taking advantage of this architecture , development of a pathology - specific module ( pm ) for the sps application was undertaken using an agile software development process . sps - pm requirements [ table 1 ] were defined from the process map [ figure 1 ] and then further refined with stakeholder feedback on specific areas of waste or inefficiency . over a 2-month period , several development cycles ( sprints ) were undertaken during which key functionality was added to the sps - pm , tested by a resident serving as an end - user technical representative , and then formally integrated into the sps - pm module . once core functionality was present , the sps - pm was launched to end users for an initial 2-month evaluation period . the software was designed for ease of use , with a user interface paradigm consistent with typical office productivity software . an initial basic demonstration of functionality , but no specific end - user training , was provided to a cohort of residents on the frozen section service . in turn residents and fellows then served as information sources and advocates for adoption by the frozen section staff including staff pathologists , pathology assistants , and technicians . the attending head of the frozen section working group was the physician champion for the project , and one of the residents directly involved in the development of the software acted as a superuser to provide real - time support for issues and also to serve as a recipient for evaluation feedback . during the initial evaluation period , ongoing user feedback was used to improve sps - pm stability and to identify several additional key features ( case searching and an operating room visual overview ) to be added . after the review period , the sps - pm application was moved into a production environment , and additional presentations were given to key stakeholder groups ( attending physicians and physician assistants ) to demonstrate features and functionality . pathology module feature requirements associated with case review , communication of review information , and real - time intraoperative case management the sps is a. net c # windows presentation foundation application that runs on the microsoft windows operating system , version 7 or greater . it was developed using the csla.net ( http://cslanet.com/ ) and model - view - viewmodel ( mvvm ) light ( https://mvvmlight.codeplex.com/ ) frameworks , using a mvvm ( https://msdn.microsoft.com/en-us/library/hh848246.aspx ) design pattern to separate application logic from the presentation layer . listing information was maintained in sybase and ms structured query language ( sql ) databases accessible through standard sql queries . additional clinical information was retrieved from the mayo clinic electronic medical record ( ge healthcare , wauwatosa , wi , usa ) through web service application programing interface requests . access to protected health information through these systems is implemented through client authentication within a monitored hipaa - compliant environment . after 3 months of use , staff pathologists , residents , and fellows were surveyed to assess the sps - pms impact on practice workflow . study survey data were captured and managed using the research electronic data capture ( redcap ) system ( vanderbilt university , nashville , tn , usa ) . fifty - five physicians , fellows , and residents involved in the frozen section pathology processes were surveyed . there were 21 respondents ( 34 non - respondents ) , of which five had not used the sps - pm application and sixteen had used the application . the sixteen respondents included five staff pathologists , eleven trainees ( residents or fellows ) . a lean process improvement strategy was utilized to identify areas of waste in the surgical pathology list review process . a workflow process map [ figure 1 ] was developed to capture the essential inputs and outputs required to complete the list review process and then utilize the gathered information for surgical pathology diagnoses . specific areas for process improvement were identified by interviewing stakeholders ( residents , fellows , pathology assistants , and staff pathologists ) , with a focus on tasks perceived as inefficient or unproductive , and by independently observing and mapping the current processes . presurgical case preparation was identified as an area of significant potential inefficiency , with residents and fellows reporting that case review using the ehr involved several repetitive activities . abstraction of information from the ehr for pathological diagnosis can be a complex and time consuming ; in our practice , the average reported time to complete the list review and case annotation ( ~125 total cases with ~35 frozen section cases ) was 4 hours per person per day ( with single fellow and/or resident participating ) . significant time was consumed in shifting attention between a printed list of surgical patients ( used by residents and fellows to identify potential cases for review ) and the pathology , radiology , and clinical note sections within the ehr . once cases were identified , pertinent details then had to be abstracted from the ehr by manual transcription to either a printed surgical patient list or separate note sheet . in the absence of an established mechanism for collating the list review work product before morning report , residents and fellows independently reviewing the list ( typically accomplished at night and/or from home ) sometimes duplicated case review work . process flow diagram for ( a ) presurgical case review , ( b ) morning report , and ( c ) real - time case review . an accessory process for surgical listing information recipients who participate in specimen grossing is shown in tan . information flows between processes are designated by red arrows other areas identified for improvement by stakeholders were clarity and completeness of morning report ( during which information from the case review is disseminated to the frozen section laboratory team ) and situational case awareness during the intraoperative case review . interestingly , the physical size of the existing printed surgical list was identified as a potential logistical issue in case review . although only 3040 surgical cases in a day ( out of 75175 ) might require a frozen section consultation , the electronic listing system in place was only capable of printing a surgical list including all cases ( typically 3040 pages in length ) . stakeholders reported that a nontrivial amount of time was used to simply search through the printed list to locate a specific listing ( and associated case notes ) when tissue from a new case would arrive in the laboratory . after mapping the case review process in its entirety , three major subprocesses were identified [ figure 1 ] in the creation and application of the surgical list for frozen section pathology : ( 1 ) generation of the pathology notes for the surgical list ; ( 2 ) communication of those findings to the laboratory staff ; and ( 3 ) utilization of those notes to aid in the diagnostic process . furthermore , the utilization of pathology notes by other staff ( junior residents and pathology assistants ) to perform the gross dissection of frozen section specimens was identified as an important accessory process [ figure 1 ] . to increase efficiency and reduce the possibility for errors in the gathering , recording , communicating , and application of case information , it was determined that a software application should be developed . modification of an existing surgical and procedure scheduling ( sps ) software application was identified as the best approach for meeting the needs of the surgical pathology practice . the sps application is custom software developed in - house and primarily used by the mayo clinic department of surgery to create and manage the institutional surgical schedule . listing in the application contains patient demographics , procedure type , procedure indication , diagnosis ( if available ) , medication orders , patient assessments , required equipment or supply notes , and pharmacy and anesthesia requests . the sps application was designed to accommodate modules with additional functionality from other departments , such as anesthesia or pharmacy , to facilitate total care of surgical patients . taking advantage of this architecture , development of a pathology - specific module ( pm ) for the sps application was undertaken using an agile software development process . sps - pm requirements [ table 1 ] were defined from the process map [ figure 1 ] and then further refined with stakeholder feedback on specific areas of waste or inefficiency . over a 2-month period , several development cycles ( sprints ) were undertaken during which key functionality was added to the sps - pm , tested by a resident serving as an end - user technical representative , and then formally integrated into the sps - pm module . once core functionality was present , the sps - pm was launched to end users for an initial 2-month evaluation period . the software was designed for ease of use , with a user interface paradigm consistent with typical office productivity software . an initial basic demonstration of functionality , but no specific end - user training , was provided to a cohort of residents on the frozen section service . in turn residents and fellows then served as information sources and advocates for adoption by the frozen section staff including staff pathologists , pathology assistants , and technicians . the attending head of the frozen section working group was the physician champion for the project , and one of the residents directly involved in the development of the software acted as a superuser to provide real - time support for issues and also to serve as a recipient for evaluation feedback . during the initial evaluation period , ongoing user feedback was used to improve sps - pm stability and to identify several additional key features ( case searching and an operating room visual overview ) to be added . after the review period , the sps - pm application was moved into a production environment , and additional presentations were given to key stakeholder groups ( attending physicians and physician assistants ) to demonstrate features and functionality . pathology module feature requirements associated with case review , communication of review information , and real - time intraoperative case management the sps is a. net c # windows presentation foundation application that runs on the microsoft windows operating system , version 7 or greater . it was developed using the csla.net ( http://cslanet.com/ ) and model - view - viewmodel ( mvvm ) light ( https://mvvmlight.codeplex.com/ ) frameworks , using a mvvm ( https://msdn.microsoft.com/en-us/library/hh848246.aspx ) design pattern to separate application logic from the presentation layer . listing information was maintained in sybase and ms structured query language ( sql ) databases accessible through standard sql queries . additional clinical information was retrieved from the mayo clinic electronic medical record ( ge healthcare , wauwatosa , wi , usa ) through web service application programing interface requests . access to protected health information through these systems is implemented through client authentication within a monitored hipaa - compliant environment . after 3 months of use , staff pathologists , residents , and fellows were surveyed to assess the sps - pms impact on practice workflow . study survey data were captured and managed using the research electronic data capture ( redcap ) system ( vanderbilt university , nashville , tn , usa ) . fifty - five physicians , fellows , and residents involved in the frozen section pathology processes were surveyed . there were 21 respondents ( 34 non - respondents ) , of which five had not used the sps - pm application and sixteen had used the application . the sixteen respondents included five staff pathologists , eleven trainees ( residents or fellows ) . the sps - pm application was designed for three specific tasks within the list review process : ( 1 ) preconsultation case review , ( 2 ) communication of review information to frozen section laboratory staff , and ( 3 ) real - time intraoperative case management . the preconsultation case review functionality was intended to provide all pathology - relevant information on a surgical listing , while eliminating nonrelevant information ( e.g. anesthesia or nursing notes ) that slowed or obscured the review process . the listing information is presented to the user within a scrollable list [ figure 2 ] that allows all cases for a given day and surgical site to be viewed and annotated . by default , the list is organized by surgical specialty , but it can be organized by patient name , operating room , or status ( preoperative , in surgery , or postoperative ) by the user . pathology notes are shown in bold lettering at the bottom of each listing , with author identified by initials . options allow for showing cases that have been excluded and also filtering of cases by status in the operating room each sps - pm case listing includes the patient 's unique medical record number , which serves a direct link to that patient 's record within the ehr . an annotation function then allows pathology - specific notes to be added to listing information by text entry or copy - paste from the medical record . in contrast to other listing notes , pathology notes are visible only to users within the department of pathology . the sps - pm annotation screen is accessed by double - clicking on a patient 's surgical listing [ figure 3 ] . in addition to providing for entry of case notes , the system prefetches case - associated radiology , pathology , and operative reports from the ehr and presents them within a sortable field for review . once added and saved , pathology notes are visible within the main surgical listing view [ figure 2 ] . user initials appended to the end of the notes allow for identification of the note author . cases without a requirement for intraoperative pathology consultation can be excluded in this annotation screen to hide them from the active case list . these excluded cases , however , do remain in the system and are retrievable at any time . potentially , infectious cases can be designated as hood required , which adds emphasis text to a surgical listing as a visible reminder that tissue should be processed in a biohazard safety cabinet . to facilitate morning report and real - time case tracking , two print views were created : a standard print view that prints in a format similar to the old printed surgical list minus excluded cases , and a condensed list view allowing the entire surgical list to be printed on as few sheets as possible [ figure 4 ] . a note textbox can be used to enter typed or copied text . in the lower right , prefetched pathology , radiology , and operative note information can be seen and selected for review . this screen also allows a case to be excluded from the active set of listings or defined as requiring a biosafety hood print - friendly case listing view . the operating rooms included within a given column are listed for easy reference at the bottom of that column several application features were designed to facilitate intraoperative case management . cases are automatically designated as pending ( orange ) , active ( green ; time of incision noted ) , or complete ( blue ; time of closure noted ) . assessment of case status and serves as a filterable field whereby users can select only pending cases , active cases , or not yet completed cases . the list can also be organized by surgical specialty , patient name , or operating room number to facilitate different workflows within the frozen section laboratory . text search functionality is available for rapid filtering based on text matching to any portion of the surgical listing including the pathology notes . before the development of the sps - pm application , a different software tool was used to provide situational awareness in the frozen section laboratory . that tool provided a map - like overview of the operating rooms and was much prized for its ability to provide a quick overview of workload status . based on user feedback , a similar feature was added to the sps - pm application [ figure 5 ] . this feature consists of a pseudogeographic overview of the operating rooms at either hospital , with coloration and text providing essential real - time case information . the overview screen can be refreshed manually at any time , and automatically refreshes at 2 minute intervals may be enabled with a toggle button . to facilitate diagnostic workflow when using this view , a specimen jar pathology - specific notes in this view can be accessed by hovering over the specimen jar icon as a tooltip or by double - clicking on the operating room to reveal a modal dialog box [ figure 6 ] . operating room map view . a pseudotopological map representing the operating room layout at mayo clinic in rochester saint mary 's campus . case status in each operating room is color coded : entry ( orange ) , incision ( green ) , and closure ( blue ) . a specimen jar icon in the upper left indicates cases with associated pathology notes operating room map view pathology notes detail . icon reveals a case 's associated pathology notes adoption of sps - pm by residents and fellows was nearly instantaneous upon its introduction into clinical use . a redcap survey of perceptions of the sps - pm application was sent to 21 staff , fellows , and residents ; there were 16 respondents ( five staff pathologists and eleven trainees ) . the survey asked questions about three areas of practice : preconsultation case preparation , morning report , and real - time case awareness . nearly all trainees ( 10/11 ; 91% ) reported that the application both improved abstracting information from the ehr and the time required to complete case preparation . time saved by the application was a mean of 1.4 h / day ( range of 04 h ; one trainee reported no time savings ) . the sps - pm application also improved identification of potentially infectious cases ( 7/11 ; 64% ) . for morning report and surgical day performance , respondents indicated the application improved the speed ( 11/16 ; 69% ) , clarity ( 13/16 ; 81% ) , and accuracy ( 10/16 ; 63% ) of morning presurgical case review . the survey was conducted before the addition of the operating room map overview feature ; even so , a majority surveyed reported case review at the time of diagnosis ( 14/16 ; 88% ) and situational awareness of multiple ongoing cases ( 13/16 ; 81% ) was improved by the sps - pm . the sps - pm application was designed for three specific tasks within the list review process : ( 1 ) preconsultation case review , ( 2 ) communication of review information to frozen section laboratory staff , and ( 3 ) real - time intraoperative case management . the preconsultation case review functionality was intended to provide all pathology - relevant information on a surgical listing , while eliminating nonrelevant information ( e.g. anesthesia or nursing notes ) that slowed or obscured the review process . the listing information is presented to the user within a scrollable list [ figure 2 ] that allows all cases for a given day and surgical site to be viewed and annotated . by default , the list is organized by surgical specialty , but it can be organized by patient name , operating room , or status ( preoperative , in surgery , or postoperative ) by the user . pathology notes are shown in bold lettering at the bottom of each listing , with author identified by initials . options allow for showing cases that have been excluded and also filtering of cases by status in the operating room each sps - pm case listing includes the patient 's unique medical record number , which serves a direct link to that patient 's record within the ehr . an annotation function then allows pathology - specific notes to be added to listing information by text entry or copy - paste from the medical record . in contrast to other listing notes , pathology notes are visible only to users within the department of pathology . the sps - pm annotation screen is accessed by double - clicking on a patient 's surgical listing [ figure 3 ] . in addition to providing for entry of case notes , the system prefetches case - associated radiology , pathology , and operative reports from the ehr and presents them within a sortable field for review . once added and saved , pathology notes are visible within the main surgical listing view [ figure 2 ] . user initials appended to the end of the notes allow for identification of the note author . cases without a requirement for intraoperative pathology consultation can be excluded in this annotation screen to hide them from the active case list . these excluded cases , however , do remain in the system and are retrievable at any time . potentially , infectious cases can be designated as hood required , which adds emphasis text to a surgical listing as a visible reminder that tissue should be processed in a biohazard safety cabinet . to facilitate morning report and real - time case tracking , two print views were created : a standard print view that prints in a format similar to the old printed surgical list minus excluded cases , and a condensed list view allowing the entire surgical list to be printed on as few sheets as possible [ figure 4 ] . a note textbox can be used to enter typed or copied text . in the lower right , prefetched pathology , radiology , and operative note information can be seen and selected for review . this screen also allows a case to be excluded from the active set of listings or defined as requiring a biosafety hood print - friendly case listing view . the operating rooms included within a given column are listed for easy reference at the bottom of that column several application features were designed to facilitate intraoperative case management . cases are automatically designated as pending ( orange ) , active ( green ; time of incision noted ) , or complete ( blue ; time of closure noted ) . assessment of case status and serves as a filterable field whereby users can select only pending cases , active cases , or not yet completed cases . the list can also be organized by surgical specialty , patient name , or operating room number to facilitate different workflows within the frozen section laboratory . text search functionality is available for rapid filtering based on text matching to any portion of the surgical listing including the pathology notes . before the development of the sps - pm application , a different software tool was used to provide situational awareness in the frozen section laboratory . that tool provided a map - like overview of the operating rooms and was much prized for its ability to provide a quick overview of workload status . based on user feedback , a similar feature was added to the sps - pm application [ figure 5 ] . this feature consists of a pseudogeographic overview of the operating rooms at either hospital , with coloration and text providing essential real - time case information . the overview screen can be refreshed manually at any time , and automatically refreshes at 2 minute intervals may be enabled with a toggle button . to facilitate diagnostic workflow when using this view , a specimen jar pathology - specific notes in this view can be accessed by hovering over the specimen jar icon as a tooltip or by double - clicking on the operating room to reveal a modal dialog box [ figure 6 ] . operating room map view . a pseudotopological map representing the operating room layout at mayo clinic in rochester saint mary 's campus . case status in each operating room is color coded : entry ( orange ) , incision ( green ) , and closure ( blue ) . a specimen jar icon in the upper left indicates cases with associated pathology notes operating room map view pathology notes detail . adoption of sps - pm by residents and fellows was nearly instantaneous upon its introduction into clinical use . a redcap survey of perceptions of the sps - pm application was sent to 21 staff , fellows , and residents ; there were 16 respondents ( five staff pathologists and eleven trainees ) . the survey asked questions about three areas of practice : preconsultation case preparation , morning report , and real - time case awareness . nearly all trainees ( 10/11 ; 91% ) reported that the application both improved abstracting information from the ehr and the time required to complete case preparation . time saved by the application was a mean of 1.4 h / day ( range of 04 h ; one trainee reported no time savings ) . the sps - pm application also improved identification of potentially infectious cases ( 7/11 ; 64% ) . for morning report and surgical day performance , respondents indicated the application improved the speed ( 11/16 ; 69% ) , clarity ( 13/16 ; 81% ) , and accuracy ( 10/16 ; 63% ) of morning presurgical case review . the survey was conducted before the addition of the operating room map overview feature ; even so , a majority surveyed reported case review at the time of diagnosis ( 14/16 ; 88% ) and situational awareness of multiple ongoing cases ( 13/16 ; 81% ) was improved by the sps - pm . review of clinical information , prior pathology , and imaging are an essential component of pathological diagnosis in all settings . the frozen section practice at mayo clinic in rochester is unique in that its rapid turn - around time and large case volumes require that a large number of individuals take part in rendering an intraoperative consultation . as all team members require working knowledge of the relevant case information , gathering and dissemination of that information take on a vital significance . while the time - tested method of manual record abstraction from the ehr followed by verbal communication of the information to the laboratory staff was effective , it was also inefficient . development of the sps - pm application allowed many of those inefficiencies to be addressed , resulting in expected improvements in both trainee 's time required and effort expended . one immediate benefit of moving the surgical list review process to an electronic platform was the transferability of completed case annotations when a surgical date would change . before implementation of the sps - pm , trainees would usually be required to wait until the next day 's surgical schedule was finalized at 19:00 h before embarking on the several hour processes of running the list . three factors drove this behavior : ( 1 ) a requirement that the list be discussed at morning report in the correct final order ( no list changes in operating room assignments or case order ) , ( 2 ) a desire to avoid reviewing cases that might be cancelled or postponed , ( 3 ) a need to ensure all cases requiring frozen section diagnosis were identified and researched ( i.e. , no omission of late add - on cases ) . the sps - pm application has largely nullified these concerns . if the order of cases is changed or a patient 's surgery moved , the surgical list is automatically reordered with all associated pathology notes ; therefore , there was no risk of lost effort in reviewing a case . indeed , the persistence of case notes has enabled trainees to begin the list review process several days in advance of the surgical date ( as soon as a listing is made ) , thereby easing the caseload for review on the evening before the surgical day . finally , because the case list can be quickly scanned in real time with associated notes , seemingly last minute in addition , there were several unintended but beneficial outcomes associated with the sps - pm application . although not a planned feature , the visibility of notes in a central repository allowed for better coordination between trainees reviewing the same list , largely eliminating the problem of duplicate case review . furthermore , the addition of text searching within the surgical list led to organ system - specific subspecialty groups ( e.g. , hematopathology and neuropathology ) preemptively screening the day 's cases to identify those likely to require intraoperative subspecialty consultation or special handling at frozen section . finally , the recent integration of research protocol information into the sps - pm has allowed for expedited research collections , replacing the previously performed daily manual list abstraction work performed by pathology assistants and technicians . the sps - pm application was rapidly adopted into the frozen section pathology laboratory practice at mayo clinic . the survey results suggest user acceptance was , at least in part , due to the perception that the application significantly improved essential aspects of case review workflow . while it is unlikely that a software application for frozen section case review would be necessary at all institutions practicing frozen section pathology , the user - centered development process and rapid prototyping design process may be useful approaches for process improvement initiatives in many settings . in addition , this work provides a practical example of how software applications can assist pathology workflows by enabling extraction ( from the ehr ) and presentation of diagnostically relevant patient information .

background : the frozen section pathology practice at mayo clinic in rochester performs ~20,000 intraoperative consultations a year ( ~7080/weekday ) . to prepare for intraoperative consultations , surgical pathology fellows and residents review the case history , previous pathology , and relevant imaging the day before surgery . before the work described herein , review of pending surgical pathology cases was a paper - based process requiring handwritten transcription from the electronic health record , a laborious and potentially error prone process.methods:to facilitate more efficient case review , a modular extension of an existing surgical listing software application ( surgical and procedure scheduling [ sps ] ) was developed . the module ( sps - pathology - specific module [ pm ] ) added pathology - specific functionality including recording case notes , prefetching of radiology , pathology , and operative reports from the medical record , flagging infectious cases , and real - time tracking of cases in the operating room . after implementation , users were surveyed about its impact on the surgical pathology practice.results:there were 16 survey respondents ( five staff pathologists and eleven residents or fellows ) . all trainees ( 11/11 ) responded that the application improved an aspect of surgical list review including abstraction from medical records ( 10/11 ) , identification of possibly infectious cases ( 7/11 ) , and speed of list preparation ( 10/11 ) . the average reported time savings in list preparation was 1.4 h / day . respondents indicated the application improved the speed ( 11/16 ) , clarity ( 13/16 ) , and accuracy ( 10/16 ) of morning report . during the workday , respondents reported the application improved real - time case review ( 14/16 ) and situational awareness of ongoing cases ( 13/16).conclusions : a majority of respondents found the sps - pm improved all preparatory and logistical aspects of the mayo clinic frozen section surgical pathology practice . in addition , use of the sps - pm saved an average of 1.4 h / day for residents and fellows engaged in preparatory case review .

I M Process analysis Development process Technical background Survey R Application features User perceptions D C Financial support and sponsorship Conflicts of interest

PMC4301210

escherichia coli o157:h7 ( eco157 ) usually causes uncomplicated diarrhoea , but occasionally , it is responsible for causing severe forms of illness including haemorrhagic colitis , and potentially lethal haemolytic uraemic syndrome ( hus ) in humans [ 1 , 2 ] . humans can become exposed to eco157 through direct contact with an infected animal or a contaminated farm environment . the most common mode of human exposure is through the consumption of contaminated foods including meat , milk , raw vegetables and fruits . the majority of outbreaks or illnesses with eco157 in humans , either foodborne or otherwise can be traced back to cattle [ 1 , 7 , 8 ] . a large body of information on the epidemiology of eco157 in cattle has accumulated as awareness of human illness due to this pathogen has increased . studies on the presence of eco157 in cattle and their environments have revealed a seasonal pattern with increased prevalence of eco157 faecal shedding during summer and early autumn [ 1012 ] . experimental inoculation studies have identified the sites of localization of the bacterium in the gastrointestinal tract of cattle , including the jejunum , ileum , caecum , colon and recto - anal junction . the magnitude and duration of shedding and the associated risk factors have been studied under both experimental [ 13 , 14 ] and observational [ 15 , 16 ] settings . a few studies have quantified eco157 transmission in cattle using mathematical modelling [ 12 , 17 , 18 ] . while these studies have been useful in describing eco157 transmission dynamics and identified potential control strategies , the transmission rate [ often partitioned into the direct and indirect ( environmental ) rate ] used in those models and the contribution of the direct and environmental transmission routes to the overall infection spread , were largely assumed . the transmission rate , , is defined as the product of contact rate ( number of contacts per unit time ) and the probability of transmission given the contact . this is a very important quantity in modelling of infectious diseases because it determines the dynamics of the infection spread predicted by the model . environment settings that can be impacted by host population density and age - related susceptibility , pathogen strain colonization ability and environmental factors affecting any of these characteristics . therefore , in mathematical modelling studies it is important yet challenging to choose a value that closely represents the true rate of transmission . in 2004 , laegreid & keen estimated the transmission of eco157 in beef calves using seroconversion data . more recently , another group used experimental transmission data to quantify eco157 transmission in dairy calves . however , there are two major problems with the transmission rates reported in these two studies : ( i ) they did not consider how the level of environmental contamination with the bacterium may influence transmission , which is important given that eco157 is spread by the faecal oral route and ( ii ) they did not consider the possibility of a difference in transmission among strains of eco157 . a recent , experimental inoculation study has highlighted the strain - specific differences in the probability of colonization post - inoculation and in the faecal shedding pattern of colonized cattle . given that different strains of eco157 have different host colonization abilities and shedding patterns in cattle , it is likely that transmission dynamics and the transmission rate will also vary between strains of eco157 . transmission experiments under controlled settings provide a valuable means to study the effect of a few selected factors ( e.g. strain type ) in which the confounding variation due to other factors is minimized . the objective of this study was to estimate and compare the transmission rates for three different strains of eco157 in steers under controlled experimental conditions that involved joining susceptible ( non - inoculated ) steers and steers inoculated with a three - strain mixture of eco157 . the hypotheses to be tested were : ( i ) that different strains of eco157 have different rates of transmission from infectious to susceptible animals , and ( ii ) that the rate of transmission in cattle is affected by the level of eco157 environmental contamination . in an effort to explain the mechanism by which environmental contamination influences eco157 transmission in animals , an in vitro experiment was conducted to test survival of the three eco157 strains in bovine faeces . all procedures were approved by the university of wisconsin madison school of veterinary medicine institutional animal care and use committee ( protocol no . twenty - two holstein steers , aged 67 months , were selected for the transmission experiment . these steers had been used previously in a separate study involving single strain challenge experiments with one of three strains of eco157 ( but no infection transmission among animals ) and they were reused in the current experiment for ethical reasons ( to minimize the number of animals to be sacrificed ) . moreover , the experimental design partially controlled for the confounding effect of host factors which might have been introduced by enrolment of different animals . following the termination of the single strain challenge experiments on 23 july 2010 , the steers were cleared of residual infection with eco157 by orally dosing them with 6 g neomycin ( neomycin oral solution 200 mg / ml ; agrilabs , usa ) , once daily for 5 days . after completing the course of the antibiotic treatment , the steers were confirmed to be clear of eco157 by testing negative by culture and polymerase chain reaction ( pcr ) for at least three consecutive recto - anal mucosal swabs ( rams ) taken on alternate days . rams were obtained by inserting sterile cotton - tipped applicators 12 inches into the rectum and rubbing 34 times along the mucosal surface . out of the 22 steers , 12 were randomly selected and separated for inoculation as the infectious group and housed in two separate pens ( six steers each ) in one room . pens within a room were separated by a corridor to prevent contact of animals between groups . the remaining 10 steers were housed in a separate room to serve as the susceptible contacts in two replicates of the transmission experiment . pen environment was made free of eco157 by disinfection and eco157-free status was confirmed by taking environmental samples before housing the steers . the two replicates of the transmission experiment were conducted to capture possible variations between experiments , so that a reliable estimate of transmission could be measured . more than two replicates for the experiment were not feasible due to financial and ethical constraints . on day 0 , the 12 steers in the infectious group were inoculated orally with a cocktail of three eco157 strains , each at a dose of 10 colony - forming units ( c.f.u . ) . the simultaneous inoculation of three strains was chosen in order to control for the confounding effect of host factors and under the assumption that the strains do not have any effect ( synergistic or antagonistic ) on each other . each of the 12 steers was inoculated by mixing 1 ml inoculum with ~10 ml clean tap water in a drinking cup . a procedure described previously was followed to ensure the inoculum was consumed . the infection status of the inoculated steers was determined 1 day post - inoculation ( p.i . ) by bacteriological culture of rams : the steers were considered infectious if they were shedding at least one of the three inoculated strains . the three inoculated strains were frik47 , frik1641 and frik2533 , which have been described in previous studies [ 22 , 24 ] . briefly , the three strains have different genotypes ( e.g. frik1641 and frik2533 lacked stx1 and hly933 , respectively ) and ecological histories ( frik47 was involved in a ground beef - associated human outbreak , frik1641 was recovered from a raccoon and was selected as a representative of non - bovine adapted strains , and frik2533 was obtained from water on a cattle farm ) . for brevity , from here onwards we will refer to frik47 , frik1641 and frik2533 strains as st1 , st2 and st3 , respectively . one day after confirmation of the infectious status ( i.e. day 2 p.i . ) , five randomly selected animals each from the susceptible contact group were joined with five randomly selected infectious animals and each newly formed group of 10 animals was housed in one of the two separate rooms . the two groups of steers , each with five infectious and five susceptible steers , established the two replicates of the experiment . for brevity both groups were followed for 30 days to monitor eco157 colonization status and environmental contamination over time by testing rams and environmental samples , respectively . the length of 30 days was chosen based on the expectation that the duration of eco157 infection will not exceed 30 days . shedding of eco157 in cattle is intermittent in nature , and so we adopted a frequency of sampling every other day , which was considered frequent enough to capture the intermittent shedding behaviour and was logistically feasible . the protocol for collection of rams and environmental samples is described in kulow et al . . the rams from each individual steer and samples from selected environmental locations were collected the day after mixing ( i.e. day 3 p.i . ) and on alternate days thereafter for the follow - up period , for a total of 15 sampling occasions . sampled environmental locations included : animal hides , feed troughs , drinking water , water - cup run - offs and pen floors . the details of the performed eco157 isolation and enumeration and pcr confirmation were as described previously . briefly , rams were placed into a sterile tube containing 5 ml modified e. coli broth ( mec ; remel , usa ) and novobiocin ( 002 mg / ml , mecnov ; remel ) . drinking water ( 10 ml ) was sampled directly from water cups . to collect samples of hides and feed troughs , we used sterile cellulose sponges ( 2 35 1 inch ) moistened with mecnov broth ; after sampling , a sponge was placed in a sterile whirl - pak ( nasco , usa ) containing 10 ml mecnov broth . samples from water - cup run - offs and pen floor were collected using sterile nylon ropes about 3-feet long and 8-mm thick ( ~52 g ; koch industries , usa ) . colonies suspected to be eco157 were presumptively identified using the rim e. coli o157:h7 latex test ( remel ) . for each sample containing colonies that were positive by the latex agglutination test , up to 10 randomly selected colonies were taken for strain differentiation ( i.e. to identify st1 , st2 and st3 ) using pcr . the minimum detection limit was ~2 c.f.u./ml , i.e. 10 c.f.u./rams and 10 c.f.u./environmental sample . bovine faeces collected from a local farm was processed as described previously and the water content of the faeces was reduced to a final aw between 087 and 090 by heating small aliquots ( ~2030 g wet feces ) in a glass dish until an aw of 087089 was achieved . the aw was determined using an aqualab model 4te aw meter ( decagon devices , usa ) according to the manufacturer 's instructions . the faeces were then sterilized at 121c , 15 psi , for 15 min , which did not significantly change the aw . the three bacterial strains ( st1 , st2 , st3 ) were grown from a single colony in 5 ml luria bertani ( lb ) broth at 37c for 2224 h and diluted 1:10 in 1 pbs to a concentration of ~10 c.f.u./ml . the diluted culture ( 650 l ) was mixed with 65 g dried faeces in sterile dishes and incubated at 30c for 28 days . samples were taken on days 0 , 1 , and 3 during the first week and twice a week thereafter . samples ( 05 g faeces ) were serially diluted in pbs and plated in duplicate on lb agar plates , incubated at 37c for 2224 h , and the number of c.f.u . enumerated . three independent experiments were performed . to facilitate proper analysis of the data , the infectious status of the individual animals at each sampling day was determined and categorized into one of the three groups : susceptible ( s ) , infectious ( i ) or latent ( l ) . in this study , infectious ( i ) is used to indicate the shedding state of an animal that is capable of infecting ( colonizing ) susceptible animals . the latent ( l ) class is used to indicate an infected ( colonized ) animal during its transient non - shedding periods , when the animal is considered to be non - infectious . thus , an infected animal is intermittently in either i or l class and a non - infected animal is in s class . the infection status for each steer on each of the 15 sampling days was determined under the following assumptions that reflected the intermittent shedding pattern of eco157 by the infected animals as reported in gautam et al . : ( i ) an animal testing positive was considered infectious ( i ) , ( ii ) an animal testing negative could be classified either as latent ( l ) or susceptible ( s ) depending on the timing and the number of consecutive samples that were test - negative . if an animal was negative on 4 consecutive samplings ( i.e. 8 days ) following a positive sample , it was classified as latent ( l ) , otherwise susceptible ( s ) . a steer that was introduced into the experiment as a susceptible animal was considered susceptible until it tested positive for the first time . similarly , an animal that was found negative on more than four consecutive sampling days ( i.e. > 8 days ) after the last positive test was considered recovered from colonization , and thus susceptible again . if an animal tested negative towards the end of the study and it was not possible to apply the criterion mentioned above to classify the animal into one of the two possible non - shedding classes ( l or s ) because of too few negative samples , then that particular sampling observation on the individual animal was censored . using the above information and considering that incidents of new infections ( cases ) at consecutive samplings are governed by a stochastic process , the transmission of strain - specific eco157 can be described by a modified susceptible - infectious - susceptible ( sisl ) mathematical model where the infectious state branches into a latent state ( fig . although there were three strains in the transmission experiment , st3 was only detected a few times in only one inoculated steer and was not detected in any of the other inoculated and susceptible steers in the two groups . since no new cases of cattle colonized with st3 were observed the transmission rate for this strain was considered zero ( the statistical estimation of the transmission rate was not possible because no new case was observed ) . schematic representation of the sisl transmission model of eco157 in cattle . in a closed population of size n = s+i+l = 10 , the number of susceptible individuals that may become infectious per time interval , t , depends only on the transmission rate ( the rate at which a randomly chosen susceptible animal had successful infectious contacts in the time interval t ) , the number of susceptible ( s ) animals , the number of infectious ( i ) animals and the total population size n. if the number of new cases c at the end of each time interval ( t ) is considered a stochastic process based on a binomial distribution with s possible outcomes , then the transmission rate can be estimated using a function of i , s , c , n and t and can be represented for our sisl system as follows:(1)where s is the number of susceptible s(t ) individuals at the start of the interval , i is the average number of infectious i(t ) individuals during the interval , n is the population size ( here n = 10 ) and t is the sampling interval . taking log on both sides , equation ( 1 ) can be expressed as:(2)here and onwards , log denotes the natural logarithm ( base e ) . from equation ( 2 ) , log( ) can be estimated from the transmission experiment data by using a generalized linear model ( glm ) . equation ( 2 ) was extended into a covariate effects model to control for the effect of covariates on log( ) as follows:(3)the considered covariates were strain , st ( st1 or st2 ; term 1 ) , experimental group , g ( g1 or g2 ; term 2 ) , and the strain- and group - specific environmental contamination level on the preceding sampling day ( e ; term 3 ) . the level of environmental contamination during the immediately preceding sampling day was used because contamination level on the same day of sampling is unlikely to generate a new case on that very day . lag times longer than 2 days ( i.e. the preceding sampling date ) were deemed biologically irrelevant because it has been estimated that shedding occurs at the latest 2 days after successful inoculation . the overall level of environmental contamination for a given sampling day was determined by log10 transformation of the sum of the eco157 counts from all positive environmental samples for a given strain and group on that day among the collected samples of animal hides , feed troughs , drinking water , water - cup run - offs and pen floor . the considered environmental contamination covariate was a proxy for some true , but impossible to measure , total environmental contamination on a particular day . however , because the measurements were consistently estimated for all strains , experimental groups and days , they were comparable . the above model [ equation ( 3 ) ] was implemented in r version 2.13.1 ( r development core team , 2011 ) using glm function with a complementary log - log link . the glm version of the model equation is:(4)where c represents the number of new cases , s defines the number of trials of a binomial distribution , st is the strain of eco157 ( with st1 = 0 and st2 = 1 ) , g is the experimental group ( with g1 = 0 and g2 = 1 ) , e is the pathogen load in the environment during the immediately previous sampling day ( continuous on log10 scale ) and offset = log[(i / n)*t ] . in the offset term , note that if i = 0 , this will produce an indeterminate value preventing the glm from computing the transmission rate . on several sampling days , there were no infectious individuals ( i.e. i = 0 ) for st2 in g1 . for those days , in order to facilitate the computation , we added one infectious animal to the state i , and subtracted one from the state s ( i.e. we used i+1 and s 1 ) . the implications of this assumption were assessed by comparing the model results from the full dataset with those produced for a subset of dataset after eliminating observations for st2 in g1 . in the model shown in equation ( 4 ) the coefficient 0 can be interpreted as the transmission rate on the log scale ( log( ) ) for st1 in g1 when e = 0 . the coefficient 1 is the average difference in log( ) between the two strains of eco157 after controlling for group and e , 2 is the average difference in log( ) between the two groups after controlling for strain and e , and 3 is the average increase in log( ) for a 1-unit increase in e after controlling for group and strain differences . for the specific combination of the group , strain and contamination load in e , log( ) was estimated , the exponentiation of which gives transmission rate , . the intercept - only form of the model in equation ( 4 ) was run on the subsets of data for the individual strains to estimate the actual strain - specific transmission rates in the conducted experiment . it is important to note that , as defined in this study , is frequency dependent and therefore , the term 1/n is absorbed into its parameterization . the model in equation ( 4 ) was used to test the hypotheses that ( i ) transmission rates differ for two different eco157 strains ( st1 , st2 ) and ( ii ) environmental contamination level influences the transmission rate of these two eco157 strains in cattle . these hypotheses were tested by running several glms , including models with one covariate at a time ( univariate analysis ) and models with multiple covariates considered simultaneously ( multivariable analysis ) . in these analyses we considered environmental contamination as a continuous variable ( i.e. log10-transformed total eco157 load ) or dichotomized variable ( representing whether the environment was contaminated or not with a particular strain on a particular day ) . when using environmental contamination as a continuous covariate , the validity of the linearity assumption was assessed graphically by plotting , and lowess smoothing , of complementary log - log probability of the outcome variable against the explanatory variable [ 33 , 34 ] . brown - forsythe levene 's test supported the validity of the assumption of homogeneity of variances between the strain - specific data ( p = 008 ) . the best - fitting model was selected using akaike 's information criterion ( aic = 7726 ) . model fit was further evaluated by checking the over - dispersion parameter , which is the ratio of the deviance over the degrees of freedom . the over - dispersion parameter was slightly < 1 for the final covariate model and 1 for the two strain - specific intercept models ; it was therefore concluded that the final models did not have the over- or under - dispersion problem . to compare survival of the three eco157 strains in bovine faeces , a repeated - measures anova was performed on the natural log ( base e)-transformed counts of eco157 for the three strains and the strain - specific decay rates were calculated using the standard exponential growth / decline equation : where r = decay rate , n(t ) = log(c.f.u . eco157 ) at time zero , and t = time ( in days ) . time ( t ) was the length of time between the first and the last sampling day in this experiment ( i.e. days 0 and 28 ) . the sisl system in figure 1 was solved analytically to obtain an expression for the basic reproduction number ( r0 ) . we used the next - generation matrix ( ngm ) method to derive r0 . the ngm can be derived from the transmission matrix f and transition matrix v : the ngm , h , was obtained by taking the product of matrix f and the inverse of matrix v : from the spectral radius of matrix h , r0 was determined to be /f . in the derived r0 expression 1/ represents the length of the infectious period before moving into the s compartment ( indicating recovery ) or into the l compartment ( indicating temporary cessation of infectiousness ) . the term f can be interpreted as the weighting variable to account for the fact that only a fraction , f , of animals leaving state i enter into state s while the fraction ( 1f ) temporarily stops shedding and is thus considered latent ( l ) . because f reduces the recovery rate , it extends the total duration of infectious period ( i.e. 1/f ) . previously , we estimated 1/ for st1 to be 43 [ 95% confidence interval ( ci ) 04172 ] days and for st2 to be 53 ( 95% ci 09128 ) days . we also estimated f for st1 to be 025 and for st2 to be 05 . these estimates indicate that compared to st2 , st1 has on average larger ( i.e. shorter single episode of shedding ) but smaller f ( i.e. higher probability of extending infection through the mechanism of intermittent shedding ) . for an infected individual , the total duration of effective infectious period after discounting for the time spent in l ( 1/f ) , was estimated as 172 ( 95% ci 16688 ) days for st1 and 106 ( 95% ci 18256 ) days for st2 . the strain - specific r0 was then calculated using the derived expression r0 = /f . none of the inoculated and contact animals developed clinical symptoms during the experiments . all inoculated animals in g1 and four inoculated animals in g2 were shedding at least one of the three strains of eco157 on day 1 p.i . in one of the inoculated steers , shedding was not detected until day 11 p.i . and this animal was considered susceptible until it was first detected as positive ( when it was considered a new case ) . four inoculated steers in g1 and three inoculated steers in g2 were excreting st1 on day 1 p.i . , while only two inoculated steers were shedding st2 in both groups on day 1 p.i . only one inoculated steer from g1 tested positive for st3 during the entire study period . that steer excreted st3 on days 1 , 11 , 13 and 15 p.i . at the average shedding level in positive rams of 095 log10 c.f.u./ml . none of the contact animals in either experimental group excreted st3 during the study period . overall , average numbers of positive rams between the two experimental replicates for st1 , st2 and st3 were 565 , 185 and 25 , respectively . the average ( standard deviation , median ) shedding level in positive rams for st1 was 212 ( 132 , 191 ) log10 c.f.u./ml in g1 and 237 ( 156 , 221 ) log10 c.f.u./ml in g2 . the average ( standard deviation , median ) shedding level in positive rams for st2 was 177 ( 058 , 164 ) log10 c.f.u./ml in g1 and 177 ( 112 , 176 ) log10 c.f.u./ml in g2 . the number of steers testing positive and negative for st1 and st2 in the two experimental groups for any given day is given in table 1 . table 1.experimental infection transmission data for the two strains of eco157 in each of the two experimental groups ( g1 and g2 ) . inoculated steers were joined with the susceptible group on day 1 post - inoculationsddg1 ( n = 10)g2 ( n = 10)eco157 positivemodel input dataeco157 positivemodel input dataiccciscldtsteiccciscldtste014/50/546 st1 3/50/537 st1 233/50/546001 st1 3/50/5357101 st1 453/50/537002 st1 202/50/536012 st1 0673/50/537002 st1 322/52/536222 st1 6893/51/547202 st1 264/53/5454122 st1 4810112/51/546002 st1 352/50/5753102 st1 2812132/51/536012 st1 04/50/526022 st1 2914152/52/546112 st1 323/50/546002 st1 016173/51/545022 st1 03/50/537002 st1 018193/51/545012 st1 03/50/537002 st1 020214/50/545012 st1 191/52/547202 st1 022234/51/545012 st1 01/53/5356112 st1 324253/50/555002 st1 01/52/545012 st1 026273/51/536012 st1 02/53/535022 st1 028291/51/546002 st1 01/53/555002 st1 030313/50/536122 st1 02/53/5455112 st1 18012/50/5280 st2 2/50/528 st2 230/50/528001 st2 1/51/5258101 st2 450/50/5010002 st2 01/52/5258102 st2 0670/50/5010002 st2 01/50/537002 st2 0890/50/5010002 st2 01/50/518012 s2 3710110/50/5010002 st2 02/53/5258312 st2 4112130/50/5010002 st2 02/50/5555102 st2 2714150/50/5010002 st2 02/51/526022 st2 016171/50/5.510102 st2 02/50/535022 st2 018190/50/519002 st2 02/52/5256122 st2 020210/50/5010002 st2 01/50/545012 st2 022230/50/5010002 st2 02/50/518012 st2 024251/50/5.510102 st2 01/50/528002 st2 026270/50/519002 st2 00/51/5159102 st2 028290/50/5010002 st2 01/51/518012 st2 030310/50/5010002 st2 01/50/527012 st2 12 sampling day ; days post - inoculation ; inoculated steers ; contact steers ; average number of infectious steers during the time interval before sampling . new cases were assumed to be infectious in between the time interval ; number of susceptible steers at the beginning of the time interval prior to sampling ; number of new cases per time interval ; number of intermittent non - shedding steers at the beginning of the time interval preceding sampling ; time interval between two samplings ; strain of eco157 ; number of eco157 in the environmental sample during the preceding sampling time in log10 scale . experimental infection transmission data for the two strains of eco157 in each of the two experimental groups ( g1 and g2 ) . inoculated steers were joined with the susceptible group on day 1 post - inoculation sampling day ; days post - inoculation ; inoculated steers ; contact steers ; average number of infectious steers during the time interval before sampling . new cases were assumed to be infectious in between the time interval ; number of susceptible steers at the beginning of the time interval prior to sampling ; number of new cases per time interval ; number of intermittent non - shedding steers at the beginning of the time interval preceding sampling ; time interval between two samplings ; strain of eco157 ; number of eco157 in the environmental sample during the preceding sampling time in log10 scale . the environmental samples were collected on 15 sampling days from each of the two groups ( g1 and g2 ) of animals following the mixing of infectious and susceptible steers ( table 2 ) . in g1 , st1 and st3 were recovered , respectively , on six and two out of 15 sampling occasions and st2 was never detected in any of the environmental samples . in g2 , environmental samples were positive on seven , five and zero sampling occasions for st1 , st2 and st3 , respectively . for st1 , the average load ( standard deviation , median ) of eco157 in positive environmental samples was 274 ( 067 , 293 ) log10 c.f.u./ml in g1 and 334 ( 152 , 292 ) log10 c.f.u./ml in g2 ; for st2 , the average load ( standard deviation , median ) was 238 ( 110 , 268 ) log10 c.f.u./ml in g2 ( no samples were positive in g1 ) ; and for st3 , the average load ( standard deviation , median ) was 273 ( 030 , 273 ) log10 c.f.u./ml in g1 ( no samples were positive in g2 ) . table 2.eco157 load ( expressed as log10-transformed c.f.u./ml ) in the environment during the infection transmission experiment for the two strains ( st1 and st2 ) in each of the two experimental groups by sampling day post - inoculationgroupenv . 1970001650000002840000001820000000000000000001feed st1 0000001860000000000000000000000000000000000001water st1 0002420000000000000000000000000000000000000001pen st1 000317246348000288000000070000000000n.a.0000001w.cup st1 090000000000000000000000090000000000n.a.0000002hide st1 0002874681812910000000000000000000000001180002feed st1 0000002510000000000000000000000000000000000002water st1 0000000000000000000000000000000000000000000002pen st1 000600417273000000000n.a.000300000n.a.000n.a.1592w.cup st1 000000361000000000000000000000000n.a.0001711341hide st2 0000000000000000000000000000000000000000000001feed st2 0000000000000000000000000000000000000000000001water st2 0000000000000000000000000000000000000000000001pen st2 000000000000000000000000000000000000n.a.0000001w.cup st2 000000000000000000000000000000000000n.a.0000002hide st2 0000000004120000000000000000000000000001180002feed st2 0000000000000000000000000000000000000000000002water st2 0000000000000000000000000000000000000000000002pen st2 000000372273268000000n.a.000000000n.a.000n.a.0002w.cup st2 000000000000000000000000000000000n.a.000000085 feed trough sample ; pen floor sample ; water - cup run - off sample.n.a . sample not collected because the rope was eaten by the steers ; st3 was only detected from the environment in group 1 on one occasion from the hide ( day 7 post - inoculation ) and on two occasions from the pen floor surface ( days 3 and 7 post - inoculation ) and is not shown in the table . eco157 load ( expressed as log10-transformed c.f.u./ml ) in the environment during the infection transmission experiment for the two strains ( st1 and st2 ) in each of the two experimental groups by sampling day post - inoculation feed trough sample ; pen floor sample ; water - cup run - off sample . . sample not collected because the rope was eaten by the steers ; st3 was only detected from the environment in group 1 on one occasion from the hide ( day 7 post - inoculation ) and on two occasions from the pen floor surface ( days 3 and 7 post - inoculation ) and is not shown in the table . in the univariate analysis , the effect of environmental contamination level significantly influenced the transmission rate ( p = 00002 ) , the group effect was marginally significant ( p = 0052 ) and the strain effect was not significant . all three covariates were used in the multivariable glm to assess the effect of strain on transmission rate . the average transmission rate per animal per day for st1 in experimental group g1 in the absence of bacterial contamination of the environment was 0038 ( table 3 ) . the transmission rate for st2 was on average 169 times greater than that for st1 , albeit not statistically significantly different after controlling for the experimental group and environmental contamination . table 3.transmission rate ( ) with 95% confidence interval ( ci ) estimated using the generalized linear model with covariates ( strain , group , environment)variables 95% cip valuebaseline*003800133009strain st2169065437027 st1group g215605846104 g1 e151191900006*corresponds to estimate for st1 in group 1 at log10 ( env ) = 0.eco157 load in the environment during the preceding sampling time ( i.e. 2 days prior ) in log10 scale . transmission rate ( ) with 95% confidence interval ( ci ) estimated using the generalized linear model with covariates ( strain , group , environment ) corresponds to estimate for st1 in group 1 at log10 ( env ) = 0 . eco157 load in the environment during the preceding sampling time ( i.e. 2 days prior ) in log10 scale . after controlling for the strain and group factors , contamination of the environment during the immediately preceding sampling day significantly increased the transmission rate . on average , the transmission rate increased 15 times for every 1-unit ( on log10 scale ) increase in the environmental contamination . table 4 shows the increase in the mean transmission rate and the corresponding r0 for st1 and st2 for increasing levels of environmental contamination as predicted by the model . table 5 shows the transmission rate and the corresponding r0 for the two strains describing the overall dynamics in the conducted experiments that accounts for the strain - specific levels of environmental contamination over time implicitly . the transmission rate estimated for st2 after eliminating observations from g1 slightly increased the estimated value , but did not alter the result significantly . this confirmed that adding an infectious and subtracting a susceptible animal for st2 on days when there were no detected infectious animals in g1 did not affect the conclusions of the study . table 4.transmission rate ( ) and basic reproduction number ( r0 ) with 95% confidence intervals ( ci ) for different levels of environmental contamination ( e ) represented in log10 scalestraingroupe = 0e = 1e = 3e = 5 ( 95% ci)r0 ( 95% ci ) r 0 r 0 r 0 st1 g10038 ( 0013009)065 ( 022155)005709801712940285490g20060 ( 0008042)103 ( 014722)009155027464045774all0045 ( 00180097)077 ( 031167)006811702033490338581 st2 g10064 ( 0009039)068 ( 010413)00961020288242048510g20096 ( 0005182)102 ( 0051929)01441210432458072763all0086 ( 0013046)091 ( 014488)012913703874100645684 table 5.transmission rate ( ) and basic reproduction number ( r0 ) with 95% confidence intervals ( ci ) for the two strains of eco157 estimated using the intercept - only generalized linear modelstrainmean ( 95% ci)mean r0 ( 95% ci ) st1 011 ( 006018)189 ( 103310 ) st2 014 ( 007024)148 ( 074254 ) transmission rate ( ) and basic reproduction number ( r0 ) with 95% confidence intervals ( ci ) for different levels of environmental contamination ( e ) represented in log10 scale transmission rate ( ) and basic reproduction number ( r0 ) with 95% confidence intervals ( ci ) for the two strains of eco157 estimated using the intercept - only generalized linear model the average number of c.f.u.s of st1 , st2 and st3 in faeces over a 28-day storage period is shown in table 6 and the percent survival of each strain over time is presented in figure 2 . the growth of the three strains in lb was similar which resulted in essentially equal starting numbers of c.f.u.s ( viable counts ) at day 0 , suggesting that the three strains have similar growth rates in the conditions tested . during the first 24 h post - inoculation in faeces , all three strains displayed around a 1-log reduction in viable counts with an average percent survival of 22% , 13% and 2% , respectively for st1 , st2 and st3 . after this initial decrease , the viable numbers of st1 remained relatively constant over the 28 days of incubation ( 254 10 c.f.u./g , 10% survivors ) . by contrast , st3 had the initial 1-log reduction followed by an additional 3-log reduction in viable counts during the 28 days of incubation ( 743 10 c.f.u./g , 001% survivors ) . the survival curve of st2 was in between those of st1 ( best survival ) and st3 ( poorest survival ) and had a 3-log reduction during the 28 days of incubation ( 279 10 c.f.u./g , 008% survivors ) . the mean decay rates estimated for st1 , st2 and st3 were 021 , 026 , 039 , respectively . nevertheless , the results of the repeated - measures anova and comparison of decay rates did not confirm existence of a significantly different survival in the three strains . 2.comparison of the percent survival of the three eco157 strains ( st1 , st2 , st3 ) in sterilized bovine faeces over time . count with the standard deviation ( s.d . ) , expressed per million ( 10 ) , for three eco157 strains , st1 , st2 and st3 , in bovine faeces during the survivability assayday st1 st2 st3 mean s.d . 038401740351093139502410166142445509211715835786531330320330427444546064025018024103053920680240160261419025004701201001517342476040012012017211512110100030020032420929401000400100228254359003002001001 comparison of the percent survival of the three eco157 strains ( st1 , st2 , st3 ) in sterilized bovine faeces over time . count with the standard deviation ( s.d . ) , expressed per million ( 10 ) , for three eco157 strains , st1 , st2 and st3 , in bovine faeces during the survivability assay the aim of this study was to quantify and compare the transmission rates of different strains of eco157 in steers through a combination of an infection transmission experiment and mathematical modelling . the main finding of the study strongly supports the hypothesis that the transmission rate significantly increases with increasing levels of environmental contamination . two of the three tested strains had similar transmission rates , but the third did not transmit at all , supporting the hypothesis that eco157 strains are characterized by different transmission rates in the cattle population . in the following sections , we will describe these findings in the context of existing information and discuss important implications , strengths and limitations . previously , laegreid & keen estimated r0 for eco157 in beef calves in a cow - calf operation to be 525 ( 95% ci 387664 ) based on time to seroconversion after birth , using final size of the infected population . this means that one calf could contribute to at most one case when it seroconverted for eco157 and could not account for re - infection . in our study , we allowed the animals to contribute to more than one case by permitting them to join the susceptible group immediately after recovery from infection . later , another group estimated r0 of eco157 in dairy calves to be 73 , based on the transmission experiment data under field conditions ( i.e. the calves were raised in outside pasture ) with a sampling interval of 1 day , and in dairy cattle to be 07 , based on the shedding prevalence with a sampling interval of 1 month . the sampling interval of 1 month that they used in dairy cattle is longer than the average duration of infectiousness for two strains of eco157 we estimated in a previous study ( 172 days for st1 and 106 days for st2 ) , which may have resulted in the r0 estimate of < 1 in dairy cattle . furthermore , those studies only considered the infectious animals in the transmission model and did not consider the influence of eco157 load in the environment ; neither did they consider the intermittent shedding pattern explicitly in the model . in the current study we have included the effect of environmental contamination in the estimation of transmission rate ( and showed how eco157 transmission in cattle is affected by the level of bacterial contamination in the barn environment ) and explicitly represented the intermittent shedding pattern in the model . if the duration of infectiousness is not corrected for the intermittent shedding pattern ( i.e. by discounting the period of non - shedding ) , it will lead to overestimation of r0 . the differences between our and their estimated values of r0 could be partly due to the differences in the level of environmental contamination with the bacterium , due to strain differences and/or the experimental setting . regarding intermittent shedding as modelled in our study , it is possible that the recorded intermittent shedding actually represented shedding that alternated below and above the detection level . however , the detection level in our study was relatively low ( 2 c.f.u./ml ) . therefore , even if an animal recorded as negative was actually shedding at a level of < 2 c.f.u./ml , we believe that such an animal was less infectious than the animals that tested positive and that it is appropriate to consider such an animal as non - infectious . although not significantly different , the mean transmission rate for st1 was smaller than that estimated for st2 . however , due to a substantially longer duration of infectiousness of st1 , the mean r0 values computed for this strain for several different fixed values of environmental contamination were similar to those estimated for st2 ( table 4 ) . furthermore , the overall r0 estimated for st1 was higher than that estimated for st2 ( table 5 ) , which may be explained by a higher average load of st1 in positive environmental samples ( 274 log10 c.f.u./ml in g1 and 334 log10 c.f.u./ml in g2 ) compared to st2 ( no positives in g1 and 238 log10 c.f.u . in g2 ) . according to velthuis et al . , our study had a very low power to detect a difference between st1 and st2 even if such a difference exists , but would have had a reasonable power ( up to 80% ) to detect a difference between st3 and either of the strains st1 or st2 should the data for st3 have allowed statistical comparison . that said , detecting a small difference in strains , such as that between st1 and st2 , would have a limited practical value from the control point of view and would not be of interest . the estimate of the transmission rate for the two strains when the environment is completely clean may represent the contribution of direct transmission from an infectious to susceptible animal , or somewhat less likely although not impossible , indirect transmission through environmental locations not represented in our sampling plan . when e = 0 , the corresponding r0 values for the strains were < 1 suggesting that infection would die out if there is only direct transmission present . with an increase in the contamination level , r0 quickly exceeds 1 , indicating the critical role of bacterial concentration in the environment in causing an outbreak and sustaining transmission . provided that our results are generalizable to field conditions , the identified influence of environmental contamination in the spread of eco157 indicates the importance of control strategies which either aim to decrease the frequency or rate of eco157 faecal shedding , such as vaccination or promote elimination of the pathogen from the environment such as through barn cleaning . moreover , the results indicate a major role of environmental transmission and only a minimal role of direct transmission in the epidemiology of eco157 in cattle . these results have implications for the current practice of mathematical modelling in epidemiology of eco157 in cattle . assuming that the results obtained for the eco157 strains and experimental conditions evaluated in this study hold for all eco157 strains and in field conditions , the results suggest that the infection spread should be modelled with a predominant rate of environmental transmission ( i.e. occurring through the contaminated environment ) while the rate of direct transmission should be negligible . this is important because mathematical models of eco157 in cattle have largely used assumed arbitrary values for the rate of direct and environmental transmission [ 12 , 18 , 38 ] . from previous studies [ 22 , 24 ] we know that host - to - host variation plays an important role in eco157 colonization and shedding accordingly , it was important to control for the confounding effect of host factors in the infection transmission experiment . however , it is also important to understand whether there could be any between - strain interactions ( e.g. competition ) that would mask the true colonization or transmission characteristics of the strains represented in this study . while we did not have the means to verify whether transmission could have been different if the steers were infected with a single strain , we did have the data from a separate single strain inoculation experiment [ 22 , 24 ] to compare colonization and shedding patterns of these strains on the same experimental animals . the shedding and colonization pattern for the three strains in steers inoculated with a mixture of strains compared closely with the shedding and colonization pattern when steers were inoculated with a single strain . for example , st3 was not shed by the inoculated steers during the single strain inoculation experiment and it was not shed by the steers when inoculated with a mixture of strains . st1 showed better colonization ability with more episodes of intermittent shedding than st2 in the single strain experiment and a similar pattern was observed in the steers inoculated with the mixture of strains in the transmission experiment . based on these similarities , we feel comfortable assuming that transmission characteristics would have been similar had the transmission experiment been conducted separately for st1 , st2 and st3 on these same animals ( which would have been flawed because of the effect of age of the steers on eco157 colonization and shedding patterns [ 39 , 40 ] ) . we used a cut - off of four sampling days to determine whether a steer was latent or recovered , very similar to what has been described by previous studies [ 21 , 41 ] . it is possible , but not likely , that the cut - off choice was incorrect to the extent that it would significantly alter the results . all animals were rigorously tested to ensure they were free of eco157 at the beginning of the experiment . considering that the test used could detect eco157 at a concentration of 10 c.f.u./rams , it is highly unlikely that any steer would continue to shed below this level for three consecutive sampling days ( 6 days ) without clearing from infection . false - negative results , if any , would therefore be minimal and unlikely to have affected the findings of the experiment . in an attempt to explain the results of the infection transmission experiment described here we conducted assays of survivability of the three eco157 strains in bovine faeces . the observed trend suggested that st1 survived better than st2 and even more so than st3 ( fig . 2 ) , which would explain why st1 was isolated more often than st2 ( and substantially more often than st3 ) from the environmental samples during the transmission experiment . however , despite the trends , no statistically significant difference was found in the strains which may have been due to the low number of replicates or because there truly is no difference . in summary , the results indicate that one of the three tested strains did not transmit at all in cattle while the other two strains had considerable , but indistinguishable , transmission rates . for the two transmitted strains , the main finding is that the level of environmental contamination strongly , positively influences the rate of infection transmission in animals . one of the two strains ( st1 ) had on average higher r0 values compared to the other strain ( st2 ) , which was attributed to its longer duration of infectiousness and the ability of this strain to maintain a higher load of the pathogen in the environment . this implies that the on - farm measures to reduce shedding duration and decrease the pathogen load in the environment are important in the transmission and control of eco157 in cattle . it also has implications on the practice of mathematical modelling of eco157 spread and the underlying understanding of the contribution of the direct and environmental transmission to the overall infection spread .

summarya pen infection - transmission experiment was conducted to elucidate the role of pathogen strain and environmental contamination in transmission of escherichia coli o157:h7 ( eco157 ) in cattle . five steers were inoculated with a three - strain mixture of eco157 and joined with five susceptible steers in each of two experimental replicates . faecal and environmental samples were monitored for eco157 presence over 30 days . one eco157 strain did not spread . transmission rates for the other two strains were estimated using a generalized linear model developed based on a modified susceptible infectious susceptible mathematical model . transmission rates estimated for the two strains ( 011 and 014 ) were similar . however , the rates significantly ( p = 00006 ) increased 15 times for every 1-unit increase in the level of environmental contamination measured as log10 c.f.u . depending on the level of environmental contamination , the estimated basic reproduction numbers varied from < 1 to 8 . the findings indicate the importance of on - farm measures to reduce environmental contamination for eco157 control in cattle that should be validated under field conditions .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

PMC2613436

in modern computing era , health is an important aspect for society since it involves every citizen . the current health care system is designed to reward medical doctors and medical institutions to treat people when they are sick , and not acting proactively thus preventing them from being sick . recently , in many countries the health care system is subject to reform since the rapidly growing populations of elderly combined with the increasing cost of health care services impose several challenges on health care providers , insurance companies , hospitals , and even on patients . all these suggest that first of all healthcare needs a major shift toward a more proactive direction focusing on prevention and/or early detection of acute events and afterwards more scalable and more affordable solutions should be offered . therefore , all the above - mentioned requirements have accentuated the need for pervasive and ubiquitous embedded e - health environments , given that limited financial and human resources will be committed . the modern backbone communication infrastructures , networks , and technologies like gsm , umts , wcdma , wimax , internet , wireless , and bluetooth protocols provide extraordinary high data rates , thus allowing cost - efficient and time - efficient remote delivering of medical data that have been collected from the portable , embedded devices that reside onto the end - users ( monitored patients ) toward remote medical servers , for further processing . furthermore , the pervasiveness and ubiquitous character of modern user - centric nomadic environments , in which the user is the central point surrounded by different types of embedded and small computing devices , like , for example , electrocardiograph ( ecg ) and pulse , oxygen saturation , and blood pressure sensors , add extra requirements concerning security aspects such as the protection of sensitive medical data and measurements , data integrity and confidentiality , and protection of patients privacy . in such environments , it is imperative to design and deploy efficient and effective network architectures as well as a generic , if possible , communication interface targeted to connect the external networks with the smart individual - person ( equipped with different types of smart embedded medical devices ( emds ) ) satisfying the basic security requirements for real - time , secure data communication , and protection of sensitive medical data and measurements , data integrity and confidentiality , and protection of the monitored patient 's privacy . the architectures and the interface must consider the limited resources of the interconnected embedded systems , especially in light of the significant resources required for implementing security , which , in general , are quite resource - hungry leading thus to significant technical problems . mpeg-21 is a standard that defines mechanisms and tools as means of sharing digital rights , permissions , and restrictions for digital content from content creator to content consumer . it is an xml - based standard that is designed to communicate machine - readable license information in an ubiquitous , unambiguous , and secure manner between peer entities . although mpeg-21 is a standard that is used for sharing multimedia digital content , under well - defined terms , we argue that it could be used for protection of medical data and user 's privacy in a pervasive healthcare environment , assuming that the digital content is the medical data generated by the emds . the content creator is any user under monitoring and the end - user is anyone having access to a remote medical server located in a hospital either being supervisors , trained medical stuff , or doctors . in our work , we propose a generic security framework based on the mpeg-21 standard adopted for wearable emds thus filling an existing gap which made these devices and the data acquired by them vulnerable to any kind of attacks . although the mpeg-21 standard has been adopted for delivery of multimedia content , we argue that it can be used in our architecture in order to enhance users ' privacy and to enhance security requirements that are applied in such environments . moreover , since the medical measurements that taken from patients ' emds can include different data types , besides raw data , like , for example , a snapshot of electrocardiograph data in a jpeg file , or a video file of electrocardiograph data , the mpeg-21 standard can help us in content delivery , with security enhancement toward different types of end - users . our endeavor demonstrates that no general - purpose home server is required for processing medical data before sent toward end - users , that is , doctors , medical staff , thus making the proposed architecture a good candidate for portable devices in pervasive healthcare environments exposing limited resources . we strongly believe that the outcome of this work contributes toward the establishment of a standard context - aware security environment in which parts of sensitive personal information can be viewed only by valid users even though many users can have access to them . the main characteristics of our approach , which directly affects medical safety and treatment efficacy , comprise standardization , minimalizm , and sophistication . ultimately , affects reduction of total health care cost , thus leading to a better utilization of limited healthcare resources . more specifically , section 2 gives a presentation of the relevant work , while section 3 analyzes the emerging of emds security requirements in combination with mpeg-21 standard . section 4 describes our proposed architecture and section 5 provides the conclusions and the future work projection . gialelis et al . propose a pervasive healthcare architecture into which a wearable health monitoring system is integrated into a broad telemedical infrastructure allowing high - risk cardiovascular patients to closely monitor changes in their critical vital signs and get experts feedback to help maintain optimal health status . consistent with the major challenge to provide good quality and reliable health care services to an increasing number of people utilizing limited financial and human resources , they propose a person - based health care system which consists of wearable commercial of - the - shelf nodes which are already used in the hospital environment , and they are capable of sensing and processing blood - oxygen , blood - pressure , ecgs , and other vital signs and can be seamlessly integrated into wireless personal area networks ( wpans ) for ubiquitous real - time patient monitoring . their architecture lacks safety , security , and privacy considerations , which may lead to serious breaches to architecture 's and emds functionalities or to users ' privacy . venkatasubramanian and gupta made a survey on security solutions for pervasive healthcare environments focusing on securing of data collected by emds , securing the communications between emds and investigation of mechanisms for controlling the access to medical data . they propose the use of cryptographic primitives , where measurements of physiological values are used for cryptographic keys , eliminating thus the necessity for key distribution , for securing data , and for the establishment of secure communications between two entities . concerning the access control to medical data , they survey methods that are based on role - based access control ( rbac ) , extending it for usage in pervasive healthcare environments . as far as we know , the only proposal for usage of mpeg-21 as a mechanism to access control to medical records has been proposed by brox . the author links patients records into mpeg-21 digital items and attempts to find access control mechanisms based on mpeg-21 standard . in this work , the author does not provide a clear architecture that implements the usage of mpeg-21 and also he does not use the mpeg-21 intellectual property management and protection ( ipmp ) components for the protection of medical records , but he mentions its use as a future and open research issue . the primary function of a pervasive healthcare architecture is to collect , to process , and to store medical data from different types of emds , which are located on the patient , locally into general - purpose computing devices for further handling or to remote - located servers . in order to identify the security mechanisms that should be deployed into the proposed architecture for pervasive healthcare with the utilization of emds , we must identify and classify ( i ) possible attackers and malicious users of the aforementioned environment ; ( ii ) safety , security , and privacy requirements . provide a short classification of possible malicious users and adversaries in a pervasive healthcare environment , identifying active adversaries which are malicious users that have explicit interference and physical access to an emd ( i.e. , launching probing attacks , tampering attacks),passive adversaries which are malicious users that have implicit interference with an emd , through eavesdropping the communication link between an emd and another system or through side - channel attacks , insiders which comprise the most probable adversaries in such environments . such attackers include healthcare personnel , nurses , doctors , it professionals , and even the patients themselves . security is a general requirement in modern computing environments , but in pervasive healthcare systems security is an imperative requirement because those systems handle very sensitive data like medical data and electronic personal records ( eprs ) . in order to identify the security aspects and requirements in such environments , we have to identify the threat model ; some of the most significant threats for pervasive healthcare environments are ( a ) nonauthorized access to patient 's medical data , that is , a nurse and a doctor must have different authorities and access control to medical data ; ( b ) intentional alteration of medical data , thus leading to incorrect diagnosis and patient 's treatment ; ( c ) disclosure of medical data to third parties ( e.g. , to insurance companies or any third parties that may use such records to gain profit ) , aiming to increase their revenue . in their work , halperin et al . , also , describe a generic framework for analysis , design , and evaluation of security and privacy issues in implantable medical devices ( imds ) . although our architecture does not contain any type of imds , a subset of the security and safety goals that they describe in their work applies to us , also . they identify two directions , one concerning safety and utilization goals and the other concerning security and privacy goals , and furthermore they are trying to find tradeoffs and tensions between those directions . safety and utility goals are traditional requirements in pervasive healthcare systems , involving some security aspects . such goals are ( a ) accessibility to medical data , that is , ensuring that only appropriate entities must have access to emds and their medical data ; ( b ) accuracy of measurements and data , that is , all data and measurements taken from the emd must be accurate ; ( c ) traceability and identification of emds , that is , an emd must have mechanisms that allow it to make its presence clear to authorized entities , whenever it is necessary ; ( d ) maintenance and reconfigurability , that is , authorized personnel may alter emds configuration , locally or remotely ; finally , ( e ) resources efficiency , that is , extend emds battery life , through minimization of power consumption . besides safety and utility goals , due to their nature , the modern pervasive healthcare environments have also to fulfill some security- and privacy - related goals and requirements . those goals are not different from traditional security requirements of computing systems that basically rely on the attainment of authentication and authorization , data confidentiality , data integrity , availability and protection of users ' privacy , and have to be extended and possibly reviewed in the context of pervasive healthcare environments . authentication . refers to methods and mechanisms which allow to an entity to prove to a remote end its identity , that is , in a transaction between two end - users over a possibly unsafe communication network , there must be mechanisms that assure that each part can be authenticated by a remote end . authorization , refers to access control mechanisms and to the ability of an entity to access shared resources . two basic subcategories of authorization can be distinguished ( a ) personal authorization , that is , specific people or groups of people may access patients ' data and perform specific action over it ; ( b ) role - based authorization , that is , a person may have access to medical data based on a specific role that he has , for example , a doctor , a nurse , a caregiver . mechanisms ensure that when there is an interchange of data between two peer entities , the received data and the original ones are the same , and that no intermediate alteration has occurred , for example , through interference of an eavesdropper . in a typical pervasive healthcare system , various messages and data are interchanged between different participating entities , so the integrity of the transmitted messages and data is a basic requirement . data confidentiality , it assures that stored or transmitted data are well protected from possible disclosure . it is a security requirement which implies that a malicious user may not be able to passively or actively perform a denial - of - service attack to an emd , for example , battery attack , memory overflow , jam the communication interface , thus preventing it from operating normal and smoothly . privacy , it can be defined as an entity 's ability to control how , when , and to what extent personal information about the entity will be communicated to third parties . anderson defines privacy as the secrecy for the benefit of an individual entity , where secrecy refers to generic mechanisms that do not allow unauthorized usage and access of data and resources . extending privacy in the context of pervasive healthcare environments , we refer to ( i ) emd - existence and emd - type privacy , that is , an emd should not make its presence and its type clear to an unauthorized party , since the user ( patient ) might not want to know nonauthorized users what type of emd they carry with them and what type of functions it serves . active adversaries which are malicious users that have explicit interference and physical access to an emd ( i.e. , launching probing attacks , tampering attacks ) , passive adversaries which are malicious users that have implicit interference with an emd , through eavesdropping the communication link between an emd and another system or through side - channel attacks , insiders which comprise the most probable adversaries in such environments . such attackers include healthcare personnel , nurses , doctors , it professionals , and even the patients themselves . our proposed architecture addresses authentication and authorization issues , since each user has a license that defines what he / she has access to . moreover , data confidentiality is achieved through cryptographic mechanisms , that is , sensitive data are encrypted and data integrity is achieved transparently by the doctor 's devices that have access to the data . finally , the utilization of a drm mechanism through the mpeg-21 framework helps in the protection of user 's privacy , that is , different groups of users have access to different types of medical data . mpeg-21 is a new standard which has been proposed for primary use in the context of multimedia world , allowing the seamless , transparent , and universal delivery of multimedia content to the end - user , thus solving any interoperability issues . the core notion into the standard is the digital item ( di ) that represents the digital asset ( e.g. , a movie , an audio track , or other data ) , and which contains the digital content and other related metadata ( e.g. , creator 's details , usage rules and terms , licenses , security related information ) ; the di hierarchy is represented in digital item declaration language ( didl ) which is an xml - based document . in the digital item hierarchy , see figure 1 , we identify ( i ) resources which are the individual multimedia data , that is , a picture , audio or video data , or any other data ; ( ii ) components which are collection of resources with their descriptors . a component by itself is not an item , but the components are the basic building blocks of items , and ( iii ) descriptors which are metadata that accompany a resource , containing information that concerns all or part of the specific resource . also , a container is a structure which comprises of items and containers , forming thus logical packages for interexchanges between entities . the security problems may arise from the fact that the digital item 's description , that is , its structure , contents , attributes , and metadata , is a clear xml document and it is easily visible to anyone and vulnerable to nonauthorized usage . due to that fact , the mpeg-21 includes a part named intellectual property management and protection ( ipmp ) which provide mechanisms for protection of digital item . more specifically , mpeg-21 ipmp in conjunction with the mpeg - rel rights expression language ( rel ) provide a framework that enables all users in the digital contents delivery chain to express their rights and interests in digital items and to have the assurance that those rights and interests will be persistently and reliably managed and protected across a wide range of networks and devices . the core notion in mpeg-21 ipmp is related with the ipmp tools that are used to protect the digital item . those tools are not predescribed by the standard , but each user , vendor , and so forth , may define and implement his own set of tools which perform basic security functions like encryption / decryption algorithms , authentication and data integrity mechanisms , watermarking , and fingerprinting . with the use of mpeg-21 ipmp components , we may protect the whole di or a part of it through the encapsulation of the original didl elements that we want to protect , with additional information ( ipmp info ) that refer to mechanisms and tools for the protection of the original elements . mpeg-21 ipmp defines a new set of ipmp didl elements which have the same role and semantics as an element defined in didl . the ipmpdidl : info element contains information about protection and usage rules of the digital content , which may be categorized to ( i ) information about protection of the whole digital item , which is included in the child element ipmpdidl : ipmpgeneralinfodescriptor and ( ii ) information about protection of a certain part of the digital item 's hierarchy , which is included in the child element ipmpdidl : ipmpinfodescriptor , see figure 2 . both prementioned child elements have two purposes of existence ( a ) to describe the tools that are used for digital items protection , and ( b ) to provide a set of licenses that accompany the content and define its usage rules . more specifically , the ipmpdidl : ipmpinfodescriptor element contains the following child elements ( see figure 3 ) : ( a ) the ipmpinfo : tool child element which is used to specify a tool ( or tools ) that protect the specified part of the di 's hierarchy . each tool has a unique i d and may be either referenced , if it is located in a remote ipmp tools server or it may be included in line to the digital item ( in the latter case , the base-64 encoded version of the tool is included ) ; ( b ) the ipmpinfo : rightsdescriptor child element which contains the licenses that govern the usage of contents . part 5 of mpeg-21 standard defines the mpeg rights expression language ( rel ) which a language that is used to create licenses that express usage rules and rights set by the creators of the digital items , regarding actions over them . moreover , the license can be used to convey some other sensitive data , like , for example , decryption keys for the encrypted ( protected ) contents of the digital item . the mpeg - rel defines the encryptedlicense element which contains the license 's contents in an encrypted form and it is used when the issuer wants to keep the content of the license confidential . when decrypted , the original license 's contents are unveiled . the child element ipmpdidl : contents contains the protected digital asset itself which has been protected with a set of tools ( e.g. , cryptographic tools , watermarking , hashing , digital signatures ) that have been described in the child element ipmpdidl : info . in the high - level view of the proposed architecture as depicted in figure 4 , we can identify the user at home , equipped with different portable emds that transmit their measurements into a pda which aggregates them for temporal storage and further processing ( creation of encapsulated mpeg-21 ipmp - protected metadata file ) . then , the generated file is sent to a data server which is located at the patient 's hospital and it is stored to a database . if it is necessary , the patient 's doctor may extract data from that file remotely through his pda device , acting and informing accordingly the patient . more specifically , tier 1 encompasses a number of portable emds equipped with the corresponding sensors , easy to handle and maintain , which are integrated into a wireless wearable personal area network ( wwpan ) . each of them can acquire , sample , and process one or more physiological parameters . more specifically , a small ( 5 cm 5 cm ) portable electrocardiogram device is utilized for the heart activity monitoring by providing a full 12-lead ecg by collecting signals through ten sensors and electrodes with fixed position . the ten input channels ( vla , vra , vll , vrl , v1-v6 ) are sampled at 500 hz with a 10-bit resolution analog to digital ( a / d ) converter . a small ( watch size ) portable wrist ( and arm if chosen ) blood pressure device is utilized for monitoring patient 's blood pressure upon request . oxygen saturation is monitored through a small ( 2 cm 2 cm ) portable device which its sensor fits to one of patient 's figures . pulse rate is also measured either from the portable wrist or the ecg device . tier 2 encompasses a personal server application running on a personal digital assistant , a cellular phone , a laptop , or a home pc . the personal server undertakes a number of tasks such as a transparent interface to the wwpan ( portable devices ) , an interface to the user and an interface to tier 3 ( medical server & thin client ) . the network configuration task , among other functions , supports device and sensor registration and initialization , customization , and network communication setup . after the network is configured , the personal server application is initiated in order to manage the network , that is , channel and time synchronization , data acquisition , data processing , data fusion , and so forth . furthermore , upon the availability of a communication channel , the personal server establishes a secure link toward tier 3 and transmits reports or files which are being further processed and integrated in the patient 's health record . in case the availability of a communication channel is not possible , the personal server stores the data locally until the channel becomes available . tier 3 comprises the medical server which , actually , encompasses other servers such as emergency server , healthcare server , medical records server , mail server , and the thin - client used for remote accessing of medical server applications utilizing a visualization applet under secure link . the medical server runs a large diversity of applications comprising setting up communication channel to personal servers , collecting users ' reports , integrating data into patients ' medical records data bases , processing medical records upon authorized doctors and/or specialized personnel demand , processing patients messages and other critical applications . as soon as the medical data measurements are collected by the emds , they are sent toward the pda ( tier 2 ) which is responsible for the creation of the mpeg-21-protected container that contains the medical measurements with appropriate usage licenses , as it can be seen in figure 5 . in steps 14 , medical data measurements from different user - located emds ( ecg , blood pressure and oxygen saturation , and pulse rate ) are taken and sent toward the pda , which in turn , aggregates them and creates an initial packaged data file that contains the previous data measurements . afterwards , the packaged file is amended to further processing in order to be encoded according to mpeg-21 terms , with appropriate metadata , protection information , and patient 's instructions ( relevant usage licenses ) ( step 5 ) . then , the new mpeg-21-based packaged file is sent to the hospital 's servers for further handling , that is , storage in data servers , and informing physicians and doctors about patient 's situation ( step 6 ) . in the following figures , we depict an abstract view of an mpeg-21 digital item that contains emds measurements , figure 6 , and the schematic encapsulation of them into an ipmp container , see figure 7 . as we have stated before , the mpeg-21-based encapsulation of data protects it from unauthorized view and possible malicious usage . in section 4 , we have emphasized that in such pervasive healthcare environments the protection of patient 's privacy and medical data confidentiality are key issues . so when the mpeg-21-based packaged file comes to the hospital , its structure and its contents should not be fully accessible to nonappropriate users . we identify three groups of people that should have different access rights to the protected contents ( i ) the it supervisors which are responsible for storing the received packaged files to databases , ( b ) the specialized medical personnel like , for example , the nurses , and ( c ) the doctors , who are responsible for the attendance of the patients . for example , the it stuff may just see the patient 's identification number , ( patid ) , which is unique for each patient , in order to store the packaged file in data server and update patient 's electronic record . on the other hand , a nurse or trained medical stuff must be able to see some details about the patient , for example , a part of patients ' personal data like surname , name , and maybe an initial diagnosis , which may have automatically taken part at patient 's home after the occurrences of data measurements , in order to inform the patient 's doctor for further handling of the patient 's situation . finally , the patient 's doctor may have full access to contents of mpeg-21-packaged files , that is , patients personal data , detailed medical data measurements taken by emds , time of measurements , and initial automated diagnosis . so the generated mpeg-21 container contains three different items ; the first item is unprotected and contains a text data file with the patient 's identification number , which can be used for storage of the file to a database for further handling , and some other data , like the time that the file was created at the user 's pda . the other two items are protected with the mpeg-21 ipmp components and are indented for use by the trained medical staff and the doctors , containing appropriate data . each of these items contains a license of usage , in an encrypted form , which grants access to contents of file to specific user . the license also contains the key which has been used from the patient 's pda for the encryption of the medical data file . we assume that each patient and each of the doctors and trained medical staff have a prearranged set of public - private keys ; those key pairs are assigned by the main hospital 's server and can be revoked at any time since there is continuous communication between the hospital 's server and the patient 's ( home ) server . so the license for data usage can be encrypted using the public key of the entity with which it is related , for example , the doctor with whom the patient is related . in order to access the protected items ' contents , the end - user must decrypt the license with his private key , retrieve the decryption key from the license , and decrypt the encrypted contents . following , in figure 8 , we depict a schematic view of the encapsulated mpeg-21-based data file which is transmitted from patient 's home server toward hospital data servers . finally , in figure 9 , we present an xml - view of the mpeg-21 ipmp - protected container that contains the three prementioned items . the contents of each item are protected ( encrypted ) with the use of a tool that implements a specific symmetric cryptographic algorithm , and it is remote referenced into the digital items ' description . even if one malicious user has access to the remote location of the tool , he needs some cryptographic sensitive information like , for example , a decryption key for decoding the protected contents ; the key for the decryption is included into the encrypted license that accompanies each item , which furthermore defines the usage rules of the contents . each item contains some information that identifies the user for whom this item is destined , that is , item 1 has to be processed by the it stuff , item 2 has to be taken care by a nurse , and so forth . after the reception of each item , each relevant user initiates the procedure which will decrypt his / her license with the use of his private key and then he / she will decrypt the protected contents with the symmetric key that is embedded into his / her license , thus revealing the original packaged medical information . we strongly believe that the outcome of this work contributes toward the establishment of a standard security environment in which parts of sensitive personal information can be viewed only by valid users even though many users can have access to them . the main characteristics of our approach , which directly affect medical safety and treatment efficacy , comprise standardization , minimalizm , and sophistication . ultimately , affects reduction of total health care cost , thus leading to a better utilization of limited healthcare resources . moreover , our endeavor demonstrates that no general - purpose home server is required for processing of medical data before sent toward end - users , that is , doctors , medical staff , thus making the proposed architecture a good candidate for portable devices in pervasive healthcare environments exposing limited resources . in our proposal , we use the mpeg-21 standards ' ipmp components in order to enhance users ' privacy and achieve security requirements that are applied in such environments . to our knowledge , there is no other security framework compliant with mpeg-21 that has been applied to protect such content , thus no comparison with other similar frameworks is provided . as a matter of fact furthermore , with respect to future work , we are planning to exploit some other elements of the mpeg-21 standard which , for example , would allow us to include ( in line ) into the ipmp - protected digital item the protection tools that are used to govern the protected item rather than remote referenced them , and also we could use some other elements of mpeg-21 ipmp that allow digitally authentication of the protection tools , that is , each tool carries a digital signature which validated each time the tool is used , before appliance of them to the protected contents . a lot of our research work is taken place investigating issues that are related with the security of our architecture under known attacks . moreover , as the mpeg-21 is a quite new open standard that can be used for protection of sensitive data with the use of drm , as a future work , we are investigating the identification of possible leaks and vulnerabilities of the standard , that may lead to attacks and a compromised system . we are also investigating issues that are related with the safe destruction of the medical data after their viewing ; in that context , the use of trusted platform architectures could be a direction that will lead to a solution . at this time , we assume that the hospital 's server is a trusted one , and that the devices on which those data are viewed are also safe and trusted , and that there is no possibility for data storage or alteration .

nowadays in modern and ubiquitous computing environments , it is imperative more than ever the necessity for deployment of pervasive healthcare architectures into which the patient is the central point surrounded by different types of embedded and small computing devices , which measure sensitive physical indications , interacting with hospitals databases , allowing thus urgent medical response in occurrences of critical situations . such environments must be developed satisfying the basic security requirements for real - time secure data communication , and protection of sensitive medical data and measurements , data integrity and confidentiality , and protection of the monitored patient 's privacy . in this work , we argue that the mpeg-21 intellectual property management and protection ( ipmp ) components can be used in order to achieve protection of transmitted medical information and enhance patient 's privacy , since there is selective and controlled access to medical data that sent toward the hospital 's servers .

1. Introduction 2. Related Work 3. Security RequirementsThreat Model 4. Proposed Architecture 5. Conclusions

PMC3303761

protein - protein interactions are involved in most cellular process . in order to gain a better understanding of the function of a protein , it is necessary to know how it interacts with other proteins at the molecular level . experimental techniques , such as x - ray crystallography and nuclear magnetic resonance ( nmr ) , have been used to determine an increasing number of protein structures that are deposited into the protein data bank ( pdb ) . however , determining the structures of protein complexes is more difficult by using these techniques . in the pdb , the number of protein complexes as biologically functional units is relatively small ( 11,331 ( pdb search criteria : chain type : protein ; entity type : protein ; number of entities > 1 ; october 14 , 2011 ) ) compared to the total number of protein structures ( 70,852 ) [ 1 , 2 ] . as for the computational tools , a successful protein docking method can be used to predict the three - dimensional structures of protein complexes and provide theoretical understanding of protein - protein interactions at the atomic level . the most challenging problems for the computational approaches are extensive enough conformational sampling , accurate scoring , and inclusion of the flexibility of proteins [ 3 , 4 ] . a variety of search algorithms were developed and implemented in protein docking programs , such as fast fourier transforms ( fft ) , geometric matching , and the monte carlo technique . fft - based methods , which are used in zdock , ftdock [ 5 , 6 ] , and so forth , use correlations to search the sampling space and evaluate the putative complexes with grid representation . these methods initially assess the geometrical surface complementarity and further expand to include electrostatics and hydrophobic complementarity . the monte carlo methods , which are used in rosettadock , icm [ 8 , 9 ] , and so forth , conduct stochastic rotation / translation searches from random initial structures around the known or hypothetical binding site and thus explore only certain regions of the surface . the brownian dynamics ( bd ) method , which is similar to the force - biased monte carlo approach [ 1113 ] , has been used in the past to predict protein - protein interactions [ 1420 ] . in earlier work , we used bd to successfully simulate the recognition between scorpion toxins and potassium channels [ 21 , 22 ] . recently , our previous bd prediction of the interaction between scorpion toxin lq2 and kcsa potassium channel has been verified by the potassium channel - charybdotoxin complex structure ( pdb : 2a9h ) , which was determined by nmr studies [ 21 , 23 ] . the sequence identity between the kcsa of our model and the experimental structure is 97% . scorpion toxins lq2 and charybdotoxin ( ctx ) share 78% sequence identity , and the rmsd of c atoms between lq2 and ctx is 1.62 . the rmsd of our predicted kcsa - lq2 model and experimental kcsa - ctx structure is 2.85 ( based on the backbones of lq2 and ctx only , two kcsa structures are superimposed ) , which shows excellent agreement between the bd predicted and experimentally solved complex structures . our bd results indicate that the strong electrostatic interactions between scorpion toxins and potassium channels are the main driving force for their recognition and association . in the original version of the bd program ( macrodox ) , however , for protein docking simulations it is critical to include the short - range interaction energy terms to rank the final complexes correctly . accordingly , we have introduced additional local energy minimizations after bd simulations by using a force field , which includes electrostatics , van der waals , and desolvation energy terms . we used the coulomb potential with a sigmoidal distance - dependent dielectric permittivity to model the electrostatic energy term , a 12 - 6 lennard - jones potential to model the van der waals energy term , and the general approach of wesson and eisenberg to model the desolvation term . the 12 - 6 lennard - jones potential parameters of four united atom types ( c , n , o , and s ) were obtained from the autodock program . the atomic solvation parameters were calculated based on the absolute partial charge on the atom . we previously used a similar grid - based force field for protein loop predictions . in order to systematically evaluate the performance of our newly adapted bd program for protein - protein docking simulations , we applied the bd approach on a test set of 24 crystal protein complexes from pdb , which was used by other groups for protein docking approach evaluations . the results from redocking experiments showed that the root mean square deviation ( rmsd ) between the predicted structures and the crystal structures is within 2 . we have further developed the approach to account for the flexibility of proteins , which has been successfully applied to reproduce the experimental complex structure from two unbounded proteins . these results indicate that this adapted bd protein docking approach can be useful for prediction of protein - protein interactions . the program package macrodox , version 3.2.2 , was used to assign charges on proteins , solve the linearized poisson - boltzmann equation , and run the bd simulations . the bd algorithm for this program , there are only two solute particles , the receptor and ligand proteins treated as rigid bodies . in this study , the receptor of the complex was defined as protein i and was kept fixed with its center of mass ( com ) at the origin while the ligand was defined as protein ii of which translational and rotational motions were simulated ( figure 1 ) . in each trajectory , protein ii starts with a random orientation and position on the b - surface , which was defined as the sum of the maximum radii of the two proteins plus 2 . protein ii was subjected to three forces : electrostatic , the random brownian force , and the frictional force due to solvent viscosity . during the simulation , the motion of the complex(es ) satisfying the reaction criteria for encounter complex formation is retained as an effective trajectory . within such an effective trajectory , two configurations which have lowest electrostatics interaction energy and shortest distance between protein i and protein ii , respectively , are recorded as two independent sampled conformations . the trajectory was terminated when protein ii either escaped out of the c - surface ( 5 outside of the b surface ) or was running longer than 20 ns . local energy minimizations were conducted for all putative protein complexes obtained from the bd simulations . we developed a shell - based grid force field based on our earlier work , which includes van der waals ( vdw ) , electrostatics , and hydrophobic potentials to represent protein i ( united atom types ) and solvation effect . figure 2 displays the two - shell grid force field generated from protein i of 1ay7 . the inner shell includes 948,240 grids with 0.5 resolution representing electrostatics , vdw , and hydrophobic potentials , 15 width from the surface of protein i. the outer shell includes 962,432 grids with 0.75 resolution representing electrostatics potentials , 30 width from the surface of protein i. the van der waals interactions are described by the 12 - 6 lennard - jones potentials : ( 1)(r)=c12r12c6r6 , where the c12 and c6 lennard - jones parameters of four atom types ( united atoms c , n , o , and s ) are from the autodock program . since the real vdw potential ( real stands for the standard vdw potential as defined in ( 1 ) ) is extremely sensitive to the distance r , we also used a soft potential which replaces the energy value calculated at r by the lowest value calculated from r , r 0.5 , and r + 0.5 using ( 1 ) . as shown in figure 3 , this soft potential has the effect of smoothing the potential energy surface and thus helping protein ii to cross local energy barriers during the energy minimization . coulomb potential (r ) , with a sigmoidal distance - dependent dielectric function was used to model solvent screening , based on the work of mehler and solmajer : ( 2)(r)=4(r)r,(r)=a+b1+kebr , where is partial charge and b = 0 a , 0 = 78.4 ( the dielectric constant of bulk water at 25c ) , a = 6.02944 , = 0.018733345 , and k = 213.5782 are parameters . the original parameter set has been modified to produce better results when comparing with the gb model . when the distance between two charges is < 1.32 , a dielectric constant of 8 is used . the general approach of wesson and eisenberg was used , and the atomic solvation parameters were calculated based on the absolute partial charge on the atom : ( 3)gdesolv = wdesolvi , j(sivj+sjvi)erij2/22,si = ai+k|qi| , where i is the index of atoms in the ligand , j the index of atoms in the receptor , wdesolv = 1 , linear regression coefficient or weight for the desolvation free energy term , si the solvation term for atom i , ai atomic solvation parameter , k charge - based atomic solvation parameter , qi partial atomic charge on atom i , vi the atomic fragmental volume of atom i , rij the distance between atom i and atom j ( in ) , and the gaussian distance constant = 3.5 . the parameters are obtained from the autodock4 program . only nonpolar carbon atoms ( the absolute value of charge is < 0.2 ) the rigid energy minimizations of the protein complexes are based on the downhill simplex method using the newly developed force field [ 26 , 30 ] . six variables ( three translation values and three rotation values of protein ii relative to protein i ) are allowed to change during the energy minimizations . to account for protein flexibility , we conducted molecular dynamics simulations on each individual protein complex obtained from the bd docking with low interaction energies . the complexes were subjected to 100 steps of the steepest descent ( sd ) energy minimizations followed by 2ps simulated annealing ( sa ) from 300 k to 50 k , and additional 100 steps of sd energy minimizations by using the generalized born ( gb ) solvation module in the charmm program . the optimized complex structures were rescored by the same force field we developed here , but in the explicit form ( i.e. , without resorting to the grids and without smoothing the lj potential ) with cutoff values of 8 and 15 for van der waals and electrostatic interactions , respectively . the top 200 low energy complex structures obtained from rigid body energy minimization by soft energy potential were selected to conduct the flexible protein md simulations in this study . all simulations were conducted on a sun linux beowulf cluster ( 2.6 ghz opteron and 1 tb ram ( 4 gb32 gb per node ) ) at the center for high performance computing ( chipc ) at virginia commonwealth university . computational costs are closely related to the size of both the protein receptor and ligand . for bd sampling , one million runs can be completed within 20 h / cpu for this test set ( average residue number is about 500 in the protein complexes ) . for the soft optimization , it usually takes 12 h using 40 cpus by splitting the trajectory into 40 subtrajectories . for the real optimization , only the top 500 complexes , ranked by soft interaction energies , are selected to be further calculated , which usually takes 1 h per cpu . for each complex the post - md - simulated annealing refinement process takes about 30 minutes per cpu . to evaluate the performance of our newly developed bd protein docking approach , we first applied it for a redocking experiment on a test set of 24 protein - protein complex crystal structures which was previously used by several other groups for protein docking method evaluations . the computational protocol of the bd protein docking method including conformational sampling , compact complex selection , and energy minimization is presented in figure 4 . firstly , the experimental crystal complex structures were subjected to 200 steps of the steepest descent ( sd ) followed by 2000 steps of the adopted basis newton - raphson method ( abnr ) energy minimizations using the generalized born ( gb ) solvation module in the charmm program to remove steric overlap that produces bad contacts . then bd simulations were conducted to explore the configuration space of the protein ligand relative to the protein receptor by translational and rotational motions to form the protein complexes . the intermolecular forces and torques between proteins were given by the sum of electrostatic and excluded volume forces . for each system , one million independent bd simulations were conducted that usually generated about 0.9 million protein complexes . to reduce computational costs , a distance - based criterion was introduced to filter the complexes from the bd sampling . the protein complexes whose distance between the surface of protein i and the com of the protein ii is smaller than the maximum radius of protein ii were retained . this filtering step usually yielded around 0.5 million of these compact complexes , which were further optimized using precalculated shell - based grid soft potentials ( see figure 3 ) . the top 500 complexes with the lowest interaction energies were selected and followed by structural optimization using the shell - based grid real potentials . the optimized complex structures were reranked and the one , with the lowest interaction energies were selected as the final predicted protein complexes . the predicted results from the newly developed bd protein docking approach are listed in table 1 . the rmsds from the corresponding crystal structures of all the redocked complexes are within 2 , ranging from 0.18 to 1.98 ( the rmsds are based on the c atoms of the ligands since the receptors are always fixed ) . with the single exception of 2pcb ( see section 4 ) , in each system studied , the near - native conformation was ranked first based on our scoring function . to further verify the shell - based grid force field , we also optimized the crystal structures with the real potential map ( unsmoothed vdw potential ) ( table 1 ) . one can see that the interaction energies of crystal complexes are comparable to those of our redocked complexes . on the other hand , we analyzed the correlation between rmsd and interaction energy for each complex . taking 1cho as an example , the plot of interaction energies versus rmsd is shown in figure 5 . after the optimization with the grid - based real potentials the complex with the lowest interaction energy has the smallest rmsd to the crystal structure . both aspects indicate that this force - field - based scoring function can discriminate between the crystal conformation and other decoy conformations . to further evaluate bd docking approach for real protein - protein interactions , we also conducted docking studies using the unbound structures of the two proteins in a complex . crystal structures for proteins , trypsin and bpti , are available in both bound ( trypsin / bpti , pdb : 2ptc ) and unbound ( trypsin , pdb : 5ptp ; bpti , pdb : 1bpi ) forms , which provide us an opportunity to further evaluate our bd approach . 90,079 complexes were obtained from bd docking simulations and followed by rigid body local energy minimization by the soft potential . the predicted complex with the lowest rmsd ( 2.9 ) from the crystal structure complex is ranked as the 142nd by interaction energy . it suggests that even though the bd approach is able to sample the native configuration of the complex correctly , it can not be selected based on the energy ranking . it is not surprising since the flexibility of both proteins was not considered during the bd docking . we conducted md - simulated annealing simulations for the top 200 complexes with the lowest interaction energies obtained from the bd docking simulations and followed by rescoring with the same force field we developed here ( see section 2 ) . the energy ranking for the structure with the lowest rmsd has been significantly improved to the 4th with the rmsd of 3.2 from the crystal structure . the optimized complex structure of trypsin / bpti superimposed with the crystal complex structure is shown in figure 6 . sampling of the complex conformation is the critical step in protein - protein docking process , since neither scoring function evaluation nor following optimization refinement would be enough if correct or near - native conformations can not be produced by sampling . in our docking procedure , as described in section 2 ( figure 1 ) , the radius of b - surface is 2 larger than the sum of the maximum radii of the two proteins ; c - surface is 5 from the b - surface . both are much smaller than default values in the macrodox ( default values : the radius of b - surface is 20 larger than the sum of the maximum radii of the two proteins and the radius of c - surface is 200 ) . also , 20 ns was set as the longest simulation time for each bd run . this choice of parameters significantly reduced bd simulation time per run making it possible to perform 1 million bd runs for each complex in reasonable computation time . bd approach has been used to predict the protein - protein interactions using electrostatics forces as the biasing guide in addition to brownian stochastic motions . adding electrostatics forces to the docking / diffusion process has the advantage of speeding up the encounter of the receptor and the complex formation . when the attractive electrostatics is the dominant interaction at the binding site , it can even produce the near - native conformations with only a few thousand bd runs [ 21 , 22 , 37 , 38 ] . on the other hand , when the vdw interaction or repulsive electrostatics at the binding site is the dominant force , one million bd runs would be a reasonable number based on our calculations . it could cover the entire sampling space around the receptor ( figure 7(a ) ) . from the column of sequence number / total number in table 1 one it also indicates that one million bd runs are sufficient for sampling in most cases . one exception is 1vfb where three million bd runs were needed due to the repulsive electrostatics interaction of 1.61 kcal mol between the receptor and ligand based on our calculations . cluster analysis was also conducted to illustrate the electrostatics effect on bd sampling within this test set . as shown in figures 8(a ) and 8(b ) , the more negative ( stronger ) the electrostatics interaction between the receptor and ligand is , the more of the conformations will fall into the cluster of the near native ; and the near - native conformations are produced increasingly early as well . generally when the electrostatic interaction energy is lower than 5 kcal mol , the bd sampling procedure produces the near - native conformations faster and more easily than in other cases . the grid potential maps generated from the receptor were defined in a shell around the surface of the molecule ( see section 2 , figure 2 ) . this shell - based grid map has the advantage of largely reducing the number of grids compared to the 3d box grid map . taking 1ay7 as an example , the box - shape potential map would include 1,739,781 and 1,737,808 grids for the inner and outer boxes , respectively ; while in the same condition the shell - based map only takes half the number of grids , 948,240 and 962,432 for the inner and outer shells , respectively . in practice for example , in earlier work , only part of the protein surface could be covered for sampling with the box - shape map . therefore , the binding site had to be identified with the aid of other programs or experimental data . our shell - based potential map is able to cover the whole surface area of the receptor for conducting a global search , which is necessary when clear information is unavailable for the binding sites of protein receptors . we used a force - field - based scoring function , which includes vdw , electrostatics , and hydrophobic potentials . we found that when the smoothed vdw energy term ( soft potential ) was used for the energy optimization , it was much easier for a ligand to find the global energy minima due to its smooth potential energy surface . as seen from figure 7(b ) , the rmsd of the ligands between the bd sampling and the crystal structure was 11.5 , which was reduced to 2.5 after the energy minimization by using the soft potential . moreover , although the interaction energies obtained from the soft potential function are not as accurate as the real ones , they can still be used for preranking of the predicted complexes . the top 500 preranked complexes were selected and followed by a further energy minimization using the real potential function , which was found to be adequate and effective for the predictions of native protein complexes . it was ranked the 9th , but the top 8 conformations formed a single cluster with rmsd around 30 from the crystal structure . we have checked the crystal structure of 2pcb and found an abnormal phenomenon that od2 of asp34 of cyt c peroxidase and oe2 of glu90 of cytochrome c is apart only by 2 . it was speculated that occasional crosslinking between these two proteins might account for this phenomenon . such a short distance between two negatively charged oxygen atoms may lead to higher interaction energy , and thus other decoy conformations appear to have more favorable interaction energy . in addition to the rmsds , which were calculated based on all the c atoms of ligands , we also calculated the rmsds for the ligands based on the interface c atoms of the predicted structures and compared them with the results from other groups ( table 2 ) . the ligand interface residues were defined as having at least one atom within 5 of an atom on the receptor molecule . rmsds based on interface c atoms in this test set are within 1 except for 2pcb ( 1.18 ) . 18 of 24 complex rmsds are smaller than those from the icm method . considering that protein structures in the test set were determined by x - ray crystallography , it is possible that the protein complexes could be affected by crystal packing . we reconstructed the crystal packing protein structures for all the proteins in the test set by using the swiss pdb viewer program . we found that in 12 out of the 24 crystal structures , ligands were located at the interface of multiple subunits in the crystal packing , and thus native conformations could be affected by the crystal packing . to test this possibility , we conducted a comparison study in which crystal packing information was included by adding additional related subunits . however , 2 complexes out of 12 packing structures had to be excluded because the additional subunits block the entrances of protein ligands to access the binding sites of protein receptors . thus , 10 complexes were completely redocked under the condition of crystal packing . the results ( table 3 ) show that rmsds of global c atoms range from 0.26 to 1.44 . comparing the results without crystal packing , there are no significant differences in rmsds , which indicates that the crystal packing could be ignored for protein docking method evaluations based on the current test set . when applying bd sampling approach to predict the complex structure staring from the structures of the unbound proteins trypsin and bpti , we found that bd approach was able to sample the near - native conformations , which indicates that the current bd approach is efficient and feasible for real protein complex sampling . however , the near - native poses often have unfavorable interaction energies due to minor steric clashes and therefore can not be selected based on the energy ranking . although the soft potential used in the current bd approach includes implicitly some partial protein flexibility , it is still not enough to rank the sampled conformations correctly . to achieve high - accuracy prediction , protein flexibility , including flexible side - chains and backbones , has to be considered in either the sampling stage or the postrefinement process . our new approach based on md - simulated annealing is a postrefinement process , which accounts for the flexibility of both proteins , and shows very promising results . prediction of a correct protein - protein complex relies on sampling the near - native conformation and discriminating it from other decoy conformations . our bd sampling is validated to sample the near - native conformation within 1 million runs in most cases and works best for the electrostatic attractive protein complexes . we recommend performing 3 million runs for electrostatically unfavorable systems or for any system where electrostatic information is unavailable . another critical step in our study is using md - simulated annealing method to account the protein flexibility , which can significantly improve the ranking of near native conformations . in a real case of protein - protein study , the top 500 or more conformations from the soft energy ranking list may need to be included for flexible protein refinement by the md - simulated annealing simulations . we have developed an adapted brownian dynamics method to predict the structures of protein complexes . one million independent bd simulations are conducted to explore the entire possible conformational spaces of the protein complexes . in step two , the compact complexes selection , a distance - based criterion is used to select compact complexes . in step three , local energy minimization , all compact complexes are optimized using grid - based soft potentials followed by grid - based real potentials . the complexes with the lowest interaction energies are selected as final predicted protein complexes . to reduce the computational costs for energy evaluations , a grid - based force field was developed to represent protein receptors and solvation effect . the performance of this newly developed bd protein docking approach was evaluated on a test set of 24 crystal protein complexes which was previously used by other groups . the results show that the rmsds between the predicted and the crystal structures are within 2 , which are close to the accuracy of the icm results from fernndez - recio et al . . however , compared with the icm approach , the advantage of our newly adapted bd method is its global sampling , which does not require knowledge of the binding sites of the protein receptors before conducting the bd simulations . shell - based potential maps can significantly reduce the computer memory requirements compared to rectangular box - based potential maps , which make the global sampling possible . the predicted near - native conformations have the lowest interaction energies for all complexes in the test set except the 2pcb . the results indicate that the grid - based force field scoring function we developed can discriminate the crystal conformation from other sampled conformations . however , in the case of positive electrostatics interaction between the protein receptor and ligand , more bd runs are needed . on the other hand , bd sampling has a distinct advantage when stronger electrostatic attraction ( < 5 kcal mol ) exists between the ligand and receptor proteins . in the current adapted bd protein docking method , both the protein receptor and ligand are treated as rigid bodies . the flexibility during docking is partially considered by using the grid - based soft potentials . we found that a postrefinement process based on the md - simulated annealing to account for the flexibility of proteins shows us very promising results and can be also useful if combined with other rigid docking approaches to account for protein conformational changes after the rigid docking process . in our future work , more elegant techniques , such as local move monte carlo ( lmmc ) approach for loop sampling , could also be implemented to account for the flexibility of proteins for mimicking the ligand - induced effect .

we present a newly adapted brownian - dynamics ( bd)-based protein docking method for predicting native protein complexes . the approach includes global bd conformational sampling , compact complex selection , and local energy minimization . in order to reduce the computational costs for energy evaluations , a shell - based grid force field was developed to represent the receptor protein and solvation effects . the performance of this bd protein docking approach has been evaluated on a test set of 24 crystal protein complexes . reproduction of experimental structures in the test set indicates the adequate conformational sampling and accurate scoring of this bd protein docking approach . furthermore , we have developed an approach to account for the flexibility of proteins , which has been successfully applied to reproduce the experimental complex structure from the structure of two unbounded proteins . these results indicate that this adapted bd protein docking approach can be useful for the prediction of protein - protein interactions .

1. Background 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC4484562

although facial paralysis is fairly commonly encountered by ear , nose , and throat physicians , bilateral facial paralysis is very rare . the frequency of facial paralysis is 2025 per 100,000 patients , but simultaneous bilateral facial paralysis occurs in only 1 in 5 million patients . simultaneous bilateral facial paralysis is considered present if the time elapsed from paralysis of one side to paralysis of the other side does not exceed 4 weeks . a differential diagnosis of bilateral facial paralysis must consider systematic , traumatic , neuromuscular , vascular , toxic , and infectious diseases and idiopathic causes . we present 40-year - old male patient with bilateral facial paralysis caused by blunt head trauma . the patient was followed up in our intensive care unit for 10 days , because of multiple traumas caused by the accident , and was referred to our clinic with a diagnosis of facial paralysis , hearing loss , and anosmia . he was conscious , cooperative , and oriented . on physical examination , both tympanum membranes were intact . upon house - brackmann ( hb ) classification , hb grade 6 facial paralysis was observed on the right side and hb grade 5 on the left . upon audiometric testing , a slight sensorineural hearing loss ( snhl ) was evident on the right side with advanced mixed - type hearing loss on the left . on facial maxillary computed tomography ( ct ) examination , multiple nondisplaced fracture lines were observed on the base of the right frontal sinus base , on the left frontal bone , on the zygomatic bone , and on both sides of the sphenoid bone . also , a transversely progressive fracture line was evident in both petrous temporal bones ( figures 1(a ) and 1(b ) ) . as the facial paralysis had developed immediately after the accident and as total denervation was evident upon electromyography ( emg ) examination , a transmastoid facial decompression operation was performed on the right side in the third week after trauma . the right side was graded hb 1 and the left side hb 2 at the 1-year follow - up ( figures 2(a ) and 2(b ) ) . bilateral facial paralysis caused by bitemporal bone fracture is very rare , with seven cases reported to date ( table 1 ) . unlike unilateral facial paralysis , the most important causes of bilateral facial paralysis are trauma , infectious diseases ( infectious mononucleosis , syphilis , bilateral otitis media , herpes zoster , lyme disease , and meningitis ) , neurological diseases ( multiple sclerosis , neoplasms , or stroke ) , and other diseases , the aetiologies of which are uncertain ( guillain - barre syndrome , sarcoidosis , melkersson - rosenthal syndrome , and leukaemia ) . trauma causes 5% of all facial paralysis , and 3% of cases are associated with temporal bone fractures , which can be divided into longitudinal , transverse , and mixed fractures . the fracture lines of transverse fractures are vertical , running through the squamous petrous bone ; these constitute 10% of all temporal bone fractures . a total of 3050% of patients with transverse fractures present with facial paralysis and snhl . such facial paralysis is rather serious , and the prognosis is very poor . in longitudinal fractures , the fracture lines lie parallel to the long axis of the temporal bone . such fractures constitute 90% of all temporal bone fractures and , in 1025% of cases , are accompanied by facial paralysis . , a fracture line exhibiting transverse progression was observed in both temporal bones , and facial paralysis was accompanied by snhl in the right ear and a mixed type of hearing loss in the left ear . the technique explores the extent of facial nerve activity , and denervation of over 90% has been identified when electroneuronography ( enog ) was performed within 6 days of the trauma . emg and high - resolution ct aid surgical decision - making for patients who are diagnosed late . in the present case emg test revealed that there were bilateral fibrillation potentials and no response after stimulation , which is a typical pattern of a complete degeneration of the nerve . such patients may be divided into four groups : those suffering from neuropraxia , those suffering from axonotmesis , those suffering from neurotmesis , and those suffering from nerve injuries . in patients with neuropraxia , myelin sheath damage is evident but the axon is robust . distal axons undergo wallerian degeneration after 7 days of injury , followed by axonal regeneration from intact nervous tissue . if nerve incision is evident , it is necessary to place a nerve graft in the area in which the nerve incision has caused neural function to be lost . in our case , we found that facial nerve integrity was in fact complete but that bone spicules had compressed the facial nerve . the middle cranial fossa approach is used to treat not only longitudinal fractures , but also mixed and transverse fractures , combined with a transmastoid exploration technique . we employed the latter technique in our patient ; we followed the fracture line and cleared bone spicules thereon . fisch stated that an intraneural haematoma might compress a nerve in those with delayed paralysis and recommended urgent exploration . watchful waiting for 3 - 4 weeks was suggested for patients with early - stage paralysis . this was because the nerve was readily visualised ( oedema was present ) and because haematoma regression was often evident in those with early - stage paralysis . the present case was diagnosed as complete bilateral facial paralysis at the first day of trauma . he was operated upon in the third week of trauma after 10-day follow - up in the intensive care unit . in patients with facial paralysis the use of synthetic tears and eye closure at night reduce the risk of keratitis . traumatic bilateral facial paralysis is very rare and is often accompanied by serious fractures of the skull base . as patients with such fractures exhibit high levels of mortality , death may occur before diagnosis . early diagnosis and treatment are vital in patients with bilateral facial paralysis . however , such patients may experience long stays in intensive care units , associated with delays in diagnosis , because facial asymmetry can not be evaluated if the facial paralysis is bilateral . in such situations , it may be helpful to note that a patient can not close his or her eyes completely and that the eye globes move up or out ( the bell phenomenon ) when she or he is asked to close the eyes .

bilateral facial paralysis caused by bilateral temporal bone fracture is a rare clinical entity , with seven cases reported in the literature to date . in this paper , we describe a 40-year - old male patient with bilateral facial paralysis and hearing loss that developed after an occupational accident . on physical examination , house - brackmann ( hb ) facial paralysis of grade 6 was observed on the right side and hb grade 5 paralysis on the left . upon temporal bone computed tomography ( ct ) examination , a fracture line exhibiting transverse progression was observed in both petrous temporal bones . our patient underwent transmastoid facial decompression surgery of the right ear . the patient refused a left - side operation . such patients require extensive monitoring in intensive care units because the presence of multiple injuries means that facial functions are often very difficult to evaluate . therefore , delays may ensue in both diagnosis and treatment of bilateral facial paralysis .

1. Introduction 2. Case Report 3. Discussion

PMC4851520

the most efficient way to prevent caries is by using fluoridated dental products ( 1 , 2 ) . fluoride enters the body with food , through respiration and products containing fluoride ( 3 ) . fluoride is a part of the natural environment and is therefore constantly present in people s lives . however , concentration of fluoride can vary from one region to another . from a chemical point of view , it is the most electronegative and reactive of all the elements due to its small atomic radius . since it is highly reactive , it is usually bound as inorganic fluoride and not found in its elementary state ( 4 ) . it ranks 13 in terrestrial abundance and represents 0.06 - 0.09% of weight of the earth s crust . it enters the environment through volcanic eruptions , rock dissolution and numerous human activities ( coal burning , ore processing , production and use of fertilizers , and industrial plants ) ( 4 , 5 ) . higher concentrations of fluoride in water can be present near hot springs of volcanic origin ( 5 ) . there are some plants which can accumulate a few 100 ppm of fluoride ; the best known is the tea plant ( camellia sinensis , syn . the aim of our research was to review the literature about fluoride toxicity and to inform physicians , dentists and public health specialists whether fluoride use is expedient and safe . data we used in our review were systematically searched from articles published until 2015 and collected from official web pages and documents published from different international institutions . the search was conducted between 16 and 27 march 2015 and revised between 1 and 18 december 2015 . at the end of our review topics discussed in this review are : dietary intake of fluoride , fluoride metabolism , history of fluoride use , the mechanism of fluoride action , overuse and toxicity of fluoride . dietary intake of fluoride , history of fluoride use , the mechanism of fluoride action , overuse and toxicity of fluoride . data we used in our review were systematically searched from articles published until 2015 and collected from official web pages and documents published from different international institutions . the search was conducted between 16 and 27 march 2015 and revised between 1 and 18 december 2015 . at the end of our review topics discussed in this review are : dietary intake of fluoride , fluoride metabolism , history of fluoride use , the mechanism of fluoride action , overuse and toxicity of fluoride . dietary intake of fluoride , history of fluoride use , the mechanism of fluoride action , overuse and toxicity of fluoride . even though fluoride is generally present in our everyday life , we consume it in small amounts . in general , it can be found in meat , fish , and cereals ( 2 , 5 ) . in higher concentrations , it can also be found in canned anchovies , canned fruits , ground chicken meat products ( with a higher percentage of ground bones ) , chocolate milk and some baby dietary supplements ( 8) . table 1 shows the average concentrations of fluoride in different types of food ( 8) . fluoride concentrations for different types of food . for instance , teflon cookware is a great source of fluoride ions ( 9 ) . the most important factor for fluoride presence in alimentation is fluoridated water ( 8) . the reason behind this are preventive programs for fluoridation of drinking water ( in the usa , the healthy people 2010 objective was to increase proportion of usa population served by community water systems with optimally fluoridate water to 75% ) ( 10 ) . these methods are especially important in regions where other preventive programs are not available ( 11 ) . in certain countries , fluoridated salt has been widely used in germany , france and switzerland since 1955 . nowadays , 30 to 80% of marketed salt is fluoridated ( 12 ) . salt usually contains 250 ppm of fluoride ( 11 ) , whereas milk contains 2.5 ppm or up to 5 ppm of fluoride ( 11 ) . however , this method is becoming less suitable , because of modern guidelines for low salt diets ( 7 ) . according to different evidence based research , there is insufficient evidence that fluoridated milk has caries - protective effects ( 13 ) . products used for oral hygiene are also an important means of systemic fluoride intake . a certain amount of toothpaste is consumed during brushing . it is therefore recommended that children use toothpaste with a lower fluoride concentration in small amounts ( table 2 ) ( 14 ) . source : european academy of paediatric dentistry ( eapd ) , 2009 . about 90% of fluoride is absorbed in the gastrointestinal tract after consumption ( up to 25% in the stomach and around 77% in proximal part of the small intestine ) . after absorption , fluoride is transported into the bloodstream and is distributed through the organism ( 4 ) . the mean time of the peak concentration is 20 - 60 minutes after consumption ( 15 ) . in the plasma , fluoride ions adults retain around 36% of fluoride , whereas children retain approximately 50% of fluoride ; 99% of that is contained in mineralized tissues ( bone and teeth ) and 1% can be found in soft tissue ( 15 ) . the remaining part of the absorbed fluoride is excreted through the kidneys into the urine ; excretion through saliva and sweat is negligible ( 4 ) . the kidneys are therefore the only human organ that helps maintain the fluoride concentration in our bodies . the most important are : acid base disorders , hematocrit , altitude , physical activity , circadian rhythm , hormones , kidney function , genetic predispositions and diet ( 15 ) . in pregnant women , uptake of fluorides in the placenta is dependent on the fluoride concentration in the mother`s bloodstream . when the concentration is low , fluoride is transmitted into the placenta ( 16 ) . on average , the concentration in the placenta is about 60% of the concentration in the mother`s bloodstream ( 17 ) . if the fluoride concentration increases over 0.4 ppm ( 17 ) , the placenta works as a barrier , preventing the fluoride from passing through and thus protecting the fetus from a high fluoride concentration ( 16 ) . fluoride can also be transmitted through the plasma into the mother s milk ; however , the concentration is low ( 18 ) . the idea of using fluoride as a beneficial agent in caries prevention is not new . they started observing and describing the effects of fluoride in the late 19 and beginning of the 20 century ( 19 ) . mckay was studying brown stains on enamel observed in the population from different parts of the usa . he listed enamel spots based on size and color and measured the degree of tooth impairment . he compared enamel changes to fluoride concentration in drinking water and made the connection between fluoride content and the quantity of enamel spots , which he termed dental fluorosis ( 23 ) . he documented dental fluorosis prevalence in the usa until 1942 and then compared it to caries prevalence in children . he noticed a strong inverse relationship ( 24 ) . after concluding his 21-city study ( 25 , 26 ) , he found that drinking water with 1 ppm of fluoride can prevent dental caries , increase tooth strength and does not have a negative impact on enamel . the first studies about positive effects were conducted in 1945 with systemic fluoridation of drinking water in four american cities ( grand rapids , evanston , brantford and newberg ) , where 1 mg of fluoride per liter was added to drinking water . they came to the conclusion that fluoride in suitable concentrations significantly affects dental caries prevalence ( 27 - 30 ) . these observations and discoveries triggered massive drinking water fluoridation , the use of fluoridated salt and milk and an increase in diet supplement production ( pills , drops , chewing gum , lozenges ) . bibby et al ( 31 ) conducted a study comparing the efficacy of fluoride - coated pills intended to be swallowed , with fluoride lozenges intended to be dissolved slowly in the mouth . research showed a lower number of new caries lesions in the research group which consumed lozenges . this research triggered different studies comparing pre- and post - eruptive effects , which consequently led to a rethinking of the theory of systemic use of fluoride and its incorporation into enamel during teeth development ( odontogenesis ) . in the last 30 years , studies have shown that the maximum anti - caries benefits of fluoride are primarily through topical use and direct contact on the tooth surface . daily use of topical supplements with suitable fluoride concentration is beneficial ( 2 ) . methods , which led to greater fluoride exposure and lowered caries prevalence , are considered to be one of the greatest accomplishments in the 20 century`s public dental health ( 32 ) . the primary and most important action of fluoride is topical , when the fluoride ion is present in the saliva ( 33 ) in the appropriate concentration . hydroxyapatite is the main mineral responsible for building the permanent tooth enamel after the development of the teeth is finished ( 34 ) . during tooth growth , the enamel is constantly exposed to numerous demineralization processes , but also important remineralization processes , if the appropriate ions are present in the saliva . the main action is inhibition of demineralization of enamel , which is carried out through different mechanisms . there are different cariogenic bacteria in the plaque fluid the most important being s. mutans . when bacteria metabolize sugars , they produce lactic acid ( 33 ) which decreases the ph in saliva . when the ph falls below the critical level of hydroxyapatite ( ph 5.5 ) , the process of demineralization of enamel takes place and caries is formed . at the beginning , the process is reversible and it is possible to reduce the formation of new lesions with appropriate preventive measures . if fluoride is present in plaque fluid , it will reduce the demineralization , as it will adsorb into the crystal surface and protect crystals from dissolution . because the fluoride ion coating is only partial , the uncoated parts of the crystal will undergo dissolution on certain parts of the tooth , if the ph falls below level 5.5 . when the ph rises above the critical level of 5.5 , the increased level of fluoride ion leads to remineralization , because it absorbs itself into the enamel and forms fluorhydroxyapatite ( 33 ) . after repeated cycles of demineralization and remineralization , the outer parts of enamel may change and become more resistant to the acidic environment due to a lowered critical ph level of newly formed crystals ( ph 4.5 ) ( 33 ) . the most important effect of fluoride on caries progression is thus on demineralization and remineralization processes . it has also been proposed , that the fluoride ion can affect the physiology of microbial cells , which can indirectly affect demineralization . one of them being a direct inhibition of cellular enzymes glycolytic enzymes , h+atpases ) . it affects cellular membrane permeability and also lowers cytoplasmic ph , resulting in a decrease in acid production from glycolysis ( 33 ) . it is caused by systemic overexposure to fluoride during the first six years of life , when the enamel of the crowns of permanent teeth is formed . clinical manifestation vary from ( quantitative ) narrow , white horizontally running lines , larger patches or yellow to light brown colored areas of porous enamel , to ( qualitative ) loss of enamel in varying degrees ( 35 ) . for the optimal effect of fluoride toothpaste , it is important to follow recommended guidelines for the use of products containing fluorides ( table 2 ) ( 7 ) . in this way , the probability for fluorosis is decreased and the protective effect of fluoride on the development of caries is significantly important ( 1 ) . just like any other substance we are exposed to in our everyday lives ( oxygen , water , table salt ) , fluoride can be toxic in certain quantities . acute toxicity can occur after ingesting one or more doses of fluoride over a short time period which then leads to poisoning . this is followed by a collapse with paleness , weakness , shallow breathing , weak heart sounds , wet , cold skin , cyanosis , dilated pupils , hypocalcaemia and hyperkalemia , and in to two to four hours even death . based on research papers and some overdose cases , the probable toxic dose ( ptd ) was defined at 5 mg / kg of body mass . the ptd is the minimal dose that could trigger serious and life - threatening signs and symptoms and requires immediate treatment and hospitalization ( 36 ) . example : ptd in a 20 kg child would be achieved at ingesting 100 g ( 75 ml ) of toothpaste which contains 1000 - 1500 ppm of fluorides or 100 pills , that contain fluorides ( 0,5 - 1 mg fluoride ) ( 36 ) . despite the widespread presence of fluoride in our life and the seriousness of the conditions associated with its toxicity , the number of cases of acute toxicity today , compared to the first half of twenty first century , is very rare . at that time , sodium fluoride was used as a pesticide and rat poison . because of its appearance it was often mistaken for flour , powdered sugar or any other white powder product , which is used in the kitchen . the most notable example is from the oregon state hospital from 1942 . during the preparation of scrambled eggs powdered milk was replaced by sodium fluoride which caused 467 cases of acute poisoning of which 47 were fatal . today , poisoning is mainly due to unsupervised ingestion of products for dental and oral hygiene and over - fluoridated water ( example : hooper bay , alaska , 1992 ) ( 37 ) . the european academy of paediatric dentistry ( eapd ) recommends to everybody , including pregnant women , the preventive use of fluoride toothpaste as a primary preventive measure against caries ( 7 ) . parents should start brushing children s teeth with fluoride toothpaste as soon as the first tooth erupts in the concentration and quantity recommended by the eapd ( table 2 ) ( 7 ) . parents should use the recommended amount of toothpaste and assist or supervise their children with tooth brushing at least up to the age of 7 . even though fluoride is generally present in our everyday life , we consume it in small amounts . in general , it can be found in meat , fish , and cereals ( 2 , 5 ) . in higher concentrations , it can also be found in canned anchovies , canned fruits , ground chicken meat products ( with a higher percentage of ground bones ) , chocolate milk and some baby dietary supplements ( 8) . table 1 shows the average concentrations of fluoride in different types of food ( 8) . fluoride concentrations for different types of food . for instance , teflon cookware is a great source of fluoride ions ( 9 ) . the most important factor for fluoride presence in alimentation is fluoridated water ( 8) . the reason behind this are preventive programs for fluoridation of drinking water ( in the usa , the healthy people 2010 objective was to increase proportion of usa population served by community water systems with optimally fluoridate water to 75% ) ( 10 ) . these methods are especially important in regions where other preventive programs are not available ( 11 ) . in certain countries , fluoridated salt has been widely used in germany , france and switzerland since 1955 . nowadays , 30 to 80% of marketed salt is fluoridated ( 12 ) . salt usually contains 250 ppm of fluoride ( 11 ) , whereas milk contains 2.5 ppm or up to 5 ppm of fluoride ( 11 ) . however , this method is becoming less suitable , because of modern guidelines for low salt diets ( 7 ) . according to different evidence based research , there is insufficient evidence that fluoridated milk has caries - protective effects ( 13 ) . products used for oral hygiene are also an important means of systemic fluoride intake . a certain amount of toothpaste is consumed during brushing . it is therefore recommended that children use toothpaste with a lower fluoride concentration in small amounts ( table 2 ) ( 14 ) . about 90% of fluoride is absorbed in the gastrointestinal tract after consumption ( up to 25% in the stomach and around 77% in proximal part of the small intestine ) . after absorption , fluoride is transported into the bloodstream and is distributed through the organism ( 4 ) . the mean time of the peak concentration is 20 - 60 minutes after consumption ( 15 ) . in the plasma , fluoride ions adults retain around 36% of fluoride , whereas children retain approximately 50% of fluoride ; 99% of that is contained in mineralized tissues ( bone and teeth ) and 1% can be found in soft tissue ( 15 ) . the remaining part of the absorbed fluoride is excreted through the kidneys into the urine ; excretion through saliva and sweat is negligible ( 4 ) . the kidneys are therefore the only human organ that helps maintain the fluoride concentration in our bodies the most important are : acid base disorders , hematocrit , altitude , physical activity , circadian rhythm , hormones , kidney function , genetic predispositions and diet ( 15 ) . in pregnant women , uptake of fluorides in the placenta is dependent on the fluoride concentration in the mother`s bloodstream . when the concentration is low , fluoride is transmitted into the placenta ( 16 ) . on average , the concentration in the placenta is about 60% of the concentration in the mother`s bloodstream ( 17 ) . if the fluoride concentration increases over 0.4 ppm ( 17 ) , the placenta works as a barrier , preventing the fluoride from passing through and thus protecting the fetus from a high fluoride concentration ( 16 ) . fluoride can also be transmitted through the plasma into the mother s milk ; however , the concentration is low ( 18 ) . the idea of using fluoride as a beneficial agent in caries prevention is not new . they started observing and describing the effects of fluoride in the late 19 and beginning of the 20 century ( 19 ) . mckay was studying brown stains on enamel observed in the population from different parts of the usa . he listed enamel spots based on size and color and measured the degree of tooth impairment . he compared enamel changes to fluoride concentration in drinking water and made the connection between fluoride content and the quantity of enamel spots , which he termed dental fluorosis ( 23 ) . he documented dental fluorosis prevalence in the usa until 1942 and then compared it to caries prevalence in children . he noticed a strong inverse relationship ( 24 ) . after concluding his 21-city study ( 25 , 26 ) , he found that drinking water with 1 ppm of fluoride can prevent dental caries , increase tooth strength and does not have a negative impact on enamel . the first studies about positive effects were conducted in 1945 with systemic fluoridation of drinking water in four american cities ( grand rapids , evanston , brantford and newberg ) , where 1 mg of fluoride per liter was added to drinking water . they came to the conclusion that fluoride in suitable concentrations significantly affects dental caries prevalence ( 27 - 30 ) . these observations and discoveries triggered massive drinking water fluoridation , the use of fluoridated salt and milk and an increase in diet supplement production ( pills , drops , chewing gum , lozenges ) . consequently , caries prevalence was successfully decreased . in 1950 , bibby et al ( 31 ) conducted a study comparing the efficacy of fluoride - coated pills intended to be swallowed , with fluoride lozenges intended to be dissolved slowly in the mouth . research showed a lower number of new caries lesions in the research group which consumed lozenges . this research triggered different studies comparing pre- and post - eruptive effects , which consequently led to a rethinking of the theory of systemic use of fluoride and its incorporation into enamel during teeth development ( odontogenesis ) . in the last 30 years , studies have shown that the maximum anti - caries benefits of fluoride are primarily through topical use and direct contact on the tooth surface . methods , which led to greater fluoride exposure and lowered caries prevalence , are considered to be one of the greatest accomplishments in the 20 century`s public dental health ( 32 ) . the primary and most important action of fluoride is topical , when the fluoride ion is present in the saliva ( 33 ) in the appropriate concentration . hydroxyapatite is the main mineral responsible for building the permanent tooth enamel after the development of the teeth is finished ( 34 ) . during tooth growth , the enamel is constantly exposed to numerous demineralization processes , but also important remineralization processes , if the appropriate ions are present in the saliva . the main action is inhibition of demineralization of enamel , which is carried out through different mechanisms . there are different cariogenic bacteria in the plaque fluid the most important being s. mutans . when bacteria metabolize sugars , they produce lactic acid ( 33 ) which decreases the ph in saliva . when the ph falls below the critical level of hydroxyapatite ( ph 5.5 ) , the process of demineralization of enamel takes place and caries is formed . at the beginning , the process is reversible and it is possible to reduce the formation of new lesions with appropriate preventive measures . if fluoride is present in plaque fluid , it will reduce the demineralization , as it will adsorb into the crystal surface and protect crystals from dissolution . because the fluoride ion coating is only partial , the uncoated parts of the crystal will undergo dissolution on certain parts of the tooth , if the ph falls below level 5.5 . when the ph rises above the critical level of 5.5 , the increased level of fluoride ion leads to remineralization , because it absorbs itself into the enamel and forms fluorhydroxyapatite ( 33 ) . after repeated cycles of demineralization and remineralization , the outer parts of enamel may change and become more resistant to the acidic environment due to a lowered critical ph level of newly formed crystals ( ph 4.5 ) ( 33 ) . the most important effect of fluoride on caries progression is thus on demineralization and remineralization processes . it has also been proposed , that the fluoride ion can affect the physiology of microbial cells , which can indirectly affect demineralization . one of them being a direct inhibition of cellular enzymes glycolytic enzymes , h+atpases ) . it affects cellular membrane permeability and also lowers cytoplasmic ph , resulting in a decrease in acid production from glycolysis ( 33 ) . it is caused by systemic overexposure to fluoride during the first six years of life , when the enamel of the crowns of permanent teeth is formed . clinical manifestation vary from ( quantitative ) narrow , white horizontally running lines , larger patches or yellow to light brown colored areas of porous enamel , to ( qualitative ) loss of enamel in varying degrees ( 35 ) . for the optimal effect of fluoride toothpaste , it is important to follow recommended guidelines for the use of products containing fluorides ( table 2 ) ( 7 ) . in this way , the probability for fluorosis is decreased and the protective effect of fluoride on the development of caries is significantly important ( 1 ) . just like any other substance we are exposed to in our everyday lives ( oxygen , water , table salt ) , acute toxicity can occur after ingesting one or more doses of fluoride over a short time period which then leads to poisoning . this is followed by a collapse with paleness , weakness , shallow breathing , weak heart sounds , wet , cold skin , cyanosis , dilated pupils , hypocalcaemia and hyperkalemia , and in to two to four hours even death . based on research papers and some overdose cases , the probable toxic dose ( ptd ) was defined at 5 mg / kg of body mass . the ptd is the minimal dose that could trigger serious and life - threatening signs and symptoms and requires immediate treatment and hospitalization ( 36 ) . example : ptd in a 20 kg child would be achieved at ingesting 100 g ( 75 ml ) of toothpaste which contains 1000 - 1500 ppm of fluorides or 100 pills , that contain fluorides ( 0,5 - 1 mg fluoride ) ( 36 ) . despite the widespread presence of fluoride in our life and the seriousness of the conditions associated with its toxicity , the number of cases of acute toxicity today , compared to the first half of twenty first century , is very rare . at that time , sodium fluoride was used as a pesticide and rat poison . because of its appearance it was often mistaken for flour , powdered sugar or any other white powder product , which is used in the kitchen . the most notable example is from the oregon state hospital from 1942 . during the preparation of scrambled eggs powdered milk was replaced by sodium fluoride which caused 467 cases of acute poisoning of which 47 were fatal . today , poisoning is mainly due to unsupervised ingestion of products for dental and oral hygiene and over - fluoridated water ( example : hooper bay , alaska , 1992 ) ( 37 ) . the european academy of paediatric dentistry ( eapd ) recommends to everybody , including pregnant women , the preventive use of fluoride toothpaste as a primary preventive measure against caries ( 7 ) . parents should start brushing children s teeth with fluoride toothpaste as soon as the first tooth erupts in the concentration and quantity recommended by the eapd ( table 2 ) ( 7 ) . parents should use the recommended amount of toothpaste and assist or supervise their children with tooth brushing at least up to the age of 7 . the european academy of paediatric dentistry ( eapd ) recommends a preventive topical use of fluoride supplements because of their cariostatic effect . you do nt have to brush all your teeth , just the ones you want to keep .

introduction : appropriate oral health care is fundamental for any individual s health . dental caries is still one of the major public health problems . the most effective way of caries prevention is the use of fluoride.aim:the aim of our research was to review the literature about fluoride toxicity and to inform physicians , dentists and public health specialists whether fluoride use is expedient and safe.methods:data we used in our review were systematically searched and collected from web pages and documents published from different international institutions.results:fluoride occurs naturally in our environment but we consume it in small amounts . exposure can occur through dietary intake , respiration and fluoride supplements . the most important factor for fluoride presence in alimentation is fluoridated water . methods , which led to greater fluoride exposure and lowered caries prevalence , are considered to be one of the greatest accomplishments in the 20th century`s public dental health . during pregnancy , the placenta acts as a barrier . the fluoride , therefore , crosses the placenta in low concentrations . fluoride can be transmitted through the plasma into the mother s milk ; however , the concentration is low . the most important action of fluoride is topical , when it is present in the saliva in the appropriate concentration . the most important effect of fluoride on caries incidence is through its role in the process of remineralization and demineralization of tooth enamel . acute toxicity can occur after ingesting one or more doses of fluoride over a short time period which then leads to poisoning . today , poisoning is mainly due to unsupervised ingestion of products for dental and oral hygiene and over - fluoridated water.conclusion:even though fluoride can be toxic in extremely high concentrations , it`s topical use is safe . the european academy of paediatric dentistry ( eapd ) recommends a preventive topical use of fluoride supplements because of their cariostatic effect .

1. INTRODUCTION 2. AIM 3. METHODS 3.1 Sources and search strategy 3.2 Reviewed fields 4. RESULTS AND DISCUSSION 4.1 Dietary intake of fluoride and fluoride supplements 4.2 Fluoride metabolism 4.3 Historical overview of fluoride use 4.4 The mechanism of fluoride action 4.5 Overuse and toxicity of fluoride 4.6 European directive for the use of fluorides 5. CONCLUSION

PMC5279936

a new understanding of energy homeostasis initially proposed that osteocalcin ( ocn ) , a bone - derived hormone , is released into the circulation in response to insulin - mediated bone resorption , and activates gprc6a , a member of the family c , g - protein coupled receptors , to stimulate insulin and testosterone secretion in - and leydig - cells , respectively , , , . the existence of this novel ocn - gprc6a endocrine network regulating energy metabolism and sex hormone production was supported by mouse genetic studies . in this regard , both ocn and gprc6a mice exhibit impaired glucose tolerance , insulin resistance , obesity , hepatosteatosis , and low circulating testosterone ( t ) , , ; compound double heterozygous gprca and ocn mice show additive effects on these metabolic abnormalities , and conditional deletion of gprc6a in pancreatic -cells , and testicular leydig cells , prevents ocn stimulation of insulin and t production , respectively . based on an expanded understanding of gprc6a expression and functions , an updated schema proposes a more complex network , where gprc6a is activated by multiple ligands , and has broader functions in many more tissues than originally envisioned . in addition to the ligand ocn , gprc6a mediates the rapid , non - genomic signaling responses to t and is also activated by basic amino acids and cations . gprc6a is expressed in liver hepatocytes , skeletal muscle myocytes , and possibly adipocytes , in which direct effects of this receptor to regulate glucose and fat metabolism have been described . activation of gprc6a stimulates the production and secretion of key , metabolically active hormones in addition to insulin from islets , , and t from leydig cells , . gprc6a also stimulates release of glucagon - like peptide-1 ( glp-1 ) from intestinal cells , , , adiponectin from adipocytes , , and interleukin 6 ( il-6 ) from myocytes , to create novel inter - organ communications ( figure 1 ) . gprc6a is expressed in the prostate , upregulated in prostate cancer cells and tumors , and implicated in prostate cancer ( pca ) risk and progression gprc6a may provide a molecular basis for the well - known association between metabolic syndrome ( mets ) and the risk of pca ( figure 1 ) . more importantly , evolutionary divergence of gprc6a in humans suggests that this receptor has undergone important changes in its biological functions . modern humans , neanderthals , and denisovans ( all genus homo ) have a common polymorphism of gprc6a that substitutes a .. y amino acid sequence at position 744 in the third 3rd intracellular loop for a highly evolutionarily conserved rklp sequence present in all other vertebrates ( figure 2a ) . in the 1000 human genomes project , the uniquely human k .. y allele is present in 99.4% of the east asian descent population , 91.8% of the european descent population , 83.4% of the american hispanic population , and 59.9% of the persons of african descent ( figure 2b ) . in contrast , the ancestral rklp sequence in 3rd intracellular loop of gprc6a , which is evolutionarily conserved in all pre - human species , is present only in a minority of humans , and is considered a variant ( rs386705086 ) that is most commonly found in african - derived populations ( figure 2c ) . the emergence of the k .. y polymorphism only in hominids and its preservation during evolution indicate a selection pressure that modifies the physiological function of this allele . on the other hand , the persistence of the ancestral rklp sequence and its predominant expression in humans of african descent ( the oldest humans ) suggest that this allele may contribute to the racial disparities in diseases associated with gprc6a , including mets , and pca . this schema is controversial in part , because it is difficult to conceive of a physiological purpose for a single receptor to sense so many disparate ligands and influence numerous metabolic hormones and organ - specific metabolic processes . some remain committed to the original simple view of ocn activation of gprc6a and do not attempt to incorporate either the effects of basic amino acids and testosterone to activate this receptor or the broad effects of gprc6a to regulate diverse tissue functions beyond tissue involved in energy metabolism . others posit that only basic amino acids , and neither ocn nor t , play physiological roles to activate gprc6a , a nihilist view that gprc6a has no function in humans , . thus , progress to date places gprc6a on the precipice of either being a master regulator of metabolic processes or , alternatively , on the list of loss - of - function tolerant gene variants that have little clinical impact in humans . which of these suppositions is correct will determine if gprc6a participates in the pathogenesis of human diseases and is a valid target to develop agonists and antagonists to respectively treat mets and pca . though there are several issues that need to be resolved , the majority of evidence points to an important role for gprc6a in mice , and a likely role of gprc6a in modulating the risks of high - fat - diet induced mets , and racial disparities in the risk of mets and pca in humans . as noted , gprc6a is activated by multiple ligands , including cations , such as calcium , zinc , and magnesium , the basic amino acids l - arginine ( l - arg ) , l - lysine ( l - lys ) , and l - ornithine ( l - orn ) , the bone - derived peptide ocn , and t. gprc6a mediates the non - genomic effects of testosterone . are all of these physiologically relevant ligands for gprc6a , and , if so , what is the structural basis for such ligand diversity , and how do these work together or separately to regulate gprc6a tissue functions ? there is a general consensus that l - arg activates gprc6a in vitro , but the ability of l - arg administration to regulate metabolic process through activation of gprc6a in vivo has produced conflicting results , . l - arg , derived from dietary intake or protein catabolism , might be expected to regulate metabolic functions through gprc6a , but to date a link between gprc6a and metabolic effects of protein intake have not been established . there is an intriguing and yet - to - be validated possibility that gprc6a may function intracellularly to sense lysosomal amino acids ( vide infra ) . to date , there have been no demonstrable in vivo divalent cation sensing functions of gprc6a , however , the trivalent cation aluminum , which activates gprc6a in vitro , have been shown to modulate aluminum - induced cytokine activation by monocytes in vitro and in vivo through gprc6a . many studies show that ocn , , , , , and t , , activate gprc6a in different in vitro model systems , including heterologous cells that gain ocn and t sensing function when transfected with gprc6a cdna and cells with endogenous expression of gprca that lose ocn and t activation with knock - down or inhibition of gprc6a signaling . in addition to its published effects on -cells , leydig cells , muscle cells , enteroendocrine l - cells , and prostate cancer cells , ocn acting through gprc6a also protects adipocytes and muscle from autophagy and er - stress and restores insulin sensitivity in high fat diet induced models of insulin resistance . in contrast , several investigators have been unable to demonstrate effects of either ocn or t to activate gprc6a in cell culture models , . these few negative in vitro studies logically can not disprove the many positive findings ; the disparate results likely represent differences in cell models and cdna constructs used in studies involving transfection of the receptor , . nevertheless , the physiological role of ocn in regulating energy and glucose metabolism remains controversial . indeed , questions regarding the functions of ocn as a ligand for gprc6a have also been raised . first , ocn is a dual functioning protein that undergoes post - translational -carboxylation of three glu to gla residues for anchoring to bone mineral and is de - carboxylated in the acidic environment of bone resorption to generate a ligand for gprc6a . the domains in ocn predicted to bind to gprc6a are the 3-helical tertiary structure of bi - carboxylated ocn and a 6-amino acid c - terminal region ( 7 ) . during dissolution of bone mineral , however , undercarboxylated ocn , -carboxylated ocn , and peptides including a 6-aa c - terminal ocn peptide lacking a carboxylation site are generated . initially , either recombinant ocn or chemically de - carboxlyated native ocn were shown to activate gprc6a ( 34 ) . in other studies , only a small difference in efficacy of uncarboxylated and -carboxylated forms of ocn were observed . in prostate cancer cells , thus , the function of ocn as a ligand can be regulated by post - translational de - carboxylation and cleavage , potentially in a tissue - specific fashion . second , ocn has low overall sequence homology across species , with human ocn having a 57.1% and 75.5% sequence identity with mouse and rat ocn , respectively , suggesting that its function may not be conserved across species . indeed , there are differences in the phenotype of rat and mouse ocn knockouts , suggesting that the functions of ocn may not be conserved across species . in contrast to the bone and metabolic phenotypes observed in ocn mice , , ocn rats have a bone but no metabolic phenotype . the rat ocn gene structure is closer to humans , and the rat strategy to delete gprc6a only required targeting a single gprc6a locus . in contrast , the mouse has a more complex ocn locus that required deletion of two bone expressed ocn gene copies and an interspersed adiponectin receptor , paqr6 , , raising the possibility that the metabolic phenotype in ocn - deficient mice is due to loss of paqr6 . doubts arising from these inconsistencies are counterbalanced by the pharmacological effects of parenteral administered ocn to stimulate insulin secretion , , improve glucose tolerance and peripheral insulin sensitivity , decrease fat mass , and protect against hepatosteatosis in wild - type mice , . ocn stimulated gprc6a functions in -cells to regulate insulin secretion and in muscle to regulate exercise capacity were lost with organ - specific deletion of gprc6a . however , in spite of these positive results in animal models , there has been little translational progress in developing ocn as a valid therapeutic . orally administered ocn can also have effects on insulin release and glucose utilization and protect against the adverse metabolic effects of high fat diets in animals , . the effect of orally administered ocn is likely mediated by gprc6a - dependent effects of ocn to stimulate the release of the incretin hormone glp-1 from enteroendocrine l - cells . unexpectedly , however , male mice administered oral ocn paradoxically exhibited glucose intolerance and insulin resistance . the basis for sex - dependence in response to oral and parenteral ocn treatment remains unclear , but t also activates gprc6a and complex interactions between ocn , t , and sex hormone binding globulin ( shgb ) might occur ( vide infra ) . more recently , molecular modeling of gprc6a provides a structural basis for understanding diverse ligand binding to this receptor . gprc6a consists of a venus fly trap ( vft ) motif that is attached to a prototypic 7-transmembrane ( 7-tm ) domain ( figure 2a ) . there is a general consensus that basic amino acids , such as l - arginine ( l - arg ) , l - lysine ( l - lys ) , and l - ornithine ( l - orn ) activate gprc6a through binding to the vft motif . binding sites for ocn are predicted to be in the 7-tm domain and mutations of these sites block the ability of ocn to activate gprc6a . the 6-aa carboxyl terminal of ocn can also activate gprc6a by binding to the 7-tm site , and amino acid substitutions in this region abrogate activation by ocn , but not by l - arg . t is also predicted to bind to epitopes in the 7-tm domain of gprc6a , and mutagenesis of these sites disrupts t activation of gprc6a as well . interestingly , shbg , which binds t , has homologies to ocn and is also proposed to bind to gprc6a and modulate the response to t . compounds containing 2-phenyl indoles and calcimimetics , which activate the related calcium sensing receptor , casr , also activate gprc6a . in addition , mutation of phe 783 to ser , which is homologous to an amino acid change that creates a constitutively active casr , also leads to enhanced activation of gprc6a in response to l - arg ( unpublished observations ) . crystal structures of gprc6a are needed to confirm and expand these computational predictions regarding ligand interactions with gprc6a . in addition to the full - length gprc6a , two other isoforms of gprc6a have been identified that have alternative splicing of exon 3 encoding amino acids 270445 and exon 4 encoding 445516 , altering the extracellular domain of this receptor . there is limited information regarding the effects of these extracellular domain alterations on ligand functions , but the full - length form of gprc6a is the most highly expressed in metabolic tissues , except the kidney , which expresses equal amounts of isoform 2 . the possibility that gprc6a represents a receptor that can sense multiple , disparate ligands , that potentially interact as positive and negative allosteric modulators , is novel and suggests that this receptor serves as a point of integration of diverse extracellular signals and cellular responses in multiple tissues to create new endocrine networks . indeed , gprc6a is widely expressed , and our current understanding of its tissue - specific functions largely comes from analysis of the effects of ocn administration and mouse genetic studies . ocn administration acts through gprc6a to increase pancreatic -cell number , insulin release , and insulin sensitivity , . ocn administration also stimulates human islets in vitro and in xenotransplants to increase -cell number and insulin release . ocn signaling through gprc6a in muscle enables increased adaptation to exercise by stimulating fatty acid and glucose uptake and utilization , as well as regulating muscle mass ; and aged mice treated with ocn increase muscle mass . mouse genetic studies show that gprc6a is a recessive gene , since heterozygous mice have no demonstrable phenotype . however , gprc6a null mice exhibit direct organ - specific defects in glucose and fat metabolism , as well as indirect metabolic effects resulting from gprc6a regulation of an assortment metabolically active hormones in the mouse . for example , gprc6a is expressed in pancreatic islet , liver , skeletal muscle , and fat tissue , , , in which activation of gprc6a regulates metabolic processes , including stimulation of insulin secretion and -cell proliferation , , , enhancement of peripheral tissue insulin sensitivity , catabolism of glucose and fatty acids in myocytes , , , and incompletely understood effects to regulate glucose and fatty acid metabolism in liver and adipose tissues . gprc6a knockout , -cell and muscle specific gprc6a knockout mice do not respond to ocn , , indicating that gprc6a is required for ocn effects . null mice lacking either ocn or gprc6a have diminished exercise capability , , , while a knockout of gprc6a decreases muscle mass . conditional deletion of gprc6a in muscle results in impaired glucose uptake and release of interleukin-6 , a myokine that promotes adaptation to exercise . in addition , the ligand for gprc6a , ocn , is increased by exercise and may regulate muscle metabolism and muscle mass through gprc6a , , . in contrast , using another gprc6a null mouse model , others found ablation of gprc6a increased voluntary wheel running in mice . although gprc6a null mice exhibit an increased susceptibility to high fat diet induced obesity , further studies are needed to assess if this is due to direct effects of gprc6a in adipocytes . however , ocn has been shown to directly regulate glucose transport as well as tnf- and adiponectin secretion in primary adipocytes . as noted above , gprc6a stimulates glp-1 from intestinal cells , , , but the gprc6a ligand l - arg moderately stimulated glp-1 although the stimulation did not reach statistical significance , in gprc6a null mice , indicating other mechanisms of l - arg actions . gprc6a is also expressed in the brain , where existing data suggests that it has central nervous system functions , including regulation of myelin production and seizure thresholds ( unpublished data ) and lh secretion , but there is disagreement if loss of gprc6a signaling mediates the neurological abnormalities in ocn mice . in addition , t also stimulates gprc6a to regulate proliferation in skin keratinocytes , and the activation of gprc6a by t is likely to have other effects attributed to the non - genomic actions of t . there are other tissues , such as the salivary gland , that highly express gprc6a and for which the function of this receptor is not known . gprc6a is also expressed in bone and gprc6a mice exhibit osteopenia and impaired osteoblast mediated bone formation , and uncarboxylated ocn stimulates osteoblast differentiation in murine osteoblasts , , ; however , another study using a different gprc6a knockout mouse model failed to show a bone phenotype . gprc6a may also regulate immune responses . mets is associated with inflammation , and gprc6a , which is expressed in monocytes , has been shown to regulate nlrp3 inflammasome responses in response to aluminum in vitro and in vivo . gprc6a is expressed in the myocardium , undercarboxylated ocn protects against diabetic cardiomyopathy , and gprc6a mice develop a dilated cardiomyopathy ( unpublished observations ) . as noted above , gprc6a is expressed in the prostate , is increased in human prostate cancer ( pca ) cells , and regulates prostate cancer progression in mice . thus , there are a myriad of physiological functions potentially regulated by gprc6a and its ligands . in contrast to the metabolic phenotypes reported in mouse models in which gprc6a was disrupted , , , , , , , , neither glucose intolerance nor other phenotypes attributed to gprc6a were reported in two other gprc6a knockout mouse models , . different strategies were used to generate gprc6a deficient mice , including targeting exons in the extracellular domain , encoding the 7tm domain of gprc6a , the entire gprc6a locus . deletion of the 7tm , which leaves intact the extracellular domain of gprc6a , may act as a dominant negative factor , as reported for a calcium sensing receptor ( casr ) knockout model , but this does not explain the absence of a metabolic phenotype in the model that completely lacks gprc6a . genetic background differences may confound the findings , since the susceptibility to mets is mouse strain dependent . a more intriguing possibility is that gprc6a is a recessive gene that confers susceptibility to high fat diets . indeed , gprc6a null mice that exhibited no metabolic phenotype on a normal diet exhibit greater increases in basal plasma glucose levels and impairment of oral glucose tolerance and insulin resistance when compared to wild - type mice when placed on a high fat diet , . additional studies are needed to determine if gprc6a represents a susceptibility gene for high fat diet induced metabolic complications in humans , as suggested by these animal studies . others have also questioned whether gprc6a is directly activated by ocn and t. in contrast to studies showing the ability of ocn and t to activate gprc6a , another group found that gprc6a is activated only by basic amino acid , but not ocn and t . ocn , t , shbg , basic amino acids and cations are simultaneously present in the physiological milieu . at present the vft and 7-tm binding sites create the potential for both positive interactions and allosteric modulation of gprc6a activity by ligands binding to these different sites , as well as antagonist effects of ligands competing for the same sites ( figure 3 ) . k .. y polymorphism , which is present in 84% of humans , is activated by l - arg , but not by t and ocn , , suggesting that this polymorphism may have altered the ligand sensing functions of gprc6a to favor basic amino acids over ocn and t. studies have also variably shown coupling of gprc6a to gs- , gi- and gq - dependent signaling pathways , ( figure 4 ) . this suggests that gprc6a may have cell - type specific co - factors that modify its functions . gprc6a erk , camp , pi3k / akt / mtor and ampk signaling , , , . some investigators have purported that the k - y allele is non - functional due to faulty cellular trafficking leading to endosomal localization . k .. y changes in the 3rd il might affect -arrestin binding and the kinetics of membrane trafficking , leading to alterations in the magnitude and/or duration of gprc6a signaling and/or gain - of - lysosomal signaling due to preferential intracellular localization . a precedent for this comes from the recently discovered endosomal signaling of the pth gpcr through -arrestin - dependent mechanism . thus , the k .. y polymorphism in human gprc6a may have evolved to alter cell trafficking that preferentially leads to activation of mtorc1 intracelluar signaling functions . the highly conserved amino acid signaling through mtorc1 occurs in the lysosome , and is mediated by complex cascades involving rag gtpases , ragulator , and vacuolar h - atpase . it is currently unclear , however , how amino acid sufficiency or limitation is sensed in mammalian cells . this creates an intriguing possibility that humans have evolved changes in gprc6a as a way to regulate this intracellular amino acid sensing pathway ( figure 4 ) . however , even if the k .. y polymorphism proves to be non - functional , the ancestral rklp variant ( rs386705086 ) should have functions and might still explain racial disparities in mets and pca . although the individual metabolic functions of the multiple hormones stimulated by gprc6a are well known , the idea that there is a coordinated release of insulin , , , t , , glp-1 , , , adiponectin , and il-6 , in response to gprc6a activation is a novel concept . a surprising number of inter - organ communications and feedback loops are being recognized that are created by gprc6a - mediated release of hormones , , , , , , , . for example , ocn released from bone would directly stimulate insulin , glp-1 , and t release and have direct and indirect effects on muscle , liver , fat , and testes . t activating gprc6a in leydig cells would further stimulate its own production and also have peripheral tissue effects to regulate glucose and fat metabolism . t may also act centrally through gprc6a to suppress lh secretion and disrupt the ocn - t positive endocrine loop , as evidenced by paradoxical stimulation of lh by t in gprc6a mice . insulin and glp-1 are connected in the fed state , with glp-1 enhancing insulin release , slowing gastric emptying , and inhibiting glucagon release . adiponectin administration increases insulin sensitivity , an effect complementary to the increased insulin and glp-1 induced by ocn activation of gprc6a . gprc6a stimulation in exercising skeletal muscle releases il-6 , which has a feed forward effect to release additional ocn from bone . moreover , since gprc6a has direct effects on many of the same organs and activates the same signaling pathways that are targeted by these hormones , including erk , camp , pi3k / akt / mtor , and ampk , , , , there is potential cross talk between direct and indirect effects of gprc6a ( figure 4 ) . for example , in the liver , there is overlap between gprc6a signaling pathways and those regulated by insulin and glucagon . gprc6a is positioned to impact both insulin signaling through pi3k / akt and glucagon pathways through activation of camp , as well as additional effects through erk and ampk activation . thus , gprc6a might modulate both pathways , thus serving as a context - dependent enhancer of liver metabolism . the functions of ocn and gprc6a in humans remain to be established . with regards to ocn , clinical studies show an inverse correlation between undercarboxylated ocn levels and obesity , glycemic status , insulin resistance , triglycerides , and leptin and positive correlations with adiponectin , , . genome wide association studies have linked single nucleotide polymorphisms in the coding and non - coding region of the ocn gene with body composition in humans , ; a snp resulting in a r43q change ( rs34702397 ) in ocn that is predicted to affect binding to gprc6a is associated with insulin sensitivity in african americans . the rs34702397 snp in ocn is unevenly distributed in the world population , with 4.65% of people with african , 0.26% of latino , < 0.01% of european , and 0% of asian descent having the mutant allele ( http://exac.broadinstitute.org/variant/6-117150008-g-a ) . with regards to the gprc6a gene , others have questioned if the predicted metabolic functions of gprc6a are relevant to humans , citing the failure to demonstrate membrane location and/or function of the human gprc6a and high number of snps in gprc6a predicted to inactivate the gene , and the apparent lack of disease associations between these polymorphisms and mets in humans . indeed , there are no confirmed mendelian inherited diseases caused by inactivating mutations of gprc6a in humans . although mutations in gprc6a have been implicated in familial partial lipodystrophy type i ( fpld1 ) , the implicated k774-l - p polymorphism ( snp ) and an r57x stop mutation found in fpld1 is present in 8% of the general population ( http://exac.broadinstitute.org/variant/6-117150008-g-a ) . the evolutionary changes in gprc6a provide the strongest evidence for a function of gprc6a in humans . the sequence rklp in the 3rd intracellular loop of gprc6a is present in mice , in which its function has been established , and conserved in all mammalian species except humans , in whom k .. y replaces the rklp sequence in the majority of people ( 60% in african , 85% in caucasians , and 99% in asian descent ) . the rklp sequence is defined as a variant ( rs38670586 ) in humans and is most commonly found in african - derived populations ( 40% versus 15% in caucasians and 1% in asian descent ) . more recently , genome - wide association studies ( gwas ) show that the p91s ( rs2274911 ) snp is associated with insulin resistance but not obesity , , , the p91s and f464y snps are associated with reduced sperm count and cryptorchidism , , and the gprc6a genetic locus is associated with c - reactive protein levels , , cardiovascular diseases , , and the risk of pca in humans , , . this schema is controversial in part , because it is difficult to conceive of a physiological purpose for a single receptor to sense so many disparate ligands and influence numerous metabolic hormones and organ - specific metabolic processes . some remain committed to the original simple view of ocn activation of gprc6a and do not attempt to incorporate either the effects of basic amino acids and testosterone to activate this receptor or the broad effects of gprc6a to regulate diverse tissue functions beyond tissue involved in energy metabolism . others posit that only basic amino acids , and neither ocn nor t , play physiological roles to activate gprc6a , a nihilist view that gprc6a has no function in humans , . thus , progress to date places gprc6a on the precipice of either being a master regulator of metabolic processes or , alternatively , on the list of loss - of - function tolerant gene variants that have little clinical impact in humans . which of these suppositions is correct will determine if gprc6a participates in the pathogenesis of human diseases and is a valid target to develop agonists and antagonists to respectively treat mets and pca . though there are several issues that need to be resolved , the majority of evidence points to an important role for gprc6a in mice , and a likely role of gprc6a in modulating the risks of high - fat - diet induced mets , and racial disparities in the risk of mets and pca in humans . as noted , gprc6a is activated by multiple ligands , including cations , such as calcium , zinc , and magnesium , the basic amino acids l - arginine ( l - arg ) , l - lysine ( l - lys ) , and l - ornithine ( l - orn ) , the bone - derived peptide ocn , and t. gprc6a mediates the non - genomic effects of testosterone . are all of these physiologically relevant ligands for gprc6a , and , if so , what is the structural basis for such ligand diversity , and how do these work together or separately to regulate gprc6a tissue functions ? there is a general consensus that l - arg activates gprc6a in vitro , but the ability of l - arg administration to regulate metabolic process through activation of gprc6a in vivo has produced conflicting results , . l - arg , derived from dietary intake or protein catabolism , might be expected to regulate metabolic functions through gprc6a , but to date a link between gprc6a and metabolic effects of protein intake have not been established . there is an intriguing and yet - to - be validated possibility that gprc6a may function intracellularly to sense lysosomal amino acids ( vide infra ) . to date , there have been no demonstrable in vivo divalent cation sensing functions of gprc6a , however , the trivalent cation aluminum , which activates gprc6a in vitro , have been shown to modulate aluminum - induced cytokine activation by monocytes in vitro and in vivo through gprc6a . many studies show that ocn , , , , , and t , , activate gprc6a in different in vitro model systems , including heterologous cells that gain ocn and t sensing function when transfected with gprc6a cdna and cells with endogenous expression of gprca that lose ocn and t activation with knock - down or inhibition of gprc6a signaling . in addition to its published effects on -cells , leydig cells , muscle cells , enteroendocrine l - cells , and prostate cancer cells , ocn acting through gprc6a also protects adipocytes and muscle from autophagy and er - stress and restores insulin sensitivity in high fat diet induced models of insulin resistance . in contrast , several investigators have been unable to demonstrate effects of either ocn or t to activate gprc6a in cell culture models , . these few negative in vitro studies logically can not disprove the many positive findings ; the disparate results likely represent differences in cell models and cdna constructs used in studies involving transfection of the receptor , . nevertheless , the physiological role of ocn in regulating energy and glucose metabolism remains controversial . indeed , questions regarding the functions of ocn as a ligand for gprc6a have also been raised . first , ocn is a dual functioning protein that undergoes post - translational -carboxylation of three glu to gla residues for anchoring to bone mineral and is de - carboxylated in the acidic environment of bone resorption to generate a ligand for gprc6a . the domains in ocn predicted to bind to gprc6a are the 3-helical tertiary structure of bi - carboxylated ocn and a 6-amino acid c - terminal region ( 7 ) . during dissolution of bone mineral , however , undercarboxylated ocn , -carboxylated ocn , and peptides including a 6-aa c - terminal ocn peptide lacking a carboxylation site are generated . initially , either recombinant ocn or chemically de - carboxlyated native ocn were shown to activate gprc6a ( 34 ) . in other studies , only a small difference in efficacy of uncarboxylated and -carboxylated forms of ocn were observed . in prostate cancer cells , thus , the function of ocn as a ligand can be regulated by post - translational de - carboxylation and cleavage , potentially in a tissue - specific fashion . second , ocn has low overall sequence homology across species , with human ocn having a 57.1% and 75.5% sequence identity with mouse and rat ocn , respectively , suggesting that its function may not be conserved across species . indeed , there are differences in the phenotype of rat and mouse ocn knockouts , suggesting that the functions of ocn may not be conserved across species . in contrast to the bone and metabolic phenotypes observed in ocn mice , , ocn rats have a bone but no metabolic phenotype . the rat ocn gene structure is closer to humans , and in contrast , the mouse has a more complex ocn locus that required deletion of two bone expressed ocn gene copies and an interspersed adiponectin receptor , paqr6 , , raising the possibility that the metabolic phenotype in ocn - deficient mice is due to loss of paqr6 . doubts arising from these inconsistencies are counterbalanced by the pharmacological effects of parenteral administered ocn to stimulate insulin secretion , , improve glucose tolerance and peripheral insulin sensitivity , decrease fat mass , and protect against hepatosteatosis in wild - type mice , . ocn stimulated gprc6a functions in -cells to regulate insulin secretion and in muscle to regulate exercise capacity were lost with organ - specific deletion of gprc6a . however , in spite of these positive results in animal models , there has been little translational progress in developing ocn as a valid therapeutic . orally administered ocn can also have effects on insulin release and glucose utilization and protect against the adverse metabolic effects of high fat diets in animals , . the effect of orally administered ocn is likely mediated by gprc6a - dependent effects of ocn to stimulate the release of the incretin hormone glp-1 from enteroendocrine l - cells . unexpectedly , however , male mice administered oral ocn paradoxically exhibited glucose intolerance and insulin resistance . the basis for sex - dependence in response to oral and parenteral ocn treatment remains unclear , but t also activates gprc6a and complex interactions between ocn , t , and sex hormone binding globulin ( shgb ) might occur ( vide infra ) . more recently , molecular modeling of gprc6a provides a structural basis for understanding diverse ligand binding to this receptor . gprc6a consists of a venus fly trap ( vft ) motif that is attached to a prototypic 7-transmembrane ( 7-tm ) domain ( figure 2a ) . there is a general consensus that basic amino acids , such as l - arginine ( l - arg ) , l - lysine ( l - lys ) , and l - ornithine ( l - orn ) activate gprc6a through binding to the vft motif . binding sites for ocn are predicted to be in the 7-tm domain and mutations of these sites block the ability of ocn to activate gprc6a . the 6-aa carboxyl terminal of ocn can also activate gprc6a by binding to the 7-tm site , and amino acid substitutions in this region abrogate activation by ocn , but not by l - arg . t is also predicted to bind to epitopes in the 7-tm domain of gprc6a , and mutagenesis of these sites disrupts t activation of gprc6a as well . interestingly , shbg , which binds t , has homologies to ocn and is also proposed to bind to gprc6a and modulate the response to t . compounds containing 2-phenyl indoles and calcimimetics , which activate the related calcium sensing receptor , casr , also activate gprc6a . in addition , mutation of phe 783 to ser , which is homologous to an amino acid change that creates a constitutively active casr , also leads to enhanced activation of gprc6a in response to l - arg ( unpublished observations ) . crystal structures of gprc6a are needed to confirm and expand these computational predictions regarding ligand interactions with gprc6a . in addition to the full - length gprc6a , two other isoforms of gprc6a have been identified that have alternative splicing of exon 3 encoding amino acids 270445 and exon 4 encoding 445516 , altering the extracellular domain of this receptor . there is limited information regarding the effects of these extracellular domain alterations on ligand functions , but the full - length form of gprc6a is the most highly expressed in metabolic tissues , except the kidney , which expresses equal amounts of isoform 2 . the possibility that gprc6a represents a receptor that can sense multiple , disparate ligands , that potentially interact as positive and negative allosteric modulators , is novel and suggests that this receptor serves as a point of integration of diverse extracellular signals and cellular responses in multiple tissues to create new endocrine networks . indeed , gprc6a is widely expressed , and our current understanding of its tissue - specific functions largely comes from analysis of the effects of ocn administration and mouse genetic studies . ocn administration acts through gprc6a to increase pancreatic -cell number , insulin release , and insulin sensitivity , . ocn administration also stimulates human islets in vitro and in xenotransplants to increase -cell number and insulin release . ocn signaling through gprc6a in muscle enables increased adaptation to exercise by stimulating fatty acid and glucose uptake and utilization , as well as regulating muscle mass ; and aged mice treated with ocn increase muscle mass . mouse genetic studies show that gprc6a is a recessive gene , since heterozygous mice have no demonstrable phenotype . however , gprc6a null mice exhibit direct organ - specific defects in glucose and fat metabolism , as well as indirect metabolic effects resulting from gprc6a regulation of an assortment metabolically active hormones in the mouse . for example , gprc6a is expressed in pancreatic islet , liver , skeletal muscle , and fat tissue , , , in which activation of gprc6a regulates metabolic processes , including stimulation of insulin secretion and -cell proliferation , , , enhancement of peripheral tissue insulin sensitivity , catabolism of glucose and fatty acids in myocytes , , , and incompletely understood effects to regulate glucose and fatty acid metabolism in liver and adipose tissues . gprc6a knockout , -cell and muscle specific gprc6a knockout mice do not respond to ocn , , indicating that gprc6a is required for ocn effects . null mice lacking either ocn or gprc6a have diminished exercise capability , , , while a knockout of gprc6a decreases muscle mass . conditional deletion of gprc6a in muscle results in impaired glucose uptake and release of interleukin-6 , a myokine that promotes adaptation to exercise . in addition , the ligand for gprc6a , ocn , is increased by exercise and may regulate muscle metabolism and muscle mass through gprc6a , , . in contrast , using another gprc6a null mouse model , others found ablation of gprc6a increased voluntary wheel running in mice . although gprc6a null mice exhibit an increased susceptibility to high fat diet induced obesity , further studies are needed to assess if this is due to direct effects of gprc6a in adipocytes . however , ocn has been shown to directly regulate glucose transport as well as tnf- and adiponectin secretion in primary adipocytes . as noted above , gprc6a stimulates glp-1 from intestinal cells , , , but the gprc6a ligand l - arg moderately stimulated glp-1 although the stimulation did not reach statistical significance , in gprc6a null mice , indicating other mechanisms of l - arg actions . gprc6a is also expressed in the brain , where existing data suggests that it has central nervous system functions , including regulation of myelin production and seizure thresholds ( unpublished data ) and lh secretion , but there is disagreement if loss of gprc6a signaling mediates the neurological abnormalities in ocn mice . in addition , t also stimulates gprc6a to regulate proliferation in skin keratinocytes , and the activation of gprc6a by t is likely to have other effects attributed to the non - genomic actions of t . there are other tissues , such as the salivary gland , that highly express gprc6a and for which the function of this receptor is not known . gprc6a is also expressed in bone and gprc6a mice exhibit osteopenia and impaired osteoblast mediated bone formation , and uncarboxylated ocn stimulates osteoblast differentiation in murine osteoblasts , , ; however , another study using a different gprc6a knockout mouse model failed to show a bone phenotype . mets is associated with inflammation , and gprc6a , which is expressed in monocytes , has been shown to regulate nlrp3 inflammasome responses in response to aluminum in vitro and in vivo . gprc6a is expressed in the myocardium , undercarboxylated ocn protects against diabetic cardiomyopathy , and gprc6a mice develop a dilated cardiomyopathy ( unpublished observations ) . as noted above , gprc6a is expressed in the prostate , is increased in human prostate cancer ( pca ) cells , and regulates prostate cancer progression in mice . thus , there are a myriad of physiological functions potentially regulated by gprc6a and its ligands . in contrast to the metabolic phenotypes reported in mouse models in which gprc6a was disrupted , , , , , , , , neither glucose intolerance nor other phenotypes attributed to gprc6a were reported in two other gprc6a knockout mouse models , . different strategies were used to generate gprc6a deficient mice , including targeting exons in the extracellular domain , encoding the 7tm domain of gprc6a , the entire gprc6a locus . deletion of the 7tm , which leaves intact the extracellular domain of gprc6a , may act as a dominant negative factor , as reported for a calcium sensing receptor ( casr ) knockout model , but this does not explain the absence of a metabolic phenotype in the model that completely lacks gprc6a . genetic background differences may confound the findings , since the susceptibility to mets is mouse strain dependent . a more intriguing possibility is that gprc6a is a recessive gene that confers susceptibility to high fat diets . indeed , gprc6a null mice that exhibited no metabolic phenotype on a normal diet exhibit greater increases in basal plasma glucose levels and impairment of oral glucose tolerance and insulin resistance when compared to wild - type mice when placed on a high fat diet , . additional studies are needed to determine if gprc6a represents a susceptibility gene for high fat diet induced metabolic complications in humans , as suggested by these animal studies . others have also questioned whether gprc6a is directly activated by ocn and t. in contrast to studies showing the ability of ocn and t to activate gprc6a , another group found that gprc6a is activated only by basic amino acid , but not ocn and t . ocn , t , shbg , basic amino acids and cations are simultaneously present in the physiological milieu . at present , it is not clear how these ligands may interact to regulate gprc6a functions . the vft and 7-tm binding sites create the potential for both positive interactions and allosteric modulation of gprc6a activity by ligands binding to these different sites , as well as antagonist effects of ligands competing for the same sites ( figure 3 ) . k .. y polymorphism , which is present in 84% of humans , is activated by l - arg , but not by t and ocn , , suggesting that this polymorphism may have altered the ligand sensing functions of gprc6a to favor basic amino acids over ocn and t. studies have also variably shown coupling of gprc6a to gs- , gi- and gq - dependent signaling pathways , ( figure 4 ) . this suggests that gprc6a may have cell - type specific co - factors that modify its functions . gprc6a erk , camp , pi3k / akt / mtor and ampk signaling , , , . some investigators have purported that the k - y allele is non - functional due to faulty cellular trafficking leading to endosomal localization . k .. y changes in the 3rd il might affect -arrestin binding and the kinetics of membrane trafficking , leading to alterations in the magnitude and/or duration of gprc6a signaling and/or gain - of - lysosomal signaling due to preferential intracellular localization . a precedent for this comes from the recently discovered endosomal signaling of the pth gpcr through -arrestin - dependent mechanism . thus , the k .. y polymorphism in human gprc6a may have evolved to alter cell trafficking that preferentially leads to activation of mtorc1 intracelluar signaling functions . the highly conserved amino acid signaling through mtorc1 occurs in the lysosome , and is mediated by complex cascades involving rag gtpases , ragulator , and vacuolar h - atpase . it is currently unclear , however , how amino acid sufficiency or limitation is sensed in mammalian cells . this creates an intriguing possibility that humans have evolved changes in gprc6a as a way to regulate this intracellular amino acid sensing pathway ( figure 4 ) . however , even if the .. y polymorphism proves to be non - functional , the ancestral rklp variant ( rs386705086 ) should have functions and might still explain racial disparities in mets and pca . although the individual metabolic functions of the multiple hormones stimulated by gprc6a are well known , the idea that there is a coordinated release of insulin , , , t , , glp-1 , , , adiponectin , and il-6 , in response to gprc6a activation is a novel concept . a surprising number of inter - organ communications and feedback loops are being recognized that are created by gprc6a - mediated release of hormones , , , , , , , . for example , ocn released from bone would directly stimulate insulin , glp-1 , and t release and have direct and indirect effects on muscle , liver , fat , and testes . t activating gprc6a in leydig cells would further stimulate its own production and also have peripheral tissue effects to regulate glucose and fat metabolism . t may also act centrally through gprc6a to suppress lh secretion and disrupt the ocn - t positive endocrine loop , as evidenced by paradoxical stimulation of lh by t in gprc6a mice . insulin and glp-1 are connected in the fed state , with glp-1 enhancing insulin release , slowing gastric emptying , and inhibiting glucagon release . adiponectin administration increases insulin sensitivity , an effect complementary to the increased insulin and glp-1 induced by ocn activation of gprc6a . gprc6a stimulation in exercising skeletal muscle releases il-6 , which has a feed forward effect to release additional ocn from bone . moreover , since gprc6a has direct effects on many of the same organs and activates the same signaling pathways that are targeted by these hormones , including erk , camp , pi3k / akt / mtor , and ampk , , , , there is potential cross talk between direct and indirect effects of gprc6a ( figure 4 ) . for example , in the liver , there is overlap between gprc6a signaling pathways and those regulated by insulin and glucagon . gprc6a is positioned to impact both insulin signaling through pi3k / akt and glucagon pathways through activation of camp , as well as additional effects through erk and ampk activation . thus , gprc6a might modulate both pathways , thus serving as a context - dependent enhancer of liver metabolism . the functions of ocn and gprc6a in humans remain to be established . with regards to ocn , clinical studies show an inverse correlation between undercarboxylated ocn levels and obesity , glycemic status , insulin resistance , triglycerides , and leptin and positive correlations with adiponectin , , . genome wide association studies have linked single nucleotide polymorphisms in the coding and non - coding region of the ocn gene with body composition in humans , ; a snp resulting in a r43q change ( rs34702397 ) in ocn that is predicted to affect binding to gprc6a is associated with insulin sensitivity in african americans . the rs34702397 snp in ocn is unevenly distributed in the world population , with 4.65% of people with african , 0.26% of latino , < 0.01% of european , and 0% of asian descent having the mutant allele ( http://exac.broadinstitute.org/variant/6-117150008-g-a ) . with regards to the gprc6a gene , others have questioned if the predicted metabolic functions of gprc6a are relevant to humans , citing the failure to demonstrate membrane location and/or function of the human gprc6a and high number of snps in gprc6a predicted to inactivate the gene , and the apparent lack of disease associations between these polymorphisms and mets in humans . indeed , there are no confirmed mendelian inherited diseases caused by inactivating mutations of gprc6a in humans . although mutations in gprc6a have been implicated in familial partial lipodystrophy type i ( fpld1 ) , the implicated k774-l - p polymorphism ( snp ) and an r57x stop mutation found in fpld1 is present in 8% of the general population ( http://exac.broadinstitute.org/variant/6-117150008-g-a ) . the evolutionary changes in gprc6a provide the strongest evidence for a function of gprc6a in humans . the sequence rklp in the 3rd intracellular loop of gprc6a is present in mice , in which its function has been established , and conserved in all mammalian species except humans , in whom k .. y replaces the rklp sequence in the majority of people ( 60% in african , 85% in caucasians , and 99% in asian descent ) . the rklp sequence is defined as a variant ( rs38670586 ) in humans and is most commonly found in african - derived populations ( 40% versus 15% in caucasians and 1% in asian descent ) . more recently , genome - wide association studies ( gwas ) show that the p91s ( rs2274911 ) snp is associated with insulin resistance but not obesity , , , the p91s and f464y snps are associated with reduced sperm count and cryptorchidism , , and the gprc6a genetic locus is associated with c - reactive protein levels , , cardiovascular diseases , , and the risk of pca in humans , , . if gprc6a has similar functions in humans as are observed in mice , drugs that activate gprc6a theoretically would target several abnormalities that cause t2 dm , including disordered -cell function , insulin resistance , and obesity . indeed , direct effects on -cells to attenuate loss of -cell mass , increase insulin and glp-1 secretion and enhance glucose uptake and insulin sensitivity in peripheral tissues addresses many of the abnormalities underlying mets . either an oral or more likely an injectable ocn , or an orally bioavailable gprc6a chemical agonist , might be used alone or in combination with existing treatments for t2 dm . potential effects of gprc6a to ameliorate adverse effects of high - fat diet - induced fatty liver disease and obesity could potentially transform treatments for non - alcoholic fatty liver diseases ( nafld ) . on the other hand , activation of gprc6a if so , gprc6a may be a biomarker for pca progression and antagonism of gprc6a could be a novel approach to treat castrate resistant prostate cancer , albeit with the potential side effect of causing mets . gprc6a is implicated in the pathophysiology of human diseases ranging from metabolic syndrome ( mets ) to prostate cancer ( pca ) and could provide a molecular understanding of the observed link between mets and pca . gprc6a is a potential therapeutic target in humans , but we remain a long way from developing drugs to activate or antagonize gprc6a . the therapeutic potential of gprc6a requires confirmation that gprc6a 's function in mice can be extrapolated to humans , finding ways to selective target this receptor in specific tissues , and establishing the efficacy and safety of drugs that activate or inhibit gprc6a in pre - clinical trials in animal models of human diseases .

backgroundgprc6a , a widely expressed g - protein coupled receptor , is proposed to be a master regulator of complex endocrine networks and metabolic processes . gprc6a is activated by multiple ligands , including osteocalcin ( ocn ) , testosterone ( t ) , basic amino acids , and various cations.scope of reviewwe review the controversy surrounding gprc6a functions . in mice , gprc6a is proposed to integrate metabolic functions through the coordinated secretion of hormones , including insulin , glp-1 , t , and il-6 , and direct effects of this receptor to control glucose and fat metabolism in the liver , skeletal muscle , and fat . loss - of - gprc6a results in metabolic syndrome ( mets ) , and activation of gprc6a stimulates proliferation of -cells , increases peripheral insulin sensitivity , and protects against high fat diet ( hfd ) induced metabolic abnormalities in most mouse models . bone , cardiovascular , immune , and skin functions of gprc6a have also been identified in mice . expression of gprc6a is increased in prostate cancer ( pca ) cells , and inhibition of gprc6a attenuates pca progression in mouse models . the function of gprc6a in humans , however , is not clear . during evolution , a unique polymorphism of gprc6a emerged mainly in humans of asian and european decent that has been proposed to alter membrane trafficking and function . in contrast , the ancestral allele found in all other species is retained in 1% , 15% , and 40% of people of asian , european and african descent , respectively , suggesting gprc6a gene variants may contribute to the racial disparities in the risk of developing mets and pca.major conclusionsif the regulatory functions of gprc6a identified in mice translate to humans , and polymorphisms in gprc6a are found to predict racial disparities in human diseases , gprc6a may be a new gene target to predict , prevent , and treat mets , pca , and other disorders impacted by gprc6a .

Introduction Knowledge gaps and controversies Molecular basis for multiple ligand activation of GPRC6A ( Diverse tissue functions directly regulated by GPRC6A GPRC6A coupling to multiple signal transduction pathways Novel endocrine networks created by GPRC6A and its ligands Clinical relevance of GPRC6A in humans Conclusions Conflict of interest

PMC4089103

hiv / aids is a chronic infectious disease causing profound immunosuppression in form of decrease in cd4 cell count . introduction of antiretroviral therapy ( art ) has improved the cd4 count and transformed the disease from a fatal to manageable disease . major problem with art in resource - limited settings is the emergence of drug - resistant viral strains due to poor drug adherence and the spread of these resistant viral strains in the population . a very high levels of drug adherence ( > 95% ) are required for increasing the long - term effectiveness of art and to prevent the emergence of resistant viral strains . the goal of national art program in india is also to attain > 95% individual drug adherence rate . but adherence to art is difficult , and suboptimal art regimens may result in rapid development of drug resistance . the definition of adherence has been also expanded that include retention in care , which includes attending regular follow - up visits . since the inception of national art program in 2004 , naco has reported 7% loss to follow - up cases and 3% patients who had missed treatment at some point of time . optimizing adherence and minimizing loss to follow - up are two major challenges for the art roll - out program in india . it is evident that adherence to treatment and follow- up are dependent on patients own will and health - seeking behavior which needs to be studied within their own sociocultural context . since a large number of patients are on art in india , whose disease will progress if adherence is suboptimal , research is urgently needed to determine patient - level barriers to these issues . to study the level of drug adherence in patients accessing art through the national program and factor influencing drug adherence . a hospital - based cross - sectional exploratory study was carried out in art center of sir sunder lal hospital , banaras hindu university , varanasi , india . the study was approved by the ethics committee of the institute of medical sciences bhu . hiv positive patients enrolled in the art center and started on art , were included in the study after informed consent . subjects were started on art as per naco guidelines , that is , cd4 count < 200/l or stage 4 disease . they were given 1 month therapy and followed - up every month for 6 months . patients were asked to bring back the remaining pills in the pill box at every visit which was counted by the counselor . at every visit , drug adherence was estimated by patients self - reporting , pill count , and by using visual analogue scale ( vas ) . statistical software spss for windows ( version 15.0 ) was used for data management and analysis . statistical software spss for windows ( version 15.0 ) was used for data management and analysis . during this period , 102 newly diagnosed hiv positive patients enrolled in the art center , and only 79 patients were started on art . a total of 95% cases were in the age group of 20 - 49 years . females ( mean age : 33.75 years ) were younger than males ( mean age : 34.09 ) [ table 1 ] . majority of patients ( 60.8% ) did not know any factor which increases the drug compliance . increase awareness ( 11.7% ) , come 2 day before for taking drug ( 13.7% ) , taking drug at fixed time ( 9.8% ) are important factor which increases the drug compliance . twenty - eight patients missed the dose in 1 follow - up , nine patients missed in 2 follow - up , eight patients missed in 3 follow - up . three patients lost follow - up in 2 follow - up , three patients further lost follow - up in 3 follow - up [ table 2 ] . running out of pills ( 40.0% ) , side effect ( 15.5% ) , and family problem ( 13.3% ) , poor transport facility for taking drug ( 8.9% ) , and forgetfulness ( 11.1% ) a total of 19% patients ( 15 ) in first follow - up did not know that missing the dose make treatment ineffective , but this % decreases with follow up ( 2 : 2.6% , 3 : 1.4% ) . patients lost the follow - up were not included in this analysis . in female patients , drug adherence ( 69% ) was initially less than male patients ( 76% ) but latter on female patients ( 96.3% ) had better adherence than males ( 95.2% ) . drug adherence was initially poor in joint family ( 71% vs. 82% ) but after follow up , it becomes better than nuclear family ( 96.3% vs. 93.3% ) . distribution of cases by age and sex number of missed dose in last 7 days causes of those patients who missed the dose ( open question ) nonadherence is associated with increased rate of hospitalization and longer hospital stays . in this study , concerns related to maintenance of optimal adherence to art among hiv - infected patients receiving free art . for hiv - infected patients treated with art , adherence to art is a significant determinant of survival . adherence is second only to the cd4 cell count as a predictor of progression to aids and death . for most of the hiv patients > 95% adherence is necessary to achieve complete and durable viral suppression . keeping these issues in mind , the present study include the level of drug adherence and the various factors affecting the drug adherence to art among hiv - infected patients receiving free art . after starting the art , a total of 28 patients ( 35.4% ) missed at least one dose of art in last 7 days during first follow - up . during second follow - up , 11.4% missed the dose and three patients ( 3.8% ) lost the follow - up . in third follow - up , 10.2% patient missed the dose and further three patients lost the follow - up [ table 2 ] . thus , we can say drug adherence increases with subsequent follow - up but 3 - 4 patients ( 3.8%-5.06% ) regularly lost follow - up due to some reason . opposite to our study , some study showed that rates of adherence are known to decline over time . this might be due to proper counseling of the patients . for assessing the cause of missed dose [ table 3 ] , patient missed the dose due to running out of pills ( 40.0% ) , side effect ( 15.5% ) , family problem ( 13.3% ) , and poor transport facility for taking drug ( 8.9% ) , forgetfulness ( 11.1% ) , economic problem ( 6.7% ) , and ignorance ( 4.4% ) . drug adherence by vas was > 95% in first follow - up ( 73.4% ) second follow - up 92.2% , third follow - up 93.2% . drug adherence < 70% was noted in four patients ( 5.1% ) in first follow - up which become two patients ( 2.6% ) in 2 follow - up . in third follow - up these findings are also similar to result of some other studies . along with that , in our study the adherence rate is better than some older studies . a total of 81.0% patients know that missing the dose leads to treatment ineffective in first follow - up , but this decreases with subsequent follow - up , ( 2 : 2.6% , 3 : 1.4% ) patients lost follow - up was not in clucked . in present study , female patients drug adherence > 95% were initially lower ( 69.0% ) than male patient but in subsequent follow - up female patients show better adherence than males ( 96.3% vs. 95.2% ) . drug adherence ( > 95% ) was initially better in nuclear family ( 82.6% ) than joint family ( 71.0% ) but latter on drug adherence ( > 95% ) become better in joint family ( 96.3% ) than nuclear family ( 93.3% ) . this improvement in adherence is most probably due to social support by family members after proper counseling . an another study has also showed that an educational intervention along with proper counseling improves adherence to antiretroviral regimens and health status . this study suggest that in resource - limiting countries , proper counseling and close monitoring of the patients can help in increasing drug adherence and decreasing loss of follow - up , which may have a great role in preventing the drug resistance and art response .

background : majority of hiv / aids patients who are on highly active anti retroviral therapy ( haart ) , are not aware about drug adherence and its importance which is the most important factor for drug adherence.objectives:to study the level of drug adherence in patients accessing antiretroviral therapy ( art ) through the national program and factor influencing drug adherence.materials and methods : in present study , we enrolled 102 newly diagnosed patients , among them in 79 patients , art was started . to study the drug adherence a pretested , semistructured questionnaire was formed and patients were followed up for 6 months of the study . pretest and posttest counseling was done to all such patients.results:a total of 28 patients missed the dose in 1st follow - up , nine patients missed in 2nd follow - up , eight patients missed in 3rd follow - up . three patients lost follow - up in 2nd follow - up , three patients further lost follow - up in 3rd follow - up . running out of pills ( 40.0% ) , side effect ( 15.5% ) , and family problem ( 13.3% ) , poor transport facility for taking drug ( 8.9% ) and forgetfulness ( 11.1% ) are five major causes related to miss dose . in females patients , drug adherence ( 69% ) was initially less than male patients ( 76% ) but latter on female patients ( 96.3% ) had better adherence than males ( 95.2%).conclusion : this study suggest that drug adherence can be increased by proper counseling and close monitoring of the patients which may have a great role in preventing the drug resistance and art response .

INTRODUCTION MATERIALS AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC3113492

some synchrotron - specific skills are difficult to learn without intensive hands - on training , but not all research groups can provide comprehensive training for students and postdoctoral staff . the rapidata course is designed to provide this training , thereby increasing the capabilities of research groups who visit the x - ray beamlines at the national synchrotron light source to perform diffraction measurements . supported largely by a grant from the national institutes of health ( nih ) national center for research resources , macromolecular crystallography resource at the nsls , funded also by the department of energy ( doe ) , office of biological and environmental research . in this course we develop the ideas behind conventional methods for diffraction - data collection , we describe several different structure - solving methods , and we give students practical experience working on their own specimens . rapidata was intended to complement the long - running annual cold spring harbor laboratory ( cshl ) course in macromolecular crystallography ( mx ) created by jim pflugrath and others , which is given each fall . the cshl course treats all of mx , from growing crystals to preparing drawings of the refined structures . we hold rapidata in the spring , and we focus on the synchrotron - based component of modern mx : measuring the best possible diffraction data and then obtaining phase information to create an interpretable electron - density map . we first presented the course in april 1999 , and have given it annually since then . by now there are about 600 alumni . at first , our admission policies aimed to promote a high initial level of readiness , and we required that our students demonstrate a substantial background in the theory and practice of crystallography . however , this was at a time when the paradigm was shifting towards users with an increasingly modest crystallography background because many new crystallography laboratories were forming where the thrust of the research , and the background of the investigator , were very biological . they had relatively meager mx background . to help fill the gap in background we added a day - long lecture course on the fundamentals of crystallography to sharpen the skills of the students , preparing them for a rigorous two days of lectures on data collection and structure solving , then two more days of tutorials and data collection . we also softened our admission policies for theoretical background , and we began to favor applicants from laboratories that demonstrated an emerging project - driven need for increased experience in the theory and practice of mx . in 2002 , the us national committee for crystallography helped us launch a latin american initiative , complementing that of the american crystallographic association . they gave us seed money for a few travel scholarships to students in latin american crystallography laboratories . by 2005 , we were able to win a solid grant from the international union of crystallography for the same purpose , and we have been able to renew that each year since . to win the scholarship the applicants must be working in mx , must be under 40-years old , and must both demonstrate the need for the course and promise that they have sufficient background to profit from it . we have funded about half a dozen students each year ; most are graduate students , some hold postdoctoral positions and a few are junior faculty . the main rapidata course begins on a monday morning , in april , following an optional five - hour sunday lecture course on the fundamentals of crystallography . there are lecture courses ( and fine evening meals ) on monday and tuesday . then starting tuesday evening data collection begins , running until breakfast time on friday . at the synchrotron we are on beamline rations : bagels and fruit for breakfast , sandwiches for lunch , pizza or heroes for supper , and lots of coffee and snacks all other times . tutorials , run by the lecturers and often a helper , run essentially continuously ( day and night ) through the two full days of data collection . entry to the course is by application , open from mid - december to early february . each student submits appropriate personal data , and then a statement of what his / her preparation for the course is in terms of courses and laboratory experience , and what he / she hopes to gain from it . in addition , each student s supervisor / mentor must submit a supporting letter , giving his / her view of the student s preparation and capabilities to be gained , and how this student s attending the course would help the project leader s research program . all students are encouraged to bring their own specimens for data collection , and to bring stubborn data that can be tamed during the data - reduction and structure - solving tutorials . every section comprises some students with crystals and some without . students who want to bring a project must describe it , warts and all . typically one - third will be each of these : us citizens ; those who come from other countries , essentially the rest of the world ; and those of non - us origin who are living and working in the us . we also have a few junior faculty who are just starting out and want to sharpen their skills before they run a group , some are senior scientists wanting to find out what it is all about , and the rare , treasured candidate is the tenured nmr investigator who is ready to turn to the dark side ( really the side of enlightenment ) . we have been able each year to allocate about six nsls dipole beamlines to the 12 sections . each section will get a total of about 30 h on a beamline . in recent years we have made our two high - brightness undulator beamlines available for especially demanding projects . these beamlines , which run at nearly the pace of some of the hottest beamlines at third - generation synchrotrons , give students a taste of the feeling of drowning in data that has become typical these days . on the other hand , ordinary dipole beamline has at least two tutors to help students puzzle out whether their crystals are good enough for data collection , how best to get the data , and then how to reduce and scale the data . those without crystals will have a period of several hours to practice on sample specimens they will have mounted themselves . in addition to conventional cryo - mounting , students may consider employing automounting and use of an optical spectroscopy system for colored crystals . anywhere between five and 12 structures are typically solved during the course , from the crystals that roughly half of the students bring . each of these could be a publishable result , and typically one or two publications each year attribute rapidata as having been the source of their data . on wednesday and thursday afternoons during the data - collection portion of the course , and then on the final friday morning , the entire course , students and instructors , gather for about a hour to discuss their results and to talk about things learned . stimulated by h. klei , one of our regular helpers , and of course made possible by the remarkable advances in crystallographic structure - solving and display software , the reports of results often are mini - lectures , with an introduction to the subject and pictures to show the result and the quality of the data . the goal for the lectures on monday and tuesday has been to teach a few fundamental concepts that students might not encounter in other places , then to teach the nuts and bolts of data reduction and structure solving . here are typical topics , with the names of those who commonly present the lecture in square brackets . ( a ) some issues in data collection [ r. sweet ] : how does the rotation method work ? what is collimation ? ( b ) modern ccd - based and pixel - array x - ray detectors ( phillips et al . , 2002 ) [ c. nielsen , w. phillips ] : what is inside the box and how does it work ? what are the limitations ? what can we expect for the future ? ( c ) tactics in data collection , and x - ray damage to crystals ( dauter , 1999 ) [ z. dauter , e. garman ] : how far do i have to rotate ? ( d ) special properties of synchrotron radiation [ l. berman ] : where do x - rays come from ? what is an undulator ? what does the future hold ? ( e ) specimen preparation ( garman & schneider , 1997 ) [ e. garman , d. thiel , s. parkin ] : what are all the tricks in cryopreservation ? ( f ) data reduction with d*trek ( molecular structure corporation , 1997 ) [ j. pflugrath ] . ( g ) data reduction with the mosflm ( leslie , 1992 ) package [ f. von delft , m. szebenyi ] . ( h ) data reduction with the hkl ( otwinowski & minor , 1997 ) suite [ w. minor , z. otwinowski ] . multiple- and single - wavelength anomalous diffraction [ h. robinson ] : how does one tune the data collection to get the best phases ? ( j ) structure solving with shelx ( sheldrick , 2008 ) [ b. schierbeek , m. benning ] . ( k ) structure solving with solve ( terwilliger , 2004 ) and phenix ( adams et al . , 2010 ) [ t. terwilliger ] . ( l ) structure solving with autosharp ( vonrhein et al . , 2007 ) ( m ) structure solving with molecular replacement methods [ p. fitzgerald , p. adams ] . ( n ) automatic control of data collection with the edna ( incardona et al . , 2009 ) system [ a. soares , g. winter ] . ( o ) ( p ) operation of beamline software : cbass , pxdb ( skinner et al . we provide over 20 computer seats around the floor of the nsls , most in quiet but sometimes crowded rooms . all computers are networked to the central disk farm , which also carries all of the data measured during the course . for each tutorial room there is some provision for a large display that a group of up to ten might be able to follow . most of the tutors will provide a sign - up sheet so that students can reserve a space in advance . typically the tutorials are packed tuesday night , then from morning to well into the night on wednesday and thursday . the cryopreparation lecturer will hold court with microscopes , dewars , safety glasses , and assorted clamps , loops and other gizmos . students may practice mounting either their own crystals or crystals that our staff have prepared . in recent years we have dedicated a nearby dipole beamline to this tutorial , so that each student can evaluate their specimen preparation and cryo - search efforts using before - and - after diffraction patterns . there is also an automounter at that station for those who want to give it a try . there have been ad - hoc tutorials in the use of coot ( emsley et al . , 2004 ) [ a. hroux ] and writing fortran jiffy programs [ m. rould ] . it was a surprise to us during the 1999 course that a structure was actually solved during the course itself . although we now try not to emphasize any race to get the first structure , there is an informal competition between the several software packages each for data reduction and structure solving to have a role in each structure solved . students , to their credit , will sometimes take their data from tutor to tutor to learn how best to use each tool to do their work . the pxrr beamlines x8-c , x12-b , x12-c , x25 , x26-c and x29 have been operated by a. soares , a. hroux , d. schneider , a. saxena , h. robinson , a. orville , m. becker , h. lewis and d. stoner - ma . the new york structural biology center beamlines at x4-a and x4-c have been operated by j. schwanof , r. abramowitz and c. ogata . the case western reserve beamlines at x9/x3 have been operated by b. ramagopal , w. shi and k. rajashankar . the course would simply not exist without the administrators who have put each course together : s. sclafani , d. kranz robertson , a. baittinger and a. emrick . others who have helped in core lecturing or operations but are not named above have been h. klei , j. birktoft , l. hung , r. grosse - kunstleve , l. flaks , b. mcgrath , k. hlzer , k. schroer , w. nolan , p. dunten , j. berendzen , c. lukacs , t. radhakannan , j. jiang , m. allaire , r. jayaraman , q. liu , y. chen , m. coincon , m. echols , r. edayathumangalam , m. garcia - diaz , p. jeffrey , e. enemark , g. leonard , m. mcmillan , r. page , n. silvaggi , n. nassar , r. jayaraman and q. liu . local workers , whose enthusiastic help with setup , operation , emergency problem solving and final clean - up have been critical , are m. cowan , c. dropp , r. jackimowicz , t. langdon , j. lara , s. myers , g. shea - mccarthy , n. whalen , r. buono , m. carlucci - dayton , s. vadai and r. mcnally . many students arrive with little or no experience in crystallography , but they leave knowing how to obtain and process real data , as well as how to locate and fix problems as they arise . that is the advantage of having such a hands - on program . it is crucial to have the lecture section of the course operate like clockwork : every lecture starts on time , so everyone knows what to expect and no - one is ever late for a meal . on the other hand , the 60 h of data collection and tutorials are flexible , with each student having a chance to try many things . being able to dedicate so many beamlines to the effort gives every student a chance to try whatever comes to mind . having so many world - class scientists as lecturers and tutors provides a critical mass of high - level scientific thinking that really defines the course , and the software designers / lecturers / tutors take rapidata as the opportunity to try out new things and to test them on new data . we do provide many teachers , tutors and helpers there are over 35 on the roster for each course . a feature of rapidata that we adopted from the cold spring harbor course , which we believe contributes a lot to the success of both courses , is that the students and teachers all have meals together . the value of this small feature , in stimulating interactions , can not be overestimated . we see them at meetings all over the world , and we see their names on papers and mx bulletin boards . many rapidata alumni have now blossomed into crystallography experts , and they continue to refer their less - experienced colleagues to bnl to polish their skills . finally , one of the great sources of satisfaction for us is that at the end of the course the teachers come to ask us if they may come again next year . the course is funded in part by students fees , which until now have paid only for their room , board and supplies , and by grants from nih s national center for research resources and the doe s office of biological and environmental research . in addition to the fees and the doe and nih funding , a special grant was provided by the international union of crystallography to assist latin american students in attending the course . la roche and area detector systems corporation , and important and enduring in - kind support from hampton research corporation , rigaku americas corporation , bruker axs , bristol - myers squibb , global phasing and mitegen . finally , the employers of all the visiting teachers are donating a week of these people s time , and we warmly acknowledge that gift . for more information about rapidata , go to http://www.bnl.gov/rapidata . for more information on the pxrr , go to http://www.px.nsls.bnl.gov/.

rapidata provides two days of high - level lectures , then two more of experimental work on several beamlines of the national synchrotron light source , for about 50 students . this article provides details about the organization of the course and tells some of the reasons for its success .

Objectives of the course and some history The structure of the course The lectures The tutorials The relationship between the data collection and the tutorials The beamlines, their operators and other helpers Our view of the impact of RapiData Funding

PMC1142203

this lecture was directed at those participants who were new in the field of veterinary ap and also to refresh the knowledge of other participants before the more specific lectures . he concluded that we needed to explain the scientific basis of ap for the successful treatment of allergies and other immune disorders . he stressed the emerging immune - related diseases in animals as well as humans , and the possibility of countering them with a judicious approach including alternative therapies . she stressed the need for a tailor - made exercise regime to supplement any treatment intervention in geriatric patients for better results . dr silim recapitulated the roles of interleukins , t - helper cells , cytokines and other key compounds involved in immunology . richard tan ( usa : balance method applied to immune mediated diseases ) discussed his complex balancing methods , used to diagnose and treat external and internal syndromes . he also explored the relationships of the channels to the hexagrams of the i - ching . jean guilherme joaquim ( brazil : a comparison between ap surgery and surgery + ap treatment in dogs with thoracolumbar disk disease an immunological approach ) . stelio luna gave this paper on behalf of his colleague , who could not attend . he analyzed a comparison of the results obtained after ap , surgical or surgical + ap treatment . his research team found that surgery + electro - ap at st36 , gb34 , bl18 , bl23 , ki03 and bl60 was efficient to treat the western pattern of neurological diseases associated with thoracolumbar disk disease in dogs . however , dr luna mentioned pre - trial administration of corticoids by veterinarians before referral to the university hospital and lack of a significant number of cases as limitations of the reliability of the conclusions . he also spoke from his experience about the limitations of conducting trials in alternative medicine in institutions of poorer countries . jen - hsou lin ( taipei : determination of the safe depth of back acupoints in cats ) . his team discussed their research in relation to safe needling depths of back acupoints in cats . using magnetic resonance imaging ( mri ) , the safe depths of some important acupoints on the bladder and governing vessel channel was determined in three groups of cats with different body index ( thin , medium and fat ) . the safety depths of ap differed significantly in the three different groups , as did the difference of depths in different acupoints in the same group . the maximum safe needling depth of ap in the back area was derived by multiple regression analysis . the derived empirical formula was : y = b0 + b1x1 + b2x2 + b3x3 , where y = safe depth ( in mm ) , x1 = weight ( kg ) , x2 = age ( years ) , x3 = body length ( cm ) and b0b3 were the computed variables . variation of safety depths in different acupoints and cats with different body index was noted . it was concluded that as a precaution , needling depths beyond 1 cm should be treated with extra caution until the expertise is developed in needling techniques . shigeo hara ( japan : optimization of frequency and voltage in electro - ap stimulation of dogs ) concluded in his study that an optimum condition for electro - ap was found by gradually increasing the stimulation voltage while keeping the stimulation frequency at a low level or by gradually increasing the stimulation frequency while keeping the voltage stimulation at a low level . bruce ferguson ( usa : the formation and circulation of wei qi in tcm ) discussed the role of wei qi in the immune system and the process of its formation and circulation . he states that according to traditional chinese medicine ( tcm ) context , t - lymphocytes comes from the liver and cellular immunity is derived from the lower burner ( kidney and liver ) . citing the example of a castle surrounded and defended by water , he stressed the need for maximum tonification of the kidney for a correct immune system . however , he did emphasize the right approach of enhancing both the superficial and deep aspect of francesco longo ( italy : ap therapy in reproductive problems in mares ) gave interesting case reports of successful treatment of > 300 mares with endometrial inflammations , ovaric syndrome , prolonged winter anestrus , prolonged spring anestrus and other endocrine disruption syndromes with ap . marco testa ( italy : clinical use of extraordinary channels in horses ) discussed the clinical use of qi mai ( extraordinary channels ) in horses . the speaker concluded that by means of these key points , it was possible to treat cases of pathological courses not only by rebalancing physiological , psychological and behavioral functions , but also by converging both acquired and cosmic , primordial energies in one process . uwe peterman ( germany : ap in acute and chronic immunodeficiency ) began by saying that immunodeficiency is caused by an exhaustion of kidney energy . his presentation of several case studies demonstrating immunodeficiency caused by the pertuberative field principle was very informative . in conclusion , the speaker stressed the identification and removal of factors that disturb the pathological condition and cause a never - ending circle of chronic disease by successful intervention with ap treatment . jeffrey yuen ( usa : divergent channels and their application to immune - mediated diseases ) discussed the qualities , pathways , implications , functions and clinical application of the divergent channels , with emphasis on immunomodulation . kwang - sup soh ( seoul : kwan : characteristic features of intravascular bongham duct ) described the features of the bonghan duct ( bd ) system , named after the original discoverer , bonghan kim ( korea ) . dr kim claimed the discovery of the anatomical basis of the acupoints and channels in the early 1960s ; he reported the classical channels as part of the new bd circulatory system distributed throughout the body including the surface of internal organs even inside blood and lymph vessels . dr soh showed video clips of their dissection techniques for the special surgery of live animal models . the presence of large quantities of mucopolysaccharides in the bd and the recent finding of some researchers such as h. l. langevin and j. a. yandow ( 2002 ) ( relationship of acupoints and channels to connective tissue planes ) and their role in the effect of ap were discussed . it was unclear if the bds at the surface of the body where we trace channels may have lost their conformity to integrate in the tissue and yet have kept their characteristic features including toti - potent stem cell flow , one of the physiological functions of the bd . marcia scognamillo - szabo ( brazil : effect of ap on the hypersensitivity response induced by rhipicephalus sanguineus tick antigens in dogs and guinea pigs ) concluded that ap at st36 , si01 , li11 , yin tang and er jian acupoints reduced experimental tick antigen - induced hypersensitivity reaction in dogs . ying - ling wu [ taipei : functional magnetic resonance imaging ( fmri)investigation of cardiac function in cats ] . the representing speaker introduced the group 's work on the effect of ap at neiguan ( pc06 ) on cardiac functions of cats monitored by employing cardiac mri techniques . it was reported that electro - ap treatment at pc06 significantly reduced blood pressure and shortened the duration of recovery from administered anesthetics . the established cardiac functional parameters were also found to be improved in the electro - ap treatment groups of cats compared with the controls . are simeon thoresen ( norway : recent work on cancer using the ko - cycle ) discussed his work on ap in cancer treatment . taichong - lv03to treat mammary cancer , and to report back to him in 1 year . this brings some hope when some us scientists have ruled out the use of tamoxifen in breast cancer treatment because of its notorious side effects . dr thoresen said that the primary aim and most important function of holistic medicine is to stimulate the body 's own control mechanism , via the classical ko cycle in tcm : fire metal wood earth water fire . one must bring control to unregulated growth and development of cells that can induce cancerous cells , and , at the same time , avoid stimulating the wrong processes that may stimulate tumor growth . in using the classical ko cycle for example , for cancer that starts in the stomach ( earth ) , or along the course of the st channel ( such as the mammary gland or the lateral canthus of the mouth ) , the controlling channel is lv ( wood ) . any lv point will help , but he found lv03 to be best . for cancer that starts in the lung or large intestine , or along the course of the lu or li channels ( metal ) , the controlling channel is ht ( fire ) . any ht point will help . in treating multiple skin cancers , or sarcoids , identify the exact channel(s ) on ( or near ) which the first lesion appeared . for example , if a melanoma or sarcoid appears first at the medial canthus of the eye ( bl channel ; water ) , any point on the sp channel ( earth ; ko controller of water ) will help . he also said that there is a second ko cycle , in the form of a cross , whereby the n , s , e and w phases are fire , water , metal and wood , respectively , with earth in the centre . in that ( cross ) cycle , a point on th channel is best to bring control in prostate or cervical cancer ( water ) . the key point touches the lateral edge of metacarpal 4 ( ring finger ) , 25 mm distal to th02yemen mary battistella ( usa : human and small animal scalp acupoint comparison ) briefly discussed the different human scalp zone systems of ap and their possible inclusion in small animal medicine . mariana c. galdino [ brazil : effect of continuing stimulation of acupoint cv5 ( shimen ) in domestic female cat fertility ] reported that prolonged ap stimulation of cv05 is a potential method of achieving contraception in female cats ( queens ) . elena petrali ( canada : treatment of lumbar spine in canine using a combination of balance method , extra points and motor points ) discussed ap treatment of motor points of muscles related to the vertebral segment in a 5-year - old bitch with suspected mild spondylosis . in conjunction with ap , she advised incorporation of massage or manipulative techniques , plus acupressure , and stretching and strengthening exercise at home . james skoien ( switzerland : tcm view of immunology ) discussed in great detail the immune system from a tcm viewpoint . he stressed the importance of the fundamental substances ( qi - xue , ying - wei , jin - ye and jing ) . his vast knowledge of chinese herbal medicine and how to select herbs to treat specific tcm syndromes related to various immune - related disorders and cancer therapy was the highlight of the lecture . sagiv ben - yakir [ israel : bio - zoo ap ( b - zap ) ] discussed the b - zap concept and explained the veterinary applications of hirudotherapy and apitherapy . the basics of such treatment , precaution and his clinical case references were an interesting topic that generated many queries and questions from the audience . he revealed interesting observations on leeches attaching to an acupoint during hirudotherapy , adding ap therapy to the blood sucking . he also mentioned the properties of bee venom including melittin with a potent anti - inflammatory and analgesic property . he demonstrated the technique of apitherapy and shared his experience of treating stubborn cases of traumatic peripheral neuropathies , degenerative myelopathy and invertebral disk disease with the same . annica nygren thoresen ( norway : report from our work to re - establish where ap channels travel and where the acupoints are located in the horse ) are thoresen on behalf of his wife ( who could not attend for medical reasons ) discussed their methods to detect the acupoints and channels of special relevance in treating qi xu ( energetic deficiency ) in horses . bruce ferguson ( usa : the circulation and exteriorization of wei qi in immune disorders and cancer in tcm ) concluded that cancer cells are in a relative state of hyperactive cellular replication which is yang and correlated with heat . if the heat of the cancer process attacks the blood , there are many methods to cool it . thus approach to treatment of cancers should first focus to stimulate the zone of transit of wei qi by stimulating various acupoints for different parts such as gv20 for the head , cv17 for the thorax , back for shu points , ki16 , st25 for the abdomen , and st30 , bl57 for lower jiao . there must also be exteriorizing of wei , an increase of the secondary circulation and formation of wei . to control heat , the ying should be circulated , increase the activity of the zong qi , cool the blood , calm the mental state , stimulate the mental activity of the specific organ in which there is heat and use xi - disintegration points to dissolve blood obstruction and stagnation , particularly sp08 for abdominal masses . his results showed a significant decrease in somatic cell count ( scc ) and neutrophils , and an increase in number of macrophages in the treatment group over the control groups . c. p. ferreira ( brazil : effect of micro - doses of pgf2 to induce luteolysis in nelore cows ) concluded that leutolysis is achieved in nelore cows ( bos taurus indicus ) even at 50% of the recommended doses of luprisotol for dairy cattle , regardless of the route of administration [ intramuscular or intravulvosubmucosal ( i.v.s.m . ) ] . however , the i.v.s.m . route as reported by previous researchers does seem to be more effective then systemic administration of leutolytic agents . the administration of lower doses of leutolytic agent at the bai hui acupoint had no or a negligible leutolytic effect in nelore cows . anna hielm - bjorkman ( finland : the veterinary ap clinic at helsinki university animal hospital : what can we look back on after 6 years ? ) dr hielm discussed her experiences of running a veterinary ap clinic at helsinki university . she discussed the dos and don'ts necessary to establish the practice of alternative medicine in university hospitals . most academic veterinarians are skeptical or negative towards alternative medicine , and careful research and clinical work is necessary to gain their trust . roman skarda [ usa : the effects of ap and electro - ap ( 2 hz , 100 hz , and 2 and 100 hz alternating frequencies ) on cutaneous and rectal analgesia and immunoreactivity of methionine enkephalin and dynorphin in venous plasma of horses a comparative study ] discussed ap , electro - ap ( at low and high alternating frequencies ) on cutaneous and rectal pain threshold and their relationship to powerful endogenous opioids , methionine enkephalin ( mk ) and dynorphin ( dn ) . he concluded that mk and dn play an important role in production of cutaneous and rectal pain relief in horses treated with electro - ap at bilateral bl18 , 23 , 25 and 28 . stelio luna ( brazil : immunomediated thrombocytopenia treated with ap and chinese herbs ) concluded that immunomediated thrombocytopenia disease in dogs can be successfully treated with acupuncture and chinese herbs . krishna kaphle ( nepal / taiwan : trophin - like action of some selected herbal extracts ) discussed research on herbal extracts for potential trophin ( gonadotrophin / adrenocorticotrophin)-like action . the speaker mentioned the need for more scientific research in medicinal herbs , and explained the constraints and limitations in conducting scientific research with herbal formulae and extracts in living tissues . the speaker concluded that herbs with potent steroidogenic activity act differently from phytosterols . the possible stimulating and inhibiting actions of various herb extracts involve action in different key enzymes and intracellular signaling mechanisms . steroidogenic acute regulatory ( star ) protein and the intracellular local immune system of the steroidogenic cells are the main targets for active compounds of various herbs having stimulatory action . dirk verhoeven ( belgium ) noted an editing error in the table of herbs in the preceding lecture . his questions on the role of such herbs in gonadless and neutered animals were thought - provoking . he had satisfactory results using epimedium macranthum to treat lazy and depressed animals ( deficiency in tcm terms ) . linda boggie , ivas president , stressed the need for scientific research in the different fields of alternative medicine . she promised the full support of ivas in the quest for scientific validation of ap . this is essential if alternative therapy is to gain wider acceptance and establish itself as an indispensable part of treatment in humans and animals . she promised to work even harder to overcome the hurdles ahead and extended an invitation to all to participate in the 31st annual congress of ivas ( 2005 ) , scheduled for park city , ut , usa .

more than 155 participants from 25 countries attended the 30th annual ivas congress , september 811 , 2004 in oostende , belgium . the focus was on veterinary acupuncture ( ap ) and immunology , and the event was sponsored by the international veterinary acupuncture society ( ivas ) . ivas is a non - profit organization dedicated to promoting excellence in the practice of veterinary ap as an integral part of the total veterinary health care delivery system . the society endeavors to establish uniformly high standards of veterinary ap through its educational programs and accreditation examination . ivas seeks to integrate veterinary ap and the practice of western veterinary science , while also noting that the science of veterinary ap does not overlook allied health systems , such as homeopathy , herbology , nutrition , chiropractic , kinesiology , etc . ( ) .

DAY 1 DAY 2 DAY 3 DAY 4

PMC4297882

migraine is a central nervous system disease traditionally characterized by acute painful attacks ( goadsby et al . , 2002 ) . this definition , however , does not capture the effects of migraine that extend beyond episodic attack ( ictal ) periods ( goadsby et al . , 2002 ; stewart et al , 1992 ) , including lasting alterations in brain structure and function ( lakhan et al . , 2013 ; noseda & burstein , 2013 ; sprenger & borsook , 2012 ) that closely mirror the effects of chronic pain conditions . more broadly , migraine also appears to share physiological ( boyer et al . , 2014 ) and clinical profiles ( blumenfeld et al . , 2004a ; mccracken & iverson , 2001 ) , migraine has been linked to impaired cognitive function . pain demands and depletes cognitive resources , which is thought to contribute to cognitive impairments in chronic pain broadly defined ( eccleston & crombez , 1999 ; nes et al . , 2009 ) . the behavioral effect of migraine on cognitive function , however , has been debated within the literature , largely due to mixed results ( calandre et al . , 2002 ; pearson et al . , 2006 ; suhr & seng , 2012 ; waldie et al . , 2002 ; zeitlin & oddy , 1984 ) . a recent review noted that significant effects of migraine on cognitive function depended largely on study recruitment , such that patients recruited from neurology clinics show mild impairment across several cognitive domains , whereas migraine patients recruited from the community were less likely to show cognitive differences from healthy controls ( de arajo et al . , 2012 ) . therefore , cognitive impairment in migraine may be dependent on disease severity ( e.g. disease duration , migraine frequency , migraine pain intensity ) . pain cognition interactions in neural response have been demonstrated among young healthy populations ( petrovic et al . , 2000 ; seminowicz & davis , 2007a ) , such that cognitive engagement typically reduces pain - related activity . cognitive - related activity , on the other hand , does not tend to be altered by simultaneous pain stimuli among healthy volunteers ( seminowicz & davis , 2007a ) . however , whether these patterns are preserved among people with migraine is not known . chronic pain appears to alter the functional organization of brain networks involved in pain and cognitive processes ( farmer et al . , 2012 ) . initial evidence suggesting that cognitive modulation of pain processing may be altered or weakened in migraine patients indicated that , compared to healthy controls , patients with chronic and episodic migraine show deficient cognitive task - related suppression of evoked potential amplitude during acute pain stimulation ( de tommaso et al . , 2003 ) recently , the association between migraine and brain white matter lesion load ( kurth & tzourio , 2009 ; swartz & kern , 2004 ) has supported the hypothesis that prolonged suffering with migraine may lead to cognitive decline ( paemeleire , 2009 ) . however , a longitudinal study did not find such a relationship ( rist & kurth , 2013 ) . still , others have found correlations between structural differences between migraine patients and controls , and impairments on some cognitive tasks ( schmitz et al . , 2008 ) lending support to the hypothesis that cognitive decline in migraine may be due to structural brain changes over time . while little is known about functional neural response to cognitive challenge among migraine patients , hypotheses can be drawn from evidence of abnormal brain activity in response to cognitive tasks within other chronic pain populations including chronic low back pain ( seminowicz et al . , 2011 ) and temporomandibular disorder ( weissman - fogel et al . , additionally , studies have reported abnormal functional resting state connectivity in migraine patients between brain regions associated with affective pain processing and cognitive networks ( schwedt et al . , 2013 ; one recent study demonstrated that migraine patients also display greater evoked pain - related activity in areas thought to be associated with cognitive aspects of pain processing , including the contralateral premotor cortex ( pm ) , right dorsolateral prefrontal cortex ( dlpfc ) , and ipsilateral hippocampus ( schwedt et al . , 2014 ) . taken together , these findings suggest that changes in functional cognitive brain activity may be due to integration of pain and cognitive networks and pain cognition interactions . in the current study we examine neural response to cognitive challenge , and pain cognition interactions during concurrent pain stimulation , among migraine patients and healthy volunteers . we hypothesized that there would be group differences in task - related neural response , where migraine patients would have to recruit greater brain resources to perform the cognitive task equally well as healthy controls , and that task - related suppression of acute pain - related activity ( pain cognition interactions ) would also be altered among migraine patients . fourteen migraine patients ( 11 females , mean 40.8 years old ( sd = 11.9 ) ) and 14 healthy controls ( 11 females , mean 38.9 years old ( sd = 12.5 ) ) participated in this study and were compensated for their time . migraine patients were recruited from the johns hopkins university ( jhu ) campuses and local headache clinics , and through community advertisements . migraine patients also participated in a subsequent longitudinal study , but the procedures described here were all completed at baseline , before any intervention . evaluation of prospective 3-month headache diaries revealed that ten patients met diagnosis criteria for chronic migraine ( range : 1428 headache days per 28 days ) and four for episodic migraine ( range : 7.813.7 headache days per 28 days ) . diagnosis was determined by the study physician ( m.g . ) using the international classification of headache disorders - ii ( ichd - ii ) criteria ( headache classification subcommittee of the international headache society , 2004 ; olesen & steiner , 2004 ) . we did not exclude any patients in our primary analysis for a number of reasons . first , all patients were recruited based on reported chronic migraine . additionally , within our migraine group we examined migraine frequency as a continuous variable . though separable diagnostic criteria , chronic migraine and episodic migraine have a common underlying pathophysiology ( pietrobon & moskowitz , 2013 ) . clinical and behavioral differences between chronic and episodic migraine patients have been demonstrated in factors that vary linearly with increasing frequency , and the demarcation created for diagnostic purposes is somewhat arbitrary ( borsook et al . , 2012 ; katsarava et al . , 2012 ; silberstein et al . , furthermore , others have examined chronic and episodic migraine patients together , which has led to a number of recent advances in the understanding of migraine ( e.g. anttila et al . , 2013 ; hamedani et al . , 2013 ; hunter et al . , 2014 ) . nonetheless , to ensure that our findings were not dependent on the episodic migraine patients , we also report the main results excluding the four episodic migraine patients ( supplementary results ) . patients had migraine for an average of 10.9 years ( se = 2.0 , range : 127 years ) and rated their average migraine pain intensity during the last month as a 5.4 on a 0 ( no pain ) to 10 ( worst pain imaginable ) scale ( se = 0.6 , range : 210 ) . patients had an average of 20.1 headache days per 28 day month according to prospective headache diaries ( se = 2.0 , range : 7.828 days ) and retrospectively reported an average migraine frequency of 9.6 in the previous month ( se = 2.1 , range : 230 migraines ) . exclusion criteria included an unstable psychiatric disorder , pregnancy , illicit drug use and severe alcoholism . this study was approved by the umb and jhu institutional review boards , and informed written consent was obtained from each participant prior to any study procedures . the cognitive task and thermal stimuli were administered simultaneously according to a full 2 ( task difficulty ) 3 ( thermal stimulus intensity ) factorial design , such that while participants completed either the difficult or easy cognitive task , they experienced a concurrent moderately painful , mildly painful , or non - painful thermal stimulus [ i.e. , ( difficult / moderate pain ( t1p2 ) , difficult / mild pain ( t1p1 ) , difficult / no pain ( t1p0 ) , easy / moderate pain ( t0p2 ) , easy / mild pain ( t0p1 ) , easy / no pain ( t0p0 ) ] . participants performed a modified attentional network test ( ant ) ( fan et al . , 2002 ) , which involved identifying the direction of a central arrow , while ignoring the direction of flanking arrows . difficult tasks involved distracting , incongruent flankers , easy tasks involved congruent flankers . during both the pre - scan practice session and the mri scan sessions , participants indicated the direction of the arrow using a simple button press with their right hand . an fmri - compatible thermal stimulator delivered thermal stimuli via a contact probe ( 30 30 mm medoc pathway ats peltier device ; medoc advanced medical systems ltd . , ramat yishai , israel ) to the left volar surface of participants ' forearms . the peak temperature ( moderate pain , p2 ) used throughout the study was determined at the beginning of the pre - scan session using a simple ramp - and - hold procedure as the temperature at which participant rated a 56 on a 0 ( no pain ) to10 ( most intense pain imaginable ) pain intensity scale . the mild pain ( p1 ) stimulus stimuli were presented for a variable duration of 812 s with a ramp time of 1.6 s and variable ramp rate based on peak stimulus temperature . stimuli were separated by intervals ( 48 s ) of baseline temperature ( 32 c ) . the same temperature was used for each participant throughout the pre - scan and mri scan sessions with the exception of one patient who required the peak stimulus temperature to be lowered 1 c for the second functional run . prior to scanning , then , the individualized peak thermal stimulus ( p2 ) was determined , and participants were trained to a point where they performed with > 90% accuracy on the cognitive task . participants then performed the full experimental paradigm , with concurrent task and thermal stimulation , identical to that experienced in the mri session . pain intensity and unpleasantness ratings were obtained on a 010 numerical rating scale for each trial , and reaction time and accuracy were recorded . participants also completed retrospective reports of migraine frequency and migraine pain intensity and the pain catastrophizing scale ( pcs ) ( sullivan et al . pain catastrophizing is a cognitive coping strategy associated with increased rumination , magnification , and helplessness toward pain . we included this measure because there is some evidence that migraine patients catastrophize more than healthy controls ( hassinger et al . , 1999 ) , and pain catastrophizing increases preferential attention to pain ( sullivan et al . , 1995 ) . high catastrophizers , therefore , may be less able to redirect their attention away from their pain , which may inhibit task - related suppression of pain . the scan session included two functional runs ( run 1 : 9 min 20 s , run 2 : and 9 min 32.5 s ) containing 27 trials each . there were six trial types consisting of all possible combinations of task difficulty and thermal stimulus intensity : difficult / moderate pain ( t1p2 ) , difficult / mild pain ( t1p1 ) , difficult / no pain ( t1p0 ) , easy / moderate pain ( t0p2 ) , easy / mild pain ( t0p1 ) , and easy / no pain ( t0p0 ) . participants provided a real - time response to the cognitive task during each trial using a fiber optic mri - compatible button box in the scanner . presentation of visual stimuli for the cognitive task was delivered via a desktop computer running e - prime 2.0 ( psychology software tools , http://www.pstnet.com ) . visual stimuli were back - projected onto a translucent screen positioned behind the scanner bore and viewed from an angled mirror mounted on the head coil positioned at subject 's eye - level . images were acquired using a siemens 3 t tim trio mri scanner equipped with a 12-channel head coil . participants completed a high - resolution t1-weighted mprage anatomical scan [ 144 slices , repetition time ( tr ) 2500 ms , echo time ( te ) 3.44 ms , flip angle 9.0 , fov 230 mm , resolution 0.9 0.9 mm , matrix size 256 256 mm , slice thickness 1 mm , no gap ] and two functional runs using multi - slice t2 * -weighted , echo planar imaging sequences [ spin - echo , 224 ( run 1)/229 ( run 2 ) volumes , 36 slices , tr 2500 ms ; te 30 ms ; flip angle 90 ; fov 230 mm , resolution 1.8 1.8 , matrix size 128 128 mm , slice thickness 4 mm , no gap , oblique slices ] . the main effect of pain and group effects on pain processing are reported and discussed in a separate manuscript . a 2 ( group : patients , controls ) 2 ( task difficulty : easy , difficult ) 3 ( thermal stimulus intensity : no pain , mild pain , moderate pain ) repeated - measures anova was used to analyze all behavioral data . functional images were preprocessed and analyzed using spm8 software ( wellcome department of imaging neuroscience , london , uk ) implemented in matlab ( mathworks , cherborn , ma , usa ) . . then , the high - resolution anatomical image was coregistered onto the mean functional image and segmented into csf , white matter , and gray matter . all volumes were normalized to mni ( montreal neurological institute ) space and then spatially smoothed with an 8 mm gaussian kernel . after preprocessing , a general linear model was defined for each participant separately . each model included six regressors of interest : difficult / moderate pain ( t1p2 ) , difficult / mild pain ( t1p1 ) , difficult / no pain ( t1p0 ) , easy / moderate pain ( t0p2 ) , easy / mild pain ( t0p1 ) , and easy / no pain ( t0p0 ) . in order to model only the period where cognitive load and thermal stimulation were stable and concurrent within each trial , the first and last portions ( 1.4 s off each end of the cognitive task trial , and corresponding , but variable tails of the thermal stimulus trial ) of each trial resultant trial durations ( 5.711.8 s ) , reaction times ( parametric modulator ) , and six motion parameters ( covariates of no interest ) were modeled independently for each subject . average task - related neural response was determined using contrast images [ difficult > easy , across thermal stimulus intensity levels , t1 > t0 ] and [ difficult > easy in the absence of pain , t1p0 > t0p0 ] for each participant . to evaluate group differences in task - related activity , independent sample t - tests were also conducted on these contrasts . the full task pain interaction [ task ( difficult easy ) + pain ] [ task ( difficult easy ) + no pain ] : [ ( t1p2 t0p2 ) + ( t1p1 t0p1 ) ] 2[t1p0 t0p0 ] was examined across and within groups . additionally , we further probed this interaction by examining task - related activations during pain [ pain ( mild + moderate ) + difficult task ] > [ pain ( mild + moderate ) + easy task ] : [ ( t1p1 + t1p2 ) ( t0p1 + t0p2 ) ] as well as task - related deactivations during pain [ pain ( mild + moderate ) + easy task ] > [ pain ( mild + moderate ) + difficult task ] : [ ( t0p1 + t0p2 ) ( t1p1 + t1p2 ) ] . the latter was of particular interest as it may reflect task - related reductions in pain activity . finally , we also examined pain - related activations during the difficult task [ difficult task + no pain ] > [ difficult task + pain ( mild + moderate ) ] : [ 2t1p0 ( t1p1 + t1p2 ) ] to examine the effects of cognitive load on pain - related activity . across analyses , an initial extent threshold of p < .005 , cluster size k > 25 voxels was used , corrected for multiple comparisons at the cluster level ( p < .05 ) , unless otherwise stated . we used marsbar ( brett et al . , 2002 ) to extract beta values from regions of interest ( rois ) , defined as significant clusters identified by planned contrasts . extracted values were used in 2 ( group : patients , controls ) 2 ( task difficulty : easy , difficult ) 3 ( thermal stimulus intensity : no pain , mild pain , moderate pain ) repeated - measures anovas to probe task pain interactions and group differences in task - related activity . migraine patients had marginally higher individualized peak stimulus ( p2 ) temperatures ( mean = 47.5 c , se = .3 ) compared to healthy controls ( mean = 46.4 c , se = .5 ) , t(26 ) = 2.0 , p = .06 . as expected ( seminowicz & davis , 2007a ) a main effect of task difficulty f(1,26 ) = 195.3 , p < .001 , and a marginal effect of pain f(2,52 ) = 2.7 , p = .08 , were found on reaction times . on average , participants were slower to respond during the difficult , relative to easy task t(27 ) = 14.2 , p < .001 , and during the mild ( p1 ) pain condition relative to the moderate ( p2 ; t(27 ) = 1.8 , p = .08 ) and no pain ( p0 ; t(27 ) = 2.0 , p = .06 ) conditions . a significant task pain interaction f(2,52 ) = 4.7 , p = .01 was also seen in reaction time , such that the effect of pain on reaction time was larger during the easy task , and not significant during the difficult task ( fig . no other main effects or interactions were found . there was a main effect of task difficulty f(1,26 ) = 25.1 , p < .001 on accuracy , such that participants had lower accuracy in the difficult , relative to easy task , t(27 ) = 5.0 , p < .001 ( fig . notably , there were no main effects of group on reaction time or accuracy , suggesting that patients performed the task as well as controls . when performance was examined within each of the six individual conditions , there were still no observable differences in performance between groups ( fig . patients ( mean = 17.7 , se = 2.3 ) had significantly higher pain catastrophizing scores than controls ( mean = 6.9 , se = 2.1 ) , t(26 ) = 3.5 , p = .002 . an additional two patients and one control have partially missing reaction time and accuracy data . for these participants , all available data were included . a main effect of task difficulty f(1,24 ) = 183.7 , p < .001 , and a main effect of pain f(2,48 ) = 3.6 , p = .04 were found on reaction time , such that participants were faster to respond during the easy , relative to difficult task t(25 ) = 13.8 , p < .001 , and during moderate ( p2 ) relative to the no pain ( p0 ; t(25 ) = 2.0 , p = .06 ) and mild pain ( p1 ; t(25 ) = 2.6 , p = .02 ) conditions . a main effect of task difficulty f(1,24 ) = 23.4 , p < .001 was also found on task accuracy , such that participants had lower accuracy in the difficult , relative to easy conditions , t(25 ) = 4.7 , p < .001 . no other effects were significant and there were no group differences in performance in any of the six individual conditions ( fig . task difficulty was associated with neural activity within regions known to be associated with cognitive processing ( table 1 , fig . all participants showed task - related activations in bilateral regions of the inferior occipital gyrus ( iog ) , superior parietal lobule ( spl ) and pm cortices , and task - related deactivations within midline regions , including the medial prefrontal cortex ( mpfc ) and posterior cingulate cortex ( pcc ) , and the r pins . in order to isolate smaller regions of interest ( task rois ) from large suprathreshold clusters , we increased the cluster forming threshold to p < .001 , k > 25 for activations , and p < when the effect of task was examined only within the no pain condition , similar patterns of activation were seen in bilateral regions of the iog , and pm cortices , as well as in the left inferior parietal lobule ( ipl ) , and similar task - related deactivations were seen within midline regions , including the mpfc and pcc , as well as the r pins ( table 1 , fig . , a more stringent threshold was used to isolate separable deactivation clusters ( p < .001 , k > 25 ) . controls displayed greater activity within a left ventral region of the pm ( vpm ) cortex compared with patients ( table 1 ) . patients did not reveal any activity greater than controls that survived cluster - level correction ( see supplementary table 1 for suprathreshold clusters that did not survive correction ) . in the absence of the pain stimulus , migraine patients show less deactivation than controls within the l dlpfc as well as regions in the dorsal amcc and cerebellum during the difficult relative to easy task ( group difference task rois , table 1 , fig . patients who fell within the episodic migraine diagnostic criteria range were not outliers in this analysis ( supplementary fig . 1 ) , and the significant interaction between group and task - related deactivations remained when those with episodic migraine were excluded ( l dlpfc : f(1,22 ) = 14.6 , p = .001 ; amcc : f(1,22 ) = 26.0 , p < .001 ; r cerebellum : f(1,22 ) = 11.9 , p = .002 ) . to ensure that this group difference was not driven by structural abnormalities , we extracted gray matter volume ( gmv ) from one of these regions the dlpfc and found no difference in mean gmv between migraine patients ( m = .50 , se = .02 ) and controls ( m = .49 , se = .02 ; t(26 ) = .55 , p = .6 ) . we also found that the task - related group difference in dlpfc deactivation remained after controlling for individual gmv ( task difficulty group interaction : a significant task group interaction was found in the r amcc group difference task roi ( defined as significant clusters from the patients > controls contrast in the absence of pain , fig . patients reveal a blunted pattern of responsiveness within the r amcc such that they have decreased activation during the easy task , and decreased deactivation during the difficult task , compared to controls . however , the simple effect of group within task condition was not significant . no activation or deactivation clusters exceeded the cluster - level corrected threshold in the full task pain interaction . in the full task pain interaction , controls showed greater activity within the dorsal superior frontal gyrus ( sfg ) compared to patients ( table 2 , fig . 3 ) . patients had no suprathreshold activity greater than controls . within sample analyses showed that patients had significant task pain interaction - related deactivations within the right dorsal medial sfg , which partially overlaps with the dorsal sfg identified by the two - sample analysis above . among patients , activity within the primary somatosensory cortex ( s1 ) was associated with the task pain interactions , but this did not survive cluster - level correction ( supplementary table 1 ) . among controls , this suggests a different pattern of interaction - related activity between groups . across participants , task - related deactivations during pain [ pain ( mild + moderate ) + easy task ] > [ pain ( mild + moderate ) + difficult task ] were seen across regions associated with the sensory ( e.g. r pins , amcc , s1 ) , cognitive ( e.g. mpfc , dorsal mpfc , l ventral lateral pfc ) , and affective ( e.g. l amygdala , l anterior insula ( ains ) , l orbitofrontal cortex ( ofc ) ) components of pain ( table 3 ) , suggesting that this may reflect task - related reduction of pain activity . a more stringent threshold was used in this cross - participant analysis in order to isolate separable deactivation clusters ( p < .001 , k > 25 for all but the two sample test ) . patients revealed greater task - related deactivations during pain in the l posterior pm / primary motor ( m1 ) cortex compared to controls ( table 3 ) . within group analyses suggest that this was largely due to greater activity among controls as seen in the reverse contrast within subjects ( task - related activity while in pain ) [ pain ( mild + moderate ) + difficult task ] > [ pain ( mild + moderate ) + easy task ] . overall , more widespread reductions were observed among patients relative to controls ( fig . 4 ) , whereas more robust task - related activity was seen among controls compared to patients . there were no significant pain - related reductions in task activity [ difficult task + no pain ] > [ difficult task + pain ( mild + moderate ) ] . effects of pain were seen across task rois ( defined as significant clusters from the difficult > easy contrast across participants and conditions , fig . a main effect of pain was found in all task - related activation rois as well as some of the task - related deactivation clusters ( r s1/pcc , r pins , r dlpfc , l m1 , l lateral pfc , l s1 , l ifg / ofc ) , such that pain was associated with more activity than no pain . a significant task a main effect of pain was also found in all of the group difference task rois ( defined as significant clusters from the patients > controls contrast in the absence of pain , fig . pain rois were defined as significant clusters from the pain ( p1 + p2 ) > no pain ( p0 ) contrast across participants and conditions ( supplementary fig . r pins was the only pain roi to show significant task - related modulation ( supplementary table 2 ) . a significant pain task interaction was found in the r pins , such that across participants there was deactivation during task ( difficult compared to easy ) in the no pain ( t(27 ) = 2.4 , p = .03 ) and moderate pain ( t(27 ) = 4.0 , p < .001 ) conditions , but no task difference in the mild pain condition ( p > a main effect of task was found in the r pins such that the difficult task was related to less pain - related activity compared to the easy task . in the r thalamus , a three way interaction was seen in two of the group difference task rois ( i.e. , l dlpfc and r cerebellum ) . controls show a greater decrease in activity within these regions in the difficult , relative to easy task compared to patients , particularly in the no pain condition ( fig . when we controlled for differences in pre - scan pain ratings , all of the task pain group interactions remained significant . inferential statistics are reported in supplementary table 2 , and correlations are plotted in supplementary fig . pain catastrophizing was not correlated with activity in task rois across participants ( patients and controls ) . among patients , migraine pain intensity over the last month was positively correlated with activity in the r vpm task - roi ( supplementary fig . 3b ) , indicating that migraine pain intensity was associated with enhanced task - related activation in this cluster . migraine frequency over the last month was positively correlated with three task deactivation rois ( l middle temporal gyrus ( mtg ) , l amygdala , l posterior mtg , supplementary fig . 3c ) , suggesting that increased migraine frequency was associated with abnormal task - related deactivations and may be associated with blunted deactivations observed among patients in task - related activity . disease duration controlling for age was negatively correlated with the l s1 and l amygdala task deactivation rois , indicating that the longer patients have had migraines , the larger the magnitude of task - related deactivations in these regions ( supplementary fig . pain catastrophizing , migraine frequency , migraine pain intensity , and disease duration controlling for age , were not correlated with task - related activity in the group difference task rois . when we did not control for age , disease duration was negatively correlated with the l dlpfc , such that the longer patients have had migraines , the more they deactivate this area during the difficult task ( supplementary fig . patients show a similar relationship between l dlpfc activity and age as they do with disease duration . 3f ) , suggesting that age may not fully explain the relationship between disease duration and task - related activity in l dlpfc among migraine patients . here , we characterize brain activity associated with cognitive task performance and pain cognition interactions among patients with migraine . overall differences in cognitive networks primarily demonstrated that acute pain - related modulation of task - related deactivations present in controls is absent in migraine patients , and that migraine patients show reduced task - related deactivations and altered task robust task - related activations in task - positive ( visual , sensorimotor , and executive ) networks and deactivations in regions of the default mode network were observed across migraine patients and healthy controls . task difficulty increased activity within regions previously shown to increase as a function of cognitive load ( owen et al . , 2005 ; seminowicz & davis , 2007a ) . also consistent with prior literature , greater task difficulty was associated with greater deactivation of midline regions of the default mode network , including the mpfc and pcc ( buckner et al . , 2008 ) . this overall pattern of activations and deactivations was similar in the presence or absence of acute thermal pain . across participants , a significant pain task interaction was found in the r pins , such that task difficulty was associated with r pins deactivation , and pain intensity was associated with r pins activation . this was true whether we defined the r pins region based on the pain contrast ( i.e. , task - related modulation of pain activity ) or based on the task contrast ( i.e. , pain - related modulation of cognitive activity ) . this interaction pattern within the r pins has been demonstrated previously in healthy controls ( seminowicz & davis , 2007a ) , and suggests that the pins may be an important site of task pain integrations . while no full interaction [ task ( difficult easy ) + pain ] [ task ( difficult easy ) + no pain ] clusters survived in the whole - brain analysis , task - related reduction during pain [ pain ( mild + moderate ) + easy task ] > [ pain ( mild + moderate ) + difficult task ] was observed at the level of the whole - brain analysis across brain regions associated with the sensory , cognitive , and affective components of pain . examination of task - related reduction in pain - related regions of interest revealed significant reduction in r pins , which is consistent with prior research ( bantick et al . , 2000 ; peyron et al . , 1999 ; seminowicz & davis , 2007a ; shackman et al . , 2011 ; 2008 ) , and may reflect cognitive modulation of pain . however , pain intensity and unpleasantness ratings were not significantly attenuated by task - difficulty . activity within a region of the thalamus identified in the pain contrast was increased during the difficult task , demonstrating an opposite pattern than was observed in the r pins and mcc . the present findings are consistent with prior studies that have found posterior and mid - thalamic activity to be positively associated with pain modulation , displaying increased activation in response to concomitant pain stimulus and cognitive task ( peyron et al . , 1999 ; valet et al . , 2004 ) . pain - related modulation of cognitive activity was not observed at the level of whole - brain analyses . this is consistent with a previous study in healthy controls that reported no pain - related modulation of cognitive activity when univariate analyses were used ( seminowicz & davis , 2007a ) . however , pain was associated with greater task - related activations and decreased task - related deactivations across most cognitive regions of interest , consistent with previous findings ( seminowicz & davis , 2007b ) . as both pain and cognitive tasks engage cognitive processes ( eccleston & crombez , 1999 ) , it is possible that this increase is due to the increasing attentional demands when both the difficult task and pain are present . this is supported by evidence that increased cognitive load is often associated with greater task - related deactivation ( mckiernan et al . , 2003 ) . thus , the current findings generally support previous work on brain activity associated with pain cognition interactions . overall , aberrant task - related activity among migraine patients emerged as blunted task - related activations and deactivations . across conditions , patients revealed a blunted task - related response within cognitive regions of interest as well as at the whole brain level of analysis . during task performance in the absence of pain , migraine patients revealed blunted task - related deactivation in the l dlpfc , amcc , and cerebellum compared to controls . analysis of activity within these clusters across conditions revealed that while controls show greater deactivation in these regions during the no pain condition , patients show no significant difference in activity within these regions across the pain and no pain conditions , suggesting that effects of acute pain on reducing task - related deactivation are absent among patients . in other words , patients show a pattern of task - related activity within these regions during no pain that is similar to activity in the presence of an external pain stimulus . these regions , therefore , may be important sites of alteration in cognitive processing in migraine . importantly , taken together with prior research , these results suggest that the dlpfc , in particular , may be an important site of alteration in chronic pain . prior work also found decreased l dlpfc task - related deactivations among chronic low back patients , relative to healthy controls , in the absence of a pain stimulus ( seminowicz et al . , 2011 ) . importantly , effective pain treatment was shown to normalize l dlpfc activity , such that deactivations became stronger and more like those seen among healthy controls ( seminowicz et al . , 2011 ) . furthermore , decreased dlpfc cortical thickness has been demonstrated within other pain populations ( apkarian et al . , 2004b ; seminowicz et al . , 2011 ) , and is also partially reversible with cognitive interventions that reduce pain and pain catastrophizing ( seminowicz et al . , 2013 ) . the blunted deactivation in l dlpfc task - related activity found here may reflect relatively constant engagement of the dlpfc across conditions and perhaps chronically among migraine patients . dlpfc is involved in pain modulation ( lorenz et al . , 2003 ) , and among chronic pain populations may be chronically upregulated to increase descending modulation of pain ( seminowicz et al . , 2013 ) pcc activity was associated with the full task pain interaction among controls , whereas deactivations in the dorsal sfg were associated with interactions among patients . to further probe this interaction , we examined whole - brain task - related activations and deactivations during pain , among patients and controls . patients revealed greater and more widespread task - related deactivations within regions associated with pain processing , but less task - related activations in task - positive regions during pain , compared to controls . taken together , this suggests that cognitive effects may be more related to pain reduction in patients , and task performance in controls , and that the presence of pain appears to be disturbing cognitive processing to a greater extent in patients than it does in healthy controls . among healthy populations , attention to a task is associated with both cognitive task - specific and anti - nociceptive activations ( kucyi et al . , 2013 ) . however , among migraine patients , allocation of cognitive resources to pain suppression may be prioritized over task - specific processes . disease duration was associated with greater l s1 and l amygdala task - related deactivation , even after controlling for patient age , such that the longer patients have had migraines , the more task - related deactivation is seen in the l s1 and l amygdala . a plausible interpretation of this result is that patients have higher baseline levels of activity within these regions , resulting in greater task - related decreases , and this baseline activity increases with disease duration . migraine frequency was associated with blunted deactivation in the l mtg , l amygdala , and l pmtg . this suggests that increased migraine frequency may be leading to the abnormal task - related deactivations seen among migraine patients , and is consistent with a previous finding that cognitive impairment in migraine was related to migraine frequency ( calandre et al . , 2002 ) . pain catastrophizing was not significantly associated with task - related activity or pain task interactions . the limitations of the current study include not strictly controlling the level of ongoing headache pain during scanning , the small sample size , and the inclusion of four patients with high frequency episodic migraine . studies with larger samples powered to examine differences between clinical subtypes of migraine , including episodic migraine and chronic migraine , and the potential effects of medication overuse ( grazzi et al . , 2010 ) , will be informative . future research should also examine the effects of migraine on cognitive networks over time , as symptom severity changes , as well as potential treatment effects on these alterations . taken together , these findings suggest that migraine patients have blunted cognitive - related neural activity in brain regions associated with cognitive processing and altered patterns of activity related to pain cognition interactions . specifically , healthy controls have strong task - related deactivations in the dlpfc , amcc , and cerebellum that are decreased with acute pain , but patients have blunted task - related deactivations that are not modulated by acute pain . while no behavioral cognitive decrements were observed , blunted neural response may be reflective of broader functional restructuring as has been shown in other chronic pain populations ( baliki et al . , 2008 ; seminowicz et al . , these changes may be related to migraine pain frequency , but were not associated with pain catastrophizing or migraine pain intensity in this sample . furthermore , differential activity underlying pain cognition interactions suggests that allocation of cognitive resources to pain suppression may be prioritized over task - specific processes in migraine .

little is known about the effect of migraine on neural cognitive networks . however , cognitive dysfunction is increasingly being recognized as a comorbidity of chronic pain . pain appears to affect cognitive ability and the function of cognitive networks over time , and decrements in cognitive function can exacerbate affective and sensory components of pain . we investigated differences in cognitive processing and pain cognition interactions between 14 migraine patients and 14 matched healthy controls using an fmri block - design with two levels of task difficulty and concurrent heat ( painful and not painful ) stimuli . across groups , cognitive networks were recruited in response to a difficult cognitive task , and a pain task interaction was found in the right ( contralateral to pain stimulus ) posterior insula ( pins ) , such that activity was modulated by decreasing the thermal pain stimulus or by engaging the difficult cognitive task . migraine patients had less task - related deactivation within the left dorsolateral prefrontal cortex ( dlpfc ) and left dorsal anterior midcingulate cortex ( amcc ) compared to controls . these regions have been reported to have decreased cortical thickness and cognitive - related deactivation within other pain populations , and are also associated with pain regulation , suggesting that the current findings may reflect altered cognitive function and top - down regulation of pain . during pain conditions , patients had decreased task - related activity , but more widespread task - related reductions in pain - related activity , compared to controls , suggesting cognitive resources may be diverted from task - related to pain - reduction - related processes in migraine . overall , these findings suggest that migraine is associated with altered cognitive - related neural activity , which may reflect altered pain regulatory processes as well as broader functional restructuring .

Introduction Methods Results Discussion

PMC2997503

in sweden , a common definition of extreme tall stature is an adult height exceeding 200 cm for boys and 185 cm for girls corresponding to approximately 3 sd above the mean . however , an underlying disorder must always be considered in tall individuals as many of them need special medical attention . these include homocysteinuria , growth hormone excess , and various syndromes such as klinefelter , sotos , marfan , and beckwith - wiedemann syndrome [ 1 , 2 ] . extreme tall stature is a common reason for referral to pediatric endocrinology centers in northern european countries . patients and their families often express a strong desire for intervention aiming to reduce adult height . the treatment options for children with extreme tall stature are limited . to reduce final height ( fh ) , boys have been treated with high - dose testosterone and girls with high - dose estradiol [ 3 , 4 ] . the treatment was introduced already in 1956 , but its use has decreased dramatically in the last two decades , possibly due to a change in attitude towards tall stature and concerns of long - term complications . in a meta - analysis study , hormonal treatment was only found to reduce final height if initiated before a bone age of around 14 yrs in both genders . it was suggested that administration of sex steroids after this age causes extra growth instead of growth inhibition . undesired side effects are commonly reported in patients undergoing hormonal treatment for tall stature . for boys , other documented side effects are weight gain , muscle ache , gynecomastia , hypertrichosis , behavioural changes , and aggressiveness [ 7 , 8 ] . for girls , side effects include nausea , headache , elevated serum triglycerides , calf cramps , and weight gain [ 810 ] . studies have shown changes in coagulation parameters although no studies have been able to show an increased risk of venous thrombosis . there is also a fear of a further increased risk of breast cancer [ 4 , 9 , 13 ] and decreased fertility . one study from australia shows that women who have been on high - dose oestrogen treatment to reduce their final height are more likely to have tried for over a year to get pregnant ( relative risk 1.80 , 95% ci 1.402.30 ) and to have taken fertility drugs ( relative risk 2.05 , 95% ci 1.343.04 ) . they are also less likely to conceive in any given menstrual cycle although the possibility of conceiving at all is only slightly decreased , possibly due to the availability of fertility treatments . because of the various side effects associated with hormonal treatment and sometimes limited efficacy , an alternative treatment modality is desired . surgical epiphysiodesis performed around the knee has for many years been used to treat leg length discrepancies . in extremely tall boys , the use of bilateral percutaneous epiphysiodesis was recently reported to reduce adult height by approximately 5 cm when performed at a mean bone age of 13.9 years . however , it is important to point out that still this is a controversial treatment as it is being performed in healthy adolescents and further supportive data are lacking . the aim of our study was to determine the efficacy and safety of bilateral epiphysiodesis around the knee when performed to limit further growth in extremely tall adolescent girls and boys . we included radiological assessments of bone growth and bone angles and a reference group of untreated extremely tall subjects to document the efficacy and safety of bilateral percutaneous epiphysiodesis . all study subjects have been followed at the pediatric endocrine clinic at astrid lindgren children 's hospital , karolinska university hospital , stockholm , sweden . after approval by the local ethics committee , all tall girls and boys who fulfilled the inclusion criteria ( table 1 ) were offered to undergo bilateral percutaneous epiphysiodesis around the knee . informed consents were given by all patients who participated in the study and their parents . until january 2009 , 49 patients ( 21 boys and 28 girls ) have undergone surgery . out of these , 21 subjects ( 12 girls and 9 boys ) had reached final height , and data from all these subjects are presented in this paper . all patients had been diagnosed with constitutional tall stature except three patients with marfan syndrome and one with klinefelter syndrome . most of the patients were midpubertal at time of surgery , the girls generally slightly less advanced in their pubertal development ( range b2b4 ) than the boys ( g2g5 ) . in the constitutionally tall patients , the midparental height ( defined as the average of the parent 's heights 6.5 cm for girls and + 6.5 cm for boys ) midparental height was 177.7 1.9 cm in girls and 190.5 1.1 cm in boys ( corresponding to + 1.7 sds and + 1.5 sds , resp . ) . two patients with constitutional tall stature were excluded from this calculation , one with a parent who had received hormonal treatment for tall stature and another with a parent diagnosed with marfan syndrome . at each occasion , bone age maturation was determined by two blinded experts on an x - ray film of the left hand and wrist using the atlas of greulich and pyle . final height predictions were made according to the bailey and pinneau tables . to further verify the efficacy and safety of the treatment , hip - knee - ankle ( hka ) angles and leg lengths ( femur and tibia lengths ) were measured digitally on plain x - ray films before surgery and at final height . due to technical and practical problems , complete information for hka - angle was only available in 15 patients and for leg length in 9 patients . to validate the precision of our final height predictions , we recruited 17 untreated subjects ( 12 girls and 5 boys ) , all diagnosed with constitutional tall stature . they had been through a similar workup as the operated patients , including a hand x - ray but had never undergone treatment . at time of first visit , the mean age was 13.6 0.4 yrs in girls and 15.2 0.8 yrs in boys while the mean bone age was 12.9 0.1 yrs in girls and 14.6 0.6 yrs in boys . these reference subjects did not significantly differ from the study population ( table 3 ) . the mean height at this initial workup was 175.9 1.1 cm in girls and 188.5 5.9 cm in boys . based on evaluation of a hand and wrist x - ray film obtained , the final height predictions were 184.0 1.0 cm and 200.7 2.2 cm in girls and boys , respectively . when comparing predicted final height with achieved adult height , we found that girls were overpredicted by a mean of 0.2 0.6 cm and boys overpredicted by a mean of 1.3 1.8 cm . in addition , we recruited another group of 12 young normal height adults ( age 2040 years ; 11 females and 1 male ) that were measured twice in a day with an eight - hour interval ( at 8 am and 4 pm ) with the aim to quantify the well - known reduction of height that normally occurs throughout the day . in these subjects , the mean loss in height over 8 hours was 0.7 0.1 cm . when validating the final height predictions , we compensated for the calculated hourly loss in height over the day ( 0.092 0.012 cm / hour ) to correct for any error caused by a difference in time between measurements performed in individual subjects . if making this correction , final height predictions in the tall untreated girls were equal to the achieved adult heights for girls ( 0.1 cm ) while boys were still slightly overpredicted ( 0.5 cm ) . as the mean prediction error was so small , we decided not to correct for this when calculating the efficacy of the operation in the study population . the growth plates of the distal femur and proximal tibia and fibula were identified using an x - ray image intensifier ( c - arm ) . through one cm incisions at the lateral aspects of the femoral and tibial growth plates , epiphysiodesis was performed with a drill ( 8 mm ) and oval angulated curettes ( 57 mm ) . the growth plate of the fibula was curetted with a 3 mm straight oval curette through the tibial incision from an anterior direction to avoid damage to the peroneal nerve . between 25% and 50% of each growth plate was removed to induce the formation of bony bridges across the physis to ensure complete elimination of growth . the total operation time was approximately 45 minutes per leg . in the here reported patients , more recently , we have discontinued to apply femoral nerve blockage which has facilitated the postoperative mobilization with no increase in the reports of postoperative pain ( data not shown ) . to achieve full range of knee movement , physiotherapy and full weight bearing with crutches patients were discharged from the hospital when they were able to ambulate and bend their knees at least 90 degrees which typically occurred on the 2nd or 3rd day postoperatively . as a precaution , patients were advised to avoid strenuous physical activities for 4 weeks ( no running or jumping ) . twelve months postoperatively , total destruction of operated growth plates was confirmed on plain knee x - ray films . total height , sitting height , and arm span were measured every 12 months after surgery until adult height was reached . in addition , leg length was measured radiologically both preoperatively and when adult height had been reached . according to our definition , adult height was reached when the calculated growth velocity was less than 0.5 cm per year when assessed over a minimum of a 6-month interval . when adult height had been reached , each patient went through a work up which included clinical examination focusing on the efficacy and long - term safety of the treatment . in addition , plain digital x - ray films were taken of both legs to determine if any angulation ( hip - knee - ankle angle ) or leg length discrepancy had developed as a consequence of the previous operation . student 's t - test was applied and when normality test failed wilcoxon signed rank test was used to analyse treatment effects . average height at surgery , predicted final height , and achieved final height for the study population are shown in table 3 . mean predicted final height at time of surgery corresponded to + 3.8 0.2 sds in girls and + 3.8 0.3 sds in boys . then , the remaining predicted growth ( final predicted height subtracted by height at surgery ) was 11.9 1.2 cm in girls and 17.9 2.3 cm in boys ( figure 1 ) . in patients who underwent bilateral percutaneous epiphysiodesis , the mean achieved final height was 186.4 0.8 cm in girls and 199.1 1.5 cm in boys ( table 3 ) corresponding to + 3.1 0.1 sds and + 2.8 0.2 sds , respectively . figure 2 illustrates the distribution of height reductions versus initial predictions for all treated patients . the mean reduction of final height versus initial prediction at time of surgery was 4.1 0.7 cm in girls ( p < .001 ) and 6.4 1.2 cm in boys ( p < .001 ) corresponding to growth reductions of 33.8 3.4% and 33.6 4.2% , respectively ( table 3 ) . as expected , after surgery most of the growth occurred in the upper body segment ( figure 1 ) . the mean increment in sitting height was 7.7 0.7 cm in girls and 10.4 1.0 cm in boys , respectively , while the mean leg growth ( increase in total height subtracted by increase in sitting height ) was only 0.1 0.2 cm in girls and 1.1 0.4 cm in boys ( figure 1 ) . in the patients where leg growth was assessed radiologically , mean femoral growth was 0.4 0.2 cm while mean tibial growth was 0.7 0.2 cm . altogether , the men leg growth ( femur + tibia growth ) from surgery until final height was 1.2 0.1 cm ( n = 9 ) as measured by x - ray . in boys , final adult height was reduced by 3.910.7 cm except for one single patient who had a reduction of only 0.3 cm . in this patient , the upper body growth after surgery was 9.0 cm while leg growth was only 0.9 cm . except for one boy with marfan syndrome ( subject no . 20 ; table 2 ) , no patient showed a leg growth exceeding 1.2 cm after surgery . in this boy , leg growth was 3.3 cm while his upper body segment increased by 16.7 cm resulting in a 10.2 cm reduction of adult height compared to the initial prediction . an x - ray obtained 12 months after surgery clearly demonstrated that his distal tibia growth plates were still completely open while his operated proximal tibia and distal femur growth plates were , as expected , fused . when excluding patients with primary growth disorders ( three with marfan and one with klinefelter syndrome ) , mean leg growth after surgery was only 0.4 0.2 cm and if analyzed separately 0.0 0.2 cm in girls and 0.9 0.3 cm in boys . figure 3 shows a clear negative correlation between bone age at time of surgery and height reduction , a correlation which was slightly stronger in boys ( r = 0.818 ; p < .01 ) than in girls ( r = 0.769 , p < .01 ) . one of the female patients was operated at an early bone age ( 10.1 yrs ) . if this patient is excluded , the negative correlation between bone age and final height reduction does not remain significant in girls . all operated girls except one ( figure 4(a ) ) and all boys except two ( figure 4(b ) ) had a relative sitting height below or well below the mean for age showing that the subjects had relatively long legs at time of surgery . the relative sitting height ( sh ) increased in every treated girl ( figure 4(a ) ) and boy ( figure 4(b ) ) . in girls , the mean relative sh was 51.9 0.3% at fh versus 49.9 0.3% at operation ( p < .001 ) . in boys , the relative sh at fh was 52.3 0.4% versus 50.0 0.3% at operation ( p < .001 ) . in the reference population , the mean relative sh at fh is 52.4% in girls and 51.5% in boys . thus , in our operated patients , relative sh at fh was still below the mean in girls and for boys just slightly above the mean . for girls , average sitting height percentage was 52.1 in patients treated before and 51.8 for those treated after a bone age of 12.5 yrs . for boys , the sitting height percentage was 51.8 in patients treated before and 52.7 for those treated after a bone age of 14.0 . from time of surgery to final height , mean arm span increased 3.2 1.6 and 4.0 1.2 cm more than height in girls ( n = 9 ) and boys ( n = 7 ) , respectively . one patient had a postoperative superficial cutaneous infection which did not require treatment with antibiotics . nine patients reported postoperative pain from the site of surgery and received oral analgesics ( 214 days ) . leg length discrepancy did not change significantly between time of surgery until final height was achieved ( n = 10 ; p = .560 ) . the mean leg length difference before operation was 0.4 0.1 cm ( range 0.00.9 cm ) and at final height 0.3 0.1 cm ( 0.01.0 cm ) . to verify if any knee angulations developed after surgery , radiologic measurements of the hip - knee - ankle ( hka ) angle were performed before surgery and at final height . between these two occasions , the hka angle was increased in seven patients ( 0.55.0 degrees ) while decreased in ten ( 0.55.0 degrees ) . in four of these patients , we here report that bilateral percutaneous epiphysiodesis performed around the knee effectively inhibits further leg growth when performed in extremely tall adolescent girls and boys . previously the only report of long - term outcome of this procedure has been in boys . the height reduction in our male patient group is in agreement with what was previously reported . we have not only based our data on auxological measurements but also verified the efficacy and safety radiologically . to further strengthen the accuracy of the final height predictions , a validation cohort was recruited . in both girls and boys , bilateral percutaneous epiphysiodesis reduced the predicted remaining growth from time of surgery by approximately one third . when reviewing studies of hormonal treatment , epiphysiodesis seems to be more efficient in reducing final height . in girls treated at a bone age of 12.5 yrs , the height reduction is around 2 cm with hormonal treatment and around 4 cm with epiphysiodesis . in boys treated at a bone age of 14 yrs , epiphysiodesis results in a final height reduction by around 6 cm whereas hormonal treatment at this bone age results in no height reduction at all according to a meta - analysis . our results showed that epiphysiodesis was efficient in reducing final height in all patients except for one boy where we believe his predicted remaining growth was underestimated . this assumption is based on the fact that the growth of his upper body ( 9.0 cm ) far exceeded his leg growth ( 0.9 cm ) suggesting that the intervention was indeed effective also in this patient . with regards to safety , we monitored for short - term side effects such as postoperative infections , pain , or discomfort as well as long - term side effects including any hip - knee - ankle angulations , leg length discrepancies , or abnormal body proportions . the only side effects reported were mild to moderate postoperative pain and one superficial cutaneous infection . our findings are in agreement with a previous study of epiphysiodesis where no serious side effects were reported either . although no serious complications occurred , it should be noted that this is an invasive procedure in otherwise healthy individuals . failure of epiphysiodesis secondary to insufficient ablation of the physis has indeed been reported by others . therefore , careful diagnostic preoperative evaluation is essential , and the treatment should be performed in a center of expertise ensuring the long - term followup . regarding body proportions , relative sitting height at final height was indeed closer to the normal than before surgery when being compared to normative tables for the general population . the reason for this is that tall stature is usually characterized by relatively long legs . in most girls in contrast , most boys had a sitting height percentage which was above the normative population , and in two of them this was more pronounced , most likely linked to the fact that their sitting heights were above the mean already at the time of surgery . at final height , however , an increase in relative sitting height is to be excpected and should be considered before treatment decision is made . we do not believe that the surgical treatment will lead to any long - term side effects in contrast to hormonal treatment for which fear has been raised of side effects such as reduced fertility and breast cancer [ 11 , 13 ] . other side effects that have been associated with hormonal treatment , such as hypertension and thrombosis , were not seen in our operated patients . therefore , we regard surgery as the treatment of choice in extremely tall patients with high blood pressure and/or a family history of venous thrombosis . the most widely used method for prediction of final height was devised by bayley and pinneau ( bp method ) . this method is routinely used in clinical practise . at time of surgery , most of our patients were in midpuberty with a mean age of 13.1 0.9 yrs in girls and 14.1 1.4 yrs in boys . a previous study has shown that the mean height prediction errors ( bp method ) at these ages are 0.5 2.2 cm in girls ( 13 yrs ) and 2.7 2.9 cm in boys ( 14 yrs ) . in order to reduce any prediction errors , all x - ray images were read by two blinded observers . in an attempt to quantify the potential errors of these predictions this validation clearly demonstrated that the predication errors were so small that we could ignore it when calculating the efficacy of the treatment . it is important to point out that the untreated reference group did not differ significantly from the treated group , although not being randomly selected . it would of course have been optimal to include a randomized untreated control group , but this was not possible for technical reasons . nevertheless , the efficacy of the operation was confirmed by radiological leg measurements which is a very reliable method to assess bone growth . our data suggest that bilateral percutaneous epiphysiodesis is an efficient and safe method to reduce adult height in extremely tall adolescents . the intervention should not be performed too late , preferably at a bone age not exceeding twelve and a half years in girls and fourteen years in boys . we recommend that a treatment decision is taken before girls have reached a height of 170 cm and boys 185 cm . hereby , the expected growth reduction will exceed 5 cm as long as the predicted final height is at least 185 cm in girls and 200 cm in boys . the patients should preferably have preoperative evaluation , treatment , and followup in a specialized center with expertise in pediatric endocrinology and pediatric orthopaedic surgery . further studies are desired to document the efficacy and safety of this relatively new procedure in larger patient groups .

objective . the aim was to determine efficacy and safety of a surgical method to reduce adult height in extremely tall adolescents . methods . data for all girls ( n = 12 ) and boys ( n = 9 ) in our center subjected to bilateral percutaneous epiphysiodesis around the knee who had reached final height were included . final height predictions were based on hand and wrist x - rays before surgery . results . when compared to prediction , adult height was reduced by 4.1 0.7 cm in treated girls ( p < .001 ) and 6.4 0.7 cm in treated boys ( p < .001 ) corresponding to a 33.6 3.4% and 33.6 4.2% reduction of remaining growth , respectively . besides mild to moderate postoperative pain reported in 9 operated individuals , no other side effects were reported . postoperative x - rays confirmed growth plate closure and absence of leg angulations . conclusions . bilateral epiphysiodesis is an effective and safe method to reduce adult height in extremely tall girls and boys .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion 5. Conclusions

PMC4129384

molyneux 's question asks whether a man born blind , who can distinguish a cube and sphere by touch , could distinguish those shapes upon having his sight restored ( locke , 1690/1975 , p. 146 ) . schwenkler ( 2013 ) criticizes a 2011 experiment attempting to answer molyneux 's question and proposes a way to re - run the experiment . i argue that schwenkler 's proposed experiment is flawed , since it relies on a questionable assumption that newly sighted subjects can appreciate depth cues . in a previous i - perception article , however , schwenkler ( 2012 ) had proposed a second way to re - run the original experiment , substituting two - dimensional shapes like circles and squares for three - dimensional spheres and cubes . i argue that this two - dimensional version of the experiment successfully bypasses schwenkler 's criticism of the original experiment , and also avoids the flaw in his other proposed experiment . held and colleagues ( 2011 ) conducted an experiment to answer molyneux 's question , involving subjects who had just undergone cataract removal surgery to restore their sight . the experimenters used 20 non - identical pairs of enlarged legos as stimuli . to test whether subjects had sufficient visual ability for the experiment , they were first shown one lego , and then shown that lego along with a second , non - identical lego . asked which of the two legos was shown in the first showing , they were able to answer correctly at a mean rate of 92% . then they were visually presented with that lego along a second non - identical lego , and were asked which of the two legos they were previously touching . subjects performed this task at near chance levels , with a mean of only 58% correct . held and colleagues take the tv results to show that the answer to molyneux 's question is likely negative , since they indicate that someone with newly restored vision would likely not be able to tell , based on vision alone , which object is the cube or the sphere ( held et al . , 2011 , the problem with the experiment , according to schwenkler , is that the bar for the vv task was too low : success at it did not establish that subjects had the visual capacities necessary for passing the tv task . they could perform well in the vv task just by making crude discriminations based on low - level features like shadows and overall contours , aided by the fact that the three - dimensional objects were presented only from a single angle ( with the same shadow and the same overall contours ) . in other words , the experimenters failed to ensure that their subjects were able to form robust visual shape representations involving spatial features invariant to a shifting perspective ( schwenkler , 2013 , p. 92 ) . furthermore , another study suggests that the subjects in the held study could not have formed robust visual shape representations given the conditions of the experiment . in that study , subjects were shown three - dimensional objects like pyramids and cubes two weeks to three months after having their sight restored , yet they were unable to integrate the facets into the percept of a single three - dimensional object ( ostrovsky , meyers , ganesh , mathur , & sinha , 2009 , p. 1,486 ) . schwenkler takes this as evidence that under the conditions of the held experiment , subjects were precluded from forming robust visual shape representations . schwenkler suggests a way to re - run the experiment . in the original experiment , schwenkler writes , this kept them from accessing the richer array of spatial cues that a changing perspective on the object would have supplied , while at the same time making it especially easy to use low - level features in the vision - to - vision task , since they could be sure that they would encounter each object from the same viewing angle ( schwenkler , 2013 , p. 93 ) . he suggests the experiment be re - run , allowing either the stimuli to move , or the subjects to move , or both . according to him , this would allow subjects to access more spatial cues , enabling them to build robust representations of the three - dimensional shapes . schwenkler wants to re - run held and colleagues ' experiment so that subjects might build robust shape representations , but an alternative is to re - run the experiment so that it does not demand robust shape representations for the tv task in the first place . specifically , the experimenters should re - run the experiment with two - dimensional circle and square raised - line drawings , rather than three - dimensional shapes . since several studies indicate that raised - line drawings can be difficult to identify by touch , however , it might seem that re - running the held experiment using raised - line drawings is a non - starter ( for a survey of raised - line drawings , see picard & lebaz , 2012 ) . if subjects can not identify a shape by touch , how can they match a shape they see with one they had previously touched ? however , as picard and lebaz summarize , identifying raised - line drawings by touch is hard but not impossible it is important to note that since raised - line drawing studies typically are testing for proficient tactile identification in general , using a range of complex shapes , they are often testing at a level of difficulty unnecessary for a two - dimensional test of molyneux 's question , where what is required is just proficient tactile identification for a few simple shapes ( in particular , circles and squares ) . all of this suggests that rather than indicating that a two - dimensional test of molyneux 's question is a non - starter , the raised - line drawing studies point to constraints for such a test instead . for instance , as picard and lebaz suggest , the two - dimensional pictures should be simple and printed on high - quality paper ( picard & lebaz , 2012 , p. 430 ) . while using two - dimensional shapes departs from molyneux 's original question , note that in several prominent philosophical treatments of molyneux 's question , philosophers have deliberately changed the original question , often at the very outset of their discussion , from a question about three - dimensional cubes and spheres to a question about two - dimensional circles and squares . ) , evans ( 1985 , p. 365 ) , and van cleve ( 2007 , p. 252 ) all make this move , and give us a good reason to do so . van cleve writes , [ a ] subject gaining sight for the first time might not be able to perceive depth ( as berkeley maintained ) , in which case he could nonetheless still presumably distinguish from one another two - dimensional figures like circles and squares ( van cleve , 2007 , p. 252 ) . for diderot , van cleve , and evans , schwenkler uses the three - dimensional formulation of molyneux 's question and proposes that held and colleagues re - run their experiment , allowing either the stimuli to move , or the subjects to move , or both . this is because watching an object as it moves with respect to one'or as one moves with respect to it'can bring into view features of it that would otherwise have been hidden , and makes available more information concerning how its surfaces are oriented in depth but this is precisely what diderot , evans , and van cleve have questioned as proponents of the two - dimensional formulation of molyneux 's question : that a newly sighted man would be able to appreciate the depth cues available in visual perception ( evans , 1985 , p. 365 ) . and studies showing that a newly sighted person can appreciate depth cues a few months after surgery , which schwenkler cites , are not enough to show that they can appreciate them immediately after surgery . we are therefore left with a worry about whether subjects can appreciate depth cues immediately post - surgery . given this worry , if schwenkler 's proposed experiment were to yield a no answer to molyneux 's question , we would be unable to ensure that the reason was not just due to the inability of subjects to detect depth cues . by running an experiment based on the two - dimensional formulation of molyneux 's question , we can avoid the problem with schwenkler 's proposed experiment . in a two - dimensional molyneux experiment , given a no answer to molyneux , we can ensure that the reason was not just due to the inability of subjects to detect depth cues . depth cues are irrelevant to a two - dimensional experiment . by running an experiment based on the two - dimensional formulation of molyneux 's question , we can also avoid schwenkler 's original criticism of the held experiment . according to schwenkler , the problem was that the experimenters did not ensure that their subjects could form robust representations of visual shape , and other experimental work on the restoration of sight suggests that the subjects can not have done this within the conditions of the experiment ( schwenkler , 2013 , p. 93 ) . the other experimental work references the study where two weeks to three months after having their sight restored , subjects were shown three - dimensional objects like pyramids and cubes , but were unable to integrate the facets into the percept of a single three - dimensional object but in a two - dimensional molyneux test , subjects would not need to integrate distinct facets into a single object . so , schwenkler 's criticism of the held experiment would not apply to a two - dimensional molyneux experiment .

in a recent i - perception article , schwenkler ( 2012 ) criticizes a 2011 experiment by r. held and colleagues purporting to answer molyneux 's question . schwenkler proposes two ways to re - run the original experiment , either by allowing subjects to move around the stimuli , or by simplifying the stimuli to planar objects rather than three - dimensional ones . in schwenkler ( 2013 ) , he expands on and defends the former . i argue that this way of re - running the experiment is flawed , since it relies on a questionable assumption that newly sighted subjects will be able to appreciate depth cues . i then argue that the second way of re - running the experiment is successful both in avoiding the flaw of original held experiment , and in avoiding the problem with the first way of re - running the experiment .

Introduction Schwenkler's argument Reply to Schwenkler

PMC1469296

this paper examines the associations between average daily particulate matter less than 10 microns in diameter ( pm10 ) and temperature with daily outpatient visits for respiratory disease including asthma , bronchitis , and upper respiratory illness in anchorage , alaska , where there are few industrial sources of air pollution . in anchorage , pm10 is composed primarily of earth crustal material and volcanic ash . carbon monoxide is measured only during the winter months . the number of outpatients visits for respiratory diagnoses during the period 1 may 1992 to 1 march 1994 were derived from medical insurance claims for state and municipal employees and their dependents covered by aetna insurance . the data were filtered to reduce seasonal trends and serial autocorrelation and adjusted for day of the week . the results show that an increase of 10 micrograms / m3 in pm10 resulted in a 3 - 6% increase in visits for asthma and a 1 - 3% increase in visits for upper respiratory diseases . winter co concentrations were significantly associated with bronchitis and upper respiratory illness , but not with asthma . winter co was highly correlated with automobile exhaust emissions . these findings are consistent with the results of previous studies of particulate pollution in other urban areas and provide evidence that the coarse fraction of pm10 may affect the health of working people.imagesfigure 1 . afigure 1 . bfigure 2 . afigure 2 . bfigure 3 . afigure 3 . bfigure 4 . afigure 4 . b

Images

PMC4370927

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substantial increase in luminosity , will provide enough statistics to probe also differential distributions , thereby testing the standard model in much greater detail . in this paper we will study in a model - independent way the impact of new physics in the full angular distribution of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z{\ell } ^+{\ell } ^-$$\end{document}hz+- decay , with the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z on - shell and eventually decaying into a lepton pair . we will argue that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z{\ell } ^+{\ell } ^-$$\end{document}hz+- is a useful channel not only for spin identification [ 812 ] , but also to test nonstandard couplings : it provides a rich 4-body angular distribution with a clean 4-lepton final - state signature . for earlier work our results can be parametrized in terms of six independent dynamical form factors , which include the effects of virtual electroweak bosons ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z ) as well as heavier states , whose effects at the electroweak scale are encoded in contact interactions . since we aim at model independence , we will study the new physics contributions using the effective field theory ( eft ) scheme developed in [ 15 , 16 ] , which is the most general eft of the electroweak interactions . as opposed to particular models , the resulting set of new - physics coefficients will remain undetermined . however , their natural sizes can still be estimated with the aid of power - counting arguments . certain aspects of this decay mode have already been discussed recently [ 1719 ] , with a focus on the dilepton - mass distribution . the observation there is that mass distributions can unveil new - physics structures in an otherwise sm - compatible integrated decay rate . this , however , comes at the expense of some fine - tuning in the new - physics parameters . in contrast , by exploiting angular distributions one can identify structures that do not contribute to the integrated decay rate . thus , one can still be compatible with the sm decay rates without tuning the new - physics parameters . as opposed to loop - induced processes , such as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow \gamma z$$\end{document}hz , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- does not look a priori like a promising testing ground for new - physics effects . as we will show below , they are expected , at most , at the few \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\%$$\end{document}% level in certain observables . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- is , however , an exceptionally clean decay mode and the natural suppression of new physics can be compensated with statistics . in fact , the lhc running at 14 tev with an integrated luminosity of 3000 fb\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{-1}$$\end{document}-1 will potentially be sensitive to new - physics effects in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- . our analysis also shows that cp - odd effects in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- are expected only at the per - mille level . the remainder of this paper will be organized as follows : in sect . 2 we will derive the full angular distribution for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z{\ell } ^+{\ell } ^-$$\end{document}hz+- . expressions for the dynamical form factors in terms of eft coefficients will be given in sect . 3 , with a discussion of their expected sizes in both weakly and strongly coupled scenarios . in sect . 4 we denote the amplitude for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z \ell ^+\ell ^-$$\end{document}hz+- decay of a higgs boson by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\varepsilon ^\mu { \mathcal m}_{3,\mu } $ $ \end{document}m3, , and for the decay of an on - shell \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - boson into a lepton pair by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\varepsilon ^\mu { \mathcal m}_{2,\mu } $ $ \end{document}m2, , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\varepsilon ^\mu $ $ \end{document} is the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - boson polarization . the fully differential decay rate for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(k)\rightarrow z(p)\ell ^+(q_1)\ell ^-(q_2)$$\end{document}h(k)z(p)+(q1)-(q2 ) , followed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z(p)\rightarrow \ell ^{'+}(p_1)\ell ^{'-}(p_2)$$\end{document}z(p)+(p1)-(p2 ) , is then given , in the narrow - width approximation , by1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{d\gamma } { ds\ , d\cos \alpha \ , d\cos \beta \ , d\phi } = \frac{\lambda } { ( 2\pi ) ^5\ , 2^{10}\ , \sqrt{r}\gamma _ z } \end{aligned}$$\end{document}ddsdcosdcosd=(2)5210rzm3m2,2where we have defined2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r&= \frac{m^2_z}{m^2_h}\ , , \quad s=\frac{q^2}{m^2_h } , \nonumber \\ \lambda&= ( 1 + r^2 + s^2 - 2r - 2s - 2 r s)^{1/2 } \end{aligned}$$\end{document}r = mz2mh2,s = q2mh2,=(1+r2+s2 - 2r-2s-2rs)1/2and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ z$$\end{document}z is the total width of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z . the kinematics is further discussed in appendix a. for massless leptons the decay amplitudes can be written as ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon _ { 0123}=+1$$\end{document}0123=+1)3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&{\mathcal m}_{3,\mu } = i \frac{2^{1/4 } g^{1/2}_f r}{s - r } \cdot \bar{u}(q_2)\left [ 2 f_1 \gamma _ \mu ( g_v - g_a \gamma _ 5)\right . + \frac{q_\mu } { m^2_h } \not \ ! k ( h_v - h_a \gamma _ 5)+ \frac{\epsilon _ { \alpha \mu \beta \lambda } } { m^2_h } p^\alpha q^\beta \gamma ^\lambda ( k_v - k_a \gamma _ 5)\right ] \nonumber \\&\quad \times \,\ , v(q_1 ) \end{aligned}$$\end{document}m3,=i21/4gf1/2rs - ru(q2)2f1(gv - ga5)+qmh2k(hv - ha5)+mh2pq(kv - ka5)v(q1)and4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \mathcal m}_{2,\mu } = i \bar{u}(p_2)\gamma _ \mu ( g_v - g_a \gamma _ 5 ) v(p_1 ) . \end{aligned}$$\end{document}m2,=iu(p2)(gv - ga5)v(p1).the form of the amplitude in ( 3 ) is valid through next - to - leading order ( nlo ) of the general electroweak effective lagrangian described in and in sect . the form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a are functions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r$$\end{document}r and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s$$\end{document}s . the global normalization of the amplitude has been chosen such that in the standard model at leading order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1\equiv 1$$\end{document}f11 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v = g_v$$\end{document}gv = gv , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_a = g_a$$\end{document}ga = ga . summing over the final - state lepton polarizations gives5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left| { \mathcal m}^\mu _ 3 { \mathcal m}_{2,\mu } \right| ^2 = \sqrt{2 } g_f m^4_h \left ( \frac{r}{r - s}\right ) ^2 j(r , s,\alpha , \beta , \phi ) \end{aligned}$$\end{document}m3m2,2=2gfmh4rr - s2j(r , s,,,)where6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&j(r , s,\alpha , \beta , \phi ) = j_1\frac{9}{40}(1+\cos ^2\alpha \cos ^2\beta ) \nonumber \\&\quad + \,\,j_2\frac{9}{16}\sin ^2\alpha \sin ^2\beta + j_3 \cos \alpha \cos \beta \nonumber \\&\quad + \,\,\left ( j_4 \sin \alpha \sin \beta \right . \phi \nonumber \\&\quad + \,\,j_8 \sin ^2\alpha \sin ^2\beta \sin 2\phi + j_9 \sin ^2\alpha \sin ^2\beta \cos 2\phi . \end{aligned}$$\end{document}j(r , s,,,)=j1940(1+cos2cos2)+j2916sin2sin2+j3coscos+j4sinsin+j5sin2sin2sin+j6sinsin+j7sin2sin2cos+j8sin2sin2sin2+j9sin2sin2cos2.the previous expression factors out the angular dependence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji being dynamical functions which depend only on the invariant masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r$$\end{document}r , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s$$\end{document}s . they are given by7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } j_1&= \frac{640}{9 } f^2_1 ( g^2_v + g^2_a ) ( g^2_v + g^2_a ) r s\nonumber \\ j_2&= \frac{32}{9 } f_1 ( g^2_v + g^2_a ) \left [ 2 f_1(g^2_v + g^2_a ) ( \lambda ^2 + 2 r s)\right . + \,\,(g_v h_v + g_a h_a)\lambda ^2 ( 1-r - s)\right ] \nonumber \\ j_3&= 128 f^2_1 g_v g_a g_v g_a r s\nonumber \\ j_4&= 8 f_1 ( g_v k_a+ g_a k_v)g_v g_a \lambda \sqrt{r s } ( 1-r - s)\nonumber \\ j_5&= f_1 ( g_v k_v + g_a k_a ) ( g^2_v + g^2_a ) \lambda \sqrt{r s } ( 1-r - s)\nonumber \\ j_6&= -8f_1 g_v g_a \sqrt{r s } \left [ 8 f_1 g_v g_a(1-r - s ) \right . \nonumber \\&\left . + ( g_v h_a + g_a h_v)\lambda ^2\right ] \nonumber \\ j_7&= -f_1 ( g^2_v + g^2_a ) \sqrt{r s } \left [ 4 f_1(g^2_v + g^2_a ) ( 1-r - s)\right . j_8&= -4 f_1 ( g_v k_v + g_a k_a ) ( g^2_v+ g^2_a ) \lambda r s\nonumber \\ j_9&= 8 f^2_1 ( g^2_v + g^2_a ) ( g^2_v + g^2_a ) r s. \end{aligned}$$\end{document}j1=6409f12(gv2+ga2)(gv2+ga2)rsj2=329f1(gv2+ga2)2f1(gv2+ga2)(2 + 2rs)+(gvhv+gaha)2(1-r - s)j3=128f12gvgagvgarsj4=8f1(gvka+gakv)gvgars(1-r - s)j5=f1(gvkv+gaka)(gv2+ga2)rs(1-r - s)j6=-8f1gvgars8f1gvga(1-r - s)+(gvha+gahv)2j7=-f1(gv2+ga2)rs4f1(gv2+ga2)(1-r - s)+(gvhv+gaha)2j8=-4f1(gvkv+gaka)(gv2+ga2)rsj9=8f12(gv2+ga2)(gv2+ga2)rs.as will be explained in more detail in sect . 3 , the form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a receive leading - order contributions in the standard model , whereas \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a only arise as next - to - leading - order corrections and capture , respectively , cp - even and cp - odd contributions . in writing the expression for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji , we have therefore consistently neglected terms of second order in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a . it follows that to leading order in the standard model the observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_4$$\end{document}j4 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_5$$\end{document}j5 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_8$$\end{document}j8 , which carry the dependence on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a , are zero , as one would expect from general cp considerations . with sufficient data , a general fit to the angular distribution of the four final - state leptons could in principle extract all nine terms \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji in the fully differential decay rate ( 1 ) , ( 5 ) and ( 6 ) . from ( 7 ) we see that measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_1$$\end{document}j1 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ldots $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_6$$\end{document}j6 , for example , would determine the 6 independent combinations8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \begin{array}{l@{\quad } l } g^2_v+ g^2_a , & { } g_v g_a\\ g_v h_v + g_a h_a,&{}g_v h_a + g_a h_v\\ g_v k_v + g_a k_a,&{}g_v k_a + g_a k_v \end{array}. \end{aligned}$$\end{document}gv2+ga2,gvgagvhv+gaha , gvha+gahvgvkv+gaka , gvka+gakv.all of the six form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a could then be obtained . the remaining three observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_7$$\end{document}j7 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_8$$\end{document}j8 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_9$$\end{document}j9 give no independent information on these form factors . the coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a are not directly related to the process \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- and have to be constrained independently from properties of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z decays . with limited data , it is more efficient to extract the different \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji projecting them from ( 6 ) . integrating the distribution in ( 6 ) over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document} we are left with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_1$$\end{document}j1 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_2$$\end{document}j2 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 as the only observables . integrating in addition over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} eliminates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 . thus , the differential rate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\gamma /ds$$\end{document}d/ds , fully integrated over the angular variables , remains sensitive only to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_1+j_2$$\end{document}j1+j2 . performing the angular integrations one obtains the dilepton - mass spectrum of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- rate , multiplied by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{'+}\ell ^{'-}$$\end{document}z+- branching fraction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\ell $ $ \end{document}b. from ( 1 ) one finds9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{d\gamma } { ds}&= b_\ell \ , \frac{g_f m^3_h}{\sqrt{2}\ , 192\pi ^3 } \frac{\lambda r}{(r - s)^2}\nonumber \\&\times \,\,f_1 \left [ 2 f_1(g^2_v + g^2_a ) ( \lambda ^2 + 12 r s)\right . + \,\,(g_v h_v + g_a h_a)\lambda ^2 ( 1-r - s)\right ] \end{aligned}$$\end{document}dds = bgfmh321923r(r - s)2f12f1(gv2+ga2)(2 + 12rs)+(gvhv+gaha)2(1-r - s)where10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } b_\ell = \frac{(g^2_v + g^2_a ) m_z}{12\pi \gamma _ \end{aligned}$$\end{document}b=(gv2+ga2)mz12z.in contrast , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ldots $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_9$$\end{document}j9 have to be accessed with appropriate angular asymmetries . for instance , the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 can be extracted by integrating over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document} and forming a suitable forward backward asymmetry in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cos \alpha $ $ \end{document}cos and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cos \beta $ $ \end{document}cos. in sect . 4 we examine this and other angular asymmetries in detail . the angular distribution in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- is similar to the one in the rare \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b$$\end{document}b - meson decay \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b\rightarrow k^*\ell ^+\ell ^-$$\end{document}bk+- , which has been discussed for instance in [ 2023 ] . however , in the present case the angles \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} are on an equal footing , and accordingly the angular dependence in ( 6 ) is symmetric under the interchange of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document}. note in particular that the forward backward asymmetry term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 is proportional to the product \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cos \alpha \ , \cos \beta $ $ \end{document}coscos , thus representing a kind of correlated double asymmetry in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document}. it vanishes when either \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} are integrated over their full range . this is in contrast to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b\rightarrow k^*\ell ^+\ell ^-$$\end{document}bk+- , where a forward backward asymmetry in the single angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} exists due to the more complicated structure of the hadronic transition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b\rightarrow k^*$$\end{document}bk. in order to estimate the form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a in ( 3 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a in ( 4 ) we will work with the nonlinear effective lagrangian discussed in [ 15 , 16 ] . a subset of the relevant operators has also been discussed in [ 24 , 25 ] . in this framework , electroweak symmetry breaking is realized by spontaneously breaking a global \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$su(2)_l\times su(2)_r$$\end{document}su(2)lsu(2)r down to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$su(2)_v$$\end{document}su(2)v . the resulting goldstone modes are then collected into a matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u$$\end{document}u transforming as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_l u g_r^{\dagger } $ $ \end{document}glugr under the global group . one also defines11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } d_\mu u=\partial _ \mu u+i g w_\mu u -i g ' b_\mu u t_3 \end{aligned}$$\end{document}du=u+igwu - igbut3such that the sm subgroup \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$su(2)_l\times u(1)_y$$\end{document}su(2)lu(1)y is gauged . for convenience we will use the shorthand notation12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } l_{\mu } = iud_{\mu } u^{\dagger } , \qquad \tau _ l = ut_3u^{\dagger } \end{aligned}$$\end{document}l=iudu,l = ut3ufor the goldstone covariant derivative and the custodial symmetry breaking spurion \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_3$$\end{document}t3 . the higgs field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h is introduced as an additional light ( pseudo - goldstone ) boson , singlet under the sm gauge group . with these definitions one has at leading order [ 16 , 26 , 27]13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \mathcal { l}}_{lo}&= -\frac{1}{2}\langle w_{\mu \nu } w^{\mu \nu } \rangle -\frac{1}{4 } b_{\mu \nu } b^{\mu \nu } + i\!\sum _ { f}\bar{\psi } _ f \!\not \!\ ! f\nonumber \\&+\frac{v^2}{4}\ \langle l_\mu l^\mu \rangle f\left ( \frac{h}{v}\right ) -\frac{1}{2}h(\partial ^2+m^2_h)h - v(h).\nonumber \\ \end{aligned}$$\end{document}llo=-12ww-14bb+iffdf+v24llfhv-12h(2+mh2)h - v(h).for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z{\ell } ^+{\ell } ^-$$\end{document}hz+- the final - state fermions can be taken massless to an excellent approximation and therefore we have omitted the yukawa terms above . the main contribution to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- comes from the subprocess \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow zz^*$$\end{document}hzz , which is described by the gauge - boson mass term , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(h / v)$$\end{document}f(h / v ) can be truncated at linear order for the process of interest here:14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } f\left ( \frac{h}{v}\right ) = 1 + 2a\frac{h}{v}. \end{aligned}$$\end{document}fhv=1 + 2ahv.at next - to - leading order ( nlo ) there are eight relevant cp - even operators15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&{\mathcal { o}}_{xh1}=g^{\prime 2 } b_{\mu \nu } b^{\mu \nu } \frac{h}{v } f_{xh1}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{xh2}=g^2\langle w_{\mu \nu } w^{\mu \nu } \rangle \frac{h}{v } f_{xh2}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{xu1}=g^{\prime } gb_{\mu \nu } \langle w^{\mu \nu } \tau _ l\rangle f_{xu1}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{xu2}=g^2 \langle w^{\mu \nu } \tau _ l\rangle ^2 f_{xu2}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{v7}=-{\bar{l}}\gamma _ { \mu } l\langle \tau _ ll^{\mu } \rangle f_{v7}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{v8}=-{\bar{l}}\gamma _ { \mu } \tau _ l l\langle \tau _ ll^{\mu } \rangle f_{v8}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{v10}=-{\bar{e}}\gamma _ { \mu } e\langle \tau _ ll^{\mu } \rangle f_{v10}\left ( \frac{h}{v}\right ) , \nonumber \\&{\mathcal { o}}_{\beta _ 1}= -v^2\langle \tau _ ll_{\mu } \rangle ^2f_{\beta _ 1}\left ( \frac{h}{v}\right ) , \end{aligned}$$\end{document}oxh1=g2bbhvfxh1hv , oxh2=g2wwhvfxh2hv , oxu1=ggbwlfxu1hv , oxu2=g2wl2fxu2hv , ov7=-llllfv7hv , ov8=-lllllfv8hv , ov10=-eellfv10hv , o1=-v2ll2f1hv , and four cp - odd ones:16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \mathcal { o}}_{xh4}&= g^{\prime 2 } \epsilon _ { \mu \nu \lambda \rho } b^{\mu \nu } b^{\lambda \rho } \frac{h}{v } f_{xh3}\left ( \frac{h}{v}\right ) , \nonumber \\ { \mathcal { o}}_{xh5}&= g^2\epsilon _ { \mu \nu \lambda \rho } \langle w^{\mu \nu } w^{\lambda \rho } \rangle \frac{h}{v } f_{xh4}\left ( \frac{h}{v}\right ) , \nonumber \\ { \mathcal { o}}_{xu4}&= g^{\prime } g \epsilon _ { \mu \nu \lambda \rho } b^{\mu \nu } \langle w^{\lambda \rho } \tau _ l\rangle f_{xu4}\left ( \frac{h}{v}\right ) , \nonumber \\ { \mathcal { o}}_{xu5}&= g^2\epsilon _ { \mu \nu \lambda \rho } \langle \tau _ l w^{\mu \nu } \rangle \langle \tau _ l w^{\lambda \rho } \rangle f_{xu5}\left ( \frac{h}{v}\right ) . \end{aligned}$$\end{document}oxh4=g2bbhvfxh3hv , oxh5=g2wwhvfxh4hv , oxu4=ggbwlfxu4hv , oxu5=g2lwlwfxu5hv.some comments are in order : fermionic tensor operators are in principle also present , but they turn out to be negligible : first , they have a chiral suppression and second , they do not interfere with the standard model and thus can only appear at nnlo.for simplicity , the list above includes only fermions of the first family . the extension to include the second family is , however , trivial.the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_i(h / v)$$\end{document}fi(h / v ) above are generic functions with model - dependent coefficients . as a result , the previous operators contain all the possible powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h . in the following , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_i$$\end{document}ai and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_i$$\end{document}bi will denote , respectively , the dimensionless wilson coefficients for the pieces without \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h / v$$\end{document}h / v and linear in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h / v$$\end{document}h / v , which are the relevant ones for the process under study . fermionic tensor operators are in principle also present , but they turn out to be negligible : first , they have a chiral suppression and second , they do not interfere with the standard model and thus can only appear at nnlo . for simplicity , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_i(h / v)$$\end{document}fi(h / v ) above are generic functions with model - dependent coefficients . as a result , the previous operators contain all the possible powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h . in the following , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_i$$\end{document}ai and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_i$$\end{document}bi will denote , respectively , the dimensionless wilson coefficients for the pieces without \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h / v$$\end{document}h / v and linear in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h / v$$\end{document}h / v , which are the relevant ones for the process under study . the operators above give the most general direct contributions to the vertices of fig . 1 , but they also lead to a renormalization of the fields and parameters [ 28 , 29 ] . these effects will be consistently included in all our results . as the fundamental electroweak parameters we will employ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ { em}=e^2/4\pi $ $ \end{document}em = e2/4 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_z$$\end{document}mz and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_f$$\end{document}gf ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - standard definition ) . then the nlo corrections can finally be expressed in terms of the following effective interactions:17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \mathcal { l}}_{nlo}&= 2^{1/4 } g^{1/2}_f m_z^2f_1\ , h\ , z^{\mu } z_{\mu } + b_2 \frac{h}{v}z^{\mu \nu } z_{\mu \nu } \nonumber \\&+b_2^{\gamma } \frac{h}{v}z^{\mu \nu } a_{\mu \nu } + b_3 \frac{h}{v } \epsilon _ { \mu \nu \lambda \rho } z^{\mu \nu } z^{\lambda \rho } \nonumber \\&+b_3^{\gamma } \frac{h}{v } \epsilon _ { \mu \nu \lambda \rho } z^{\mu \nu } a^{\lambda \rho } + z_\mu { \bar{l}}\gamma ^{\mu } \left [ g_v - g_a\gamma _ 5\right ] l\nonumber \\&+ \frac{h}{v } z_\mu { \bar{l}}\gamma ^{\mu } \left [ h_v - h_a\gamma _ 5\right ] l. \end{aligned}$$\end{document}lnlo=21/4gf1/2mz2f1hzz+b2hvzz+b2hvza+b3hvzz+b3hvza+zlgv - ga5l+hvzlhv - ha5l.for convenience , we have defined18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_v&= \frac{g}{4 c_z}(\kappa _ 1 - 4 s^2_z\kappa _ 2),\nonumber \\ g_a&= \frac{g}{4 c_z}\kappa _ 1 \end{aligned}$$\end{document}gv = g4cz(1 - 4sz22),ga = g4cz1such that at leading order in the standard model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa _ i=1$$\end{document}i=1 . here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_z$$\end{document}sz ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_z$$\end{document}cz ) denotes the sine ( cosine ) of the weinberg angle in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - standard definition ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha = \alpha ( m_z)$$\end{document}=(mz))19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } s^2_z c^2_z\equiv \frac{\pi \alpha } { \sqrt{2 } g_f m^2_z } \end{aligned}$$\end{document}sz2cz22gfmz2and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g$$\end{document}g is the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$su(2)_l$$\end{document}su(2)l gauge coupling , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g s_z = e=\sqrt{4\pi \alpha } $ $ \end{document}gsz = e=4. by analogy to ( 18 ) we have defined20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } h_v&= \frac{g}{4 c_z}(\omega _ 1 - 4 s^2_z\omega _ \end{aligned}$$\end{document}hv = g4cz(1 - 4sz22),ha = g4cz1.in terms of the coefficients of ( 17 ) , the form factors read21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_v&= g_v \left ( 1-\frac{b_2}{f_1}\frac{1-r - s}{r}\right ) -\frac{b_2^{\gamma } e}{2 f_1}\frac{(1-r - s)(s - r)}{r s}\nonumber \\&+ \frac{h_v}{2 f_1 } \frac{s - r}{r},\nonumber \\ g_a&= g_a \left ( 1-\frac{b_2}{f_1}\frac{1-r - s}{r}\right ) + \frac{h_a}{2 f_1 } \frac{s - r}{r},\nonumber \\ \end{aligned}$$\end{document}gv = gv1-b2f11-r - sr - b2e2f1(1-r - s)(s - r)rs+hv2f1s - rr , ga = ga1-b2f11-r - sr+ha2f1s - rr , hv=4b2rgv+2b2es - rrs , ha=4b2rga , kv=-8b3rgv-4b3es - rrs , ka=-8b3rga.in turn , the operators in the lagrangian ( 17 ) can be expressed in terms of the basic eft operators listed before . for the coefficients this implies ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_z = s_z / c_z$$\end{document}tz = sz / cz),22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&f_1=a(1 + 2\beta _ 1 -\delta _ + \frac{t_z^{-1}}{2}b_{xu2,5}\right ) , \end{aligned}$$\end{document}f1=a(1 + 21-g)-b1,b2,3=e222tz2bxh1,4+tz-2bxh2,5-bxu1,4+tz-22bxu2,5,b2,3=e2 - 2tzbxh1,4+tz-1bxh2,5 + 12(tz-1-tz)bxu1,4+tz-12bxu2,5,and23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \kappa _ 2&\equiv 1+\frac{1}{2s^2_z } a_{v10}+\frac{\delta _ g-\beta _ 1 - a_{xu1 } e^2/s^2_z}{c^2_z - s^2_z},\nonumber \\ \omega _ 1&\equiv -b_{v7}+\frac{1}{2 } b_{v8 } + b_{v10},\nonumber \\ \omega _ 2&\equiv \frac{1}{2s^2_z } b_{v10}. \end{aligned}$$\end{document}11-av7 + 12av8+av10+1-g,21 + 12sz2av10+g-1-axu1e2/sz2cz2-sz2,1-bv7 + 12bv8+bv10,212sz2bv10.for simplicity , in ( 23 ) we have dropped the family indices , but one should keep in mind that the nlo corrections are in general different for electron and muon final states . incidentally , notice that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa _ i$$\end{document}i also contain a universal ( family - independent ) contribution , proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ 1$$\end{document}1 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ g$$\end{document}g , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{xu1}$$\end{document}axu1 , which results from taking into account the nlo renormalization effects . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ g$$\end{document}g above stands for the renormalization of the fermi constant , which includes 4-fermion operators not listed in ( 15 ) . 1different contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- different contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- for completeness we will also discuss the weakly coupled case using the eft developed in using the notation of ( for different approaches see [ 32 , 33 ] ) . the relevant operators are now24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&{\mathcal { o}}_{hb}= g^{\prime 2}b_{\mu \nu } b^{\mu \nu } h^{\dagger } h,\nonumber \\&{\mathcal { o}}_{hw}=g^2\langle w_{\mu \nu } w^{\mu \nu } \rangle h^{\dagger } h,\nonumber \\&{\mathcal { o}}_{hwb}=gg^{\prime } h^{\dagger } w_{\mu \nu } h b^{\mu \nu } , \nonumber \\&{\mathcal { o}}_{hd}=|h^{\dagger } d_{\mu } h|^2,\nonumber \\&{\mathcal { o}}_{hl}^{(1)}=(h^{\dagger } i\mathop { d}\limits ^{\leftrightarrow } _ { \mu } h ) ( { \bar{l } } \gamma ^{\mu } l),\nonumber \\&{\mathcal { o}}_{hl}^{(3)}=(h^{\dagger } i\mathop { d^a}\limits ^{\leftrightarrow } _ { \!\!\!\!\mu } h ) ( { \bar{l}}\gamma ^{\mu } \tau _ a l),\nonumber \\&{\mathcal { o}}_{he}=(h^{\dagger } i\mathop { d}\limits ^{\leftrightarrow } _ { \mu } h ) ( { \bar{e}}\gamma ^{\mu } e),\nonumber \\&{\mathcal { o}}_{h\box } = ( h^{\dagger } h)\box ( h^{\dagger } h ) , \end{aligned}$$\end{document}ohb = g2bbhh , ohw = g2wwhh , ohwb = gghwhb,ohd=|hdh|2,ohl(1)=(hidh)(ll),ohl(3)=(hidah)(lal),ohe=(hidh)(ee),oh=(hh)(hh),and ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde{x}}_{\mu \nu } = \epsilon _ { \mu \nu \lambda \rho } x^{\lambda \rho } $ $ \end{document}x~=x)25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&{\mathcal { o}}_{h{\tilde{w}}}=g^2\langle { \tilde{w}}_{\mu \nu } w^{\mu \nu } \rangle h^{\dagger } h,\nonumber \\&{\mathcal { o}}_{h{\tilde{b}}}=g^{\prime 2}{\tilde{b}}_{\mu \nu } b^{\mu \nu } h^{\dagger } h,\nonumber \\&{\mathcal { o}}_{h{\tilde{w}}b}=gg^{\prime } h^{\dagger } { \tilde{w}}_{\mu \nu } h b^{\mu \nu } \end{aligned}$$\end{document}ohw~=g2w~whh , ohb~=g2b~bhh , ohw~b = gghw~hbfor the cp - even and cp - odd sectors , respectively . the effect of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}_{h\box } $ $ \end{document}oh is to renormalize the higgs kinetic term . this shift can be absorbed by a field redefinition of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h , which then affects the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow zz$$\end{document}hzz coupling . this is of no relevance for the distributions but affects the global normalization of the decay . for comparison with the nonlinear case it is convenient to define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bar{\alpha } } _ j = v^2\alpha _ j$$\end{document}j = v2j . the result reads26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&f_1=\left ( 1+{\bar{\alpha } } _ { h\box } \right . \left . + \frac{{\bar{\alpha } } _ { hd}}{4}-\delta _ g\right ) , \nonumber \\&b_{2,3}=\frac{e^2}{2}\left ( 2t_z^2{\bar{\alpha } } _ { hb , h{\tilde{b}}}\right . \left . + t_z^{-2}{\bar{\alpha } } _ { hw , h{\tilde{w}}}+{\bar{\alpha } } _ { hwb , h{\tilde{w}}b}\right ) , \nonumber \\&b_{2,3}^{\gamma } = e^2\left ( -2t_z { \bar{\alpha } } _ { hb , h{\tilde{b}}}\right . -\frac{1}{2}(t_z^{-1}-t_z){\bar{\alpha } } _ { hwb , h{\tilde{w}}b}\right ) \end{aligned}$$\end{document}f1=1+h+hd4-g , b2,3=e222tz2hb , hb~+tz-2hw , hw~+hwb , hw~b , b2,3=e2 - 2tzhb , hb~+tz-1hw , hw~-12(tz-1-tz)hwb , hw~band27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \kappa _ 1&\equiv 1+({\bar{\alpha } } _ { hl1}+{\bar{\alpha } } _ { hl3}-{\bar{\alpha } } _ { he})- \frac{{\bar{\alpha } } _ { hd}}{4}-\delta _ g,\nonumber \\ \kappa _ 2&\equiv 1-\frac{1}{2s^2_z } { \bar{\alpha } } _ { he}+\frac{1}{c^2_z - s^2_z}\left ( \frac{{\bar{\alpha } } _ { hd}}{4}\right . \left . + e^2\frac{{\bar{\alpha } } _ { hwb}}{2s_z^2}+\delta _ g\right ) \nonumber \\ \omega _ 1&\equiv 2({\bar{\alpha } } _ { hl1}+ { \bar{\alpha } } _ { hl3}-{\bar{\alpha } } _ { he}),\nonumber \\ \omega _ 2&\equiv -\frac{1}{s^2_z } { \bar{\alpha } } _ { he}. \end{aligned}$$\end{document}11+(hl1+hl3-he)-hd4-g,21 - 12sz2he+1cz2-sz2hd4+e2hwb2sz2+g12(hl1+hl3-he),2-1sz2he.it is worth noting that , while the contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h z\ell ^+\ell ^-$$\end{document}hz+- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z \ell ^+\ell ^-$$\end{document}z+- , encoded in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ i$$\end{document}i and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa _ i$$\end{document}i , respectively , come from the same ( family - dependent ) nlo operators , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa _ i$$\end{document}i also receives a universal nlo renormalization through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}_{hd}$$\end{document}ohd , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}_{hwb}$$\end{document}ohwb , and the operators associated with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ g$$\end{document}g . therefore , the contact term contribution to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- is in general uncorrelated to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^+\ell ^-$$\end{document}z+- , even in the case of the linearly realized higgs sector . similarly , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z mass term and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow zz$$\end{document}hzz vertex come from the same lo operator but nlo corrections renormalize them differently . as a result , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta f_1\ne 0$$\end{document}f10 in ( 26 ) . 2 we pointed out that at nlo there are six independent form factors entering the dynamical functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji . with high enough statistics one can fit the full distribution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j$$\end{document}j to experimental data . however , at least in the first stages of the run 2 at the lhc , where statistics will be rather limited , it is more efficient to devise a set of observables that can project out the different form factor combinations through angular asymmetries . a possible strategy is to extract \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_vg_a$$\end{document}gvga from the forward backward asymmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} , after integration over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}:28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } a_{\alpha \beta } & = \left ( \frac{d\gamma } { ds}\right ) ^{-1 } \int \limits _ { -1}^{1 } d\!\cos \alpha \ , { \mathrm { sgn}}(\cos \alpha ) \nonumber \\&\int \limits _ { -1}^{1}d\!\cos \beta \ , { \mathrm { sgn}}(\cos \beta ) \displaystyle \frac{d\gamma } { ds\ , d\!\cos \alpha \ , d\!\cos \beta } \nonumber \\&= \frac{j_3}{j_1+j_2 } \end{aligned}$$\end{document}a=dds-1-11dcossgn(cos)-11dcossgn(cos)ddsdcosdcos=j3j1+j2and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(g_v^2+g_a^2)$$\end{document}(gv2+ga2 ) from an asymmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_\phi $ $ \end{document}a in the angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}:29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } a_{\phi } & = \left ( \frac{d\gamma } { ds}\right ) ^{-1}\int \limits _ 0^{2\pi } d\phi \ , { \mathrm { sgn}}(\cos 2\phi ) \displaystyle \frac{d\gamma } { dsd\phi } = \frac{32}{9\pi } \frac{j_9}{j_1+j_2}.\nonumber \\ \end{aligned}$$\end{document}a=dds-102dsgn(cos2)ddsd=329j9j1+j2.knowing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\phi } $ $ \end{document}a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a can be determined through the combinations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_vh_v+g_ah_a\simeq g_ah_a$$\end{document}gvhv+gahagaha and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_vh_a+g_ah_v\simeq g_ah_v$$\end{document}gvha+gahvgahv . these can be extracted , respectively , from the total rate given in ( 9 ) and the asymmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\phi $ $ \end{document}b,30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } b_{\phi } = \left ( \frac{d\gamma } { ds}\right ) ^{-1}\int \limits _ 0^{2\pi } d\phi \ , { \mathrm { sgn}}(\cos \phi ) \displaystyle \frac{d\gamma } { dsd\phi } = \frac{\pi } { 2}\frac{j_6}{j_1+j_2}.\nonumber \\ \end{aligned}$$\end{document}b=dds-102dsgn(cos)ddsd=2j6j1+j2.the observables discussed so far test new physics in the cp - even sector . cp - odd contributions are parametrized by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a , which can be determined through the structures \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_vk_v+g_ak_a\simeq g_ak_a$$\end{document}gvkv+gakagaka and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_vk_a+g_ak_v\simeq g_ak_v$$\end{document}gvka+gakvgakv . they can be extracted from 2 additional asymmetries in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}:31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } c_{\phi } & = \left ( \frac{d\gamma } { ds}\right ) ^{-1}\int \limits _ 0^{2\pi } d\phi \ , { \mathrm { sgn}}(\sin 2\phi ) \displaystyle \frac{d\gamma } { dsd\phi } = \frac{32}{9\pi } \frac{j_8}{j_1+j_2 } , \nonumber \\ d_{\phi } & = \left ( \frac{d\gamma } { ds}\right ) ^{-1}\int \limits _ 0^{2\pi } d\phi \ , { \mathrm { sgn}}(\sin \phi ) \displaystyle \frac{d\gamma } { dsd\phi } = \frac{\pi } { 2}\frac{j_4}{j_1+j_2}.\nonumber \\ \end{aligned}$$\end{document}c=dds-102dsgn(sin2)ddsd=329j8j1+j2,d=dds-102dsgn(sin)ddsd=2j4j1+j2.similar cp - odd observables have been discussed previously in the literature [ 3538 ] . in order to assess the experimental relevance of these asymmetries , we will rely on numerical estimates of new - physics effects based on general power - counting arguments . accordingly , one would naively expect the nlo coefficients given in the previous section to be generically of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(v^2/\lambda ^2)$$\end{document}o(v2/2 ) , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda \sim 4\pi v$$\end{document}4v . therefore , keeping track of the gauge couplings , we will assume \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1=a+{\mathcal { o}}(v^2/\lambda ^2)$$\end{document}f1=a+o(v2/2 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}=g_{v , a}^{(0)}+g{\mathcal { o}}(v^2/\lambda ^2)$$\end{document}gv , a = gv , a(0)+go(v2/2 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{2,3}^{(\gamma ) } \sim e^2{\mathcal { o}}(v^2/\lambda ^2)$$\end{document}b2,3()e2o(v2/2 ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}\sim g { \mathcal { o}}(v^2/\lambda ^2)$$\end{document}hv , ago(v2/2 ) . the main source of deviations from the sm comes from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1$$\end{document}f1 . this parameter measures the signal strength of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow zz^*$$\end{document}hzz , and it is currently constrained to deviate less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$20\,\%$$\end{document}20% from the sm . since our conclusions will be independent of it , we will set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a=1$$\end{document}a=1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1=1$$\end{document}f1=1 for simplicity . new - physics corrections are then naturally dominated by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta g_{v , a}$$\end{document}gv , a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta g_{v , a}$$\end{document}gv , a are constrained by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z partial width and lep data sets bounds on them at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{-3}$$\end{document}10 - 3 level [ 33 , 39 ] , which is within the eft expectation . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a are instead unconstrained , and might in principle attain values larger than the naive eft dimensional estimate because of numerical enhancements . consider , for instance , the local \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- couplings \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a to be induced by the tree - level exchange of a composite heavy vector resonance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r$$\end{document}r , mediating \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z r^*$$\end{document}hzr , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r^*\rightarrow \ell ^+\ell ^-$$\end{document}r+- . then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}\sim v^2/m^2_r\sim v^2/\lambda ^2$$\end{document}hv , av2/mr2v2/2 . if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_r$$\end{document}mr is numerically smaller than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda \approx 3\,\mathrm{tev}$$\end{document}3tev by a factor of three , say , the resulting value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a might be 510 times bigger than the naive eft estimate . this assumes consistency with other phenomenological constraints , which is plausible in view of the free parameters in this scenario . for simplicity we will consider a scenario where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}\ne 0$$\end{document}hv , a0 , with all other corrections set to zero . due to the smallness of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv in the sm , the most sensitive probes of new physics are those linear in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv , namely \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b , with corrections that can easily reach 50100 % . incidentally , notice that neither \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a nor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b are constrained by the angular distributions collected for the spin - parity analysis . this has to be compared with the mass distribution , with typical corrections of a few \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\%$$\end{document}% qualitatively , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_v$$\end{document}hv controls \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_a$$\end{document}ha affects the mass distribution . thus , one can get large corrections on the former while barely affecting the latter . the reason for this is the accidental suppression of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv relative to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_a$$\end{document}ga in the standard model by about an order of magnitude . a typical correction from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_v$$\end{document}hv therefore has a larger impact on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_a$$\end{document}ha has on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_a$$\end{document}ga ; see eq . asymmetries that are proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v } g_{a}$$\end{document}gvga are then rather sensitive to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_v$$\end{document}hv . on the other hand , the mass distribution is governed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^2_v + g^2_a$$\end{document}gv2+ga2 , which is approximately given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^2_a$$\end{document}ga2 and thus mainly affected by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_a$$\end{document}ha . whereas \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_v$$\end{document}hv and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_a$$\end{document}ha are parametrically of the same order of magnitude , we find it interesting for illustration to consider a situation where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_v$$\end{document}hv is numerically enhanced over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_a$$\end{document}ha . this could be realized in scenarios with a vector resonance that is lighter than the lowest axial vector state . in fig . 2 we illustrate such scenarios for the parameter choices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-2,0.3)$$\end{document}(hv , ha)=v2/2(-2,0.3 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-6,0.3)$$\end{document}(hv , ha)=v2/2(-6,0.3).fig . 2values for the angular asymmetries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b defined in the main text . the dashed line corresponds to the sm prediction , while the solid lines incorporate potential new - physics effects for the parameter choices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-2,0.3)$$\end{document}(hv , ha)=v2/2(-2,0.3 ) ( in blue ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-6,0.3)$$\end{document}(hv , ha)=v2/2(-6,0.3 ) ( in red ) . for comparison , the lower panel shows the differential mass distribution ( in units of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{-6}$$\end{document}10 - 6 gev ) . the plots illustrate the high sensitivity of the angular asymmetries to new physics for scenarios where the mass distribution is left almost unaffected values for the angular asymmetries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b defined in the main text . the dashed line corresponds to the sm prediction , while the solid lines incorporate potential new - physics effects for the parameter choices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-2,0.3)$$\end{document}(hv , ha)=v2/2(-2,0.3 ) ( in blue ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h_v , h_a)=v^2/\lambda ^2(-6,0.3)$$\end{document}(hv , ha)=v2/2(-6,0.3 ) ( in red ) . for comparison , the lower panel shows the differential mass distribution ( in units of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{-6}$$\end{document}10 - 6 gev ) . the plots illustrate the high sensitivity of the angular asymmetries to new physics for scenarios where the mass distribution is left almost unaffected with the lhc running at 14 tev and with an integrated luminosity of 3000 fb\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{-1}$$\end{document}-1 , one expects around 6400 reconstructed events for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- . with such statistics one could in principle reach a 12 % sensitivity in the observables that we are discussing . since the overall effects for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b lie around the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\%$$\end{document}% level , as illustrated in fig . 2 , they could be accessible at the lhc , at least in its final stage . regarding the cp - odd sector , within the range of validity of our eft , the asymmetries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_{\phi } $ $ \end{document}c and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } 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} \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(g_v^2+g_a^2)$$\end{document}(gv2+ga2 ) with higher precision than through the total decay rate . before closing this section , one should note that , strictly speaking , the form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_i , h_i , k_i$$\end{document}gi , hi , ki always appear in combination with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a in the products32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \begin{array}{l@{\quad } l } ( g_v^2+g_a^2)(g_v^2+g_a^2),&{}(g_vg_a)(g_vg_a),\\ ( g_vh_v+g_ah_a)(g_v^2+g_a^2),&{}(g_vh_a+g_ah_v)(g_vg_a),\\ ( g_vk_v+g_ak_a)(g_v^2+g_a^2 ) , & { } ( g_vk_a+g_ak_v)(g_vg_a),\\ \end{array}\nonumber \\ \end{aligned}$$\end{document}(gv2+ga2)(gv2+ga2),(gvga)(gvga),(gvhv+gaha)(gv2+ga2),(gvha+gahv)(gvga),(gvkv+gaka)(gv2+ga2),(gvka+gakv)(gvga),which account for the processes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{\prime + } \ell ^{\prime -}$$\end{document}z+- , respectively . in order to determine \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_i , h_i , k_i$$\end{document}gi , hi , ki with a certain precision , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a should be known comparably well . unfortunately , with lep data the bounds on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_a$$\end{document}ga are too loose to be informative . in contrast , the ilc could offer a clean determination of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ze^+e^-$$\end{document}ze+e- couplings , since the center - of - mass enhanced corrections to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w^+w^-$$\end{document}w+w- production can be cast entirely in terms of these corrections . as a result , they get singled out at high energies and , within the ilc energy - range , they can naturally be boosted to a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$20\,\%$$\end{document}20% correction to the production cross section . an analogous mechanism for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\mu ^+\mu ^-$$\end{document}z+- couplings could in principle be pursued in a muon linear collider through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^+\mu ^-\rightarrow w^+w^-$$\end{document}+-w+w- . we have studied , in a general and systematic way , how the decay \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- can be used to probe for physics beyond the standard model in the higgs sector . for this purpose we have employed a general parametrization of the amplitude in terms of form factors , neglecting lepton masses . in view of the large gap between the electroweak scale and the expected scale of new physics we have computed the form factors in terms of the coefficients of an effective lagrangian , which is defined by the sm gauge symmetries , a light scalar singlet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h , and the remaining sm particles , but is otherwise completely general . the main points of our analysis can be summarized as follows.we discuss the most general observables arising from the full angular distribution of the 4-lepton final state in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{'+}\ell ^{'-}$$\end{document}z+- . the nine coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji describing the angular distribution are expressed through the six form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a.interesting observables , besides the dilepton - mass spectrum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\gamma /ds$$\end{document}d/ds , can be constructed from the angular distribution . examples are : the forward backward asymmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_6$$\end{document}j6 . these quantities are strongly suppressed in the sm because of the smallness of the vectorial coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv . on the other hand , this implies an enhanced relative sensitivity to new physics . the required precision of a few \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\%$$\end{document}% might be within reach of the lhc.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_7$$\end{document}j7 or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_9$$\end{document}j9 give similar information as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\gamma /ds$$\end{document}d/ds , but should have different experimental systematics because of the characteristic angular dependence associated with them.cp violation in the coupling of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h to electroweak bosons is probed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_4$$\end{document}j4 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_5$$\end{document}j5 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_8$$\end{document}j8 , which enter the terms in the decay distribution odd in the angle between the dilepton planes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}. their effects are , however , expected at the per - mille level and thus out of reach of the lhc.the form factors are expressed in terms of the coefficients of the complete effective lagrangian at next - to - leading order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o } } ( v^2/\lambda ^2\sim 1/(16\pi ^2))$$\end{document}o(v2/21/(162 ) ) . we use the electroweak chiral lagrangian , extended to include a light higgs singlet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h , and take into account all nlo new - physics effects at tree level , including the renormalization of sm fields and parameters . the effective lagrangian for a linearly realized higgs is also considered with operators up to dimension 6.based on effective - theory power counting , the potentially dominant impact of new physics arises from the leading - order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$hzz$$\end{document}hzz coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a , which only affects the overall decay rate , but not the angular and dilepton - mass distributions . the latter can only be modified by the nlo coefficients in the lagrangian.power counting gives a typical size of the nlo coefficients of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sim v^2/\lambda ^2\sim 1\,\%$$\end{document}v2/21% , up to coupling constants and numerical factors . with this estimate in particular , the contributions of the virtual \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} , which could in principle be inferred from the profiles of the different mass distributions turn out to be at the per - mille level and therefore too small to be detected . somewhat larger effects ( up to 5 % ) may be possible in specific scenarios , for instance from enhanced \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$hz\bar{l}l$$\end{document}hzll local couplings \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a in a strongly interacting higgs sector . quantities such as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b , with their large sensitivity to np corrections , could be especially interesting in this respect.for the quantitative extraction of new - physics coefficients from data , radiative corrections have to be taken into account . to nlo ( one loop ) in the standard model they have been computed in [ 44 , 45].new - physics effects in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- decay distributions are expected to be small , even in the case of a strongly interacting higgs sector . the tree level sm contribution is the dominating effect and np can potentially show up typically at the percent level . nevertheless , this np suppression can be compensated by statistics , and we have shown that interesting opportunities exist for precision measurements , already at the lhc , which could provide valuable insight into electroweak symmetry breaking . the rich subject of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- observables should therefore be fully explored by experiment . we discuss the most general observables arising from the full angular distribution of the 4-lepton final state in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{'+}\ell ^{'-}$$\end{document}z+- . the nine coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_i$$\end{document}ji describing the angular distribution are expressed through the six form factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{v , a}$$\end{document}gv , a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{v , a}$$\end{document}kv , a . interesting observables , besides the dilepton - mass spectrum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\gamma /ds$$\end{document}d/ds , can be constructed from the angular distribution . examples are : the forward backward asymmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_6$$\end{document}j6 . these quantities are strongly suppressed in the sm because of the smallness of the vectorial coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_v$$\end{document}gv . on the other hand , this implies an enhanced relative sensitivity to new physics . the required precision of a few \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\%$$\end{document}% might be within reach of the lhc.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_7$$\end{document}j7 or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_9$$\end{document}j9 give similar information as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\gamma /ds$$\end{document}d/ds , but should have different experimental systematics because of the characteristic angular dependence associated with them.cp violation in the coupling of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h to electroweak bosons is probed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_4$$\end{document}j4 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } 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\setlength{\oddsidemargin}{-69pt } \begin{document}$$a_{\alpha \beta } $ $ \end{document}a measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_3$$\end{document}j3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\phi } $ $ \end{document}b measuring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_6$$\end{document}j6 . these quantities are strongly suppressed in the sm because of the smallness of the vectorial 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\usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}. their effects are , however , expected at the per - mille level and thus out of reach of the lhc . the form factors are expressed in terms of the coefficients of the complete effective lagrangian at next - to - leading order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o } } ( v^2/\lambda ^2\sim 1/(16\pi ^2))$$\end{document}o(v2/21/(162 ) ) . we use the electroweak chiral lagrangian , extended to include a light higgs singlet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h , and take into account all nlo new - physics effects at tree level , including the renormalization of sm fields and parameters . the effective lagrangian for a linearly realized higgs is also considered with operators up to dimension 6 . based on effective - theory power counting , the potentially dominant impact of new physics arises from the leading - order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$hzz$$\end{document}hzz coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a , which only affects the overall decay rate , but not the angular and 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\begin{document}$$\gamma $ $ \end{document} , which could in principle be inferred from the profiles of the different mass distributions turn out to be at the per - mille level and therefore too small to be detected . somewhat larger effects ( up to 5 % ) may be possible in specific scenarios , for instance from enhanced \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$hz\bar{l}l$$\end{document}hzll local couplings \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{v , a}$$\end{document}hv , a in a strongly interacting higgs sector . quantities such as \documentclass[12pt]{minimal } \usepackage{amsmath } 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^{'+}\ell ^{'-}$$\end{document}+- c.m.s . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document} is the relative angle between the normals of the decay planes , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vec { e}_x\times \vec { q}_1/|\vec { e}_x\times \vec { q}_1|$$\end{document}exq1/|exq1| and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vec { e}_x\times \vec { p}_1/|\vec { e}_x\times \vec { p}_1|$$\end{document}exp1/|exp1| , counted positive from the former to the latter in the positive direction around \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vec { e}_x$$\end{document}ex . definition of angles in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{'+}\ell ^{'-}$$\end{document}z+- the lepton momenta in the respective dilepton center - of - mass systems can then be parametrized as33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } q^\mu _ { 1}&= \frac{m_h\sqrt{s}}{2}\left ( 1,{\hat{n}}_1\right ) , \quad q^\mu _ { 2}=\frac{m_h\sqrt{s}}{2}\left ( 1,-{\hat{n}}_1\right ) , \nonumber \\ p^\mu _ { 1}&= \frac{m_h\sqrt{r}}{2}\left ( 1,{\hat{n}}_2\right ) , \quad p^\mu _ { 2}=\frac{m_h\sqrt{r}}{2}\left ( 1,-{\hat{n}}_2\right ) , \end{aligned}$$\end{document}q1=mhs21,n^1,q2=mhs21,-n^1,p1=mhr21,n^2,p2=mhr21,-n^2,with the unit vectors34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \hat{n}}_1&= ( \cos \alpha , \sin \alpha , 0),\nonumber \\ { \hat{n}}_2&= ( -\cos \beta , \sin \beta \cos \phi , \sin \beta \sin \phi ) . \end{aligned}$$\end{document}n^1=(cos,sin,0),n^2=(-cos,sincos,sinsin).the range of the kinematical variables is35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 0 \,&\le \ , s\ , \le \,(1-\sqrt{r})^2 = 0.076,\nonumber \\ 0\,&\le \ , \alpha , \beta \ , \le \ , \pi , \nonumber \\ 0 \,&\le \ , \end{aligned}$$\end{document}0s(1-r)2=0.076,0,,02.the momenta can be boosted to the higgs rest frame with the following velocities:36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \beta _ q&= \frac{\lambda } { 1-r+s},\quad \beta _ p=\frac{\lambda } { 1+r - s } \end{aligned}$$\end{document}q=1-r+s,p=1+r - swhere \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r$$\end{document}r , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s$$\end{document}s , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda $ $ \end{document} are defined in ( 2 ) . the relevant kinematical invariants are then given by37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&q_1\cdot p_1=\frac{m^2_h}{8}\big [ \left ( 1+c_{\alpha } c_{\beta } \right ) g_h+\lambda ( c_{\alpha } + c_{\beta } ) \nonumber \\&\qquad \qquad \quad -2\sqrt{r s}\ , s_{\alpha } s_{\beta } c_{\phi } \big ] , \nonumber \\&q_1\cdot p_2=\frac{m^2_h}{8}\big [ \left ( 1-c_{\alpha } c_{\beta } \right ) g_h+\lambda ( c_{\alpha } -c_{\beta } ) \nonumber \\&\qquad \qquad \quad + 2\sqrt{r s}\ , s_{\alpha } s_{\beta } c_{\phi } \big ] , \nonumber \\&q_2\cdot p_1=\frac{m^2_h}{8}\big [ \left ( 1-c_{\alpha } c_{\beta } \right ) g_h-\lambda ( c_{\alpha } -c_{\beta } ) \nonumber \\&\qquad \qquad \quad + 2\sqrt{r s}\ , s_{\alpha } s_{\beta } c_{\phi } \big ] , \nonumber \\&q_2\cdot p_2=\frac{m^2_h}{8}\big [ \left ( 1+c_{\alpha } c_{\beta } \right ) g_h-\lambda ( c_{\alpha } + c_{\beta } ) \nonumber \\&\qquad \qquad \quad -2\sqrt{r s}\ , s_{\alpha } s_{\beta } c_{\phi } \big ] , \nonumber \\&q_1\cdot q_2=\frac{m^2_h}{2 } s,\nonumber \\&p_1\cdot p_2=\frac{m^2_h}{2 } r,\nonumber \\&\epsilon _ { \mu \nu \lambda \rho } p_1^{\mu } p_2^{\nu } q_1^{\lambda } q_2^{\rho } = \frac{m^4_h}{8}\lambda \sqrt{r s}\ , s_{\alpha } s_{\beta } s_{\phi } \end{aligned}$$\end{document}q1p1=mh28[1+ccgh+(c+c)-2rsssc],q1p2=mh28[1-ccgh+(c-c)+2rsssc],q2p1=mh28[1-ccgh-(c-c)+2rsssc],q2p2=mh28[1+ccgh-(c+c)-2rsssc],q1q2=mh22s , p1p2=mh22r,p1p2q1q2=mh48rsssswhere \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_h\equiv 1-r - s$$\end{document}gh1-r - s , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_\chi \equiv \cos \chi $ $ \end{document}ccos , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_\chi \equiv \sin \chi $ $ \end{document}ssin , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon _ { 0123}=+1$$\end{document}0123=+1 .

we compute the fully differential rate for the higgs - boson decay \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\rightarrow z\ell ^+\ell ^-$$\end{document}hz+- , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z\rightarrow \ell ^{'+}\ell ^{'-}$$\end{document}z+- . for these processes we assume the most general matrix elements within an effective lagrangian framework . the electroweak chiral lagrangian we employ assumes minimal particle content and standard model gauge symmetries , but it is otherwise completely general . we discuss how information on new physics in the decay form factors may be obtained that is inaccessible in the dilepton - mass spectrum integrated over angular variables . the form factors are related to the coefficients of the effective lagrangian , which are used to estimate the potential size of new - physics effects .

Introduction Angular distribution for Form factors from effective Lagrangian Observables and form factor determination Conclusions Appendix A: 4-body decay kinematics

PMC3554295

we used data from the adult questionnaire of the 2009 california health interview survey ( chis ) , which is representative of the state s noninstitutionalized civilian population 18 years of age or older . chis is a statewide , random - digit - dial telephone survey sampling household telephone numbers assigned to both landline and cellular service ( 27 ) . one adult from each sampled household was randomly selected to complete an interview , conducted either in english , spanish , chinese , vietnamese , or korean . the overall response rate for the chis adult interview was 49% in 2009 , which is comparable to other scientific telephone surveys conducted in california ( 27 ) . data were collected on 28,186 women , who were identified based on the question are you male or female ? after excluding women who reported having doctor - diagnosed diabetes and those currently pregnant , the analytic sample included 25,433 women : 4,321 latinas ( 17.0% ) and 21,112 non - latinas ( 83.0% ) . for each of six diabetes - related behaviors , we determined the cut point for the least healthy tertile for the entire analytic sample . we used a cut point to make our analysis of behavior differences between latinas and non - latinas more understandable and useful for practitioners . we selected the tertile as the cut point because there is no established level of these behaviors at which the risk of diabetes is known to sharply increase . in the current study , the least healthy tertile was defined as the lowest tertile for physical activity behaviors and the highest tertile for the dietary behaviors examined . participants were asked two questions about walking during the past week : how many times did you walk to get some place that took you at least 10 minutes ? and how many times did you walk for at least 10 minutes for fun , relaxation , exercise , or to walk the dog ? responses to these two questions were added to make a single variable called walking , which was the total number of times per week the participant walked for at least 10 min for any purpose . participants were asked about moderate physical activity during the previous week , such as bicycling , dancing , swimming , and gardening : on how many days did you do any moderate physical activities in your free time for at least 10 minutes , other than walking ? in the same manner , participants were asked how many days in the last week they participated for at least 10 min in vigorous physical activities ( ones that take hard physical effort , such as aerobics , running , soccer , fast bicycling , or fast swimming ) . four dietary behaviors were examined : consumption of fried potatoes , ssbs , desserts , and fast food . participants were asked how many times in the last month they ate any kind of fried potatoes , including french fries , home fries , or hash browns . the monthly consumption of ssbs was assessed by combining the responses to questions about how many times they drank regular soda or pop that contains sugar , sports or energy drinks such as gatorade , red bull , and vitamin water , sweetened fruit drinks such as kool - aid , cranberry drink , and lemonade , and coffee or tea with sugar or honey added . for each of these questions , respondents were reminded not to include diet options with artificial sweeteners . monthly desserts were assessed by combining responses to the following two questions : how often did you eat cookies , cake , pie , or brownies ? and how often did you eat ice cream or other frozen desserts ? participants were instructed to not include sugar - free desserts . for fast food , include fast food meals eaten at work , home , or fast food restaurants , carryout or drive through . the following examples were provided : mcdonald s , panda express , and taco bell . participants were asked , in the past seven days , how many times did you eat fast food ? responses to this question were multiplied by 4.3 to estimate the total number of times fast food was eaten in the last month . latina ethnicity was determined by the question are you latino or hispanic ? women who responded yes were classified as latinas , and those who answered no were classified as non - latinas . we also examined seven characteristics of participants that might be related to both latino ethnicity and the diabetes - related behaviors . these characteristics , which might confound the primary relationship under study , included the following : age , household income ( expressed as percentage of the federal poverty threshold for household size ) , educational attainment , marital status , health status , smoking , and acculturation . for health status , respondents were asked would you say that in general your health is excellent , very good , good , fair , or poor ? acculturation was assessed using a six - item measure developed and validated for use with chis ( 28 ) . the six items addressed the following domains : 1 ) language ( language spoken at home , language used to complete the survey , and self - reported english proficiency ) ; 2 ) nativity ; 3 ) duration of u.s . residence ; and 4 ) citizenship status . an acculturation score was calculated according to the methods described by van wieren et al . ( 29 ) , yielding scores that ranged from 6 to 19 in our sample . we divided the subjects into three acculturation levels : low ( 610 ) , medium ( 1117 ) , and high ( 18,19 ) . all analyses were conducted using stata se , version 11.0 ( stata inc . , college station , tx ) . in all statistical testing , we used stata survey commands , which adjust variance estimates to account for the complex sample design of the chis ( 30 ) . overall , the proportion of subjects with missing data on any given variable was very low ( < 2% of sample ) . the investigators who prepared the publically available chis dataset used in our analysis used hotdeck- and regression - based methods to impute missing data for most study variables . full procedures have been described elsewhere ( 27 ) . in all analyses involving the six diabetes - related behaviors , we used binary variables ( the least healthy tertile vs. the other two tertiles ) . our key independent variable , latina ethnicity , was also binary ( latina vs. non - latina ) . using tests , we first examined the association between participant characteristics and latina ethnicity , followed by the association of diabetes - related behaviors with latina ethnicity . we then calculated the unadjusted odds of being in the least healthy tertile by each participant characteristic ( potentially confounding variables ) . using logistic regression models for each diabetes - related behavior , we calculated the odds of latinas being in the least healthy tertile . these odds were adjusted in successive models , adding groups of potentially confounding variables to the unadjusted model : model 1 ( age ) ; model 2 ( socioeconomic status ; model 1 variable + household income , education , and marital status ) ; model 3 ( health - related factors ; model 2 variables + health status and smoking status ) ; and model 4 ( acculturation ; model 3 variables + acculturation ) . age was added separately because of the large differences between latinas and non - latinas that , alone , might significantly confound the relationships under study . the variables in the socioeconomic and health groups were conceptually related and made a priori . we added these thematic groups of covariates into successive models to highlight their impact on the association between latina ethnicity and the behavioral outcomes . we used data from the adult questionnaire of the 2009 california health interview survey ( chis ) , which is representative of the state s noninstitutionalized civilian population 18 years of age or older . chis is a statewide , random - digit - dial telephone survey sampling household telephone numbers assigned to both landline and cellular service ( 27 ) . one adult from each sampled household was randomly selected to complete an interview , conducted either in english , spanish , chinese , vietnamese , or korean . the overall response rate for the chis adult interview was 49% in 2009 , which is comparable to other scientific telephone surveys conducted in california ( 27 ) . data were collected on 28,186 women , who were identified based on the question are you male or female ? after excluding women who reported having doctor - diagnosed diabetes and those currently pregnant , the analytic sample included 25,433 women : 4,321 latinas ( 17.0% ) and 21,112 non - latinas ( 83.0% ) . for each of six diabetes - related behaviors , we determined the cut point for the least healthy tertile for the entire analytic sample . we used a cut point to make our analysis of behavior differences between latinas and non - latinas more understandable and useful for practitioners . we selected the tertile as the cut point because there is no established level of these behaviors at which the risk of diabetes is known to sharply increase . in the current study , the least healthy tertile was defined as the lowest tertile for physical activity behaviors and the highest tertile for the dietary behaviors examined . participants were asked two questions about walking during the past week : how many times did you walk to get some place that took you at least 10 minutes ? and how many times did you walk for at least 10 minutes for fun , relaxation , exercise , or to walk the dog ? responses to these two questions were added to make a single variable called walking , which was the total number of times per week the participant walked for at least 10 min for any purpose . participants were asked about moderate physical activity during the previous week , such as bicycling , dancing , swimming , and gardening : on how many days did you do any moderate physical activities in your free time for at least 10 minutes , other than walking ? in the same manner , participants were asked how many days in the last week they participated for at least 10 min in vigorous physical activities ( ones that take hard physical effort , such as aerobics , running , soccer , fast bicycling , or fast swimming ) . four dietary behaviors were examined : consumption of fried potatoes , ssbs , desserts , and fast food . participants were asked how many times in the last month they ate any kind of fried potatoes , including french fries , home fries , or hash browns . the monthly consumption of ssbs was assessed by combining the responses to questions about how many times they drank regular soda or pop that contains sugar , sports or energy drinks such as gatorade , red bull , and vitamin water , sweetened fruit drinks such as kool - aid , cranberry drink , and lemonade , and coffee or tea with sugar or honey added . for each of these questions , respondents were reminded not to include diet options with artificial sweeteners . desserts were assessed by combining responses to the following two questions : how often did you eat cookies , cake , pie , or brownies ? and how often did you eat ice cream or other frozen desserts ? participants were instructed to not include sugar - free desserts . for fast food , include fast food meals eaten at work , home , or fast food restaurants , carryout or drive through . the following examples were provided : mcdonald s , panda express , and taco bell . participants were asked , in the past seven days , how many times did you eat fast food ? responses to this question were multiplied by 4.3 to estimate the total number of times fast food was eaten in the last month . latina ethnicity was determined by the question are you latino or hispanic ? women who responded were classified as latinas , and those who answered no were classified as non - latinas . we also examined seven characteristics of participants that might be related to both latino ethnicity and the diabetes - related behaviors . these characteristics , which might confound the primary relationship under study , included the following : age , household income ( expressed as percentage of the federal poverty threshold for household size ) , educational attainment , marital status , health status , smoking , and acculturation . for health status , respondents were asked would you say that in general your health is excellent , very good , good , fair , or poor ? acculturation was assessed using a six - item measure developed and validated for use with chis ( 28 ) . the six items addressed the following domains : 1 ) language ( language spoken at home , language used to complete the survey , and self - reported english proficiency ) ; 2 ) nativity ; 3 ) duration of u.s . residence ; and 4 ) citizenship status . an acculturation score was calculated according to the methods described by van wieren et al . ( 29 ) , yielding scores that ranged from 6 to 19 in our sample . we divided the subjects into three acculturation levels : low ( 610 ) , medium ( 1117 ) , and high ( 18,19 ) . all analyses were conducted using stata se , version 11.0 ( stata inc . , college station , tx ) . in all statistical testing , we used stata survey commands , which adjust variance estimates to account for the complex sample design of the chis ( 30 ) . overall , the proportion of subjects with missing data on any given variable was very low ( < 2% of sample ) . the investigators who prepared the publically available chis dataset used in our analysis used hotdeck- and regression - based methods to impute missing data for most study variables . full procedures have been described elsewhere ( 27 ) . in all analyses involving the six diabetes - related behaviors , we used binary variables ( the least healthy tertile vs. the other two tertiles ) . our key independent variable , latina ethnicity , was also binary ( latina vs. non - latina ) . using tests , we first examined the association between participant characteristics and latina ethnicity , followed by the association of diabetes - related behaviors with latina ethnicity . we then calculated the unadjusted odds of being in the least healthy tertile by each participant characteristic ( potentially confounding variables ) . using logistic regression models for each diabetes - related behavior , we calculated the odds of latinas being in the least healthy tertile . these odds were adjusted in successive models , adding groups of potentially confounding variables to the unadjusted model : model 1 ( age ) ; model 2 ( socioeconomic status ; model 1 variable + household income , education , and marital status ) ; model 3 ( health - related factors ; model 2 variables + health status and smoking status ) ; and model 4 ( acculturation ; model 3 variables + acculturation ) . age was added separately because of the large differences between latinas and non - latinas that , alone , might significantly confound the relationships under study . the variables in the socioeconomic and health groups were conceptually related and made a priori . we added these thematic groups of covariates into successive models to highlight their impact on the association between latina ethnicity and the behavioral outcomes . compared with non - latinas , latinas were younger , less educated , had lower income , and reported poorer health ( table 1 ) . latinas were less likely than non - latinas to be in the least healthy tertile for walking and dessert consumption , but they were more likely to be in the least healthy tertile for mvpa and the three other dietary behaviors : consuming fried potatoes , fast food , and ssbs ( table 2 ) . the spearman correlation coefficients between these behavior variables ( continuous measures ) were as follows : ssb and fried potato consumption ( 0.13 , p < 0.001 ) , ssb and fast food consumption ( 0.12 , p < 0.001 ) , and fast food and fried potato consumption ( 0.28 , p < 0.001 ) . participant characteristics by latina ethnicity diabetes - related behaviors by latina ethnicity increasing age was associated with poorer physical activity behaviors and improved dietary behaviors , with the exception of dessert consumption ( table 3 ) . those with lower incomes , less education , and poorer self - reported health were more likely to be in the least healthy tertile of mvpa and ssb consumption . those with lower levels of acculturation and smokers were also more likely to be in the least healthy tertile of ssb consumption . however , those with lower levels of acculturation were less likely to be in the unhealthy tertile for fried potato or dessert consumption or for walking . unadjusted odds of being in the least healthy tertile for diabetes - related behaviors by participant characteristics in unadjusted logistic regression analyses , the largest differences between latinas and non - latinas in unhealthy , diabetes - related behaviors were seen for ssb and fast food consumption ( table 4 ) . the magnitude of the association between latina ethnicity and these two dietary behaviors decreased after controlling for age ( model 1 ) . for ssbs , the association was further decreased by controlling for income , education , and marital status ( model 2 ) . controlling both associations for health status and smoking ( model 3 ) had little additional impact on the magnitude of these associations . finally , adjusting for acculturation had different impacts on each of these associations ( model 4 ) , as would be predicted by 1 ) the different relationship of acculturation to fast food and ssb consumption ( table 3 ) and 2 ) the lower levels of acculturation among latinas . in fully adjusted models ( model 4 ) , latinas had a higher risk ( odds ratio [ or ] [ 95% ci ] ) of being in the least healthy tertile of fast food ( 1.94 [ 1.632.31 ] ) , ssb beverage ( 1.53 [ 1.291.82 ] ) , and fried potato consumption ( 1.32 [ 1.181.67 ] ) . odds of latinas , compared with non - latinas , of being in the least healthy tertile for diabetes - related behaviors in fully adjusted models , latinas had lower odds of being in the least healthy tertile for two of the six diabetes - related behaviors : dessert consumption ( or 0.82 [ 95% ci 0.700.95 ] ) and mvpa ( 0.83 [ 0.700.97 ] ) . the latter association emerged only after adjusting for confounding variables . in the fully adjusted model , there was no difference between latinas and non - latinas with respect to walking ( 0.99 [ 0.851.16 ] ) . all of the adjusted differences in diabetes - related behaviors between latinas and non - latinas ( table 4 ) were larger in separate analyses comparing latinas to non - latina whites , the largest racial subgroup of non - latinas . by restricting our multivariate models to mexican - american latinas vs. non - latinas , we addressed the potential role of obesity as a confounder by controlling our final multivariate models for obesity status , and we assessed obesity as an effect modifier by running these final models separately for obese and nonobese subjects . we found no evidence that obesity was either a confounder or an effect modifier of the relationships between the health behaviors and latina ethnicity . our study shows significant differences between latinas and non - latinas with respect to all diabetes - related behaviors examined , except walking . compared with non - latinas , latinas had higher odds of being frequent consumers of fried potatoes , ssbs , and fast food . of these unhealthy dietary behaviors , although age , socioeconomic status , and acculturation confounded these relationships , the fully adjusted association of latina ethnicity with poor dietary behaviors suggests an independent effect of being latina on consuming these unhealthy food groups . latinas had lower odds of eating desserts frequently compared with non - latinas . after adjusting for all covariates , the greatest strength of this study is its inclusion of a large number of latinas , who have smaller representation in other large datasets . studying 4,321 latinas helped produce reliable estimates of this population s diabetes - related behaviors , and the association of those behaviors with latina ethnicity . using this large latina cohort , we have produced some of the first reports of the prevalence of six diabetes - related behaviors in this high - risk population . another strength of our study is the potential clinical utility of the behavioral outcomes examined here . we analyzed these diabetes - related behaviors on a scale that we believe is most meaningful and useful to clinicians , compared with more complex metrics that are difficult to incorporate into lifestyle counseling efforts . because latinos health behaviors are influenced by acculturation ( 31 ) , our use of a validated acculturation measure represents another potential strength . however , this measure does not incorporate questions about cognitive , spiritual , or emotional factors , which are part of the acculturation process . the cross - sectional nature of this study hinders our ability to draw causal inferences about the impact of latina ethnicity on diabetes - related behaviors . the data are from california , where the proportion of latinas who are mexican american ( 77% ) is higher than national estimates ( 65% ) , which may limit the ability to generalize our findings ( 26 ) . the number of latinas from other countries was too small to permit subgroup analyses in our study . in chis , the use of different time frames ( week and month ) for the dietary measures and the long recall period of one month might have resulted in inaccurate responses . however , we do not have reason to believe that these inaccuracies would have differed between latinas and non - latinas . our choice of non - latinas as a comparison group masks differences in diabetes - related behaviors among whites , blacks , and asians who comprise that group . including blacks in this group ( who exhibit high levels of physical inactivity and consumption of energy - dense , nutrient - poor foods ) ( 32 ) underestimates differences in diabetes - related behaviors between women at the highest risk ( i.e. , latinas ) and those at lowest risk ( i.e. , whites ) . our sensitivity analysis comparing behavioral outcomes between latinas and white women yielded greater differences in the same diabetes - related behaviors than those reported between latinas and non - latinas . the observed behavioral differences between latinas and non - latinas may be explained , in part , by cultural differences between these groups , which we could not measure in the current study . some have suggested that latino americans consider consuming fast food and ssbs as a status symbol , which may influence their consumption of these foods ( 33 ) . although previous studies have examined racial and ethnic differences in the same diabetes - related dietary behaviors studied here , most did not stratify analyses by sex . the only study reporting any of these dietary behaviors in latinas found that 50% of mexican - american women drink regular soda ( 13 ) . ( 14 ) estimated that mexican - american adults consume fewer calories from ssbs than whites or african americans . the different scales used to measure ssb consumption and our exclusion of latino males may help explain these differences . other national studies ( 13,15,17 ) that have explored ssb consumption in latinos found similar levels to those reported by bleich et al . these studies included only mexican americans , whereas ours included latinas from other latin american countries . however , our results were unchanged when we restricted our analysis to mexican - american women . latinos in california may have different patterns of ssb consumption than latinos in other parts of the country , which may also help explain the observed discrepancy with nationally representative studies . almost no research has focused on the differences in fast food consumption between latinos and non - latinos . one study reported a lower prevalence of fast food consumption in latinos compared with non - hispanic blacks and whites ; however the authors did not adjust for potential confounders in their analysis ( 22 ) . in our study , the association of latina ethnicity with fast food consumption was higher than for any other dietary behavior we examined . acculturation was strongly and positively associated with eating fast food in our cohort ( table 3 ) . this finding is consistent with those from the few studies that have examined acculturation and fast food consumption within latinos ( 17,34,35 ) . with respect to the other dietary behaviors studied here , we found that latinas eat desserts less frequently than non - latinas , which is consistent with previous reports ( 20,21 ) . however , our study and previous ones have not included questions about culturally specific desserts for latinas , such as dulce de leche and flan , which could lead to an underestimation of the frequency of consuming desserts among latinas . we found that latinas consume fried potatoes more frequently than non - latinas , in contrast to two previous studies ( 13,20 ) that examined french fries as the only form of fried potatoes . latinos , the physical activity literature has placed a greater focus on latinas by either restricting analyses to this group ( 19,24 ) , stratifying race / ethnicity analyses by sex ( 16 ) , or stratifying sex - based analyses by race / ethnicity ( 23 ) . in our analysis ( table 2 ) , 41% of latinas reported no mvpa , which was almost identical to previous reports ( 16,23 ) . after adjusting for covariates , however , we found that latinas were somewhat less likely to have low levels of mvpa than non - latinas . in our cohort , lower levels of income , educational attainment , health status , and acculturation were associated with less mvpa and latina ethnicity . adjusting for these factors in our analysis we found that latinas walked more frequently than non - latinas in our unadjusted analysis , which may reflect increased active transportation and occupational walking in latinas ( 36 ) , in addition to the negative association of acculturation with walking in this population ( 37 ) . after adjusting for acculturation and other covariates in our multivariate model in conclusion , our study has identified two diabetes - related behaviors ( ssb and fast food consumption ) that are more frequently performed by latinas than non - latinas , and may therefore represent important behavioral targets for diabetes prevention efforts in latinas . these specific dietary behaviors may be particularly effective targets for lifestyle counseling by diverse health professionals . given the limited time they devote to diabetes prevention with at - risk patients ( 38 ) , physicians may find such discrete dietary targets useful . as another example , certified diabetes educators and nutritionists may choose to focus on fast food and ssb consumption when taking more detailed dietary histories and making dietary recommendations for latinas . future research should assess how best to incorporate messages about reducing ssb and fast food consumption into clinical encounters and community - based diabetes prevention programs targeting latinas . effectively reducing ssb and fast food consumption among latinas will also require further qualitative research to understand the social context surrounding these diabetes - related behaviors , and the role that ssbs and fast food play in latinas lives .

objectivecertain dietary and physical activity behaviors have been associated with the risk of developing type 2 diabetes , yet little is known about the prevalence of these behaviors among latinas ( latino women ) . the purpose of this cross - sectional study was to compare the prevalence of diabetes - related behaviors in latinas and non-latinas.research design and methodsusing data from the 2009 california health interview survey , we compared self - reported diabetes - related behaviors of latinas ( n = 4,321 ) to non - latinas ( n = 21,112 ) after excluding women who were pregnant or had diabetes . for six behaviors , we determined the cut point for the least healthy tertile : walking , doing moderate to vigorous physical activity , and consuming fried potatoes , sugar - sweetened beverages ( ssbs ) , desserts , and fast food . we used logistic regression to examine the association between latina ethnicity and being in the least healthy tertile compared with the other two tertiles for each of these behaviors.resultsin multivariate models adjusted for age , income , education , marital status , health status , smoking , and acculturation , latinas had a higher risk ( odds ratio [ 95% ci ] ) of being in the least healthy tertile for the consumption of fast food ( 1.94 [ 1.632.31 ] ) , ssbs ( 1.53 [ 1.291.82 ] ) , and fried potatoes ( 1.32 [ 1.181.67 ] ) , and lower risk for desserts ( 0.82 [ 0.700.95 ] ) . latinas and non - latinas had similar physical activity levels.conclusionsdietary differences between latinas and non - latinas ( particularly in the consumption of fast food and ssbs ) may be the focus of interventions to prevent diabetes in latinas . further research among latinas is needed to understand and modify these dietary behaviors .

RESEARCH DESIGN AND METHODS Study population Diabetes-related behaviors Latina ethnicity Other participant characteristics Data analysis RESULTS CONCLUSIONS

PMC4595346

lack of exercise and sedentary lifestyle have been linked to obesity , type 2 diabetes , cardiovascular diseases , cancer , osteoporosis , and premature death . skeletal muscle is the most abundant tissue in non - obese adults , accounting for approximately 40% of the body weight . skeletal muscle adapts to mechanical , neural and humoral stimuli , and plays critical roles in physical activity , energy expenditure , and glucose disposal . exercise and anabolic hormones , e.g. , insulin , insulin - like growth factor 1 , growth hormone and testosterone , increase skeletal muscle mass ( fig . conversely , physical inactivity from aging or neuromuscular disorders , and chronic diseases , such as cancer , renal failure , respiratory failure , infection , and some endocrine disorders , e.g. , uncontrolled diabetes mellitus , hyperthyroidism and hypercortisolism , cause muscle atrophy or " sarcopenia " ( fig . sarcopenia has been linked to obesity , metabolic syndrome , and other diseases in aged populations , particularly in south asia ( fig . the concept that skeletal muscle secretes humoral factors that actively communicate with other organs was proposed many years ago . used the term " myokines " to describe cytokines and other peptides expressed and released by muscle cells . this review highlights the biological actions of myostatin and other myokines that regulate skeletal muscle mass and metabolism via autocrine , paracrine , and endocrine mechanisms . much attention has been focused on the biology of the transforming growth factor ( tgf- ) superfamily of proteins since the discovery of myostatin . myostatin , also known as growth differentiation factor 8 , is expressed and secreted predominantly by skeletal muscle and inhibits muscle growth . this function is conserved in many species , as evident by the hypermuscular phenotype resulting from inactivation of myostatin gene in mice , sheep , cattle , and human . during early postnatal development normally , the differentiation of skeletal muscle cells requires growth arrest followed by expression of muscle - specific genes . these processes are coordinated by activation of specific cyclins , cyclin - dependent kinases ( cdk ) , cdk inhibitors ( cdkis ) , and muscle regulatory factors . during the proliferation phase , myostatin up - regulates p21 ( a cdki ) , and decreases the levels of cdk2 and cdk4 , leading to cell cycle arrest . myostatin inhibits satellite cell activation by down - regulating the transcription factor pax7 , and also controls the myogenic differentiation program through inhibition of myogenic regulatory factors , such as pax3 , myod , and myf5 . studies indicate that myostatin 's inhibitory effect on muscle differentiation in the postnatal period is mediated partly by perturbation of akt / mammalian target of rapamycin complex1 signaling . in mature adult muscle fibers , the c - terminal dimer of myostatin binds to activin receptors ii ( actrii ) , mainly actriib and to a lesser degree actriia , which then recruits , phosphorylates and activates activin receptor - like kinase ( alk ) 4 and alk5 , leading to phosphorylation and activation of smad2 and smad3 . phosphorylated smad2 and smad3 form a heterodimeric complex with smad4 , which is translocated into the nucleus , and acts as a transcription factor to regulate gene expression . myostatin signaling also leads to activation of smad7 which functions as a negative feedback inhibitor . the activation of myostatin - smad pathway inhibits the translation initiation complex and protein synthesis . myostatin suppresses akt signaling and acts via forkhead box protein o1 transcription factors to promote protein breakdown through activation of the ubiquitin - proteasome system ( fig . genetic or pharmacologic inhibition of myostatin , actriib , alk4/alk5 , or smad2/3 results in skeletal muscle hypertrophy , associated with increased protein synthesis and reduced protein degradation . myostatin knockout ( mstn-/- ) mice have increased skeletal muscle mass as well as reduced body fat . myostatin - null agouti lethal yellow or leptin deficient mice have drastically reduced body fat and glucose levels raising the possibility that blockade of myostatin signaling may be useful for treating obesity and diabetes . have shown that mstn-/- mice have increased glucose utilization and insulin sensitivity . to determine whether these effects were due to a lack of myostatin signaling in muscle or adipose tissue , they compared the metabolic phenotypes of mice carrying a dominant negative actriib receptor expressed in adipocytes or skeletal muscle . the absence of myostatin signaling in adipocytes did not affect body composition or glucose homeostasis , whereas inhibition of myostatin signaling in skeletal muscle recapitulated the phenotype of mstn-/- mice , characterized by hypermuscularity , decreased body fat , and enhanced insulin sensitivity . we studied the effects of pharmacological blockade of myostatin and related peptides by treating mice on chow and high - fat diets with a soluble activin receptor type iib ( actriib - fc ) . actriib - fc treatment increased lean and skeletal muscle mass , grip strength , and contractile force , decreased body fat , and increased insulin sensitivity . mice lacking akt1 or akt2 have reduced muscle mass , grip strength and contractile force , consistent with a pivotal role of akt signaling in promoting muscle growth . contrary to in vitro studies showing that akt signaling is necessary for the ability of actriib inhibition to induce muscle hypertrophy , we found that akt1 and akt2 deficient mice responded similarly as wild type mice to actriib - fc in regard to increased muscle size , grip strength and contractile force , indicating these akt isoforms are not essential for actriib signaling . actriib - fc has also been shown to decrease diet - induced obesity and improve glucose and lipid levels in mice . importantly , actriib - fc induced the browning of white adipose tissue ( wat ) , as shown by increased expression of the thermogenic genes uncoupling protein 1 ( ucp1 ) and peroxisomal proliferator - activated receptor- coactivator 1 ( pgc1 ) . thus , the anti - obesity effect of actriib - fc is partly by increasing skeletal muscle mass as well as inducing thermogenesis in wat . other studies have confirmed that deficiency of myostatin signaling in mstn-/- mice promotes browning of wat . wat of mstn-/- mice displays features of brown adipose tissue , e.g. , increased expression of including ucp1 and pgc1 , as well as expression of beige adipocyte markers , e.g. , tmem26 and cd137 . the enhanced browning of adipose tissue appears to be mediated by irisin ( fibronectin type iii domain - containing 5 , fndc5 ) , a myokine secreted from skeletal muscle in mstn-/- mice . myostatin deficiency stimulates ampk expression and phosphorylation , which then activates pgc1 and irisin and promotes the browning of adipose tissue and thermogenesis . another study has shown that the reduction of body fat in mstn-/- mice is due to increased energy expenditure and leptin sensitivity . the cross - talk of myokines and adipokines may provide novel therapeutic tools for treating obesity , diabetes , and diseases associated with muscle atrophy . a double - blind , placebo - controlled study evaluated the safety , pharmacokinetics , and pharmacodynamics of a decoy actriib receptor ( ace-031 ) in healthy postmenopausal women randomized to receive a single dose of ace-031 ( 0.02 to 3 mg / kg subcutaneous ) or placebo . ace-031 treatment had mild adverse events and produced significant increases of lean mass and thigh muscle volume at day 29 in those receiving 3 mg / kg . moreover , ace-031 treatment increased adiponectin by 51.3% and decreased leptin by 27.7% demonstrating a favorable metabolic profile . an anti - myostatin peptibody ( amg 745/mu - s ) was evaluated in men undergoing androgen deprivation therapy for non - metastatic prostate cancer . the adverse events in amg 745 versus placebo treated groups were : diarrhea ( 13% vs. 9% ) , fatigue ( 13% vs. 4% ) , contusion ( 10% vs. 0% ) , and injection site bruising ( 6% vs. 4% ) . these preliminary results provide support for further investigation into the safety profile and of therapeutic uses of myostatin blockade to reduce sarcopenia and improve metabolism . as discussed earlier , myostatin deficiency or blockade of actriib receptor potently decreases body fat and improves metabolic outcomes in obese mice . human obesity is associated with increased myostatin expression and plasma myostatin levels . the secretion of myostatin from myotubes derived from muscle biopsies is increased in obese compared with lean women . the biological significance of these findings , and whether myostatin and other tgf- peptide superfamily can be targeted specifically for treatment of obesity and metabolic disorders requires further studies . the cytokine interleukin 6 ( il-6 ) was named a myokine because its levels increased in response to exercise and muscle contraction . evidence supporting the notion that is the source of il-6 is based on transcriptional analysis of il-6 mrna levels during exercise , in situ hybridization and immunohistochemistry of il-6 , microdialysis of contracting skeletal muscle , and measurement of arteriovenous il-6 concentrations and blood flow across an exercising leg . skeletal muscle adapts to exercise by altering glycogen content , increasing -oxidation of fatty acids , increasing intramyocellular triglyceride hydrolysis , and enhancing epinephrine - induced lipolysis . thus , the trained skeletal muscle uses fat as a substrate and is less dependent on glucose and muscle glycogen during exercise . epidemiological studies have found an inverse correlation of the amount of physical activity and plasma il-6 concentration . the basal plasma levels of il-6 are strongly associated with physical inactivity , obesity and metabolic syndrome . chronic exercise decreases the basal levels of il-6 , and the increases in plasma il-6 and muscle il-6 mrna content during acute exercise are also blunted in response to endurance training . il-6 receptor ( il-6r ) is regulated opposite to il-6 , and the basal il-6r mrna content in muscle is increased during endurance training , perhaps counteracting the reduction in il-6 . treatment of rat l6 myocytes with il-6 increases basal glucose uptake via glucose transporter 4 translocation , as well as insulin - stimulated glucose uptake . the in vitro effect of il-6 on glucose uptake is mediated , at least partly , through amp - activated protein kinase ( ampk ) activation . studies have also suggested that il-6 may stimulate fatty acid oxidation via ampk . in resting humans , acute administration of il-6 infused to achieve physiological concentrations had no effect on either endogenous glucose production or glucose disposal . in contrast , when il-6 was infused to mimic the plasma of il-6 observed during high - intensity exercise , the endogenous glucose production was markedly increased , suggesting that a muscle - liver crosstalk mediated via il-6 may have a role in regulating plasma glucose levels through endogenous glucose production during exercise . in addition to its effects on glucose metabolism , infusion of il-6 in healthy volunteers stimulates lipolysis in skeletal muscle without affecting adipose tissue . il-6 inhibits endotoxin - induced tumor necrosis factor ( tnf ) production in human monocytes , and infusion of il-6 during exercise attenuates the ability of endotoxin to increase tnf levels in healthy individuals . these anti - inflammatory properties of il-6 are associated with induction of anti - inflammatory cytokines , e.g. , il-1 receptor agonist and il-10 . il-15 was classified as an interleukin based on its 4--helical secondary structure and its ability to mimic the functions of il-2 . the plasma membrane receptor for il-15 was shown to be composed of il-2 receptor ( il-2r ) , the common gamma chain ( c ) , and a specific il-15 receptor ( il-15r ) chain . transcripts for il-15 and il-15r are widely expressed , and skeletal muscle expresses il15 and il15ra mrnas . in vitro experiments in myogenic cells suggested that il-15 was an anabolic factor for skeletal muscle ; however , increasing il-15 levels in vivo did not induce muscle hypertrophy . nonetheless , studies have revealed different locomotor phenotypes of mice lacking il-15r or il-15 or il-2r . furthermore , single nucleotide polymorphisms ( snps ) in the human il15 and il15ra genes have been associated with different muscle phenotypes , responses to resistance training , and obesity . we found that loss of il-15r leads to a remodeling of fast skeletal muscles to a more oxidative phenotype associated with increased spontaneous locomotor activity and exercise capacity , and resistance to fatigue . the molecular signature of oxidative muscle phenotype from il-15r knockout mice included altered mitochondrial biogenesis and calcium homeostasis . consistent with our observations in mice , we found a significant association between a snp in exon 3 of the il15ra gene and endurance in human athletes . a recent paper by o'connell and pistilli has shown mechanisms by which il-15r induced an oxidative skeletal muscle phenotype . muscle - specific deletion of il-15r resulted in a greater mitochondrial density and reduced twitch : tetanus ratio in extensor digitorum longus and soleus muscles indicating a oxidative shift in muscle phenotype . however , the spontaneous activity was not different in muscle il-15r deficient mice , unlike the whole body il-15r knockout mouse . thus , muscle il-15r has a role in altering contractile properties and fatigue characteristics of skeletal muscles , but the locomotor behavior is likely to be controlled by il-15r targets in brain . further studies are needed to evaluate whether il-15r can be targeted specifically for obesity treatment by increasing energy expenditure and fatty acid oxidation . bostrom et al . demonstrated that the inguinal subcutaneous wat had increased levels of ucp1 and cidea in muscle - specific pgc1 transgenic mice compared to wild - type mice . to address whether the browning of the subcutaneous wat was due to a myokine , they cultured primary murine subcutaneous adipocytes with conditioned media from pgc1 overexpressing myocytes , and found that the conditioned media increased expression of brown - fat specific genes in adipocytes . using gene array and bioinformatic methods , bostrom et al . identified fndc5 as a gene target of pgc1 , and showed that fndc5 expression was increased in muscle obtained from exercise - trained mice and humans . primary subcutaneous adipocytes treated with recombinant - fndc5 displayed an increased expression of brown adipose genes , i.e. , ucp1 , elovl3 , cox7a , and otop1 . moreover , ucp1-positive cells treated with fndc5 developed multi - loculated lipid droplets , increased mitochondrial content and oxygen consumption , consistent with a thermogenic phenotype . based on these results , the authors surmised that fndc5 induced a beige phenotype of wat in mice , and this effect was attenuated by a peroxisome proliferator - activated receptor antagonist treatment . further experiments revealed that the full - length fndc5 was a transmembrane protein , and the extracellular n - terminal portion of fndc5 was secreted and was highly homologous between mouse and humans . plasma irisin levels were shown to be increased in mice and humans after short - term exercise . adenoviral expression of fndc5 in liver increased plasma irisin levels which led to the browning of subcutaneous wat , increased energy expenditure , and protection against obesity and insulin resistance . however , questions have been raised about the biology of fndc5 expression and plasma irisin levels . it is unclear whether irisin is truly a myokine since human wat is capable of expressing fndc5 and secreting irisin . some studies indicate that neither acute nor chronic exercise consistently increases expression fndc5 and/or irisin in humans . however , others have shown associations of plasma irisin and aging , obesity , physical activity , and metabolic outcomes . these controversies surrounding the role of irisin may arise from different exercise regimens and assays for measuring irisin , suboptimal storage of tissue samples , as well as differences in the function of irisin in mouse versus human . myokines are proposed to play important roles in mediating the beneficial effects of skeletal muscle mass and exercise on health . myokines have been implicated in the pathogenesis of obesity , substrate oxidation , lipid partitioning , insulin sensitivity , and inflammation . while the list of putative myokines keeps growing , important questions that need to be answered for a presumed myokine include whether skeletal muscle is the main or only source , how the local and systemic concentrations of the myokine are regulated , whether there are biological differences among species , and what specific signaling mechanisms mediate the biological effects of the myokine in various organs . a better understanding of the actions of myokines may identify novel therapies for obesity , diabetes , cardiovascular diseases , cancer , and other diseases known to be improved by exercise .

skeletal muscle is the largest organ of the body in non - obese individuals and is now considered to be an endocrine organ . hormones ( myokines ) secreted by skeletal muscle mediate communications between muscle and liver , adipose tissue , brain , and other organs . myokines affect muscle mass and myofiber switching , and have profound effects on glucose and lipid metabolism and inflammation , thus contributing to energy homeostasis and the pathogenesis of obesity , diabetes , and other diseases . in this review , we summarize recent findings on the biology of myokines and provide an assessment of their potential as therapeutic targets .

INTRODUCTION MYOSTATIN INTERLEUKIN 6 INTERLEUKIN 15 IRISIN CONCLUSIONS

PMC3605499

it has been hypothesized that , in many areas of the brain , having different brain functionality , repeatable precise spatiotemporal patterns of spikes play a crucial role in coding and storage of information . temporally structured replay of spatiotemporal patterns have been observed to occur during sleep , both in the cortex and hippocampus ( nadasdy et al . 2009 ) , and it has been hypothesized that this replay may subserve memory consolidation . the sequential reactivation of hippocampal place cells , corresponding to previously experienced behavioral trajectories , has been observed also in the awake state ( awake replay ) ( diba and buzsaki 2007 ; davidson et al . awake replay may reflect trajectories through either the current environment or previously , spatially remote , visited environments . a possible interpretation is that spatiotemporal patterns , stored in the plastic synaptic connections of hippocampus , are retrieved when a cue activates the emergence of a stored pattern , allowing these patterns to be replayed and then consolidated in distributed circuits beyond the hippocampus ( carr et al . cross - correlogram analysis revealed that in prefrontal cortex the time scale of reactivation of firing patterns during post - behavioral sleep was compressed five- to eightfold relative to waking state ( euston et al . 2007 ; schwindel and mcnaughton 2011 ) , a similar compression effect may also be seen in primary visual cortex ( ji and wilson 2007 ) . internally generated spatiotemporal patterns have also been observed in the rat hippocampus during the delay period of a memory task , showing that the emergence of consistent pattern of activity may be a way to maintain important information during a delay in a task ( pastalkova et al . 2008 ) , i.e. patterns with precise relative phases of the spikes of neurons participating to a collective oscillation , or precise phases of spikes relatively to the ongoing oscillation . first experimental evidence of the importance of spike phases in neural coding was observed in experiments on theta phase precession in rat s place cells ( okeefe and recce 1993 ; okeefe and burgess 2005 ) , showing that spike phase is correlated with rat s position . recently , the functional role of oscillations in the hippocampal - entorinal cortex circuit for path - integration has been deeply investigated ( okeefe and recce 1993 ; lengyel et al . 2006 ) , showing that place cells and grid cells form a map in which precise phase relationship among units plays a central role . in particular it has been shown ( burgess et al . 2007 ; blair et al . 2008 ; welday et al . 2011 ) that both spatial tuning and phase - precession properties of place cells can arise when one has interference among oscillatory cells with precise phase relationship and velocity - modulated frequency . further evidence of phase coding comes from the experiments on spike - phase coding of natural stimuli in auditory and visual primary cortex ( montemurro et al . 2008 ; kayser et al . 2009 ) , and from experiments on short - term memory of multiple objects in prefrontal cortices of monkeys ( siegel et al . these experimental works support the hypothesis that collective oscillations may underlie a phase dependent neural coding and an associative memory behavior which is able to recognize the phase coded patterns . the importance of precise timing relationships among neurons , which may carry information to be stored , is supported also by the evidence that precise timing of few milliseconds is able to change the sign of synaptic plasticity ( markram et al . the dependence of synaptic modification on the precise timing and order of pre- and post - synaptic spiking has been demonstrated in a variety of neural circuits of different species . many experiments show that a synapse can be potentiated or depressed depending on the precise relative timing of the pre- and post - synaptic spikes . this timing dependence of magnitude and sign of plasticity , observed in several types of cortical ( markram et al . 2001 ) and hippocampal ( bi and poo 1998 ; magee and johnston 1997 ; sjostrom et al . 1998 ; bi and poo 2001 ) neurons , is usually termed spike timing dependent plasticity ( stdp ) . the role of stdp has been investigated both in supervised learning framework ( legenstein et al . 2005 ) , in unsupervised framework in which repeating patterns are detected by downstream neurons ( masquelier et al . 2011 ) , cortical development ( song and abbot 2001 ) , generation of sequences ( fiete et al . 2010 ; verduzco- flores et al . 2011 ) and polychronous activity ( izhikevich 2006 ) , and in an associative memory framework with binary units ( scarpetta et al . however , this is the first time that this learning rule has been used to make a if network to work as associative memory for phase - coded patterns of spike , each of which becomes a dynamic attractor of the network . notably , in a phase coded pattern not only the order of activation matters , but the precise spike timing intervals between units . we therefore present a possibility to build a circuit with stable phase relationships between the spikes of a population of if neurons , in a robust way with respect to noise and changes of frequency . the first important result of the paper is the measurement of the storage capacity of the model , i.e. the maximum number of distinct spatiotemporal patterns that can be stored and selectively retrieved , since it has never been computed in a spiking model for spatiotemporal patterns . . 2001 , and references therein ) have focused on storage capacity of binary model with static binary patterns ( hopfield 1982 ) , and much efforts have been done to use more biophysical models and patterns ( gerstner et al . 1996 , 1993 ; hopfield 1995 ; de almeida et al . 2007 ; amit and treves 1989 ; battaglia and treves 1998 ; anishchenko and treves 2006 ; borisyuk and hoppensteadt 1998 ; leibold and kempter 2006 ; memmesheimer and timme 2006 ; olmi et al . 2010 ; scarpetta et al . 2002 , 2011b ) , but , up to our knowledge , without any calculation of the storage capacity of spatiotemporal patterns in if spiking models . notably , by introducing an order - parameter which measures the overlap between phase coded spike trains , we are able quantitatively measure of the overlap between the stored pattern and the replay activity , and to compute the storage capacity as a function of the model parameters . another important result is the study of the different regimes observed by changing the excitability parameters of the network . in particular , we find that near the region of the parameter space where the network tends to become unresponsive and silent there is a regime in which the network responds selectively to cue presentation with a short transient replay of the phase - coded pattern . differently , in the region of higher excitability , the patterns are replayed persistently and selectively , and eventually with more then one spike per cycle . the paper is organized as follows : section 2 introduces the leaky - integrate - and - fire ( if ) neuronal model ; section 3 describes the stdp learning rule used to design the connections ; in section 4 we study the emergence of collective dynamics and introduce an order parameter to measure the overlap between the collective dynamics and the stored phase coded patterns ; section 5 reports on the storage capacity of the network , i.e. the maximum number of patterns that can be stored and selectively retrieved in the network ; the parameter space and the different working regions are also investigated in section 5 ; in section 6 we study the robustness of the retrieval dynamics wrt noise and heterogeneity ; section 7 reports on the implication of this model in the framework of oscillatory interference model of path - integration ; summary and discussion are outlined in section 8 . we consider a recurrent neural network with n(n 1 ) possible connections jij , where n is the number of neural units . the connections jij are designed during the learning mode , when the connections change their efficacy according to a learning rule inspired to the stdp . after the learning stage , the connections values are frozen , and the collective dynamics is studied . this distinction in two stages , plastic connection in the learning mode and frozen connections in the dynamics mode , is a useful framework to simplify the analysis . it also finds some neurophysiological motivations in the effects of neuromodulators , such as dopamine and acetylcholine ( hasselmo 1993 , 1999 ) , which regulate excitability and plasticity . the single neuron model is a leaky integrate - and - fire ( if ) ( gerstner and kistler 2002 ) . this simple choice , with few parameters for each neuron , is suitable to study the emergence of collective dynamics and the diverse regimes of the dynamics , instead of focusing on the complexity of the neuronal internal structure . we use the spike response model ( srm ) formulation ( gerstner and kistler 2002 ; gerstner et al . 1993 ) of the if model , which allows us to use an event - driven programming and makes the numerical simulations faster with respect to a differential equation formulation . in this picture , the postsynaptic membrane potential is given by : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ h_i(t)=\sum\limits_{j } j_{ij } \sum\limits _ { { \hat t}_j > { \hat t}_i } \epsilon\left(t-{\hat t}_j\right ) , \label{if } $ $ \end{document}where jij are the synaptic connections , (t ) describes the response kernel to incoming spikes on neuron i , and the sum over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat t}_j$\end{document } runs over all presynaptic firing times following the last spike of neuron i. namely , each presynaptic spike j , with arrival time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat{t}_j$\end{document } , is supposed to add to the membrane potential a postsynaptic potential of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{ij } \epsilon(t-{\hat t}_j)$\end{document } , where 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{lll } \epsilon\left(t-{\hat t}_j\right)\nonumber\\ \quad = k \left[\ ! \exp\left(-\frac{t-{\hat t}_j}{\tau_m}\right ) - \exp\left(-\frac{t-{\hat t}_j}{\tau_s}\right ) \!\right ] \theta\left(t-{\hat t}_j\right ) \label{tre } \end{array}$$\end{document } where m is the membrane time constant ( here 10 ms ) , s is the synapse time constant ( here 5 ms ) , is the heaviside step function , and k is a multiplicative constant chosen so that the maximum value of the kernel is 1 . the sign of the synaptic connection jij sets the sign of the postsynaptic potential s change , so there s inhibition for negative jij and excitation for positive jij . when the membrane potential hi(t ) exceeds the spiking threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i$\end{document } , a spike is scheduled , and the membrane potential is reset to the resting value zero . we use the same threshold th for all the units , except in section 6 where different values \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i$\end{document } are used and the robustness w.r.t . clearly the spiking threshold th of the neurons is related to the excitability of the network , an increase of the value of th is also equivalent to a decrease of k , the size of the unitary postsynaptic potential , or , equivalently to a global decrease in the scaling factor of synaptic connections jij . numerical simulations of this dynamics are performed for a network with p stored patterns , where connections jij are determined via a learning rule described in the next paragraph . we found that a few number of spikes , given a in proper time order , are able to selectively induce the emergence of a persistent collective spatiotemporal pattern , which replays one of the stored pattern ( see section 4 ) . ( 2007 ) , the average change in the connection jij , occurring in the time interval [ tlearn,0 ] due to periodic spike trains of period t , with tlearn > > t , was formulated as follows : 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta j_{ij } = \frac{t}{t_{\rm learn } } \int\limits_{-t_{\rm learn}}^{0}d t \int\limits_{-t_{\rm learn}}^{0}d t^\prime \ , x_i(t ) a(t - t^\prime ) x_j(t^\prime ) \label{lr } $ $ \end{document}where t / tlearn is a normalization factor , xj(t ) is the activity of the pre - synaptic neuron at time t , and xi(t ) the activity of the post - synaptic one . it means that the probability for unit i to have a spike in the interval ( t , t + t ) is proportional to xi(t)t in the limit t0 . the learning window a( = t t ) is the measure of the strength of synaptic change when a time delay occurs between pre and post - synaptic activity . to model the experimental results of stdp in hippocampal neurons , the learning window a( ) should be an asymmetric function of , mainly positive ( ltp ) for > 0 and mainly negative ( ltd ) for < 0 . equation ( 3 ) holds for activity pattern x(t ) which represents instantaneous firing rate , and is suitable to use in analog rate models ( scarpetta et al . 2001 , 2002 , 2008 ; yoshioka et al . 2007 ; scarpetta and marinaro 2005 ) and spin network models ( scarpetta et al . 2010 , 2011b ) . differently , here , being interested in spiking neurons , the patterns to be stored are defined as precise periodic sequence of spikes , i.e. spike - phase coded patterns . namely , activity of the neuron j is a spike train at times \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t^\mu_j$\end{document } , 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_j^\mu(t)=\sum\limits_n \delta(t- ( t^\mu_j+ n t^\mu ) ) , \label{spiketr1 } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t^\mu_j+n t^\mu$\end{document } is the set of spikes times of unit j in the pattern with period t , and frequency = 1/t . ( 3 ) , the change in the connections jij due to the learning of the pattern when the time duration of the learning process tlearn is longer then a single period t , is simply given by 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ j_{ij}^\mu=\sum\limits_{n=-\infty}^{\infty } a(t^\mu_j - t^\mu_i+ n t^\mu ) . \label{lr2 } $ $ \end{document}the window a( ) , shown in fig . 1 , is given by 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a(\tau ) = \left \ { \begin{array}{ll } a_p e^{-\tau / t_p } - a_{\tiny d } e^{-\eta \tau / t_p } & \quad \textrm{for}\quad \tau>0 \\ a_p / t_{\tiny d } } & \quad \textrm{for}\quad \tau<0 , \end{array}\right . \label{at } $ $ \end{document}with the same parameters used in abarbanel et al . ( 2002 ) to fit the experimental data of bi and poo ( 1998 ) , namely ap = [1/tp + \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\eta / t_d]^{-1}$\end{document } , ad = [/tp + \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/t_d]^{-1}$\end{document } , with tp = 10.2 ms , td = 28.6 ms , this function satisfies the balance condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\int_{-\infty}^\infty a(\tau ) d\tau = 0$\end{document}. notably , when a( ) is used in eq . ( 5 ) to learn phase - coded patterns with uniformly distributed phases , then the property \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\int a(\tau ) dt = 0$\end{document } assures that in the connection matrix the summed excitation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(1/n)\sum_{i , j_{ij}>0 } j _ { ij}$\end{document } and the summed inhibition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(1/n)\sum_{i , j_{ij}<0 } j _ { ij}$\end{document } are equal in the thermodynamic limit , and therefore it assures a balance between excitation and inhibition . fig . 1(a ) plot of the learning window a( ) used in the learning rule ( see eqs . ( 3 ) , ( 5 ) and ( 6 ) ) to model stdp effects . the parameters of the function a( ) ( eq . ( 6 ) ) are determined by fitting the experimental data reported in bi and poo ( 1998 ) ( a ) plot of the learning window a( ) used in the learning rule ( see eqs . ( 3 ) , ( 5 ) and ( 6 ) ) to model stdp effects . the parameters of the function a( ) ( eq . ( 6 ) ) are determined by fitting the experimental data reported in bi and poo ( 1998 ) writing eqs . ( 35 ) , implicitly we have assumed that , with periodic phase - coded spike trains used to induce plasticity , the effects of all separate spike pairs sum linearly , each weighted by the same stdp window reported in fig . 1 . 1 are indeed typically measured by giving an order of 100 pairs of spikes repeatly , with fixed phase relationship , and fixed frequency in a proper range . however , in different situations , for instance if the frequency is too low or to high ( sjostrom et al . 2001 ) , or in case of few spike pairs ( wittenberg and wang 2006 ) , the timing dependence of plasticity is not well described by the bidirectional window used here , and a more detailed model is needed to account for integration of spike pairs when arbitrary trains are used ( see shouval et al . the spikes patterns used in this work are periodic spatiotemporal sequences , made up of one spike per cycle and each of which has a phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi^\mu_j$\end{document } randomly chosen from a uniform distribution in [ 0,2 ) . in each pattern , information is coded in the precise time delay between spikes of unit i and unit j , which corresponds to a precise phase relationship among units i and j. a spatiotemporal pattern represented in this way is often called phase coded pattern . pattern s information is coded in the spiking phases which , in turn , shape the synaptic connectivity responsible of the emerging dynamics and the memory formation . the set of timing of spikes of unit j can be defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t^\mu_j + n t^\mu = ( \phi^\mu_j)/(2\pi \nu^\mu ) + n/\nu^\mu$\end{document } , where is the oscillation frequency of the neurons . thus , each pattern is represented through the frequency and the specific phases of spike \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi^\mu_j$\end{document } of the neurons j = 1, .. ,n . the change in the connection jij provided by the learning of pattern is given by 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{rll } j_{ij}^\mu&=&\displaystyle\sum\limits_{n } a(t^\mu_j - t^\mu_i+n t^\mu)\nonumber\\ & = & \sum\limits_n a\left(\frac{\phi^\mu_j } { 2\pi \nu^\mu}-\frac{\phi^\mu_i}{2\pi\nu^\mu } + n/\nu^\mu \right ) . \label{conn } \end{array}$$\end{document } when multiple phase coded patterns are stored , the learned connections are simply the sum of the contributions from individual patterns , namely 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ j_{ij}=\sum\limits_{\mu=1}^p j^{\mu}_{ij}. \label{connp } $ $ \end{document}note that ring - like topology with strong unidirectional connections is formed only in the case p=1 , when a single pattern is stored . when multiple patterns are stored in the same connectivity , with phases of one pattern uncorrelated with the others , bidirectional connections are possible , and the more the stored patterns , the less the ring - like is the connectivity . even in the cases when the connectivity is not ring - like the network is still able to retrieve each of the p stored patterns in a proper range of threshold values ( see storage capacity in section 5 ) . we study a recurrent network with n leaky integrate and fire units , with connections fixed to the values calculated in eqs . the results show that , within a well specified range of parameters , our if network is able to work as an associative memory for spike - phase patterns . in order to check if the network is able to retrieve selectively each of the stored patterns , we give an initial signal , made up of m n spikes , taken from the stored pattern , and we check if this initial short cue is able to selectively trigger a collective sustained activity that is the replay of the same stored pattern , i.e. checking if the sustained activity has spikes aligned to the phases \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^\mu$\end{document } of pattern . an example of successful selective retrieval process is shown in fig . 2 where , depending on the partial cue presented to the network , a different collective activity emerges with the phases of the firing neurons which resemble one or another of the stored patterns . 2examples of selective successful retrieval ( ( c ) , ( d ) ) of two stored patterns ( ( a ) , ( b ) ) . the raster plot of 50 units ( randomly chosen ) are shown sorted on the vertical axis according to increasing values of phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^1$\end{document } of the first stored pattern = 1 . the network has n = 3000 if neurons , th = 70 and connections given by eqs . ( 7 ) and ( 8) with p = 5 stored patterns at = 3 hz . two of the stored patterns used during the learning mode are shown in ( a ) , ( b ) . the dynamics emerging after a short train of m = n/10 spikes with phases similar to the pattern shown in ( a ) and ( b ) , is shown in ( c ) and ( d ) respectively . the dynamics of the network , after a transient , is periodic of period t. the spikes which belong to the trigger are shown in pink in ( c ) and ( d ) , the other different colors represent the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_i / t\!\mod 4$\end{document } , where ti is the time of the spike of the unit i during the emerging spontaneous dynamics . figure ( c ) shows that when the network dynamic is stimulated by a partial cue of pattern = 1 , the neurons oscillate with phase alignments resembling pattern = 1 , but at different frequency . otherwise , in ( d ) , when the partial cue is taken from pattern = 2 , the neurons phase relationships , even if periodic , are uncorrelated with pattern = 1 , and recall the phase of pattern = 2 examples of selective successful retrieval ( ( c ) , ( d ) ) of two stored patterns ( ( a ) , ( b ) ) . the raster plot of 50 units ( randomly chosen ) are shown sorted on the vertical axis according to increasing values of phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^1$\end{document } of the first stored pattern = 1 . the network has n = 3000 if neurons , th = 70 and connections given by eqs . ( 7 ) and ( 8) with p = 5 stored patterns at = 3 hz . two of the stored patterns used during the learning mode are shown in ( a ) , ( b ) . the dynamics emerging after a short train of m = n/10 spikes with phases similar to the pattern shown in ( a ) and ( b ) , is shown in ( c ) and ( d ) respectively . the dynamics of the network , after a transient , is periodic of period t. the spikes which belong to the trigger are shown in pink in ( c ) and ( d ) , the other different colors represent the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_i / t\!\mod 4$\end{document } , where ti is the time of the spike of the unit i during the emerging spontaneous dynamics . figure ( c ) shows that when the network dynamic is stimulated by a partial cue of pattern = 1 , the neurons oscillate with phase alignments resembling pattern = 1 , but at different frequency . otherwise , in ( d ) , when the partial cue is taken from pattern = 2 , the neurons phase relationships , even if periodic , are uncorrelated with pattern = 1 , and recall the phase of pattern = 2 in this work the cue is a stimulation with m spikes , with m = n/10 , at times \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t^\mu_i= t_{\rm stim } \phi_i^\mu$\end{document } , 0 < i < m , with tstim = 50 ms . in the example shown in fig . 2 the short stimulation ( which lasts less then 5 ms , shown in pink in all the figures ) has the effect to selectively trigger the sustained replay of pattern . note that the retrieval dynamics has the same phase relationship among units than the stored pattern , but the replay may happen on a time scale different from the scale used to store the pattern , and the collective spontaneous dynamics is a time compressed ( or dilated ) replay of the stored pattern . indeed , the period of the collective periodic pattern which emerges during retrieval stage may be different then the period of the periodic pattern used in the learning stage . in the example of fig . 2 the time scale of the retrieval dynamics ( fig . 2(c ) , ( d ) ) is faster then the time scale used to learn the patterns ( fig . in the following we will study the factors affecting the time scale during retrieval , given the time scale of the pattern used during learning . clearly , regions of the parameter space in which the network is unable to retrieve selectively the patterns also exist . in these regions the retrieval dynamics may correspond to a mixture of patterns or a spurious state , i.e. a state which is not correlated with any of the stored patterns because the number of stored patterns exceeded the storage capacity of the network . as discussed below , the storage capacity , defined as the maximum number of encoded and successfully retrieved patterns , depends on the frequency used during the learning stage ( which affects connectivity ) , and on the spiking threshold of the units ( which affects excitability and network dynamics ) . 3(b ) the network has too low excitability and the response is not persistent , while in fig . 3(a ) the emerging dynamics is not correlated with any of the stored patterns . fig . 3example of neural response in two case of failure of retrieval . a spurious state emerge in ( a ) , while a short transient response emerges in the case shown in ( b ) . n = 3000 and = 3 hz as in previous picture , while the values th and p are th = 10 , p = 5 in ( a ) and th = 95 , p = 5 in ( b ) . the dynamics emerging after a short train of m = n/10 spikes with phases equal to the stored pattern ( pattern shown in fig . for clarity , the raster plot of only 50 ( randomly chosen ) units are shown , sorted according to increasing value of phase i of the stored pattern example of neural response in two case of failure of retrieval . a spurious state emerge in ( a ) , while a short transient response emerges in the case shown in ( b ) . n = 3000 and = 3 hz as in previous picture , while the values th and p are th = 10 , p = 5 in ( a ) and th = 95 , p = 5 in ( b ) . the dynamics emerging after a short train of m = n/10 spikes with phases equal to the stored pattern ( pattern shown in fig . 2(a ) ) , is not a self - sustained retrieval of the pattern . for clarity , the raster plot of only 50 ( randomly chosen ) units are shown , sorted according to increasing value of phase i of the stored pattern to measure quantitatively the success of the retrieval , in analogy with the hopfield model , we introduce an order parameter , which estimates the overlap between the network collective activity during the spontaneous dynamics and the stored phase - coded pattern . this quantity is 1 when the phases j of neurons j coincides with the stored phases \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_j^\mu$\end{document } , and is close to zero when the phases are uncorrelated with the stored ones . therefore , we consider the following dot product |m(t)| = < (t)| > where is the vector having components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ e^{i \phi_j^\mu}$\end{document } , namely : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ |m^\mu(t)|= \left|\frac{1}{n}\sum\limits_{\stackrel{j=1,\ldots , n}{\scriptscriptstyle t - t^*<t_j^*<t } } e^{-i 2 \pi t_j^*/t^ * } e^{i \phi_j^\mu}\right| \label{nn } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_j^*$\end{document } is the spike timing of neuron j during the spontaneous dynamics , and t is an estimation of the period of the collective spontaneous periodic dynamics . ( 9 ) is equal to 1 when the phase - coded pattern is perfectly retrieved ( i.e. same sequence and phase relationships among spikes , even though on a different time scale ) , while is of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\simeq 1/\sqrt{n}$\end{document } when phases of spikes are uncorrelated to the stored phases . the order parameter |m| allow us to measure the network storage capacity in the space of parameters th and . note that the value of m(t ) between two periodic spike trains measures the similarity in the sequence of spiking neurons and in the phase lag between the spikes , being invariant by a simple change in time scale . this is a suitable choice especially when the replay of a spatio temporal pattern has to be detected independently from the compression of the time scale . note that if we have a spike train that is not periodic , we can not define the period , however we can define the order parameter eq . . this can be useful in the case when one looks for a short replay hidden in a not - periodic spike train , such in many experimental situations . the value of m(t ) after a transient converges to a stable value which is close to one when pattern is retrieved ( for example in fig . 2(c ) at large times m = 1 , and m = 0.01 ) while m(t ) is of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\simeq 1/\sqrt{n}$\end{document } for all in the case of failure of retrieval . 3(a ) m(t ) after the transient has values in the range 0.010.02 for all because the emerging dynamics is a spurious state not correlated with any of the stored phase - patterns , while m(t ) is zero in fig . , the storage capacity of the model is analyzed considering the maximum number of patterns that the network is able to store and selectively recall . in particular we investigate the role of two model parameters : the frequency of the stored patterns , and the spiking threshold th affecting the excitability of the network . numerical simulations of the if network with n = 3000 neurons were performed by systematically changing the value of the spiking threshold th , the connections jij , and for different number of patterns p and frequency . here we propose results for a unique value of the spiking threshold th for all neurons , however the behavior is also robust with respect to a variability in the threshold values among neurons , as reported in the next section . network storage capacity is defined as c = pmax / n , where n is the number of neurons and pmax is the maximum number of patterns that can be stored and successfully retrieved with an overlap |m| larger than a certain value , which measures the degree of similarity . given that in our simulations the overlap |m(t)| at large times has mostly two possible values , close to one ( success ) or close to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt n$\end{document } ( failure ) , we fixed the desired similarity value to 0.5 , since the whole storage capacity analysis is very robust with respect to this parameter ( since the transition between low values and high values of |m(t)| as a function of p is sharp ) . patterns with random phases were extracted and used to define the network connections jij with the rule eq . after the stimulation with a short train of m = n/10 spikes taken at times ti from the first pattern , the dynamics is simulated and the overlap defined in eq . if the overlap |m(t)| , averaged over 50 runs , is greater than 0.5 at time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t>\bar t$\end{document } ( where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar t= 600$\end{document } ms for all the simulations ) , then we consider the retrieval successful for that pattern . the maximum value of p , for which the network is able to successfully replay each of the stored patterns , defines the storage capacity of the network . the storage capacity as a function of the spiking threshold th and storing frequency is reported in fig . the largest capacity is achieved when the frequency of the stored patterns during learning is 8 hz and the spiking threshold of the units during retrieval is th 130 , which provides a capacity max = pmax / n = 0.016 . 4(a ) storage capacity in a network of n = 3000 units , as a function of the spiking threshold th and oscillation frequency of stored patterns . the maximum number of patterns successful retrievable pmax is shown in color - coded legend , the value grows from pmax = 0 ( dark blue ) to pmax = 50 ( strong red ) . ( b ) the storage capacity pmax as a function of the frequency of stored patterns , once fixed the threshold th to the optimal value for each frequency ( a ) storage capacity in a network of n = 3000 units , as a function of the spiking threshold th and oscillation frequency of stored patterns . the maximum number of patterns successful retrievable pmax is shown in color - coded legend , the value grows from pmax = 0 ( dark blue ) to pmax = 50 ( strong red ) . ( b ) the storage capacity pmax as a function of the frequency of stored patterns , once fixed the threshold th to the optimal value for each frequency in fig . 4(b ) we show the storage capacity pmax as a function of the frequency once fixed the value of the threshold th to the optimal value , corresponding to highest capacity for each frequency . the optimal storing frequencies and threshold values depend on the time constants of the model , such as the s,m of the if units and the temporal shape of the learning kernel a( ) , whereas different shapes of a( ) may subserve to different storing frequency ranges . in this work s,m and a( ) are set to the values described in section 2 , and the emergent collective dynamics is studied as a function of the other network parameters . 4(b ) shows that for the learning kernel a( ) used here , there is peak in the storage capacity around 8 hz , in the range 220 hz . figure 4(a ) also proves that , for each stored frequency , a large interval of spiking threshold values th exists for which the network is still able to work properly as associative memory for phase - coded patterns . the associative memory properties as a function of the spiking threshold are reported in fig . 5 , when the oscillation frequency of the patterns stored during learning is = 3 hz . 5 corresponds to cases in which the retrieval is successful and the cue is able to selectively activate the self - sustained replay of the stored pattern ( with an order parameter m larger than 0.5 ) . when spiking threshold changes in the range 10 < th < 90 the storage capacity changes between pmax / n = 1/3000 and pmax / n = 29/3000 . fig . 5(a ) storage capacity at = 3 hz : the region of successful retrieval as a function of spiking threshold and number of patterns is marked in green . the region with persistent activity not correlated with any of the stored pattern is marked in red ( spurious states ) , and the region in which the network responds with only a short transient and then becomes silent is marked in blue ( see examples in fig . ( b ) the probability that the size stot of the network response , measured as the number of the spikes that follow the cue stimulation , is larger than n , with n = 0,n/2,n , is shown as a function of spiking threshold th , in a network with = 3 hz and p = 1 . as always in this paper the number of units is n = 3000 . the figure shows that near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^{\rm crit } \simeq 90$\end{document } there s a transition from a region of persistent replay to a region of silence ( a ) storage capacity at = 3 hz : the region of successful retrieval as a function of spiking threshold and number of patterns is marked in green . the region with persistent activity not correlated with any of the stored pattern is marked in red ( spurious states ) , and the region in which the network responds with only a short transient and then becomes silent is marked in blue ( see examples in fig . ( b ) the probability that the size stot of the network response , measured as the number of the spikes that follow the cue stimulation , is larger than n , with n = 0,n/2,n , is shown as a function of spiking threshold th , in a network with = 3 hz and p = 1 . as always in this paper the number of units is n = 3000 . the figure shows that near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^{\rm crit } \simeq 90$\end{document } there s a transition from a region of persistent replay to a region of silence outside the green region the number of patterns exceeds the storage capacity and the retrieval fails . when the number p of patterns exceeds the storage capacity pmax , the network responds with a self - sustained activity that is not correlated with any of the stored patterns , i.e. a spurious state . in this regime , 5(a ) , the order parameter m(t ) is of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt n$\end{document } for all the stored patterns ( see also raster plot in fig . on the contrary , in the high th regime , the network tends to become silent and unresponsive . indeed , in the region marked with blue color , the network responds to the initial cue stimulation with a short transient and then became silent . in this case the value of m(t ) is zero because there is no self - sustained activity at time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t>\bar t$\end{document } , meaning that the stored attractors become unstable when th is too high ( see raster plot in fig . for values of the threshold greater than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^{\rm crit}$\end{document } , independently from p , the network activity is never persistent , as reported in fig . 5(a ) where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^{\rm crit}=90$\end{document}. at thresholds close to this critical value the network responds with a transient activity that is a short replay of the stored pattern , but not a persistent replay . the size stot of the network response , measured as the number of spikes that follow the cue stimulation , is reported in fig . 5(b ) as a function of th , for a network with = 3 hz , n = 3000 and p = 1 . in we focused on the dependence of the frequency of collective oscillations during replay on the model parameters . figure 6(a ) shows the collective frequency of replay as a function of the frequency of the patterns stored in the learning stage with n = 3000 . the red dots in the figure refer to the frequencies of oscillations observed during retrieval at the optimal spiking threshold ( where the maximum storage capacity occurs ) , while the bar indicates the available range of frequencies of replay , accessible through a change in the spiking threshold . important to note is that , in most of the cases , the frequency of the stored pattern and the collective replay frequency do not coincide , since the pattern is replayed compressed ( or dilated ) in time , on a time scale dependent on the network parameters . we observe that for the chosen parameters m , s of the network , and the given shape of a( ) , the replay occurs on a compressed time scale for all stored patterns of frequency lower then 25 hz , while the two time scale coincide when 25 hz . the dependence of the frequency of the collective oscillations on the spiking threshold is shown in fig . besides , for low stored frequencies ( 14 hz ) the frequency of the replay is very sensitive to the threshold value , changing from 6 hz at high spiking threshold to 30 hz at low threshold . 6(a ) frequency of the collective dynamics during replay as a function of the frequency of stored patterns in the network with n = 3000 units . ( b ) frequencies of the collective dynamics during replay as a function of the spiking threshold and for different stored frequencies ( see colors legend ) . pattern is replayed on a time scale which becomes faster if we decrease threshold th for the most of the frequencies . the dependence is much stronger for 4 hz . ( c ) the number of spikes per cycle as a function of the spiking threshold th in networks with different frequency of stored pattern . ( d ) frequency of the dynamics during replay as a function of ratio between spiking threshold and network size n. red dots are results for a network with n = 10000 units , while blue squares are results for a network with n = 1000 units . size of the symbols refers to the stored frequency , small symbols ( on the top of the picture ) correspond to stored frequency = 10 hz , medium size symbols correspond to stored frequency = 3 hz , and large symbols ( bottom ) correspond to stored frequency = 1 hz ( a ) frequency of the collective dynamics during replay as a function of the frequency of stored patterns in the network with n = 3000 units . ( b ) frequencies of the collective dynamics during replay as a function of the spiking threshold and for different stored frequencies ( see colors legend ) . pattern is replayed on a time scale which becomes faster if we decrease threshold th for the most of the frequencies . the dependence is much stronger for 4 hz . ( c ) the number of spikes per cycle as a function of the spiking threshold th in networks with different frequency of stored pattern . ( d ) frequency of the dynamics during replay as a function of ratio between spiking threshold and network size n. red dots are results for a network with n = 10000 units , while blue squares are results for a network with n = 1000 units . size of the symbols refers to the stored frequency , small symbols ( on the top of the picture ) correspond to stored frequency = 10 hz , medium size symbols correspond to stored frequency = 3 hz , and large symbols ( bottom ) correspond to stored frequency = 1 hz we also investigate the frequency s dependence on network size n. in fig . 6(d ) red dots are results for a network with n = 10000 units , while blue squares are results for a network with n = 1000 units . if what counts is only the time lag between the single units consecutive in the sequence , then one expects that result with 1 hz stored at n = 10000 would be similar to 10 hz stored at n = 1000 , and this is not the case . we see that when we use a storage frequency equal to 10 hz ( which corresponds to different time lag between cells depending on n ) , then the oscillation frequency during replay is around 30 hz in both networks ( both n = 1000 , and n = 10000 ) , while , on the other hand , if we have a storage frequency equal to 1 hz the oscillation frequency during replay may span a large range ( 5 hz25 hz ) in both networks . figure 6(d ) also shows that frequency of replay depends on the ratio between spiking threshold and network size n , and that the high sensitivity on spiking threshold value holds , when stored frequency is low ( 14 hz ) , also at different values of the network size . this open the possibility to govern the oscillation frequency of the collective replay activity via neuromodulators which change the excitability and therefore the spiking threshold of the neurons . since in our model ( see eqs . ( 1 ) and ( 2 ) ) a change of the threshold is equivalent to a change in the scale factor of all synaptic connections , a similar effect might be achieved also by simply driving the cells more due to increased synaptic input . importantly , the sensitivity of collective oscillation frequency on spiking threshold is not a sensitivity of the single unit but of the collective behavior , since , as discussed in section 6 , if we change the spiking threshold of few units the collective rhythm is still unique for the whole population . the replay frequency depends on the average threshold among units , but all the units have the same oscillation frequency during replay . moreover , for networks with 10 hz , whose replay frequency does not considerably change with spiking threshold , the replay dynamics is still affected by the spiking threshold . indeed , in this case , the number of spikes per cycle increases with lowering of the spiking threshold . the raster plots show the same pattern replayed in three networks having different values of the spiking threshold th : a burst of activity takes place within each cycle , with phases aligned with the pattern , with a number of spikes per cycle dependent on the value of th . 6(c ) where the number of spikes per cycle is reported as a function of spiking threshold , at different values of stored frequencies . therefore , by lowering the spiking threshold the replay activity occurs with more than one spike per cycle , or on a faster time scale ( see fig . 7modulation of spiking threshold changes the number of spikes per cycle , keeping preserved the phase relationship among units . recall of the pattern = 1 for networks of n = 3000 units , = 20 hz and different values of spiking threshold th = 80,65,40 is shown respectively in ( a)(c ) . depending on the value of the spiking threshold th , the phase - coded pattern is replayed with a different number of spikes per cycle . spike of the cue stimulation are shown in pink , while the response of the network in black . for clarity , the raster plot of only 50 ( randomly chosen ) units are shown , sorted according to increasing value of phase i of the stored pattern modulation of spiking threshold changes the number of spikes per cycle , keeping preserved the phase relationship among units . recall of the pattern = 1 for networks of n = 3000 units , = 20 hz and different values of spiking threshold th = 80,65,40 is shown respectively in ( a)(c ) . depending on the value of the spiking threshold th , the phase - coded pattern is replayed with a different number of spikes per cycle . spike of the cue stimulation are shown in pink , while the response of the network in black . for clarity , the raster plot of only 50 ( randomly chosen ) units are shown , sorted according to increasing value of phase i of the stored pattern the behavior of the output oscillation frequency suggests that a parameters region exists where the network always responds with one spike per cycle . in this region an increase of the excitability produces a growth of the frequency of oscillation up to a plateau value . differently , for higher excitability the frequency does not increase while the number of spikes per cycle grows . this means that the different frequencies , in addition to the information coded in the phase relationship , can code other information in relationship with the level of spiking threshold : at high frequency the threshold changes the number of spikes per cycle , while at low frequency the threshold changes the frequency of the collective oscillations during the replay . this open the possibility to have a coding scheme in which , while the phases encode pattern s information , a change in frequency or a change in rate in each cycle represents the strength and saliency of the retrieval or it may encode another variable ( lengyel and dayan 2007 ) . the recall of the same phase - coded pattern with a different number of spikes per cycle is particularly interesting at the light of recent observations of huxter et al . ( 2003 ) in hippocampal place cells , showing the occurrence of the same phases with different rates . the authors prove that the phase of firing and firing rate are dissociable and can represent two independent variables , e.g. the animal location within the place field and its speed of movement through the field . notably , the recall of the same phase coded pattern with different frequencies of oscillation is also relevant and accords well with the need to have stable precise phase relationship among cells with frequency of oscillation modulated by parameters such as the speed of the animal ( blair et al . the value of the frequency of collective activity during the replay clearly is related not only to the threshold and the stored frequency , but also to the shape of the learning window a( ) and on the two characteristic times of the model s , m . a systematic study of the dependence of the replay time scale on the shape of stdp and the characteristic times of the neuron model has not yet done in a spiking model , however a dependence on the asymmetry of a(t ) has been analytically found in a simple model with analog neurons and a single characteristic time ( yoshioka et al . while in the hopfield model the patterns are static , and information is coded in a binary pattern \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bold s^\mu= s_1^\mu,\ldots , s_n^\mu$\end{document } , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_i^\mu \in \{\pm 1\}$\end{document } , here , in this study , the patterns are time dependent , and information is coded in the phase pattern \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bold{\phi}^\mu=\phi_1^\mu, ... ,\phi_n^\mu$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^\mu \in [ 0,2\pi]$\end{document } , where the value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^\mu /(2\pi\nu^\mu)$\end{document } represents the time shift of the spike of unit i with respect to the collective rhythm , i.e the time delay among units . however , as for the hopfield model , the patterns stored in the network are attractors of the dynamics , when p do not exceeds storage capacity , and the dynamics during the retrieval is robust with respect to noise . input noise , i.e when a poissonian noise i(t ) is added to the postsynaptic potential hi(t ) given in eq . . the total postsynaptic potential of each neuron i is then given by 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ h_i(t)=\eta_i(t)+\sum\limits_{j}j_{ij}\sum\limits_{{\hat t}_j > { \hat t}_i } \epsilon\left(t-\hat{t_j}\right ) $ $ \end{document}where i(t ) is modelled as 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \eta_i(t)=j_{\text{noise}}\sum\limits_{\hat{t}_{\text{noise}}>\hat{t}_i } \epsilon\left(t-\hat{t}_{\text{noise}}\right ) . $ $ \end{document}the times \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat{t}_{\text{noise}}$\end{document } are randomly extracted for each neuron i , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{\text{noise}}$\end{document } are random strengths , extracted independently for each neuron i and time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat{t}_{\text{noise}}$\end{document}. the intervals between times \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat{t}_{\text{noise}}$\end{document } are extracted from a poissonian distribution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(\delta t ) \propto e^{-\delta t/(n\tau_{\text{noise}})}$\end{document } , while the strength \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{\text{noise}}$\end{document } is extracted from a gaussian distribution with mean \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar j}_{\text{noise}}$\end{document } and standard deviation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma(j_{\text{noise}})$\end{document}. the network dynamics during the retrieval of a pattern in presence of noise is shown in fig . 8 with different levels of noise ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tau_{\text{noise}}=10$\end{document } ms , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar j=0 $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma(j_{\text{noise}})=0,10,20,30$\end{document } in a , b , c and d , respectively ) . results show that when the noise is not able to move the dynamics out of the basin of attraction , the errors do not sum up , and the phase relationship is preserved over time ( see fig . 8(a)(c ) ) . if the input noise is very high , as in the example of fig . raster plots show that , when the pattern retrieval is triggered , network s spikes continue to have phase alignments resembling the pattern even in presence of noise . errors do not sum up until the system is in the basin of attraction of the phase - coded pattern , as in ( a)(c ) . different levels of noise are used in ( a)(d ) ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma(j_{\text{noise}})=0,10,20,30$\end{document } respectively ) , and pattern is triggered with m = n/10 as in previous cases . only in ( d ) the level of noise is too high and the system goes out of the basin of attraction . ( e ) , ( f ) for comparison , the dynamics , when the retrieval is not triggered ( m = 0 ) , is shown in subplot ( ( e ) and ( f ) ) in presence of the same noise used in ( c ) and ( d ) . figure ( e ) shows that the noise used in ( c ) usually affects strongly the dynamics of the network , however if the collective oscillation is retrieved the system is robust wrt noise . thresholds in all figures are th = 80 , n = 3000 , and synaptic connections jij are build learning p = 2 phase - patterns at = 3 hz ( a)(d ) robustness wrt noise . raster plots show that , when the pattern retrieval is triggered , network s spikes continue to have phase alignments resembling the pattern even in presence of noise . errors do not sum up until the system is in the basin of attraction of the phase - coded pattern , as in ( a)(c ) . different levels of noise are used in ( a)(d ) ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma(j_{\text{noise}})=0,10,20,30$\end{document } respectively ) , and pattern is triggered with m = n/10 as in previous cases . only in ( d ) the level of noise is too high and the system goes out of the basin of attraction . ( e ) , ( f ) for comparison , the dynamics , when the retrieval is not triggered ( m = 0 ) , is shown in subplot ( ( e ) and ( f ) ) in presence of the same noise used in ( c ) and ( d ) . figure ( e ) shows that the noise used in ( c ) usually affects strongly the dynamics of the network , however if the collective oscillation is retrieved the system is robust wrt noise . thresholds in all figures are th = 80 , n = 3000 , and synaptic connections jij are build learning p = 2 phase - patterns at = 3 hz in order to see the effects of input noise level used in fig 8(c ) , ( d ) , we report in fig . 8(e ) , ( f ) the network dynamics when the pattern retrieval is not initiated ( m = 0 ) . in particular , fig . 8(c ) is strong enough to generate spontaneous random activity in absence of the initial triggering , but is not sufficient to destroy the attractive dynamics during a successful retrieval . as in the hopfield model , errors do not sum up and the dynamics spontaneously goes back to the retrieved phase - coded pattern for all the perturbations that leave the system inside the basin of attraction . this analysis can be carried out by using a different value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i$\end{document } of spiking threshold for each neuron i : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \theta_{\rm th}^i= ( 1 + z \zeta_i ) \theta_{\rm th } $ $ \end{document}where i is a random number extracted from a uniform distribution in [ 1,1 ] , and z is the degree of heterogeneity . even with high degree of heterogeneity , the emergence of the retrieval collective dynamics forces all neurons to have exactly the same frequency of oscillation and to keep a precise phase relationship , in a very robust manner . figure 9(a ) , ( b ) shows the dynamics with threshold heterogeneity z = 0.2 , 0.5 respectively , while the remaining parameters are set to the values of fig . raster plots show that , when the pattern retrieval is triggered , units participate to the network collective oscillation , showing all the same frequency and phase alignments resembling the pattern , even in presence of threshold values heterogeneity among units . spiking thresholds of neuron i are distributed according to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i=\theta_{\rm th } ( 1+z \zeta)$\end{document } with average th = 80 and z = 0.2,0.5 in ( a ) and ( b ) respectively . 8(a ) ( n = 3000 , m = n/10 , and synaptic connections jij are build learning p = 2 phase - patterns at = 3 hz ) robustness wrt heterogeneity of spiking threshold values . raster plots show that , when the pattern retrieval is triggered , units participate to the network collective oscillation , showing all the same frequency and phase alignments resembling the pattern , even in presence of threshold values heterogeneity among units . spiking thresholds of neuron i are distributed according to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i=\theta_{\rm th } ( 1+z \zeta)$\end{document } with average th = 80 and z = 0.2,0.5 in ( a ) and ( b ) respectively . 8(a ) ( n = 3000 , m = n/10 , and synaptic connections jij are build learning p = 2 phase - patterns at = 3 hz ) the above analysis shows that a unique collective frequency emerges , which is the frequency corresponding to the mean value of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\theta_{\rm th}^i$\end{document}. this is also evident by comparing fig . = 0.5 it can be seen an additional small phase shift proportional to the value of of each neuron , but collective activity is preserved . clearly if z is too high and threshold values are distributed out of the region of successful retrieval the network is unable to retrieve the pattern and failure happens , as already discussed in section 5 . recently , path - integration system and the hippocampal - entorinal cortex circuit have been deeply investigated ( okeefe and recce 1993 ; okeefe and burgess 2005 ; burgess et al . 2007 ; jeewajee et al . 2008 ; blair et al . 2008 ; euston et al . 2007 ; geisler et al . 2007 ; mcnaughton et al . 2006 ) , showing that place cells and grid cells form a map in which precise phase relationship among units play a central role to generate the spatial tuning . a number of models of the spatial firing properties of place and grid cells were offered . generally two main categories are distinguished : models which focus on continuous attractor mechanisms and models which use interference between oscillators at dynamically modulated frequencies ; however , deeper computational principles may exist that unify the different cases of neural integration ( issa and zhang 2012 ) . in oscillatory interference models ( okeefe and burgess 2005 ; 2008 ; welday et al . 2011 ; zilli and hasselmo 2010 ) ( see also giocomo et al . 2011 , for a review ) the total synaptic input to a neuron ( such as a grid cell or a place cell ) is a weighted sum of the activities of n oscillatory inputs , whose oscillation frequency is modulated by the rat velocity and head direction . grid and place cells , according to these models , derive their temporal and spatial properties simply by detecting synchrony among such velocity - modulated oscillatory inputs . the oscillatory interference theory is one possible hypothesized mechanism of path integration and it has not been conclusively accepted or rejected . it is supported by recent studies suggesting that the predicted velocity - modulated oscillators exist as theta cells ( interneurons found throughout the septo - hippocampal circuit ) whose inter - burst frequency shows a cosine modulation by running direction and a linear increase with running velocity ( welday et al . such velocity - modulated oscillatory input was hypothesized to come from single oscillatory neurons ( okeefe and burgess 2005 ) , or networks such as subcortical ring attractors generating velocity - modulated theta oscillations ( blair et al . the properties of modulation and stability of frequency , and stability of multiple phase relationships , make our circuit a possible mechanism to build the velocity - modulated oscillators of the oscillatory interference theory . indeed our circuit has a collective oscillation frequency , which depends on the frequency stored in the connectivity matrix , and that can be modulated by changing the parameters such as th . each neuron in the circuit has a phase determined by its position in that network , i.e. determined by the phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi_i^\mu$\end{document } of the stored phase - pattern . if the parameter th is modulated by animal speed , then the collective oscillation frequency of the circuit is modulated by the animal s speed , while the neurons preserve stable phase relationship among them . the other persistent - firing models ( blair et al . 2008 ; zilli and hasselmo 2010 ; welday et al . 2011 ) of the oscillators needed by the oscillatory interference theory suffer from problems related to robustness , as those encountered by the single - cell oscillatory models ( zilli et al . 2009 ) , due to the variability in the frequency of persisting spiking ( giocomo et al . 2011 ) . indeed the oscillatory interference models impose strict constraints upon the dynamical properties of the velocity - modulated oscillatory inputs , which have to preserve robust velocity - modulated frequency and stable phase relationships among them on relevant time scales in a manner robust to noise ( many seconds , or dozens of theta cycle periods ) ( blair et al . 2008 ; welinder et al . 2008 ; zilli et al . 2009 ; giocomo et al . notably , in our model , since connections jij among units in the circuit are fixed by the learning rule ( eq . ( 7 ) ) , dynamics is a retrieval process and neurons preserve stable precise phase relationship among units and stable frequency even in noisy conditions , at least when the dynamics is in the basin of attraction of that phase relationship . even in the more recent spiking models ( zilli and hasselmo 2010 ; welday et al . 2011 ) of the oscillatory interference principle , in which many problems related to noise are solved , the heterogeneity of parameters of the cells which participate to the ring oscillator is not taken into account . here we show that the circuit level interactions among units make the oscillation frequency and the phase - relationship of the system robust even with respect to heterogeneity of the spiking thresholds of the units ( see section 6 ) . this robustness , due to the proposed coupling which forces all the units of the circuit to have exactly the same period of oscillation and to have precisely the same phase relationships of stored pattern , may be useful in all cases of sequence coding . moreover , our circuit can be easily programmed to cycle in different phase orders , by storing more than one phase - pattern as attractors . the circuit is a robust phase - shuffling ring oscillator , since the network has the capability of shuffling the order in which its neurons fire , by storing a variety of different phase - patterns within the connectivity . if the oscillators predicted by the interference theory can generate more than one phase sequence , as in the model presented here , then this could provide a potential mechanism to explain the phenomenon of hippocampus remapping ( colgin et al . one of the more interesting discovery of place cells behavior is indeed the remapping of the place cell representation of space in response to a changes in sensory or cognitive inputs , i.e. place cells change their firing properties ( place cells can appear disappear or move to other unpredictable locations ) . this change may be abrupt and similar to the switch from one attractor to another ( wills et al . if the place cell will fire at a specific place where its inputs become synchronized ( euston et al . 2007 ; blair et al . 2011 ) , by recalling a different phase - coded pattern among the ones stored in our circuit , it will change the phases of theta cells that are the inputs of the place cell , and it will change the specific place where the inputs become synchronized , and therefore it changes the place cell representation of the space . finally we note that even thou our model is not a continuous attractor , it shares many similarity with such a class of models . our model is a circuit with many distinct attractors , one for each phase - relationship stored in the network , and the number of different attractor states is set by the maximal storage capacity studied here . furthermore each attractor is a phase - coded pattern , replayed with a collective resonant frequency that can be modulated by changing for example the spiking threshold of the units . even thou during exploration the activity of place cells may be explained by the superposition of velocity - controlled oscillators inputs , the recurrent connections inside the place cells network may have anyway a relevant role . during sleep , in absence of external input , the role of recurrent connections increases , probably due to an increase of excitability via neuromodulators or other mechanisms , and the spontaneous activity of the network show temporarily short replay of stored patterns , probably initiated simply by noise . so the pattern activated repeatly during experience , is stored in the connectivity , and then activated during sleep when the network is near a critic point and noise is able to initiate short replay sequences . replay of phase - coded patterns of neural activity during sleep has been observed in hippocampus and neocortex ( schwindel and mcnaughton 2011 ) . notably in our model the time scale of reactivation is different from the time scale of storing , depends on the collective frequency which emerges from the connectivity , and may be accelerated or slowed down changing parameters such as spiking thresholds . therefore , our model might be also relevant for replay in prefrontal cortex ( pfc ) or other cortical areas in which replay is accelerated with respect to awake activity . in the hippocampus , spikes representing adjacent place fields occur in rapid succession within a single theta cycle during behavior . therefore , relative to this within theta cycle rate , reactivation during sleep in hippocampus is not accelerated . however , reactivation in rat pfc is clearly compressed five to eight times relative to the waking state ( euston et al . 2007 ; schwindel and mcnaughton 2011 ) . indeed , while in hippocampus one may think that the coding sequence is the within - theta cycle , in prefrontal cortex it is clearly seen that the cross - correlation among cells during sleep replay is time compressed compared with the cross - correlation during waking state . the playback speed declines over time as does the strength of the replay , which is consistent for example with a simple increases of spiking threshold in our model . we studied the temporal dynamics , including the storage and replay properties , in a network of spiking integrate and fire neurons , whose learning mechanism is based on the spike - timing dependent plasticity . the temporal patterns we consider are periodic spike - timing sequences , whose features are encoded in the relative phase shifts between neurons . the importance of oscillations and precise temporal patterns has been pointed out in many brain structure , such as cortex ( buzsaki and draguhn 2004 ) , cerebellum ( dangelo et al . 2009 ; dangelo and de zeeuw 2009 ) , or olfactory system ( gelperin 2006 ) . the proposed associative memory approach , with selective replay of stored sequence , can be a method for recognize an item , by activating the same memorized pattern in response to a similar input . another possibility is to have a way to transfer a memorized item to another area of the brain , such as for memory consolidation during sleep . during sleep , indeed , few spikes with the right phase relationship may initiate the retrieval of one of the patterns stored in the network and this reactivation may be useful for memory consolidation . the stored pattern is an attractor of the network dynamics , that is the dynamics spontaneously goes back to the retrieved phase - coded pattern for all the perturbations which leave the system within the basin of attraction . therefore phase errors do not sum up , and the phase relationships may be transferred and kept stable over long time scales . the time scale of the pattern during retrieval , i.e. the period of oscillation 1/ , depends on ( 1 ) the time constants of the single neuron m and s , ( 2 ) the spiking threshold th of the neurons , and ( 3 ) the connectivity , through the stdp learning shape a( ) and the time scale of the pattern during learning mode 1/. different areas of the brain may have different shape of stdp to subserve different oscillation frequencies and different functional role . here we fix the shape of a( ) to the one observed in hippocampal cultures ( see fig . 1 and bi and poo 1998 ) and focus on the dependence on the spiking threshold th of the neurons . the spiking threshold can modulate the frequency of the collective oscillation , leaving unaffected the phase relationships among the units . this opens a possible way to govern the frequency of collective oscillation via neuromodulators , and to encode information ( such as velocity of the animal ) in the frequency of the oscillations , in addition to the information encoded in the phase relationships . notably , in a particular range of frequencies , the spiking threshold does not affect the frequency of oscillation but changes the number of spike per cycle during the retrieval dynamics . this means that information can be encoded via the number of spikes per cycle , independently from the information coded in the phase relationship among units , in agreement with the observations of independent rate and phase coding in hippocampus ( huxter et al . important to note , the phase relationships and the frequency of the collective oscillation are both robust with respect to noise and to heterogeneity of the spiking threshold of the units . a systematic study of the retrieval capacity of the network is proposed as a function of two parameters of the model : the frequency of the input pattern and the spiking threshold . the storage capacity , evaluated as pmax / n , is always lower than the storage capacity of the hopfield model . however , the information content of a single pattern in our dynamical model with n units is higher than the information content of a pattern in the hopfield model with n - units . indeed , an hopfield pattern is a set of n binary values while our phase - coded pattern is a set of n real number \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi^\mu_j \in [ 0 , 2\pi]$\end{document}. the role of stdp in the formation of sequences has been recently investigated in verduzco - flores et al . these studies have shown how it s possible to form long and complex sequences , but they did not concern themselves on how it s possible to learn and store not only the order of activation in a sequence , but the precise relative times between spikes in a closed sequence , i.e. a phase - coded pattern . in our model not only the order of activation is preserved , but also the precise phase relationship among units . the tendency to synchronization of units is avoided in our model , without need to introduce delays or adaptation , due to the balance between excitation and inhibition that is in the connectivity of large networks when the phase coded pattern with random phases is learned using the rule in eqs . all the connections , both positive and negative , scale with the time of presentation of patterns , keeping always a balance . indeed , since ( 1 ) the stored phase are uniformly distributed in [ 0,2 ) and ( 2 ) the learning window has the property \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\int a(\tau ) dt = 0$\end{document } , then the connectivity matrix in eq . ( 7 ) has the property that the summed excitation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(1/n)\sum_{i , j_{ij}>0 } j _ { ij}$\end{document } and the summed inhibition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(1/n)\sum_{i , j_{ij}<0 } j _ { ij}$\end{document } are equal in the thermodynamic limit ( indeed they are of order unity , while their difference is of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt{n}$\end{document } ) . under this conditions , we investigate how multiple phase - coded patterns can be learned and selectively retrieved in the same network , as a function of time scale of patterns and network parameters . the task of storing and recalling phase - coded memories has been also investigated in lengyel et al . ( 5 ) in a network of if neurons , the paper lengyel et al . ( 2005b ) studies this problem from a normative theory of autoassociative memory , in which real variable xi of neuron i represents the neuron i spike timing with respect to a reference point of an ongoing field potential , and the interaction h(xi , xj ) among units is mediated by the derivative of the synaptic plasticity rule used to store memories . the model proposed here is a mechanism which combines oscillatory and attractor dynamics , which may be useful in many models of path - integration , as pointed out in section 7 . our learning model offers a if circuit able to keep robust phase - relationship among cells participating to a collective oscillation , with a modulated collective frequency , robust with respect to noise and heterogeneities . notably the frequency of the collective oscillation in our circuit is not sensible to the single value of the threshold of each unit , but to the average value of the threshold of all units , since all units participate to a single collective oscillating pattern which is an attractor of the dynamics . recently there is renewed interest in reverberatory activity ( lau and bi 2005 ) and in cortical spontaneous activity ( ringach 2009 ; luczak and maclean 2012 ) whose spatiotemporal structure seems to reflect the underlying connectivity , which in turn may be the result of the past experience stored in the connectivity . similarity between spontaneous and evoked cortical activities has been shown to increase with age ( berkes et al . interestingly , in our if model , in order to induce spontaneous patterns of activity reminiscent of those stored during learning stage , few spikes with the right phase relationship are sufficient . it means that , even in absence of sensory stimulus , a noise with the right phase relationships may induce a pattern of activity reminiscent of a stored pattern . therefore , by adapting the network connectivity to the phase - coded patterns observed during the learning mode , the network dynamics builds a representation of the environment and is able to replay the patterns of activity when stimulated by sense or by chance . this mechanism of learning phase - coded patterns of activity is then a way to adapt the internal connectivity such that the network dynamics have attractors which represent the patterns of activity seen during experience of environment .

we study the collective dynamics of a leaky integrate and fire network in which precise relative phase relationship of spikes among neurons are stored , as attractors of the dynamics , and selectively replayed at different time scales . using an stdp - based learning process , we store in the connectivity several phase - coded spike patterns , and we find that , depending on the excitability of the network , different working regimes are possible , with transient or persistent replay activity induced by a brief signal . we introduce an order parameter to evaluate the similarity between stored and recalled phase - coded pattern , and measure the storage capacity . modulation of spiking thresholds during replay changes the frequency of the collective oscillation or the number of spikes per cycle , keeping preserved the phases relationship . this allows a coding scheme in which phase , rate and frequency are dissociable . robustness with respect to noise and heterogeneity of neurons parameters is studied , showing that , since dynamics is a retrieval process , neurons preserve stable precise phase relationship among units , keeping a unique frequency of oscillation , even in noisy conditions and with heterogeneity of internal parameters of the units .

Introduction The model Designing the connections of the network Emerging of collective patterns in the neural dynamics of the network Storage capacity Effects of noise and robustness of collective oscillation frequency and phase relationships Relationship of this model with theories of path-integration Discussion

PMC4782681

ecg is used to measure the rate and regularity of heartbeats , the presence of any damage to the heart , and the effects of drugs or devices used to regulate the heart ( such as a pacemaker ) . normally , the frequency range of ecg signal is 0.05100 hz and its dynamic range is 110 mv . the ecg signal as depicted in figure 1 is characterized by five peaks and valleys labeled by the letters p , q , r , s , and t. the performance of ecg analyzing system depends mainly on the accurate and reliable detection of the qrs complex , as well as t- and p - waves . the p - wave represents the activation of the upper chambers of the heart , the atria , while the qrs complex and t - wave represent the excitation of the ventricles or the lower chamber of the heart . the detection of the qrs complex is the most important task in automatic ecg signal analysis . once the qrs complex has been identified a more detailed examination of ecg signal including the heart rate and the st segment can be performed [ 1 , 2 ] . most of the modern 12-lead ecg monitoring systems are based on einthoven 's triangle , wilson central terminal , and goldberger augmented leads . the developed ecg device is implemented on the principle of einthoven 's triangle and used lead - ii configuration , as it is known as a monitoring lead , given in figure 2 . based on above facts , such efforts are primarily led by the academia but extending deeply into the industries . however , most research efforts have been focusing on either the vital sign monitoring aspect or the ecg feature extraction using standard databases both falling short of expectation . having analyzed the existing solutions , this work vows to bridge the two major research efforts and bring out a more realizable product to directly benefit the consumers in the medical field . this research work offers the following contributions to the produced system ; foremost is the portable ecg monitoring platform based on a 3-lead system and a design under the ni daq card ( 6008 ) . the ecg data was collected through the daq card to the pc / laptop and then transmitted to the end user ( physician ) through smtp to analyze the patient condition . the hardware part comprises instrumentation amplifier ( ad620 ) , some passive components , operational amplifier ( lm741 ) , daq card , and laptop whereas labview is used as software . the software is used to exchange the data from analog to digital form , to perform the calculations , and to produce the ecg waveform onto the monitor . each of the components in this block diagram is explained in detail in the following subsections . the principle of the electrodes is to convert a physical parameter into an electrical output . the function of the transducer is to convert biological information into a quantifiable electrical signal . the most preferable electrode is ag / agcl , as it reduces the impedance while using it and the gel is used for the proper contact in between the surface of the skin and electrode . after receiving raw ecg signal from the subject through electrode , it has to be processed in order to bring the signal in visible form and to limit the bandwidth of the signal . to do so , the instrumentation amplifier was used to amplify the tiny signal whereas passive and active components are used to design filter and to amplify it . figure 4 illustrates the constructive circuit diagram of signal conditioning . the circuit has been designed and tested in multisims to get the appropriate output of signal . the voltage gain of the instrumentation amplifier is calculated using the following equation:(1)g=1 + 49.4 krg , g=1 + 49.4 k1 k=1 + 49.4=50.4 . the voltage gain of the operational amplifier is estimated using given formula as the used one is noninverting amplifier:(2)g=1+r3r2,g=1 + 200 k1 k=1 + 200=201 . the output of the instrumentation amplifier is fed into the passive ac coupling with a cutoff frequency , as in ( 3 ) , of 0.02 hz such that high - pass filters ( 3)fhigh - pass12r1c1=123.145.61031000106,fhigh - pass53.1456,fhigh - pass0.02 hz . the second - stage amplified signal is fed into a low - pass filter with a cutoff frequency , as in ( 4 ) , of 160 hz for removing high frequency noise or movement artifacts : ( 4)flow - pass12r4c2=123.14101031106,flow - pass10006.28=159.2 hz . the output of the signal conditioning circuit should be sent to the ni daq card for the conversion of signal from analog to digital as it has the inbuilt analog - to - digital converter . the given figure ( figure 5 ) gives the idea about the block diagram of ni usb-6008 which is a simple and low - cost multifunction i / o device from national instruments . the ni usb-6008 card has 8 differential analog input channels and 12-bit analog - to - digital converter running at a sampling frequency up to 300 hz , which can be increased up to 200 khz . it is a software package developed to build programs with symbols ( icons ) rather than writing out lines and lines of programming text . it uses symbols , terminology , and formats that are familiar to technicians , scientists , and engineers . labview is programmed to act as an interface , helping pieces of hardware communicate with each other . moreover , labview offers built - in libraries that allow the user to work over the internet and use different programming formats and systems . figure 6 shows the step of smtp of how the data is processed throughout the different stages and transferred to client . it is a part of the application layer of the tcp / ip protocol . using a process called store and forward , it works closely with something called the mail transfer agent ( mta ) to send your communication to the right computer and e - mail inbox [ http://support.aycontrol.com/ ] . the approach that has been followed in the designing of device is included in figure 7 . there were several stages like finding the appropriate electrodes to acquire the signal from body of the subject and analog signal conditioning circuit includes filtering and amplifying stage , daq card , and digital signal processing and display system . the ecg system that has been developed is used for the continuous acquisition and chart recording of single input channels . it allowed user to record and save buffered analog ecg data from one or more individuals , which is continuously acquired into a circular buffer at the same time in that data previously retrieved from the buffer is plotted [ 68 ] . a common reason to read data while the acquisition is in progress is to process and display the data in virtual - real time . the analog signal is acquired from the bread board and converted to the digital one through daq . digital signal processing is done accurately as needed in order to produce high signal - to - noise ratio so that the heart diagnostic system is precise . here , the digital filter is done to remove the power line interferences and other artifacts available in the signal . the purpose of these indicators is to provide some feedback to the user ( i.e. , physician ) regarding the patient 's average heart rate . the first part shows how the data have been acquired using data acquisition card ( 6008 ) ; immediate in next step , the retrieved data has been processed by band pass filter as well as notch filter to remove the artifacts from the signal [ 9 , 10 ] . the following given equation is used to calculate the heart rate and to identify the arrhythmia condition [ 11 , 12]:(5)heart rate=60t2t1,with t1 being occurrence of first r wave and t2 being occurrence of second r wave . after processing raw ecg signal , the signal has been displayed onto the laptop as shown in figure 9 . finally , the developed smtp tool is used to send image file of acquired ecg signal through email express vi , shown in figure 10 that represents the front panel and figure 11 that represents the block panel developed using labview which allows sending the data quickly through e - mails from labview to a health care centre or a physician . the given system is based on the principle of heart rate monitoring ; it is able to produce the results shown in figure 12 , which show accurate ecg signals and the correct calculation of both a resting and an elevated heart rate . initially , there was an issue with the system regarding doctor notification subsystem which has been short by validating the proposed concept by running the vi at home , which sent both text and e - mail to our doctor notifying them that there was a detected problem with the patient 's heart . it brings the freedom for the physician as well as for the doctor to check up on patients hearts from time to time by seeing real - time waveforms as shown in figure 12 . the proposed device is functioning well once all the hardware connected properly to meet the criteria for the proposed idea . as a whole most of the work has been done based either on hardware or on software . in the case of hardware , to transmit the ecg data , transmitter and receiver had been used , which increases the cost of the device , whereas the developed one integrated with hardware along with software to transmit the data anywhere using smtp technology . in this paper , the low - cost biomedical measurement system with the ability of storage in digital format as well as sending the data to the remote area has been presented . the hardware implementations using commercially available devices and the software written in labview program for continuously monitoring ecg data have been described . the proposed measurement system is also capable of sending the data through smtp to the physician or health care centre with no time .

this paper leads to developing a labview based ecg patient monitoring system for cardiovascular patient using simple mail transfer protocol technology . the designed device has been divided into three parts . first part is ecg amplifier circuit , built using instrumentation amplifier ( ad620 ) followed by signal conditioning circuit with the operation amplifier ( lm741 ) . secondly , the daq card is used to convert the analog signal into digital form for the further process . furthermore , the data has been processed in labview where the digital filter techniques have been implemented to remove the noise from the acquired signal . after processing , the algorithm was developed to calculate the heart rate and to analyze the arrhythmia condition . finally , smtp technology has been added in our work to make device more communicative and much more cost - effective solution in telemedicine technology which has been key - problem to realize the telediagnosis and monitoring of ecg signals . the technology also can be easily implemented over already existing internet .

1. Introduction 2. Material and Method 3. Designing Strategies 4. Designed Labview Interface 5. Result 6. Discussion 7. Conclusion and Future Work

PMC3312332

tumour necrosis factor alpha ( tnf - alpha ) is one of the most frequently studied pro - inflammatory cytokines . it drives the activation and recruitment of inflammatory cells , amplifies the production of other pro - inflammatory cytokines , and activates nuclear transcription factors , thereby promoting and maintaining the inflammatory response . tnf - alpha is likely to be a key cytokine in several autoimmune diseases such as rheumatoid arthritis ( ra ) , inflammatory bowel disease , systemic sclerosis , and systemic lupus erythematosus [ 13 ] . a very large number of studies have been performed investigating the importance of tnf - alpha in arthritis , especially ra . tnf - alpha has also attracted interest in recent years for its possible role in skeletal muscle damage . increased protein degradation , as well as decreased body weight and food consumption , was demonstrated when tnf - alpha was administered to rats via a catheter into the external jugular vein . crush injury in mice leads to elevated tnf - alpha levels in skeletal muscle tissue and there is an increase in tnf - alpha serum levels in response to repetitive strain injuries . for example , an experiment in which tnf - alpha was administered to mice via an osmotic pump led to accumulation of inflammatory cells in skeletal muscles but no signs of atrophy or injury . furthermore , the results of studies on tnf receptor knockout and tnf - alpha antibody - neutralized mice indicate that tnf - alpha can actually be involved in the recovery of muscle function after traumatic muscle injury . therefore , it might be that the role of tnf - alpha in muscle injury varies with the type , severity , and stage of the injury . in humans , tnf - alpha is known to be intimately involved in cachexia , a complex condition characterised by progressive muscle loss that affects up to 13% of patients with ra . nevertheless , a recent study showed acute elevation of tnf - alpha not to affect markers of systemic or skeletal muscle turnover in healthy humans . remarkably little data is available on the role of tnf - alpha in situations where there is a pronounced infiltration of inflammatory cells in the muscle tissue , that is , myositis . from studies in tissue other than muscle , it is known that macrophages and other immunoactive cells such as monocytes , mast cells , and neutrophils are responsible for tnf - alpha production [ 1316 ] . data addressing a possible tnf - alpha production by inflammatory cells in myositis comes almost entirely from studies of patients affected by a group of diseases known as idiopathic inflammatory myopathies these autoimmune diseases include mainly the subgroups inflammatory myopathic polymyositis , dermatomyositis , and inclusion body myositis . in these conditions , tnf - alpha is also expressed in the inflammatory cells in crush - injured and transplanted muscle autografts in mice . finally , blockade of tnf - alpha in the dystrophic ( mdx ) mouse , which is the most frequently used model of duchenne 's muscular dystrophy , reduces tnf - mediated adverse responses to exercise - induced muscle damage [ 20 , 21 ] . however , without further information , it is difficult to reach conclusions on the importance of tnf - alpha and the possible usefulness of tnf - blocking in muscle disorders , including in myositis . furthermore , it should be stressed that the majority of information on the tnf system for skeletal muscle tissue has come from studies of cultured myoblasts ( e.g. , ) . animal models are needed to advance our understanding of the disease mechanisms of tnf - alpha that are involved in myositis . our laboratory has developed a rabbit model of marked muscle ( m. triceps surae ) and tendon overuse that , when combined with injections of substances eliciting pro - inflammatory effects , results in significant myositis . this model causes myositis that morphologically resembles that seen in inflammatory myopathies [ 17 , 18 ] but without having an apparent autoimmune origin . the model leads to a muscle pathology that to some extent resembles the morphology seen in overuse musculoskeletal disorders ( see , for a review , see ) . in studies using this model , we noted evidence of local glutamate signaling in the cells of the inflammatory infiltrates within the muscle tissue . we have taken advantage of this model in order to examine the tnf - system during myositis development . thus , the aim of this study was to examine the pattern of tnf - alpha expression in one segment of the triceps surae muscle ( the soleus muscle ) affected by myositis using immunohistochemistry and in situ hybridization in order to get an insight into the possible usefulness of this model for further studies on the importance of tnf - alpha in myositis . the animals had an average weight of 4 kg and ranged in age from 6 to 9 months . six of the animals corresponded to control nonexercised animals ( subgroup 1 ) and 22 were assigned to an exercise protocol leading to marked overuse of the triceps surae muscle ( subgroups 25 ) . in order to increase the muscle affection , including the degree of inflammation , the muscle overuse was combined with paratendinous injection treatment ( cf . the following ) . as a control for this , five of the exercised animals ( subgroup 2 ) were given control substance ( nacl ) just outside the tendon of the triceps surae muscle ( i.e. , the achilles tendon ) . 17 of the exercised animals ( subgroups 35 ) were in parallel to being subjected to marked overuse , given local injections of pro - inflammatory substance ( substance p and/or endopeptidase inhibitors ; captopril , dl - thiorphan ) outside the achilles tendon . substance p was given as this neuropeptide has well - known pro - inflammatory effects and the injections of the endopeptidase inhibitors were given in order to diminish endopeptidase activities and thereby lead to more pronounced effects of substance p. for clarification of all the various animal groups , see table 1 . the animals were exposed to an exercise procedure designed to cause marked overuse of the triceps surae muscle and the associated tendon ( the achilles tendon ) . the procedure is performed according to previously described procedures , with some modifications . throughout the experiment , injections of diazepam ( 5 mg / ml ; 0.2 ml / kg ) and fentanylfluanison ( 0.2 - 0.3 ml / kg ) . fentanylfluanison ( 0.1 ml / kg ) was injected each 3045 min during the experiment in order to maintain the anaesthesia . mg / kg ) was given subcutaneously ( s.c . ) after each experiment session . the experiment was repeated every second day for one week ( 4 exercise sessions in total ) . an apparatus ( kicking machine ) was used to achieve passive repetitive flexions and extensions of the right ankle joint ; a pneumatic piston attached to the right foot produced the movements . during the plantar flexion , an active contraction was furthermore induced by electrical stimulation via surface electrodes ( pediatric electrode 40 426 a , hewlett packard , andover , ma , usa ) placed 2 cm apart over the triceps surae muscle of the right leg . the stimulation was synchronized with the plantar flexion movement of the piston by a microswitch , which trigged the stimulator unit ( disa stimulator type 14e10 , disa elektronik a / s , herlev , denmark ) . an impulse of 0.2 ms duration was delivered 85 ms after the initiation of the plantar flexion at an amplitude of 3550 v. the movement frequency was 150 repetitions per minute . the pelvis was strapped down and there were no ankle movements on the left side . one day after the final exercise session , the animals were sacrificed by an overdose of pentobarbital . for further details about injections were given into the loose connective tissue around the achilles tendon , that is , in the paratenon region . the substances injected were ( a ) nacl ( 0.91% w / v , volume : 1 ml ) ( subgroup 2 ; n = 5 ) , ( b ) substance p ( 10mol / ml ) and captopril ( sigma ) ( c4042 , 30 mol / kg ) both in distilled water ( volume : 1 ml ) and dl - thiorphan ( n-[(rs)-2-benzyl-3-mercaptopropanoyl]-glycine ) ( sigma ) ( 500 g / ml ; 0.02 ml ) ( subgroup 3 ; n = 5 ) , ( c ) captopril ( sigma ) ( c4042 , 30 mol / kg , dissolved in distilled water , volume 1 ml ) + dl - thiorphan ( sigma ) ( 500 g / ml , 0.02 ml ) ( subgroup 4 ; n = 6 ) , and ( d ) captopril ( sigma ) alone ( c4042 , 30 mol / kg , dissolved in distilled water , volume 1 ml ) ( subgroup 5 ; n = 6 ) . , unpublished observations ) and as recently reported , it has become obvious that subgroups 35 develop myositis . therefore , these subgroups were grouped together and further on referred to as the myositis group . as there are minimal or no signs of myositis in the nacl - treated subgroup ( subgroup 2 ) , the animals in this group were grouped together with the nonexercised animals ( subgroup 1 ) , comprising the non - myositis group . muscle samplesafter the animals were sacrificed , the right triceps surae muscle was dissected out and immediately transported on ice to the laboratory . samples conforming to the soleus muscle part ( 58 10 mm ) were dissected out and fixed by immersion overnight at 4c in an ice - cold solution of 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.0 ) . the samples were thereafter thoroughly washed in tyrode 's solution containing 10% sucrose at 4c overnight , mounted on thin cardboard in oct embedding medium ( miles laboratories , naperville , ill , usa ) , frozen in propane chilled with liquid nitrogen , and stored at 80c . the sections were mounted on slides precoated with chrome - alum gelatine and were then processed for immunohistochemistry . after the animals were sacrificed , the right triceps surae muscle was dissected out and immediately transported on ice to the laboratory . samples conforming to the soleus muscle part ( 58 10 mm ) were dissected out and fixed by immersion overnight at 4c in an ice - cold solution of 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.0 ) . the samples were thereafter thoroughly washed in tyrode 's solution containing 10% sucrose at 4c overnight , mounted on thin cardboard in oct embedding medium ( miles laboratories , naperville , ill , usa ) , frozen in propane chilled with liquid nitrogen , and stored at 80c . the sections were mounted on slides precoated with chrome - alum gelatine and were then processed for immunohistochemistry . other sections were processed for morphology or in situ hybridization . as a reference , human ra synovial tissue was analyzed . the tissue was fixed and further processed in the same way as were the rabbit specimens ( cf . one section from all specimens was stained in harris haematoxylin solution for 2 min . these sections were then rinsed in distilled water , dipped in 0.1% acetic acid for a few seconds , and then washed in running water . the sections were pretreated with acid potassium for 2 min , a procedure found to enhance specific immunofluorescence reactions . thereafter followed incubation for 20 min in a 1% solution of triton x-100 ( kebo lab , stockholm ) in 0.01 m phosphate buffer saline ( pbs ) , ph 7.2 , containing 0.1% sodium azide as preservative , and three 5 min washes in pbs . the sections were then incubated for 15 min in 5% normal donkey serum ( code no : 017 - 000 - 121 , jackson immune research lab . inc . ) in pbs . next , incubation with the primary antibody , diluted in pbs ( ph 7.4 ) , occurred in a humid environment for 60 min at 37c . after incubation with specific antiserum , and three 5 min washes in pbs , another 15 min incubation in normal donkey serum followed . next , the sections were incubated with either of these donkey antigoat iggs for 30 min at 37 : fitc-(fluorescein isothiocyanate- ) conjugated affinipure donkey antigoat igg ( jackson immunoresearch lab inc , dilution 1 : 100 ) or alexa fluoro 488 donkey antigoat ( invitrogen , dilution 1 : 300 ) . the sections were thereafter washed in pbs and then mounted in vectashield mounting medium ( h-1000 ) ( vector laboratories , burlingame , ca , usa ) . examination was carried out in a zeiss axioscope 2 plus microscope equipped with epifluorescence optics and an olympus dp70 digital camera . to clarify the tnf - alpha immunoreaction pattern in relation to that of white blood cells , double stainings were made . as it is frequently emphasized that macrophages [ 13 , 16 ] show tnf - alpha expression , double stainings for tnf - alpha / macrophage marker ( cd68 ) were performed . double stainings for tnf - alpha / t - cell - neutrophil marker were also performed . alexa fluoro donkey antigoat was used as secondary antibody for tnf - alpha immunolabelling , and tritc ( tetramethylrhodamine isothiocyanate- ) conjugated rabbit antimouse antibody was used for stainings for cd68 and t - cell / neutrophil marker . for detailed information about the staining procedures for tnf - alpha , when doing double stainings for cd68 and t - cell / neutrophil marker , 5% normal rabbit serum ( code no : x0902 , dako cytomation , glostrup , denmark ) , diluted in 0.1% bsa ( bovine serum albumin ) in pbs , was used as normal serum , and tritc - conjugated rabbit antimouse antibody ( r0276 , dako cytomation ) , diluted 1 : 40 in 0.1% bsa in pbs , as the secondary antibody . an antibody against tnf - alpha produced in goats was used ( af-210-na ; r&d systems ) . various dilutions were trialled to achieve the optimal fluorescence to background ratio , with a dilution of 1 : 50 found to be optimal . the supplier reports that this antibody is directed against e. coli - derived recombinant human tnf - alpha . the tnf - alpha - specific igg was purified by human tnf - alpha affinity chromatography . it is described to be specific via having the ability to neutralize the biological activity of recombinant human tnf - alpha . of note , the tnf - alpha amino acid sequence homology between species is reported to be highly conserved and tnf - alpha dna sequence comparison shows an overall high sequence homology between various species ( including rabbit ) . in control stainings , preabsorption of the primary antibody with tnf - alpha antigen ( t6674 ; sigma ; 20 g / ml antiserum ) was performed overnight at 4c . a macrophage ( cd68 ) antibody ( m0814 ) from dako cytomation ( glostrup , denmark ) was used . it is an affinity purified mouse monoclonal antibody and was used at a dilution of 1 : 100 in 0.1% bsa in pbs . the antigen for this antibody is glycosylated transmembrane glycoprotein , which is mainly located in lysosomes . a mouse antirabbit t - cell and neutrophil antibody ( mca805 g ) from abd serotec ( oxford , uk ) was furthermore used . it is an affinity purified mouse monoclonal antibody against a cell surface antigen , which is expressed by a subset of t - cells , thymocytes , neutrophils , and platelets in rabbits . stainings performed in a parallel project on rabbit soleus muscle were used as a reference ( control ) for the current double stainings . in that project , double stainings were performed using the same cd68 and t - cell / neutrophil marker antibodies as in the current double stainings . in this previous study , double stainings were made against an antibody produced in goats ( against vglut2 ; santa cruz ) , that is , being of the same type as the tnf - alpha antibody used in the current study . in these reference stainings , the same types of secondary antibodies as described previously were utilized . in situ hybridization was used as a complementary method to detect the expression of tnf - alpha , namely , at the mrna level . a digoxigenin-(dig ) hyperlabeled oligonucleotide probe ( ssdna ) for detection of rabbit tnf - alpha mrna was used on sections from myositis ( 4 specimens ) and nonmyositis ( 1 specimen ) groups ( gd1001-ds custom designed ; genedetect , new zealand ) . the procedures were performed according to an established protocol , using an alkaline phosphatase - labeled anti - dig antibody for detection . the probe for tnf - alpha mrna the tissue specimens were cut into 10 m thick fresh cryosections using a cryostat ( with a knife washed in 70% etoh in depc [ diethylpyrocarbonate]-h2o ) and mounted onto super frost plus slides ( nr.041200 , menzel glser ) . the protocol that thereafter followed was that previously used in our laboratory for detection of mrna for other substances ( e.g. , [ 3436 ] ) . an alkaline phosphatase-(ap ) labelled anti - dig antibody ( roche , germany , 11 093 274 910 ) was used for detection . the corresponding sense dig - hyperlabeled ssdna probe was used as a negative control . as a positive control probe , a -actin antisense probe ( gd5000-op ) was used , comparisons being made with sense -actin probe ( genedetect , new zealand ) . myositis was observed in subgroups 35 , and these are now collectively being referred to as the myositis group ( cf . above ) . the most noteworthy feature was the presence of an inflammatory infiltrate ( figure 1 ) , although muscle fiber changes were also observed , including muscle fiber necrosis ( cf . ) . variations in the levels of myositis were observed between the different subgroups , as well as between different animals within the subgroups . the inflammatory infiltrates were seen in some parts of the specimens . in the other groups ( subgroups 1 and 2 ) , there were no or very marginal changes seen and these are now collectively referred to as the nonmyositis group . it was considered relevant to examine a reference tissue regarding the demonstration of tnf - alpha . human ra synovial tissue was therefore analysed to provide reference information for the particular tnf - alpha antibody used , as it a well - known fact that there is a marked tnf - alpha expression in the inflammatory infiltrates in the synovial tissue of patients with ra . we observed that mononuclear - like cells of the human synovial tissue exhibited immunoreactions when incubated with the tnf - alpha antiserum ( figures 2 and 3 ) . the reactions were in high magnification seen in the form of intracellular granular reactions ( cf . figures 2 and 3 ) . the specificity of the reactions was confirmed via preabsorption with synthetic antigen ( figure 2 ) . the cells occurred as parts of immune cell aggregates or as isolated cells in the synovial tissue . pronounced tnf - alpha immunoreactions were observed in cells of the inflammatory infiltrates ( figures 4 and 5 ) in all subgroups in the myositis group . it was noteworthy that the immunoreaction patterns seen in the cells resembled those observed for the mononuclear - like cells of the human ra synovial tissue . thus , the reactions showed a granular pattern in high magnification ( figures 4 and 5 ) . in lower magnification , fibroblasts in the connective tissue did also to some extent display tnf - alpha immunoreactions , however the reactions were very faint . no specific tnf - alpha reactions were noted for blood vessel walls , the nerve fascicles , muscle spindles , and the muscle fibers ( not shown ) . in order to clarify the patterns of cellular reactions for tnf - alpha in the inflammatory infiltrates , double stainings for tnf - alpha / cd-68 and tnf - alpha / t cells and neutrophil marker were performed . it was found that cd68 coexisted with tnfalpha in cells in the inflammatory infiltrates ( figure 6 ) . on the other hand , colocalization between tnf - alpha and t cells and neutrophil marker was not observed ( not shown ) . in the reference studies ( cf . section 2 ) using the same secondary antisera and the same white blood cell markers but a primary goat antibody not directed against tnf - alpha , completely different colocalization patterns were noted . reactions for tnf - alpha mrna were revealed for white blood cells of the inflammatory infiltrates in the myositis specimens ( figure 7 ) . reactions were also seen for fibroblasts ( figure 8) and sometimes for blood vessel walls ( figure 9 ) and muscle fibers ( figure 10 ) . the muscle fibers and blood vessels for which reactions were seen were located in the regions with inflammatory infiltrates . it was noted that the muscle fibers with reactions for tnf - alpha mrna were often infiltrated by inflammatory cells . the majority of the muscle fibers and blood vessels in the tissue of the myositis samples had no demonstrable reaction . there were no reactions at all in the musculature and the blood vessel walls in the nonmyositis samples . no reactions were noted for nerve fascicles and muscle spindles in any of the specimens from the myositis or non - myositis groups . it is well known that tnf - alpha is highly involved in arthritis , notably in ra . accordingly , in our reference studies in the present investigation we found that tnf - alpha was expressed in mononuclear - like cells in the ra synovial tissue . detection of tnf - alpha reactions was thus clarified from the methodological point of view , and verifications were obtained via preabsorption stainings . with this as a basis , studies on tnf - alpha in myositis were performed . a unique model for the production of myositis in rabbit musculature ( the soleus muscle ) was utilized . the main finding was that cells in the inflammatory infiltrates in the myositis muscles were found to express tnf - alpha at both at the mrna and protein levels . expression of tnf - alpha in macrophages has previously been noted in other situations ( e.g. , [ 13 , 16 ] ) , including inflammatory myopathies . in contrast , in our recent studies using the current myositis model , expression of the vesicular glutamate transporter vglut2 was noted in white blood cells in the inflammatory infiltrates other than macrophages . a further main finding was that the muscle fibers and blood vessel walls in areas showing inflammatory infiltration exhibited tnf - alpha mrna and that fibroblasts also were seen to exhibit tnf - alpha mrna . from a methodological point of view , it was clear that muscle fibers , blood vessel walls , and fibroblasts exhibited tnf - alpha mrna but that no reactivity ( muscle fibers , blood vessel walls ) or very weak reactivity ( fibroblasts ) was noted at the protein level . the production level in these locations is therefore likely to be low , which precluded clear detection with our immunohistochemical methods . it is also possible that our in situ hybridization method detects very small quantities of tnf - alpha mrna . nevertheless , it has previously been shown that tnf - alpha can be expressed not only in inflammatory cells but also in injured muscle fibers and fibroblasts in response to muscle injury ( crush - injury ) as well as in muscle fibers and cells in the connective tissue in inflammatory myopathies [ 17 , 18 ] . the patterns of morphologic appearances of the inflammatory infiltrates and other morphologic changes seen resembled the appearances that can be seen in the muscle tissue in inflammatory myopathies . nevertheless , preliminary analysis using elisa detecting anti - jo-1 antibodies , which are known to correlate with disease activity for patients with inflammatory myopathy , does not lend proof to the theory that the myositis in our model is autoimmune in origin ( unpublished observations ) . however , further studies on this aspect are warranted . as noted previously , tnf - alpha immunoreactions in inflammatory cells invading muscles affected by myositis have previously only been documented in biopsies from patients with inflammatory myopathies [ 17 , 18 ] and in muscle of mice in response to crush - injury . thus , in combination with this previous work , our results imply that tnf - alpha is intimately involved in the inflammatory process in myositis . indeed , it has been suggested that tnf - alpha may have a role in the pathogenesis of the myositis in the inflammatory myopathies [ 40 , 41 ] and that a marked inflammatory response involving tnf - alpha may be directly responsible for damaging muscle fibres in myopathic conditions . whether or not the tnf - alpha produced by the cells of the inflammatory infiltrates is entirely responsible for pro - inflammatory and damaging effects remains open to speculation . it is well known that tnf - alpha administration can have pro - inflammatory and detrimental effects , for example , leading to various catabolic changes as seen in studies on cultured skeletal muscle cells [ 4 , 43 , 44 ] . however , there is a marked discrepancy in the literature regarding the effect of tnf - alpha on the musculature . some studies on myoblast cell culture show that tnf - alpha administration does not have catabolic effects ( e.g. , ) , and other studies documenting accumulations of inflammatory cells in skeletal muscle in response to tnf - alpha administration show no decrease in skeletal muscle proteins and no signs of muscle atrophy or injury . perhaps these discrepancies reflect a dual role of tnf - alpha where in some circumstances inflammatory cell derived tnf - alpha can play a protective role and also be involved in the recovery of muscle function after traumatic injury and in muscle regeneration . the discrepancies may also reflect the fact that different methods have been used in the studies that have been performed . the results in preliminary studies on inflammatory myopathies suggest that tnf blocking might be useful , but it is also emphasized that further studies are needed in order to clarify if this type of treatment is indeed useful . results of in vitro studies suggest that targeting tnf - alpha might be worthwhile in myositis [ 49 , 50 ] and studies on dystrophic mdx mice subjected to wheel exercise indicate that tnf blockade can reduce myofiber necrosis [ 20 , 21 ] . the use of anti - tnf treatment in studies on a rat model of repetitive reaching and grasping leads to an improvement in grip strength and attenuated task - induced increases in inflammatory cytokines , including tnf - alpha . although the use of other animal models have shown inflammation in muscle tissue in response to various forms of exercise [ 50 , 52 ] , the myositis model used in the current experiment is clearly distinguishable from these models . thus , in contrast to these models , it leads to a marked presence of inflammatory infiltrates in the muscle tissue , that is , a morphology resembling that seen in inflammatory myopathies . in fact , no experimental myositis model exists which resembles the one used here and in which a marked presence of inflammatory infiltrates becomes present in the muscle tissue . those for which such an infiltration has been demonstrated are the model of crush - injury described above and models designed to help understand the mechanisms of inflammatory myopathies that occur in man . in these latter cases , myositis is induced by various infectious agents [ 53 , 54 ] , immunization with muscle components , for example , myosin [ 55 , 56 ] , and intraperitoneal injections with lipopolysaccharide . the tnf system has not been examined in any of these myositis models replicating the inflammatory myopathies seen in man . interestingly , there is evidence indicating a relationship between the inflammatory myopathies and another condition , muscular dystrophy , in the form of complex interactions between immunological and nonimmunological features of the diseases . a noteworthy aspect with the currently used model is that marked overuse is applied in the procedures . nevertheless , a limitation of the present study is that the relative contributions of the exercise protocol and the injections of the proinflammatory substances to the observed myositis are unclear . forthcoming studies will clarify this issue . in any case , the model will , as it is currently used , provide the opportunity to evaluate the effects of interference with tnf - alpha actions in myositis development . an animal model in which the importance of tnf - alpha for myositis development can be followed has previously been lacking . using a newly established rabbit model of myositis development , a marked tnf - alpha expression has here been shown for the cells of the inflammatory infiltrates within damaged muscle . there was thus a clear evidence of local tnf - alpha production via infiltrated inflammatory cells , presumably leading to secondary inflammation - modifying effects . using in situ hybridization , it was also seen that tnf - alpha mrna was detected for muscle fibers and blood vessel walls in regions of inflammatory infiltrates . the current model can be used for further studies on the importance of tnf - alpha in the development of myositis and to document the expression patterns of other cytokines and signal substances in this condition .

the importance of tnf - alpha in arthritis is well documented . it may be that tnf - alpha is also markedly involved in muscle inflammation ( myositis ) . an animal model where this can be investigated is needed . a newly developed rabbit myositis model involving pronounced muscle overuse and local injections of substances having proinflammatory effects was therefore used in the present study . the aim was to investigate the patterns of tnf - alpha expression in the developing myositis and to evaluate the usefulness of this myositis model for further tnf - alpha research . human rheumatoid arthritis ( ra ) synovial tissue was examined as a reference . tnf - alpha immunoexpression and tnf - alpha mrna , visualized via in situ hybridization , were detected in cells in the inflammatory infiltrates of the affected muscle ( soleus muscle ) . coexistence of tnf - alpha and cd68 immunoreactions was noted , suggesting that the tnf - alpha reactive cells are macrophages . expression of tnf - alpha mrna was also noted in muscle fibers and blood vessel walls in areas with inflammation . these findings demonstrate that tnf - alpha is highly involved in the myositis process . the model can be used in further studies evaluating the importance of tnf - alpha in developing myositis .

1. Introduction 2. Material and Methods 3. Processing for In Situ Hybridization 4. Results 5. Discussion 6. Conclusions

PMC2756467

in the image restoration framework , the regularization term is crucial to incorporate knowledge concerning a priori acceptable solutions and to constrain the solution of this ill - posed inverse problem . in this paper , we are concerned with image restoration issues using information from a different modality ( intermodality ) for this fusion - based regularization term . more precisely , we herein propose an original extension of the nas - rif restoration technique allowing the nas - rif inverse filtering to be constrained from an intermodality registration , that is , a registration between anatomical and the functional medical images to be restored ( from the anatomical structure of the same patient ) . in our application , anatomical information is extracted from a high - resolution anatomical procedure such as magnetic resonance imaging ( mri ) or computed tomography ( ct ) and this high spatial resolution modality is exploited to improve the contrast of functional spect images . the contrast and signal - to - noise ratio displayed by brain single photon emission computed tomography ( spect ) images is rather limited , when compared with that from anatomical techniques ( mri , ct scanning ) . for instance , it is not easy to differentiate low tracer uptake due to a functional deficit , where brain tissue still is anatomically intact , from low uptake generated by focal atrophy , where tissue is lost and replaced by cerebrospinal fluid ( csf ) . up to now , several methods have been proposed to improve directly or indirectly the spatial resolution of spect images . these methods can be split into two major classes : those using restoration techniques during the reconstruction process from projections , and those where the restoration is performed on the already reconstructed images . in this paper , we are describing a posttomographic reconstruction process , an approach which has the advantage of being essentially independent from the physical features of the scanner . in this category , we can cite where rajabi et al . compared four widely used filters ( i.e. , hanning , butterworth , metz and wiener ) for myocardial tc - sestamibi spect perfusion studies . in a nonnegativity and support constraints recursive inverse filtering ( nas - rif ) algorithm proposed by kundur and hatzinakos , was extended to the 3d spect imaging restoration context . the nas - rif blind deconvolution technique is relevant to any situations in which a finite object of interest is imaged against a uniformly grey ( or noisy ) background . this method can thus be efficiently exploited in brain spect imaging since the true undistorted rcbf map of a human brain consists of a finite support imaged against a noisy background ( the background depending of both the poisson noise phenomenon inherent to imaging with radioactive elements and to other , nonpoisson sources of background in the images such as electronic noise from the scanner ) . the only information required for this deconvolution procedure is the nonnegativity of the true image and the support of the object to be restored . in , this support was accurately determined by an unsupervised 3d markovian segmentation technique applied to the spect volume . using the same strategy , but this time during the reconstruction process , we can cite bayesian tomographic reconstruction methods [ 5 , 6 ] which use structural information on the presence and location of important anatomical landmarks ( such as local discontinuities or extended homogeneous regions as seen for instance on an mr anatomical image ) to control noise without smoothing edges . these models usually express that , within a detected and segmented uniform anatomical region , neighboring pixels in the functional image should have similar grey level values ( local homogeneity ) or follow a gaussian distribution with a unique mean value ( global homogeneity ) [ 7 , 8 ] . in this paper , we propose to extend the method presented in by introducing into the nas - rif algorithm a new spatially - adaptive regularization term for spect image deconvolution . this regularization term allows to efficiently include anatomical information extracted from a high - resolution anatomical mri image while stabilizing the solution of the nas - rif inverse filter by preventing noise amplification and ringing artifacts . in our application , this structural anatomy - based regularization term exploits the result of an unsupervised markovian segmentation obtained after a preliminary registration step between the mri and spect volumes from the same patient . the proposed regularization term is quadratic and the nas - rif procedure thus involves recursive filtering of the degraded image to minimize a newly convex objective cost function with a conjugate gradient method . our restoration method was tested on 30 pairs of brain mri and spect images from different patients and on hoffman and jaszczak spect phantoms and compared with a standard supervised deconvolution / restoration approach using a classical metz filter . section 2 briefly describes the proposed 3d anatomical constraint version of the nas - rif deconvolution technique . we then show some of our experimental results on phantom and real brain spect volumes and validate the proposed model in section 5 . in our application , and as proposed in , we assume that 3d spect images are degraded according to the following , widely - used linear model : ( 1)g(x , y , z)=f(x , y , z)*h(x , y , z)+n(x , y , z ) , in which g(x , y , z ) , f(x , y , z ) , and h(x , y , z ) , respectively , denote the degraded 3d image , the true image and the point spread function ( psf ) . n(x , y , z ) represents the additive noise and designates the 3d discrete linear convolution operator . the 3d blind deconvolution problem consists then in determining f(x , y , z ) and h(x , y , z ) ( or its inverse ) given the blurred observation g(x , y , z ) . in the 3d extended version of the nas - rif deconvolution strategy , the output of the fir filter u(x , y , z ) of dimension nxu nyu nzu gives an estimate of the true image f^(x , y , z ) . each resulting estimation is passed through a nonlinear filter which uses a nonexpansive mapping to project the estimated 3d image into the space representing the known characteristics of the true image ( expressing in fact that the image is assumed to be nonnegative with a known support ) . the difference between this projected image f^nl and f^ , e(x , y , z ) , is used as the error signal to update the variable filter u(x , y , z ) . in the 3d context , the cost function used in the deconvolution procedure of the 3d image is defined as ( 2)j(u)=j1(u)+j2(u)+ j3(u ) with ( 3)j1(u)=(x , y , z)f^2(x , y , z)(1sgn ( f^(x , y , z))2),j2(u)=(x , y , z)(f^(x , y , z)lb)2,j3(u)=((x , y , z)u(x , y , z)1)2 , where f^(x , y , z)=g(x , y , z)*u(x , y , z ) , and sgn(f ) = 1 if f < 0 and sgn(f ) = 1 if f 0 . is the set of all pixels of g(x , y , z ) inside the region of support , and is the set of all pixels outside the region of support . the first term , j1(u ) , is used to penalize negative voxels in the support in order to keep the image estimate nonnegative . the second term j2(u ) penalizes voxels located outside the support with values which deviate significantly from the background average lb . when the background of the true image is black , that is , lb = 0 , the third term , j3(u ) , is used to avoid a trivial all - zero minimum solution ( being a positive constant ) . the authors have shown in that the above equation is convex in the 2d case with respect to u. this property remains true in the 3d case so that convergence of the algorithm to the global minimum is ensured using the conjugate gradient minimization routine . the major shortcoming of the nas - rif technique is its noise amplification at low snr . this is due to the high - pass property of the inverse filter u(x , y , z ) which amplifies high - frequency noise . as a result , the solution at convergence may not be the best estimate of the original object in the presence of noise . in order to solve this problem , a solution has been suggested by kundur and hatzinakos , which consists in halting the iterative restoration process through visual inspection . in practice , this requires a strong supervision and , even in this case , it is not so easy to determine which is the optimal iteration for termination ( different parts of the image may converge at different rates ) , making this method unreliable . in this work , we propose an alternative regularization approach for the nas - rif algorithm which can also be viewed as a way to incorporate anatomical information extracted from a ( high - resolution ) anatomical mri ( or ct ) image into the spect data . the proposed regularization term also allows stabilization of the inverse solution by preventing noise amplification , does not require supervision ( parameters tuning or stopping criterion ) and is capable of introducing better constraints on the solution of our restoration problem . this strategy consists in applying , over predetected and segmented anatomical regions , a piecewise smoothness constraint on the functional spect image to be recovered . to this end , our regularization term exploits the result of a preliminary registration step between the mri and spect images as well as the result of a segmentation of the mri image into anatomical classes . in our model , the new cost function related to the deconvolution of the 3d image is now defined as ( 4)j(u)=j1(u)+j2(u)+ j3(u)+ j4(u ) with : ( 5)j4(u)=i=13 (x , y , z)ri(f^(x , y , z)ri)2 , where the first summation is made on the three main anatomical types ( tissues ) found in the brain , that is , white matter ( rwm ) , grey matter ( rgm ) , and cerebro - spinal fluid ( rcsf ) and ri designates the mean , in grey levels , of the ith region and is a weighting factor between this anatomical constraint and the hard constraints of the nas - rif procedure . in this context , = rwm rgm rcsf and ri=(1/nri)(x , y , z)rif^(x , y , z ) where nri is the number of voxels in the region ri . j4(u ) is proportional to the sum of variance within each anatomical region ( for each transversal slice ) of the spect image . this term expresses that , within a detected and segmented anatomical region , pixels in the functional image in general tend to have similar grey level values . this regularization term is edge - preserving since it allows to apply a smoothness constraint , while preserving ( anatomical ) discontinuities . furthermore , the introduction of this regularization term j4 does not affect the convexity of the nas - rif cost function , and therefore a unique solution to the problem is still guaranteed . figure 2 shows the structure of this scheme . a coregistered spect and ( high - resolution ) mri datasets along with the result of a segmentation of the mri volume into anatomical classes the first derivative of the cost function in ( 4 ) is shown in ( 7 ) . the gradient vector of j with respect to u is : ( 6)j(u)=(j(u)u(0,0,0 ) j(u)u(i , j , l ) j(u)u(nxu1,nyu1,nzu1))t , where each entry is expressed as : ( 7)j(u)u(i , j , l ) = 2(x , y , z)f^(x , y , z)(1sgn ( f^(x , y , z))2 ) g(xi , yj , zl ) + 2(x , y , z)(f^(x , y , z)lb)g(xi , yj , zl ) + 2 ( (x , y , z)u(x , y , z)1 ) + 2n=13 (x , y , z)rn(f^(x , y , z)1nrn(x , y , z)rnf^(x , y , z ) ) (g(xi , yj , zl)1nrn(x , y , z)rng(xi , yj , zl ) ) . a gradient - based iterative restoration algorithm or its conjugate version can be efficiently applied to minimize this convex cost function . besides , since the proposed criterion is quadratic , many other optimization methods can be used . the initial inverse fir filter required by the nas - rif algorithm is the kronecker delta function ; the size of this inverse filter is set to 3 3 3 pixels . furthermore , we have used = 0 because the background of spect images is not completely black . finally , the convergence criterion of the proposed algorithm is the stability of the cost function to be minimized , that is , ( 8)j(u[l+1])j(u[l])j(u[l]) , where is a threshold , typically set in our application to 10 , with the upper - script denoting the iteration number . figure 3 shows the evolution of the cost function value along the iterations of the gradient descent process for the image restoration presented in figure 8 . in order to define our anatomically based regularization term j4 , we exploit the result of a 3d registration step between the mri and spect input volumes ( from the same patient ) [ 11 , 12 ] and then an unsupervised markovian segmentation of the ( registered ) mri 3d image into anatomical classes ( see section 3.2 ) . in order to define our anatomically based regularization term j4 , we exploit the result of a 3d registration step between the mri and spect input volumes ( from the same patient ) and then an unsupervised markovian segmentation of the ( registered ) mri 3d image into anatomical classes . the 3d registration method used in our application is based on mutual information ( mi ) and is fully described in . the mi registration criterion c( ) between the input mri and spect volumes describes the amount of information in the joint histogram of the images ; hence its maximization results in the best match of intensity correspondences between the images for registration . the optimal set of registration parameters optimal is then found by maximizing c( ) , where the vector is simply estimated by the powell 's method . the images are smoothed slightly in order to make the cost function c( ) as smooth as possible to give faster convergence and less chance of finding bad local minima ( related to a wrong registration ) . the code used to register the mr image to the spect image is mainly inspired from the software package statistical parametric mapping ( spm ) . ( see figure 7 ) . to this end , we consider two random fields ( x , g ) , where g = { gs , s s } represents the field of observations located on the 3d lattice s of sites s ( voxels ) and x = { xs , s s } the label field ( related to the class labels xs of a segmented 3d image ) . each aforementioned label is associated to a specific brain tissue category or region on the 3d image . csf and extra - cerebral tissues are combined in a single class , corresponding to tissue without ( significant ) tracer uptake . although skin and other structures outside the brain actually have a nonzero tracer presence ( they have blood flow ) , we assume this to be negligible here . the csf area designates regions within the brain and immediately around it that are actually devoid of activity ( intracerebal ventricles and peri - cerebral cysterns ) . the white matter and grey matter ( brightest region ) are associated with lower and higher levels of blood flow , respectively , . each gs takes its value in { 0 , , 255 } ( 256 grey levels ) , and each xs in { e1 = csf , e2 = whitematter , e3 = greymatter}. the distribution of ( x , g ) is defined , firstly , by a prior distribution px(x ) , assumed to be markovian and secondly , by the site - wise conditional data likelihoods pgs / xs(gs / xs ) whose shape and parameter vector (xs ) depend on the concerned class label xs ( gs designates the grey level intensity associated to the site s ) . estimation stepin order to determine = ( (e1 ) , (e2 ) , (e3 ) ) , we use the iterative conditional estimation ( ice ) algorithm and three different gaussian laws for the likelihood . in order to determine = ( (e1 ) , (e2 ) , (e3 ) ) , we use the iterative conditional estimation ( ice ) algorithm and three different gaussian laws for the likelihood . segmentation stepbased on the estimates given by the ice procedure , we can compute an unsupervised 3d markovian segmentation of the mr volume . in this framework , the markovian segmentation can be viewed as a statistical labeling problem according to a global bayesian formulation in which the posterior energy has to be maximized : ( 9)u(x , g)=ssln pgs xs(gs xs)u1(x , g)+s , tst ( 1(xs , xt))u2(x ) where u1 expresses the adequacy between observations and labels , and u2 represents the energy of the a priori pott model ( which tends to favor homogeneous regions with no privileged orientation ) . we use the deterministic iterated conditional modes ( icm ) algorithm to minimize this global energy function . for initialization of this algorithm , we use the segmentation map obtained by a maximum likelihood ( ml ) segmentation . the support is then determined simply by the set of pixels belonging to csf , white and grey matter classes . based on the estimates given by the ice procedure , we can compute an unsupervised 3d markovian segmentation of the mr volume . in this framework , the markovian segmentation can be viewed as a statistical labeling problem according to a global bayesian formulation in which the posterior energy has to be maximized : ( 9)u(x , g)=ssln pgs xs(gs xs)u1(x , g)+s , tst ( 1(xs , xt))u2(x ) where u1 expresses the adequacy between observations and labels , and u2 represents the energy of the a priori pott model ( which tends to favor homogeneous regions with no privileged orientation ) . we use the deterministic iterated conditional modes ( icm ) algorithm to minimize this global energy function . for initialization of this algorithm , we use the segmentation map obtained by a maximum likelihood ( ml ) segmentation . the support is then determined simply by the set of pixels belonging to csf , white and grey matter classes . the spect images were acquired with a triple - head gamma camera ( picker prism , cleveland , oh , usa ) equipped with low - energy , high - resolution parallel - holes collimators . the spect projections were acquired over 360. 90 projections of 50 seconds each were obtained . mri images were acquired on a siemens magnetom avanto 1.5 t scanner using a 3d - fisp with a radial trajectory in k - space . the scanning parameters were tr = 9.2 ms , te = 2.2 ms with n slices of 512 512 voxels with voxel dimensions of 0.5 0.5 1.0 mm , and n [ 130 , 150 ] . these 3d mri images were further processed to isolate the brain from other tissues , using the brain extraction tool ( bet ) of the mricro software by adjusting bet 's fractional intensity threshold . two imaging phantoms were considered in order to validate the accuracy of our spect images restoration method for spect imaging , the hoffman 3d brain phantom was scanned while containing 148 mbq of activity . the phantom was positioned so that the orientation of the slices within the phantom would match as much as possible those from the mri image . phantom spect data included a set of 61 slices of 128 128 voxels with voxel dimension of 1.85 1.85 1.85 mm . the mri phantom data contained 209 slices of 256 256 voxels with 1 mm isotropic voxels . the other phantom contains 6 spheres of different sizes ( diameters # 1 : 9.5 mm , # 2 : 12.7 mm , # 3 : 15.9 mm , # 4 : 19.1 mm , # 5 : 25.4 mm , # 6 : 31.0 mm ) . in one condition , the spheres were filled with a radioactive solution and the rest of the cylinder ( background ) with a less concentrated solution of the same radioisotope , giving an activity concentration ratio ( spheres / background ) of approximately 2.7 : 1 ; in another condition , the cylinder was filled with a low - activity solution and the spheres were filled with nonradioactive water . phantom spect data included a set of 93 slices of 128 128 voxels with voxel dimensions of 1.85 1.85 1.85 mm . the phantom mri data contained 224 slices of 256 256 voxels with 1 mm isotropic voxels . simple visual examination is an easy method for evaluation of the restorative power of a technique , but it is obviously an insufficient approach . a better evaluation approach consists in computing a performance measure based on the improvement in signal - to - noise ratio ( isnr ) , expressed in decibels ( db ) , using both the degraded phantom , the ground truth ( or the original undegraded image given by the mri image ) , and the restored phantom images . the isnr is defined by , ( 10)isnr=10 log 10(ioriideg 2ioriires2 ) , where ideg is a given degraded phantom image , iori is the corresponding original ( ground truth ) phantom image and ires is the restored phantom image . || obviously , this metric can only be used with knowledge of the original object ; in our case this came from the mri phantom and knowledge of the radioactivity concentration within each subcompartment of the phantom . in addition , restored images were also evaluated by the specific evaluation criteria proposed in [ 22 , 23 ] , based on the estimation of the four following measures : the global contrast of the image , defined by cg = ( 1 mwm / mgm ) , where mwm and mgm are the means of the pixel value in the white and grey matter areas respectively . the local contrast of the image , defined by cl = ( ri bj)/bj , where ri represents the mean grey level value inside the ith sphere and bj represents the mean grey level value outside the ith sphere ( in a circle centered around the sphere and whose radius d is half the distance from one sphere center to the next in the image . ) the image mottle mwm in the white matter region , defined by mwm = wm / mwm , where wm is the standard deviation of pixel values in this area . the image mottle mgm in the grey matter region , defined by mgm = gm / mgm , where gm is the standard deviation of pixel values in this area . these two metrics mwm and mgm allow us to measure the amplification of noise and/or to measure the presence of undesirable artifacts that could be created by the restoration procedure in a uniform region of the spect volume . due to the different number of pixels belonging to each brain anatomical tissue category , we considered the total mottle measure given by m = wmmwm + gmmgm , with wm and gm designating the proportion of pixel belonging to the white and gray matter area respectively . a reliable spect image restoration method is one that enhances image contrast with little increase in the mottle . inversely , for a given maximal mottle measure , we can measure if the contrast enhancement is significant . we have compared our blind and unsupervised deconvolution approach to a classical deconvolution technique using a metz filter . the metz filter is a supervised deconvolution ( restoration ) procedure which assumes knowledge of the point spread function ( psf ) of the imaging system . this filter allows deconvolution of spect images while attenuating very high frequencies ( i.e. , noise which could be induced by inverse filtering ) . please remember that the metz filter has two parameters to be adjusted , the full width at half maximum ( fwhm ) related to the filter size and p ( which is the order of the filter ) . restoration of clinical data was performed on pairs of mri and spect images from 30 epileptic patients . each spect data set contained n slices of 128 128 voxels with voxel dimensions of 1.85 1.85 1.85 mm , and n [ 69 , 103 ] . mri data sets contained m slices of 512 512 voxels with voxel dimensions of 0.5 0.5 1.0 mm and m [ 130 , 150 ] . in this section , isolation of the brain ( in mri ) from other tissues was made off - line prior to the registration and restoration steps . this value was chosen empirically after a series of test runs which were visually evaluated . figure 4 shows the variation in isnr of the processed phantom images with respect to the observations for a range of fwhm parameter and for the best value of p. by examining the isnr for different values of fwhm , we noticed that the optimal restoration value was for fwhm = 9.85 mm , p = 40 corresponding to isnr = 0.42 db . average contrast and total mottle were first quantified on a set of ( human brain ) degraded and restored spect images ( with and without our anatomical - based regularization term j4(u ) ) and compared to the metz filter . our proposed algorithm with j4(u ) resulted in an increase of global contrast by 1.87 and of mottle by an acceptable factor of 1.17 . the metz filter increased global contrast by 1.52 and mottle by a factor of 1.08 . for spect phantoms , for all the tested experiments , we used = 0 because the background of spect images is not completely black and we chose 25 for the weighting factor for j4 . this value was chosen empirically after a set of experiments by varying in the interval [ 10 , 100 ] . spect images of the cold spheres phantoms were also restored using partially incorrect anatomic information ( i.e. , cylindrical phantom mri with 5 different sphere sizes instead of 6 ; one sphere was removed in the mri segmented image ) . table 2 shows that the local contrast measured on this restored spect image ( with our anatomical - based regularization term j4(u ) ) remained similar to the local contrast obtained with correct anatomic information . local contrast was quantified on individual spheres of spect degraded and restored images ( with and without our anatomical - based regularization term j4(u ) ) and compared to those obtained with the metz filter . table 3 shows that our algorithm with j4(u ) resulted in a average value of the increase in local contrast of 2.07 . the metz filter increased , in average , the local contrast by only 1.53 . figures 8 and 9 show , respectively , examples of the hoffman phantom and brain spect volumes obtained with our restoration method . the reconstructed whole brain volume converged to a very good estimate of the solution without a priori information about the psf and allowed to noticeably improve the contrast of the original , unrestored spect volume . this restoration should allow more efficient detection of small , localized singularities associated with different types of lesions ( tumors , epileptogenic foci , etc . ) that often are not clearly visible in the original blurred image . the metz filter has two parameters to be adjusted , fwhm and order p. we have used the values of 4.5 , 5 , 5.5 , 6 , 6.5 , 7 , 8 , 9 , 10 and 11 mm for fwhm and 10 , 20 , 30 , 40 and 50 for order p , a total of 50 combinations of both parameters . all the above values were tested and only the best one ( in terms of isnr ) was selected for comparison with our algorithm . in addition , the 30 pairs of brain mri and spect acquisitions from different subjects have been anonymised using jim 's dicom anonymiser tool . ( this tool allows the removal from image files of information that may jeopardise patient 's or physician 's privacy . ) our restoration procedure was not highly sensitive to the bet 's fractional intensity threshold value when this was visually set by an experimented user ( e.g. , the value of contrast was stationary when the threshold went from 0.45 to 0.55 ) . therefore , this parameter was set to 0.50 for all thirty pairs of mri images and seemed to be optimal in all tested cases . notice that the degradation model used by our restoration method assumes that noise is additive while in reality it is a multiplicative poisson process . nevertheless , the results with real spect / mri data are visually impressive . it seems that at the high count level used in spect , the difference between ( multiplicative ) poisson and ( additive ) gaussian noise does not affect significantly the efficiency of the algorithm . however it would be meaningful to conduct clinical studies using roc analysis to better assess this restoration procedure . when compared with a classical restoration approach using a metz filter , our method performed better , in terms of signal - to - noise ratio . the slight increase in mottle with j4 is a limited price to pay given the gains in global contrast , isnr , and local contrast measures , which from a clinical perspective ensure better detection of focal anomalies , a task at which spect is usually notoriously poor . the tests also showed that the restored image is constrained ( or guided ) by our prior knowledge but not wrongly biased by this information while avoiding noise amplification inherent to each deconvolution process . the computational time of our technique was approximatively 6.33 minutes as compared to 0.24 minutes for the metz filter on a 2.0 ghz pc workstation running linux . our method is computationally demanding but remember that it is an unsupervised one which does not require any psf parameter . another disadvantage of our method is the need to get an mri scan ; however , many of the subjects submitted to a spect rcbf study actually require one from a clinical perspective . importantly , the accuracy of the registration procedure is crucial for the final restoration result , fortunately many highly accurate registration procedures are widely available elsewhere . in this paper , we have presented a robust restoration method using anatomical and functional information fusion with application to spect images . this method improves the quality of human brain 3d spect images and should thus be helpful to physicians interpreting such studies . our approach takes advantage of the anatomical information contained in the mri study of each subject ( or any other high - resolution image such as ct ) . the proposed constraint term allowed both stabilization of the inverse solution of the nas - rif procedure by prevention of noise amplification and the generation of a better constraint on the solution of our restoration problem . in the regularization framework , this term allowed smooth regions to be reconstructed in the spect image , where such homogeneous anatomical regions are found in the high - resolution mri images of the patient , after those were registered to the subject 's spect volume . this method was tested on a number of spect / mri pairs , demonstrating its efficiency and robustness . this 3d blind restoration technique is completely data driven and automated , therefore it could be implemented to process large numbers of 3d spect studies .

image restoration is usually viewed as an ill - posed problem in image processing , since there is no unique solution associated with it . the quality of restored image closely depends on the constraints imposed of the characteristics of the solution . in this paper , we propose an original extension of the nas - rif restoration technique by using information fusion as prior information with application in spect medical imaging . that extension allows the restoration process to be constrained by efficiently incorporating , within the nas - rif method , a regularization term which stabilizes the inverse solution . our restoration method is constrained by anatomical information extracted from a high resolution anatomical procedure such as magnetic resonance imaging ( mri ) . this structural anatomy - based regularization term uses the result of an unsupervised markovian segmentation obtained after a preliminary registration step between the mri and spect data volumes from each patient . this method was successfully tested on 30 pairs of brain mri and spect acquisitions from different subjects and on hoffman and jaszczak spect phantoms . the experiments demonstrated that the method performs better , in terms of signal - to - noise ratio , than a classical supervised restoration approach using a metz filter .

1. Introduction 2. 3D Anatomical Constraint NAS-RIF Algorithm 3. Registration and Segmentation 4. Validation 5. Experimental Results 6. Discussion 7. Conclusion

PMC2233883

ion channels gated by cyclic nucleotides are widely distributed throughout eukaryotes ( kaupp and seifert , 2002 ; robinson and siegelbaum , 2003 ) . as the molecular hardware linking changes of intracellular cyclic nucleotide levels occurring in sensory transduction to cellular electrical responses , these proteins are especially important in the coherent functioning of electrically excitable tissues . they have been most widely studied in sensory neurons , where they carry out essential tasks in the detection of photons or odorants , in cardiac myocytes , where they underlie pacemaking activity , and in cerebellar neurons , where they contribute to fine tuning in motor - coordination circuits . all such channels belong to the s4 superfamily ( yellen , 2002 ; yu and catterall , 2003 ) , which also includes voltage - dependent ca , na , and k channels ; these proteins are tetramers of identical or similar subunits , or of honorary subunits inhabiting the same polypeptide in tandem , with each subunit having six transmembrane sequences and a pore - forming sequence positioned between the fifth and sixth of these . the cyclic nucleotide - modulated channels additionally carry a cyclic nucleotide - binding domain ( cnbd ) in the cytoplasmic cooh - terminal region . these channels fall operationally into two broad subclasses , denoted cng ( cyclic nucleotide gated ) and hcn ( hyperpolarization and cyclic nucleotide activated ) . channels of the first class depend absolutely on cyclic nucleotides for opening , gate in a voltage - independent manner , and permit conduction only by monovalent cations , with little ionic selectivity among the group ia cations . in contrast , hcn channels are merely modulated by cyclic nucleotides in their primary mode of voltage - dependent gating , and , while also conductive to monovalent cations , favor k over na by a factor of approximately four . at the molecular level , both cng and hcn channels show the arginine - decorated fourth transmembrane sequence characteristic of the s4 family , and both carry similar cnbd sequences . in keeping with their k selectivity , pore - forming selectivity sequence unique and common to k channels ( heginbotham et al . , 1992 ) , while cng channels lack this sequence . the recent high - resolution structures of several k channels ( doyle et al . , 1998 ; jiang et al . , 2002 , 2003 ) provide an outline of molecular architecture in this protein family but leave many questions specific to the cyclic nucleotide - binding subfamily unaddressed . are these , like k channels , built as fourfold symmetric structures , as in the structure of an isolated cnbd of an hcn channel ( zagotta et al . , 2003 ) , or as dimers - of - dimers , as suggested by functional experiments on cng and hcn channels ( liu et al . , 1998 ; ulens and siegelbaum , 2003 ) ? what accounts for the hyperpolarization - dependent opening of hcn channels , a voltage dependence with polarity opposite to that of conventional kv - type k channels ( mannikko et al . , 2002 the 80-residue sequence connecting the cnbd to the sixth transmembrane helix , mediate between cyclic nucleotide binding and channel opening ( gordon and zagotta , 1995 ) ? so much less k selective than other k channels , which typically show 1001,000-fold selectivity for k over na ? the only productive avenue toward high - resolution ion channel structure is currently provided by bacterial homologues , which in some cases allow overexpression in escherichia coli of properly assembled proteins amenable to purification and crystallization . for this reason , we searched prokaryotic databases for s4-type ion channels containing cyclic nucleotide - binding domains . we now describe the overexpression , purification , and functional reconstitution of an s4-superfamily homologue from mesorhizobium loti , with a k selectivity sequence and a cnbd . the channel 's chromatographic behavior in detergent micelles and its ionic flux properties in liposomes respond to micromolar levels of cyclic nucleotides , and its selectivity for k is higher than that of either cng or hcn subtypes . a putative bacterial channel protein homologous to eukaryotic cyclic nucleotide - gated channels was identified by a homology search of the tigr bacterial database . the coding sequence was pcr cloned from m. loti genomic dna and inserted into an e. coli expression vector ( pask - iba2 ; sigma - genosys ) with a cooh - terminal hexahistidine tag . e. coli jm83 were transformed with this construct , and after plating on lb - amp , cells were harvested and grown in terrific broth . expression of protein was induced for 90 min with 0.2 mg / l anhydrotetracycline at a cell density ( a550 ) of 1.0 . cells were pelleted , resuspended in 100 mm kcl , 50 mm tris - hcl , ph 7.5 ( 50 ml for 13 liter culture ) , and lysed by sonication on ice in the presence of protease inhibitors ( complete edta - free , roche , and pmsf 0.17 mg / ml ) . membrane protein was extracted 2 h by adding 50 mm n - decylmaltoside ( dm ; anatrace ) , and the extract , clarified by centrifugation , was applied to a ni affinity column ( qiagen ) . after washing off nonspecifically bound proteins with running buffer ( 100 mm kcl , 20 mm tris - hcl , 5 mm dm , ph 7.6 ) supplemented with 40 mm imidazole , the protein was eluted by raising imidazole to 400 mm , concentrated to 815 mg / ml in amicon ultracentrifugal concentrators , and chromatographed on a superdex 200 size exclusion column ( amersham biosciences ) in running buffer . when needed , cyclic adenosine monophosphate or cyclic guanosine monophosphate ( camp , cgmp , fluka ) , 50200 m unless otherwise specified , was added to the bacterial suspension before sonication and maintained during all purification and reconstitution steps . in experiments in which cyclic nucleotide concentration was varied , protein was always prepared in high cyclic nucleotide , and the desired final concentration was introduced in the reconstitution step . eluted protein was analyzed by sds - page gel electrophoresis and the proteins visualized by coomassie staining . the purified protein was immediately reconstituted into liposomes at a concentration of 0.150 g protein / mg lipid in 20 mg / ml e. coli polar lipids ( avanti polar lipids , inc . ) in the presence or absence of camp or cgmp . detergent was removed either by overnight dialysis against 1,000 volumes of 400 mm kcl , 10 mm hepes , 5 mm n - methyl glucamine ( nmg ) , ph 7.6 , or by chromatography on sephadex g-50 fine , equilibrated in this same solution . the rb flux assay has been described in detail ( heginbotham et al . , 1998 ) . in brief , liposomes were thawed , sonicated , and passed over g-50 spin - columns ( 100 l liposome , 1.5 ml column ) equilibrated in uptake buffer ( 400 mm sorbitol , 50 m kcl , 10 mm hepes , 5 mm nmg , and the desired concentration of camp or cgmp ) to establish a k gradient across the liposome membrane . uptake of rb was initiated by mixing the liposomes with two to six volumes of uptake buffer containing trace amounts of rb ( 0.1 ci / ml ) , as depicted in the inset of fig . , extraliposomal rb was removed by passing a 100 l aliquot over a 1.5-ml cation - exchange dowex column ( 50wx4 - 100 ; sigma - aldrich ) converted to the nmg form , and radioactivity was measured in a liquid scintillation counter . valinomycin ( 1 g / ml ) was added at the end of each experiment to assay maximum rb uptake into all liposomes present . to follow proton uptake into electrically polarized liposomes , protein - free or reconstituted vesicles were thawed and supplemented with 50 mm kpi , ph 7.3 , and were then sonicated 510 s to trap the buffer in the internal space . for each assay , 100 l of vesicles was applied to a 1.5-ml spin column equilibrated with proton uptake solution ( 350 mm nacl , 10 mm kcl , 2 mm kpi , ph 7.3 ) to exchange the external solution and establish a k gradient . the liposomes ( 100 l ) were immediately diluted into a stirred vial containing 1.9 ml of proton uptake solution , in which a ph electrode was positioned . the ph of the solution was recorded on chart paper on a high - sensitivity scale ( 0.06 ph units full scale ) . fccp and valinomycin were used to permeabilize the liposomes to h and k , respectively . a putative bacterial channel protein homologous to eukaryotic cyclic nucleotide - gated channels was identified by a homology search of the tigr bacterial database . the coding sequence was pcr cloned from m. loti genomic dna and inserted into an e. coli expression vector ( pask - iba2 ; sigma - genosys ) with a cooh - terminal hexahistidine tag . e. coli jm83 were transformed with this construct , and after plating on lb - amp , cells were harvested and grown in terrific broth . expression of protein was induced for 90 min with 0.2 mg / l anhydrotetracycline at a cell density ( a550 ) of 1.0 . cells were pelleted , resuspended in 100 mm kcl , 50 mm tris - hcl , ph 7.5 ( 50 ml for 13 liter culture ) , and lysed by sonication on ice in the presence of protease inhibitors ( complete edta - free , roche , and pmsf 0.17 mg / ml ) . membrane protein was extracted 2 h by adding 50 mm n - decylmaltoside ( dm ; anatrace ) , and the extract , clarified by centrifugation , was applied to a ni affinity column ( qiagen ) . after washing off nonspecifically bound proteins with running buffer ( 100 mm kcl , 20 mm tris - hcl , 5 mm dm , ph 7.6 ) supplemented with 40 mm imidazole , the protein was eluted by raising imidazole to 400 mm , concentrated to 815 mg / ml in amicon ultracentrifugal concentrators , and chromatographed on a superdex 200 size exclusion column ( amersham biosciences ) in running buffer . when needed , cyclic adenosine monophosphate or cyclic guanosine monophosphate ( camp , cgmp , fluka ) , 50200 m unless otherwise specified , was added to the bacterial suspension before sonication and maintained during all purification and reconstitution steps . in experiments in which cyclic nucleotide concentration was varied , protein was always prepared in high cyclic nucleotide , and the desired final concentration was introduced in the reconstitution step . eluted protein was analyzed by sds - page gel electrophoresis and the proteins visualized by coomassie staining . the purified protein was immediately reconstituted into liposomes at a concentration of 0.150 g protein / mg lipid in 20 mg / ml e. coli polar lipids ( avanti polar lipids , inc . ) in the presence or absence of camp or cgmp . detergent was removed either by overnight dialysis against 1,000 volumes of 400 mm kcl , 10 mm hepes , 5 mm n - methyl glucamine ( nmg ) , ph 7.6 , or by chromatography on sephadex g-50 fine , equilibrated in this same solution . the rb flux assay has been described in detail ( heginbotham et al . , 1998 ) . in brief , liposomes were thawed , sonicated , and passed over g-50 spin - columns ( 100 l liposome , 1.5 ml column ) equilibrated in uptake buffer ( 400 mm sorbitol , 50 m kcl , 10 mm hepes , 5 mm nmg , and the desired concentration of camp or cgmp ) to establish a k gradient across the liposome membrane . uptake of rb was initiated by mixing the liposomes with two to six volumes of uptake buffer containing trace amounts of rb ( 0.1 ci / ml ) , as depicted in the inset of fig . , extraliposomal rb was removed by passing a 100 l aliquot over a 1.5-ml cation - exchange dowex column ( 50wx4 - 100 ; sigma - aldrich ) converted to the nmg form , and radioactivity was measured in a liquid scintillation counter . valinomycin ( 1 g / ml ) was added at the end of each experiment to assay maximum rb uptake into all liposomes present . to follow proton uptake into electrically polarized liposomes , protein - free or reconstituted vesicles were thawed and supplemented with 50 mm kpi , ph 7.3 , and were then sonicated 510 s to trap the buffer in the internal space . for each assay , 100 l of vesicles was applied to a 1.5-ml spin column equilibrated with proton uptake solution ( 350 mm nacl , 10 mm kcl , 2 mm kpi , ph 7.3 ) to exchange the external solution and establish a k gradient . the liposomes ( 100 l ) were immediately diluted into a stirred vial containing 1.9 ml of proton uptake solution , in which a ph electrode was positioned . the ph of the solution was recorded on chart paper on a high - sensitivity scale ( 0.06 ph units full scale ) . fccp and valinomycin were used to permeabilize the liposomes to h and k , respectively . in a blast search for s4-type channels containing cnbds , we noticed , among four other hits , a putative k channel homologue in the plant symbiont m. loti , an organism commonly used for genetic studies of rhizobial symbiosis and nitrogen fixation . np104392 ) encodes a 355-residue polypeptide of 38 kd with six putative transmembrane sequences ( s1s6 ) , where s4 bears three basic residues ( two arginines , one lysine ) , a k selectivity sequence ( ttgygd ) between s5 and s6 , and a cooh - terminal cnbd ( fig . the pore sequence for this protein , which we call mlok1 , is homologous to the corresponding region of other k channels such as kcsa ( 60% identity ) and shaker ( 50% ) , and its cnbd displays convincing similarity ( 30% ) to those of eukaryotic cng and hcn subtypes . unlike its eukaryotic counterparts , mlok1 does not contain the four - helix c - linker domain , which connects the end of s6 to the cnbd and is thought to transmit the cyclic nucleotide binding signal to the channel 's gate ( gordon and zagotta , 1995 ; ; johnson and zagotta , 2001 ; wang et al . , 2001 ) . in mlok1 , mlok1 in the s4 ion channel superfamily . predicted transmembrane segments shown as cylinders , cnbd in yellow , pore region in cyan . the completely conserved arg is shown in green and the adjacent conserved hydroxy amino acid in red . asterisk marks the position of acidic residues in cng channels ( varnum et al . , 1995 ) channel abbreviations , with ncbi accession nos . , are as follows : hcn1 , mouse hcn ( o88704 ) , hcn2 , human hcn ( q9ul51 ) , cnga1 , bovine cng ( q00194 ) , cnga2 , bovine cng ( q03041 ) , spih , sea urchin hcn ( np999729 ) . the channel protein , engineered with a cooh - terminal his6 tag and placed behind a tetracycline promoter , is efficiently expressed in e. coli ( fig . 2 ) , with an induction - dependent band of 35 kd visible on sds - page even in the whole - cell lysate . after extraction of the cells in detergent , high purity is achieved in a single pass over a ni - chelate column with a yield of 3 mg / l culture . the chromatographic behavior of the affinity - purified protein on gel filtration is cyclic nucleotide dependent ( fig . much of the protein elutes as an aggregate in the void volume , with some tetrameric material ( 150 kd ) following , along with several other peaks . however , with 50 m camp present , most of the material runs as a single tetrameric peak , as though the protein population is monodisperse under this condition . although gel filtration chromatography does not rigorously determine particle size , we are reasonably confident of our measurement here , since the column was calibrated with six membrane proteins of known size , run under identical detergent conditions . we note that these experiments were performed on two separate preparations processed in parallel in which the protein was extracted , purified , and chromatographed in the presence or absence of camp . this result implies that occupancy of the cnbd by ligand promotes native folding and perhaps stabilizes the channel during the purification procedure ; we have found ( unpublished data ) that the tetrameric peak purified without camp and maintained in its absence remains tetrameric for at least 7 d at 4c . protein samples were run on a 10% polyacrylamide sds gel stained with coomassie blue as follows : lane 1 , protein ladder with molecular weights indicated ; lanes 2 and 3 , pre- and post - induction whole e. coli lysates ; lane 4 , clarified membrane extract of induced cells ; lane 5 , ni column flow - through of membrane extract ; lane 6 , nonspecifically bound protein wash of ni column ; lane 7 , ni column eluted mlok1 ( 7 g ) ; lane 8 , gel filtration column - purified mlok1 ( 7 g ) . preparations of ni column purified ( solid curve ) or gel filtration purified ( dashed curve in b ) mlok1 were applied to a superdex gel filtration column monitored at 280 nm in the absence ( a ) or presence ( b ) of 50 m camp . dashed lines mark the tetrameric mlok1 peak and arrows indicate the void volume and molecular weights of two calibrated membrane proteins run under identical conditions : mthk k channel tetramer ( 250 kd ) and clc - ecl cl transporter dimer ( 92 kd ) . according to a calibration using five membrane proteins of known sizes run under identical conditions , the elution volume of mlok1 corresponds to 150 kd . scale bar for absorbance at 280 nm represents 20 mau for a and 100 mau for b. does mlok1 function as a cyclic nucleotide - gated ion channel ? we reconstituted the protein into liposomes and examined its ability to catalyze transmembrane movement of rb , a convenient radioactive k analogue . influx was followed under conditions wherein liposomes loaded with high kcl are suspended in low kcl solution containing radioactive tracer , a circumstance that leads to concentrative accumulation of radioactivity . under these conditions , significant tracer uptake can occur only in liposomes that are permeable to k but not to cl ( heginbotham et al . , 1998 ) . 4 a illustrates a typical influx time course , in which rb becomes trapped within the liposomes with approximately exponential time dependence characterized by a half - time of 30 min . however , even on the long , 2-h time scale of this experiment , the influx does not reach completion , and so the equilibrium level of trapped rb remains uncertain . we measure the maximum capacity of rb uptake by adding valinomycin , an ionophore which instantly permeabilizes all liposomes to k and rb . thus , in this experiment , mlok1 catalyzes rb uptake into 35% of the liposome space in 2 h , but because of the slowness of the time course , we can not claim that this represents the fraction of liposomes carrying functional mlok1 ; it does place a lower limit on this fraction , however . henceforth we normalize influx time courses to the maximum uptake measured by valinomycin to compare results obtained from different reconstitution conditions , as in fig . 4 b. here , the effect of camp on influx is shown . over the 1-h time course reconstitution of mlok1 increases the initial rate of uptake , and a maximal concentration of camp ( 200 m ) approximately doubles this . these results show that mlok1-mediated ion transport is not absolutely dependent on camp , but that the ligand enhances the protein 's intrinsic activity . ( a ) representative time course of accumulation of rb into liposomes reconstituted with mlok1 ( 10 g / mg lipid , 200 m camp ) . each point represents radioactivity of a single 100-l sample removed from the reaction mix at the time point indicated . after the 2-h sample was collected , valinomycin was added ( arrow ) and a sample was taken 2 min later . ( b ) valinomycin - normalized rb uptake in liposomes reconstituted with mlok1 ( 10 g / mg lipid ) in the presence of camp ( 200 m , black circles ) or in its absence ( gray circles ) . inset , cartoon of rb uptake assay , with liposomes loaded with high k ( 400 mm ) immersed in low k solution ( 50 m ) containing tracer rb ; this leads to a high negative intraliposomal potential , which the rb tracer follows ( heginbotham et al . the rate of rb uptake increases with the density of channels in the reconstituted membranes ( fig . ( < 2 g protein / mg lipid ) , uptake increases linearly , as liposomes in excess are titrated with channel protein . eventually , uptake levels off at high protein density > 5 g / mg ( fig . , this effect can not be quantified , as has been done by poisson statistics in other liposome systems ( goldberg and miller , 1991 ; heginbotham et al . , 1998 ; maduke et al . , 1999 ) , since the final steady level of rb uptake can not be accurately determined here ; nonetheless , the value of the half - saturation density ( 1 g / mg ) indicates that a large fraction of the protein is functionally active , since it is at this density that the number of liposomes is roughly equal to the number of protein molecules present in the reconstitution mix ( heginbotham et al . , 1998 ) . ( a ) valinomycin - normalized rb uptake time course for 0 ( white ) , 0.1 ( light gray ) , 1 ( dark gray ) , and 5 ( black ) g mlok1/mg lipid in the presence of 50 m camp . each time point represents rb accumulation after 45 min in liposomes containing mlok1 at the indicated concentrations and in the presence of 50 m camp . the appearance of a k selectivity sequence ( ttgygd ) between s5 and s6 suggests strongly that mlok1 should show a familiar k selectivity profile , permitting permeation by k and rb and effectively barring na and li . to test this expectation , we adapted the concentrative uptake assay to assess ion selectivity , comparing rb uptake into liposomes loaded with different test cations in the presence of camp ( fig . uptake is similar in k and rb , while neither na nor li supports significant uptake . these experiments do not allow precise estimates of ionic permeability ratios , but it is clear that rb influx rate is close to the k rate , and na and li permeate , if at all , at much lower rates . each bar represents rb uptake into liposomes reconstituted with 5 g protein / mg lipid measured after 45 min of uptake and normalized to the total amount of radioactivity in the sample . reconstituted liposomes were formed in the presence of the 400 mm of the cl salts of the indicated cations , along with of 50 m camp . cyclic nucleotides bias the open closed equilibrium of eukaryotic cng and hcn channels toward open conformations . this behavior is mirrored in mlok1 , although the maximum stimulatory effect of camp is only twofold ( fig . 4 ) . to examine the ligand dependence of this effect , we reconstituted the channel into liposomes and used the uptake assay to determine dose response curves for camp and cgmp . rb flux time courses in the presence of varying cyclic nucleotide concentrations are shown in fig . 7 ( a and b ) . both camp and cgmp stimulate the uptake , and both give similar approximately twofold maximal stimulation . response curves were derived from these time courses by normalizing the uptake values measured at 15 or 45 min to the corresponding isochronal uptake at maximal ligand concentration . these follow a simple langmuir - type concentration dependence ( hill coefficient of unity ) , with camp being 10-fold more effective than cgmp ( k1/2 = 60 and 600 nm , respectively ) . flux time courses into liposomes reconstituted with 5 g mlok1/mg lipid in the presence of camp ( a ) and cgmp ( b ) , at 0 ( white ) , 0.1 ( light gray ) , 1 ( dark gray ) , and 10 ( black ) m concentration . uptake values at 15 min ( squares ) and 45 min ( circles ) are normalized to both maximum ( saturating concentration of cnmp ) and minimum ( no cnmp ) values . smooth curves are drawn according to single - site binding functions , with k1/2 = 60 nm for camp ( black ) and 600 nm for cgmp ( gray ) . the uptake experiments above demonstrate that mlok1 mediates membrane transport for rb and k but do not distinguish between electrically conductive ion movement , as would be expected for a channel homologue , and an electroneutral mechanism . the preferred way of demonstrating a conductive mechanism is to reconstitute the protein into planar lipid bilayers and record electrical currents ; however , we have been so far unable to do this the method employs proton uptake to report on the electrical potential set up across the liposome membrane by a k gradient . 8 a ; liposomes loaded with high k are suspended in low k solution , and the ph of the suspension is recorded . a k - selective conductance present in the membrane produces a negative - inside electrical potential that attracts protons into the intraliposomal space . the proton - impermeable liposome membrane does not allow protons to follow this potential , but treatment with a proton ionophore removes this kinetic constraint and allows protons to enter , as may be observed by a ph increase in the external solution . addition of the weak - acid uncoupler fccp , which permeabilizes the membranes specifically to protons , has no effect on the ph of the suspension by itself . addition of the k - specific ionophore valinomycin polarizes the membrane and produces an immediate rise in ph , as protons are taken up against a ph gradient but down their electrical gradient . when this experiment is repeated on liposomes reconstituted with mlok1 , however , fccp causes prompt h influx without addition of valinomycin ( fig . thus , mlok1 substitutes functionally for valinomycin in setting up a negative internal membrane potential , and is therefore conductive , not electroneutral . the experiment further shows that mlok1 is selective for k over both na and h. subsequent addition of valinomycin causes only a small additional ph increase , as though < 25% of the liposomes in the population are free of functional mlok1 protein . these ph responses are completely abolished in the absence of a k gradient ( unpublished data ) . liposomes are loaded with high k and suspended in low k , with a k - specific conductance present in the membrane . under these conditions , if a proton ionophore is present to circumvent the impermeability of the lipid bilayer , protons will be drawn into the liposomes to follow the electrical potential set up by the k gradient . vesicles ( 20 mg / ml ) loaded with high k ( 450 mm ) were diluted 20-fold into lightly buffered low k ( 10 mm ) medium , and ph was recorded . at asterisk , fccp ( 0.050.5 g / ml ) , and at arrow , valinomycin ( 0.5 g / ml ) were added . horizontal scale bar , 30 s ; vertical scale bar , 13 nmol h , 0.003 ph units . all structures of ion channels determined so far have been prokaryotic in origin , mainly because of the empirical and tragic fact that overexpression of eukaryotic integral membrane proteins in e. coli has been so reliably unsuccessful . for this reason , if we wish to know what a eukaryotic channel protein looks like , we are currently forced into the unsatisfactory but practical necessity of seeking bacterial or archeal homologues of the channel of interest . in such a search we found five putative s4-type ion channels of the cyclic nucleotide - binding subclass . in striking contrast to the abundance of other channel families among bacterial genomes , such as kv and kir k channels and clc cl channels , these cnbd - containing homologues are rare , so far found only in five bacterial genomes , three of which are rhizobial symbionts . in no case is the biological function of any of these prokaryotic channels known . we chose to focus on mlok1 , which expresses well in e. coli and may be readily purified in quantity . to maintain the protein in a homogenous tetrameric state , it is necessary to keep cyclic nucleotide present throughout all steps of the purification procedure . the purified protein reconstituted into liposomes catalyzes rb uptake in a cyclic nucleotide - stimulated fashion . these two phenomena , along with the low concentration range in which the ligands act , imply that the cooh - terminal cnbd is properly folded and functionally competent . furthermore , the tetrameric nature and k - like ionic selectivity of conductive fluxes are as expected for a k channel . for these reasons , we consider that the purified mlok1 protein , in fulfilling the expectations of its sequence , is correctly folded and functional . to which subtype of cyclic nucleotide binding channel does mlok1 belong : cng or hcn ? it is certainly not of the cng subtype , since it is k selective and shows a k signature sequence . we are tempted to assign it to the hcn subfamily , on the basis of this k selectivity , as well as the substantial basal activity in the absence of cyclic nucleotides . but in fact we can not definitively assert that the channel is of hcn subtype . since we have not been able to record electrical currents from mlok1 , we can not gauge its voltage dependence , a fundamental defining characteristic of hcn channels . moreover , the high selectivity of mlok1-mediated flux for k over na would not be expected for a conventional hcn channel , as these are only weakly selective for k. we are therefore unwilling to assign this channel firmly to either of the categories defined in eukaryotic channels . both camp and cgmp bind to this channel , as indicated by chromatographic behavior and ion fluxes . in the flux assay , camp shows 10-fold higher potency than cgmp , with the two ligands giving similar maximal effects . several ligand - selectivity determinants have been identified in cnbds from eukaryotic channels , from both electrophysiological analysis of cng channels ( altenhofen et al . , 1991 ; varnum et al . , 1995 ) and direct structure determination of an isolated cnbd from an hcn channel ( zagotta et al . , 2003 ) . all eukaryotic cnbds , regardless of ligand preference , have a conserved arginine residue that coordinates the cyclic nucleotide phosphate ( zagotta et al . , 2003 ) , a threonine is always found adjacent to this arginine in eukaryotic channels that bind both camp and cgmp ; in the structure of hcn2 cnbd , the hydroxyl group of this threonine hydrogen bonds to the n2 atom of the guanine ring , while camp - specific channel isoforms , such as the sea urchin hcn channel ( gauss et al . , 1998 ) , lack this hydroxyl group . in mlok1 , this position uses a serine ( s308 ) , the hydroxyl of which may play an equivalent h - bonding role , permitting cgmp to bind . finally , cng channels that are highly selective for cgmp have an acidic residue closer to the cooh terminus ( fig . 1 , asterisk ) , and neutralizing this residue removes the ligand selectivity ( varnum et al . , 1995 ) ; mlok1 has an alanine residue here , in harmony with responsiveness to both camp and cgmp . on several grounds , therefore , the ligand dependence of mlok1 is consistent with expectations based on molecular determinants in its cnbd . one feature of mlok1 , however , is unexpected from precedent : the absence of a c - linker sequence , a motif found in all eukaryotic cng and hcn channels . the c - linker domain has been proposed to transmit the ligand - binding signal to the channel 's gate in s6 . this proposal arose from mutagenesis studies in cng and hcn channels ( gordon and zagotta , 1995 ; zong et al . , 1998 ; paoletti et al . , 1999 ; johnson and zagotta , 2001 ; wang et al . , 2001 ) and was strengthened by high - resolution structures of an isolated cnbd from an hcn isoform ( zagotta et al . , 2003 ) , where most of the intersubunit contacts in the tetramer are formed by the c - linker , which is composed of four -helices . this structure suggested that cyclic nucleotide binding drives a rearrangement of the c - linker domains , which opens the channel 's ion permeation pathway . our results refute this proposal , at least as a necessary , general mechanism of channel modulation by cyclic nucleotides , and they suggest that a minimal linking sequence is able to substitute for this function in the prokaryotic homologue . finally , we should ask whether the results here prove that mlok1 is a k channel . in fact to be sure , the gross functional characteristics of the reconstituted protein are consistent with a channel mechanism : electrically conductive transport with k - like ionic selectivity . but without a direct measurement of unitary transport rate , it is impossible to rigorously distinguish channel from transporter mechanisms , and the liposome system on which we rely does not permit an estimate of this . we note that mlok1-mediated rb uptake is 10100-fold slower than the rate expected for an always - open channel with a typical unitary conductance in the range 10100 ps , or than that observed under similar conditions for kcsa ( heginbotham et al . , 1998 ) . this low uptake rate might reflect a small unitary conductance or a low open probability , even when maximally activated by cyclic nucleotide , or a combination of both . in the absence of direct single - channel recording , this point must remain unresolved , but it is worth noting that eukaryotic hcn channels have extremely low single - channel currents ( difrancesco and mangoni , 1994 ) . in light of its tetrameric architecture , k - selective cyclic nucleotide - modulated ion fluxes , and unambiguous membership in the s4 superfamily of ion channels ,

a search of prokaryotic genomes uncovered a gene from mesorhizobium loti homologous to eukaryotic k+ channels of the s4 superfamily that also carry a cyclic nucleotide binding domain at the cooh terminus . the gene was cloned from genomic dna , and the protein , denoted mlok1 , was overexpressed in escherichia coli and purified . gel filtration analysis revealed a heterogeneous distribution of protein sizes which , upon inclusion of cyclic nucleotide , coalesces into a homogeneous population , eluting at the size expected for a homotetramer . as followed by a radioactive 86rb+ flux assay , the putative channel protein catalyzes ionic flux with a selectivity expected for a k+ channel . ion transport is stimulated by camp and cgmp at submicromolar concentrations . since this bacterial homologue does not have the c - linker sequence found in all eukaryotic s4-type cyclic nucleotide - modulated ion channels , these results show that this four - helix structure is not a general requirement for transducing the cyclic nucleotide - binding signal to channel opening .

INTRODUCTION MATERIALS AND METHODS Biochemical Flux Assays RESULTS DISCUSSION

PMC3108703

insomnia is characterized by inadequate or poor - quality sleep because of difficulty falling asleep ( sleep - onset insomnia ) , difficulty staying asleep ( sleep maintenance insomnia ) , or early morning awakening , and is classified as transient or chronic ( diagnostic and statistical manual of mental disorders , edition iv , text revision ) . transient insomnia is common and generally occurs as a result of anxiety , stress , environmental factors ( ie , noise , extreme temperatures , change in the surrounding environment ) , sleep wake schedule alterations , and medication - related side effects.1 chronic insomnia affects approximately 10% of the adult population in the us and is more complex , often associated with a combination of comorbid factors , including medical , psychiatric , and sleep disorders.2,3 both types of insomnia are common in older adults , and the prevalence increases with advancing age.35 because of changes in sleep architecture , older adults are more likely than younger adults to experience increased nocturnal awakenings , interrupted sleep , and decreased sleep efficiency.6 a study by the national institute of aging of noninstitutionalized older adults indicated that 28% of respondents had difficulty falling asleep , whereas another 42% of respondents reported having difficulty falling asleep and staying asleep.7 the treatment of insomnia includes both behavioral ( eg , cognitive behavioral therapy ) and pharmacological therapies ( sedating antidepressants , long - acting and short - acting benzodiazepines , nonbenzodiazepine hypnotics , and a selective melatonin receptor agonist ) . although benzodiazepines effectively promote sleep , they have a relatively long duration of action , which can be problematic , as the therapeutic action of these medications can extend into the waking hours and cause daytime sedation.8 use of these medications is associated with an increased risk of adverse events such as physical disability , falls , and motor vehicle accidents in older adults.9 studies suggest that older adults who are prescribed a benzodiazepine ( either long - acting or short - acting ) have an increased risk of falling at least once within 90 days of initiating treatment and are at greater risk for motor vehicle accidents.10,11 nonbenzodiazepine hypnotics are used specifically for the management of insomnia and were developed to reduce the potential for side effects associated with the benzodiazepine medications.12 although each of the nonbenzodiazepine hypnotic medications has a different pharmacokinetic profile , research suggests that falls , fractures , and motor vehicle accidents are also a concern with these medications , with serious economic consequences.1315 off - label use of sedating antidepressants , particularly trazodone , is also common in the treatment of insomnia . although studies have supported the efficacy of trazodone in improving sleep in patients with both primary and comorbid insomnia , trazodone can produce sedation , orthostatic hypotension , and blurred vision , all of which increase the risk of falls.16 recent studies suggest that the newest class of insomnia medications , the selective melatonin receptor agonist , has a sleep - promoting action that does not appear to cause balance impairments , rebound insomnia , or next - day cognitive or psychomotor effects that may lead to increased accident risks in older adults.1720 the increased risk of accidental events associated with the use of insomnia medications is particularly troublesome in older adults because they are much more likely to suffer injuries from falls , fractures , and motor vehicle accidents.2123 the use of insomnia medications may compound this risk . however , the assessment of risk is complex and may be confounded by factors associated with untreated insomnia , which by itself has been shown to increase the risks of an accidental event.24,25 the goal of the current study was to examine the risk of accidental events in a population of older adults ( 65 years of age ) prescribed common insomnia medications . a retrospective cohort analysis comparing the risk of accidental events in patients prescribed common insomnia medications was conducted based on the thomson medstat marketscan 2000-q2 2006 medicare supplemental and coordination of benefits databases . these data ( representing both the employer - paid and medicare - paid components of care ) include the health insurance claims across the continuum of care ( inpatient , outpatient , outpatient pharmacy , and enrollment data ) for retirees covered by their previous employer through privately insured fee - for - service , point - of - service , or capitated health plans . this information comes from administration claims and there is no independent validation of the data using medical charts . however , claim entries are fully adjudicated by the payer , and any data that appear to be erroneous ( ie , claims with costs that exceed reasonable limits ) are excluded on an individual basis . cost thresholds for exclusion of claims are in line with what other administration claims databases use , including those used by the healthcare cost and utilization project , which is developed by the agency for healthcare research and quality using hospital claims data . depending on the year , the medicare supplemental databases include between 2.5 and 3.1 million individuals , and these data are projectable to the us population with medicare supplemental insurance ( 12.7 million retirees ) . older adults ( 65 years ) with at least one outpatient pharmaceutical claim for an identified insomnia medication ( long - acting benzodiazepine [ lorazepam , quazepam , and flurazepam ] , short - acting benzodiazepine [ estazolam , temazepam , and triazolam ] , nonbenzodiazepine hypnotic [ zolpidem , zaleplon , and eszopiclone ] , sedating antidepressant [ trazodone ] , and selective melatonin receptor agonist [ ramelteon ] ) were included in the analysis . a 12-hour half - life was used as the cutoff between long - acting and short - acting benzodiazepines . because some of these medications , specifically lorazepam and trazodone , may have multiple indications , only patients with specific doses ( 2 mg or less for lorazepam ; 100 mg or less for trazodone ) or those taking a certain number of pills supplied per month ( no more than 30 pills per month ) were selected for study inclusion . patients with injectible formulations of lorazepam were also excluded from the study . because insomnia often is not coded on a patient s medical claim , an insomnia diagnosis was not required for study inclusion . if an insomnia diagnosis was present prior to the insomnia medication claim , it was included as a study covariate . the study population was further restricted to those patients who were considered new initiators of insomnia medication therapy ( ie , no evidence of a prescription insomnia medication during the 3-month period prior to study initiation ) and who had at least 9 months of health insurance eligibility data ( 6 months prior to the identification of the first insomnia medication claim and 3 months following the first insomnia medication claim ) . patients with evidence of nursing home admissions ( n = 2526 ; 0.5% of population ) , those with insomnia medication polytherapy ( n = 10,672 ; 2.0% of population ) during the study period , and medicare beneficiaries < 65 years of age ( n = 62,571 , 12.0% of the population ) were excluded from the analysis . clinical characteristics in this analysis were measured using the charlson comorbidity index ( deyo adaptation ) and the international classification of diseases 9th edition ( icd-9-cm ) codes from the patients medical claims data for the 6-month period preceding the initiation of treatment with insomnia medication . several factors that may contribute to accident risks were assessed because they are meaningful to patients and to the healthcare system . comorbid disorders that may have affected accident risk were recorded during the study period . these comorbidities included adjustment and stress disorders , alcohol and drug disorders , schizophrenia and other psychotic disorders , seizure disorders , abnormal movements and parkinsonism disorders , mood disorders , pain disorders , and other mental health disorders . concomitant medications that may also have affected accident risk were recorded based on the presence of at least one outpatient pharmaceutical claim during the study period . these included anticholinergic / antiparkinsonian / antispastic agents , opiate antagonists , analgesics , antidiabetic agents , antimanic agents , anxiolytics , antidepressants , benzodiazepines , and anticonvulsants ( the concomitant antidepressants , benzodiazepines , and anticonvulsant medication groups did not include those used to qualify patients for study inclusion ) . the incidence of accidental events was evaluated for two time periods : accidental events occurring during the first 30 days of insomnia therapy and events occurring during the first 3 months of insomnia therapy . accidental events ( falls , fractures , dislocations , sprains / strains , open wounds , intracranial injuries , injuries caused by cutting / piercing instruments , accidents caused by machinery , electrocution , and motor vehicle accidents ) were chosen to represent the variety of impairments that may affect daily routines and were identified using external causes of injury codes ( based on the international classification of diseases ) and routine icd-9-cm codes in the claims files . chi - square tests were used for categorical variables , and two - sided t tests were used for continuous variables . the modeled estimations of the probability of accidental events and time to accidental events were performed with stata ( statacorp , college station , tx , usa ) version 9 . to compute the regression - adjusted probability of an accidental event for the two defined time periods , multivariate analyses were conducted using probit models with variable inclusion based on a priori hypotheses . the a priori hypotheses were : 1 ) patients prescribed a selective melatonin receptor agonist would have a lower risk of an accidental event during the period immediately following the initiation of therapy , and 2 ) given the presence of an accidental event , the time to such an event would be longer for patients prescribed a selective melatonin receptor agonist . probit regression models extend the principles of generalized linear models to better treat the case of dichotomous and categorical variables in this case , the probability of an accidental event ( 0 if no event and 1 if evidence of an event ) . probit is a variant of logit modeling but is based on different data assumptions ; specifically , logit analysis is based on log odds , whereas probit uses the cumulative normal probability distribution . these multivariate models included the following covariates : gender , age , charlson comorbidity index , presence of select comorbidities , presence of an insomnia diagnosis , and presence of concomitant medications of interest . because no accidental events occurred during either the first 30 days or 3 months of insomnia therapy for patients prescribed the reference drug cohort ( a selective melatonin receptor agonist ) , model convergence was not achieved . the existence , finiteness , and uniqueness of maximum likelihood estimates for the probit regression model depend on the pattern of data points in the observation space . if the data are completely or partially separated , it may not be possible to obtain reliable maximum likelihood estimates because convergence may not occur . model nonconvergence occurs because one or more parameters in the model become theoretically infinite typically because either the model perfectly predicts the response or there are more parameters in the model than can be estimated because the data are sparse . in this particular analysis , model nonconvergence was a direct result of zero observations for the dependent variable of interest for the reference group for the drug cohort variable . in an effort to evaluate the probability of an accidental event within a multivariate framework , a random patient within the selective melatonin receptor agonist cohort was assigned a nonzero value for the dependent variable ( presence of an accidental event variable).26 this was done in an iterative manner by randomly selecting a patient in the selective melatonin receptor agonist group and assigning them a 1 value for the presence of an accidental event . we completed this process numerous times , each time selecting another random patient to be assigned the 1 value for the presence of an accidental event and re - running the probit model . model statistics , coefficients ( magnitude and direction ) and p values resulted in the same conclusions each time . a retrospective cohort analysis comparing the risk of accidental events in patients prescribed common insomnia medications was conducted based on the thomson medstat marketscan 2000-q2 2006 medicare supplemental and coordination of benefits databases . these data ( representing both the employer - paid and medicare - paid components of care ) include the health insurance claims across the continuum of care ( inpatient , outpatient , outpatient pharmacy , and enrollment data ) for retirees covered by their previous employer through privately insured fee - for - service , point - of - service , or capitated health plans . this information comes from administration claims and there is no independent validation of the data using medical charts . however , claim entries are fully adjudicated by the payer , and any data that appear to be erroneous ( ie , claims with costs that exceed reasonable limits ) are excluded on an individual basis . cost thresholds for exclusion of claims are in line with what other administration claims databases use , including those used by the healthcare cost and utilization project , which is developed by the agency for healthcare research and quality using hospital claims data . depending on the year , the medicare supplemental databases include between 2.5 and 3.1 million individuals , and these data are projectable to the us population with medicare supplemental insurance ( 12.7 million retirees ) . older adults ( 65 years ) with at least one outpatient pharmaceutical claim for an identified insomnia medication ( long - acting benzodiazepine [ lorazepam , quazepam , and flurazepam ] , short - acting benzodiazepine [ estazolam , temazepam , and triazolam ] , nonbenzodiazepine hypnotic [ zolpidem , zaleplon , and eszopiclone ] , sedating antidepressant [ trazodone ] , and selective melatonin receptor agonist [ ramelteon ] ) were included in the analysis . a 12-hour half - life was used as the cutoff between long - acting and short - acting benzodiazepines . because some of these medications , specifically lorazepam and trazodone , may have multiple indications , only patients with specific doses ( 2 mg or less for lorazepam ; 100 mg or less for trazodone ) or those taking a certain number of pills supplied per month ( no more than 30 pills per month ) were selected for study inclusion . patients with injectible formulations of lorazepam were also excluded from the study . because insomnia often is not coded on a patient s medical claim , an insomnia diagnosis was not required for study inclusion . if an insomnia diagnosis was present prior to the insomnia medication claim , it was included as a study covariate . the study population was further restricted to those patients who were considered new initiators of insomnia medication therapy ( ie , no evidence of a prescription insomnia medication during the 3-month period prior to study initiation ) and who had at least 9 months of health insurance eligibility data ( 6 months prior to the identification of the first insomnia medication claim and 3 months following the first insomnia medication claim ) . patients with evidence of nursing home admissions ( n = 2526 ; 0.5% of population ) , those with insomnia medication polytherapy ( n = 10,672 ; 2.0% of population ) during the study period , and medicare beneficiaries < 65 years of age ( n = 62,571 , 12.0% of the population ) were excluded from the analysis . clinical characteristics in this analysis were measured using the charlson comorbidity index ( deyo adaptation ) and the international classification of diseases 9th edition ( icd-9-cm ) codes from the patients medical claims data for the 6-month period preceding the initiation of treatment with insomnia medication . several factors that may contribute to accident risks were assessed because they are meaningful to patients and to the healthcare system . comorbid disorders that may have affected accident risk were recorded during the study period . these comorbidities included adjustment and stress disorders , alcohol and drug disorders , schizophrenia and other psychotic disorders , seizure disorders , abnormal movements and parkinsonism disorders , mood disorders , pain disorders , and other mental health disorders . concomitant medications that may also have affected accident risk were recorded based on the presence of at least one outpatient pharmaceutical claim during the study period . these included anticholinergic / antiparkinsonian / antispastic agents , opiate antagonists , analgesics , antidiabetic agents , antimanic agents , anxiolytics , antidepressants , benzodiazepines , and anticonvulsants ( the concomitant antidepressants , benzodiazepines , and anticonvulsant medication groups did not include those used to qualify patients for study inclusion ) . the incidence of accidental events was evaluated for two time periods : accidental events occurring during the first 30 days of insomnia therapy and events occurring during the first 3 months of insomnia therapy . accidental events ( falls , fractures , dislocations , sprains / strains , open wounds , intracranial injuries , injuries caused by cutting / piercing instruments , accidents caused by machinery , electrocution , and motor vehicle accidents ) were chosen to represent the variety of impairments that may affect daily routines and were identified using external causes of injury codes ( based on the international classification of diseases ) and routine icd-9-cm codes in the claims files . chi - square tests were used for categorical variables , and two - sided t tests were used for continuous variables . the modeled estimations of the probability of accidental events and time to accidental events were performed with stata ( statacorp , college station , tx , usa ) version 9 . to compute the regression - adjusted probability of an accidental event for the two defined time periods , multivariate analyses were conducted using probit models with variable inclusion based on a priori hypotheses . the a priori hypotheses were : 1 ) patients prescribed a selective melatonin receptor agonist would have a lower risk of an accidental event during the period immediately following the initiation of therapy , and 2 ) given the presence of an accidental event , the time to such an event would be longer for patients prescribed a selective melatonin receptor agonist . probit regression models extend the principles of generalized linear models to better treat the case of dichotomous and categorical variables in this case , the probability of an accidental event ( 0 if no event and 1 if evidence of an event ) . probit is a variant of logit modeling but is based on different data assumptions ; specifically , logit analysis is based on log odds , whereas probit uses the cumulative normal probability distribution . these multivariate models included the following covariates : gender , age , charlson comorbidity index , presence of select comorbidities , presence of an insomnia diagnosis , and presence of concomitant medications of interest . because no accidental events occurred during either the first 30 days or 3 months of insomnia therapy for patients prescribed the reference drug cohort ( a selective melatonin receptor agonist ) , model convergence was not achieved . the existence , finiteness , and uniqueness of maximum likelihood estimates for the probit regression model depend on the pattern of data points in the observation space . if the data are completely or partially separated , it may not be possible to obtain reliable maximum likelihood estimates because convergence may not occur . model nonconvergence occurs because one or more parameters in the model become theoretically infinite typically because either the model perfectly predicts the response or there are more parameters in the model than can be estimated because the data are sparse . in this particular analysis , model nonconvergence was a direct result of zero observations for the dependent variable of interest for the reference group for the drug cohort variable . in an effort to evaluate the probability of an accidental event within a multivariate framework , a random patient within the selective melatonin receptor agonist cohort was assigned a nonzero value for the dependent variable ( presence of an accidental event variable).26 this was done in an iterative manner by randomly selecting a patient in the selective melatonin receptor agonist group and assigning them a 1 value for the presence of an accidental event . we completed this process numerous times , each time selecting another random patient to be assigned the 1 value for the presence of an accidental event and re - running the probit model . model statistics , coefficients ( magnitude and direction ) and p values resulted in the same conclusions each time . the sample used to estimate the probability of an accidental event consisted of 445,329 medicare patients . a flow - chart describing the derivation of this sample the majority of patients were prescribed long - acting benzodiazepines and nonbenzodiazepine hypnotics ( 36.38% and 35.25% , respectively ) . the remaining patients were prescribed short - acting benzodiazepines ( 18.21% ) , a sedating antidepressant ( 9.94% ) , or a selective melatonin receptor agonist ( 0.22% ) . patients in each cohort except the reference cohort ( melatonin receptor agonist ) experienced at least one accidental event during the first 30 days of insomnia treatment , and the number of accidental events after 90 days was increased for all cohorts except the melatonin receptor agonist cohort ( table 2 ) . because patients in the reference cohort experienced no accidental events , the time interval to an event was right censored at 30 days for the first follow - up period and 90 days for the full study . table 3 shows the results of the probit regressions of the risk of an accidental event during the 30-day and 3-month periods following the initiation of insomnia drug therapy . the probability of an accidental event was significantly greater ( p < 0.05 ) at all time points for patients taking a long - acting benzodiazepine , a short - acting benzodiazepine , or a nonbenzodiazepine hypnotic when evaluated against the risk for patients in the reference cohort . patients taking a sedating antidepressant demonstrated a significantly increased probability of an accidental event only during the 3-month time period . at the end of the study ( 3 months ) , risks of an accidental event were 1.48 to 1.62 times greater for patients in all evaluated medication cohorts compared with those in the reference cohort . additional variables that affected the probability of an accidental event are shown in table 3 . the sample used to estimate the probability of an accidental event consisted of 445,329 medicare patients . a flow - chart describing the derivation of this sample the majority of patients were prescribed long - acting benzodiazepines and nonbenzodiazepine hypnotics ( 36.38% and 35.25% , respectively ) . the remaining patients were prescribed short - acting benzodiazepines ( 18.21% ) , a sedating antidepressant ( 9.94% ) , or a selective melatonin receptor agonist ( 0.22% ) . patients in each cohort except the reference cohort ( melatonin receptor agonist ) experienced at least one accidental event during the first 30 days of insomnia treatment , and the number of accidental events after 90 days was increased for all cohorts except the melatonin receptor agonist cohort ( table 2 ) . because patients in the reference cohort experienced no accidental events , the time interval to an event was right censored at 30 days for the first follow - up period and 90 days for the full study . table 3 shows the results of the probit regressions of the risk of an accidental event during the 30-day and 3-month periods following the initiation of insomnia drug therapy . the probability of an accidental event was significantly greater ( p < 0.05 ) at all time points for patients taking a long - acting benzodiazepine , a short - acting benzodiazepine , or a nonbenzodiazepine hypnotic when evaluated against the risk for patients in the reference cohort . patients taking a sedating antidepressant demonstrated a significantly increased probability of an accidental event only during the 3-month time period . at the end of the study ( 3 months ) , risks of an accidental event were 1.48 to 1.62 times greater for patients in all evaluated medication cohorts compared with those in the reference cohort . additional variables that affected the probability of an accidental event are shown in table 3 . in this study of commonly prescribed hypnotic medications , patients who were prescribed a benzodiazepine , nonbenzodiazepine hypnotic , or sedating antidepressant had significantly higher risks of an accidental event during the 30-day and 3-month periods following pharmacotherapy initiation compared with the reference cohort of a melatonin receptor agonist that had no observed accidental events . nonbenzodiazepine hypnotics were developed to reduce the potential for side effects associated with the benzodiazepine medications . however , this analysis suggests that the risk of an accidental event is similar among all of the sedating hypnotics . the trade - off between improved sleep with hypnotic agents and the potential risk of accidents and falls is an ongoing issue for health professionals who treat insomnia in older adults as well as regulatory agencies . previous research often focused on nursing home populations or only specific notable events ( ie , hip fractures ) . the earliest evaluations also included agents , such as barbiturates , that are no longer the standard of care . however , a recent review by glass et al described some rather compelling data associating sedative hypnotics with adverse events in the treatment of insomnia in older adults.11 using a meta - analysis , the authors demonstrate that although sedatives improve sleep quality and amount of sleep , these benefits are outweighed by the associated excessive sleepiness that may put patients at risk for falls , injurious behaviors , and motor vehicle accidents.11 the number needed to treat for improved sleep quality was 13 , whereas the number needed to harm for any adverse event was 6.11 a key literature review on the treatment of chronic insomnia in geriatric patients determined that long - term use of sedative hypnotics for insomnia lacks an evidence base and identified concerns for potential adverse drug effects , including anterograde amnesia , motor disturbances , excessive daytime sleepiness , and the potential for accidental events.9 further complicating the issue is a recent large retrospective study in michigan , usa which determined that in elderly nursing - home residents , insomnia , but not hypnotic use , is associated with a greater risk of subsequent falls.24 however , the current study used a different patient population that was substantially larger ( n = 445,329 ) and healthier ( most patients were free from comorbid conditions with no evidence of nursing home admissions).the endpoints in this study were also broader , including a variety of injuries , than previous assessments of accident risk that mostly focused on falls and fractures . overall , the current research expands on the results from previous studies and increases the understanding of accident risk associated with the use of hypnotic agents in older adults . there are several studies of accident rates both in the general population and in those with insomnia . for example , according to the national center for injury prevention and control , the rate of unintentional nonfatal injuries in the united states between 2001 and 2007 was 9305 per 100,000 people ( 0.093).27 when the data were restricted to those > 65 years , the rate was 7852.52 per 100,000 people ( 0.079).27 accident rates in people with untreated insomnia vary between studies but most show an increase in accidents among those with insomnia . in a japanese study , data from 2903 workers between the ages of 16 and 83 years showed that insomnia symptoms were associated with an adjusted odds ratio of 1.3 for the occurrence of occupational injuries.28 a study by balter and uhlenhuth in the us population showed that past - year prevalence rates for serious accidents / injuries in subjects with chronic untreated insomnia were 4.5 times higher than normal controls ( 9% and 2% , respectively).25 these findings are similar to a french study that showed patients with severe insomnia were more likely than good sleepers to experience work - related accidents over a 12-month time period ( 8% and 1% , respectively).29 in this study , the range of accident rates for older adults ( 0.28 to 0.40 ) was greater than that in the general population ( 0.079 ) , but that may have been due to several factors and not simply the presence of insomnia or hypnotics . in subjects of all ages with untreated insomnia , the rate of accidents is much higher than in normal controls.25,28,29 it is possible that the accident rates in this study , although higher than those in the overall population , may actually have been reduced compared with the potential accident rates if the patients had not been treated for insomnia . several limitations of the data and these analyses should be noted . although the prescription hypnotic medications captured in this study are consistent with those of other published studies , it is not possible to confirm that these medications were indeed used for insomnia treatment . we attempted to mitigate this possibility by eliminating doses that may be used for other conditions ( ie , trazodone at doses > 100 mg for depression treatment ) . second , there was no control group of untreated insomniacs , which may have affected the analysis due to the risk of accidental events associated with insomnia itself . it is possible that , in this study , all of the hypnotics reduced the overall accident rate associated with untreated insomnia . an overview of the accident rates in the population with and without insomnia has been provided as a comparison with the rates found in this study . third , the results of this study should be viewed in light of limitations that are inherent in retrospective claims data analysis . correct categorization of insurance database information depends on correct coding by clinicians and other medical staff . the accuracy of diagnostic coding can not be evaluated in a claims - based study . patients may also receive treatment that is not submitted to their health plan for reimbursement and thus not included in claims data . also , consideration of the methodology used to ensure that model convergence was achieved should be taken into account when evaluating the risk of accidental events . finally , this analysis included claims data from 2000 through the second quarter of 2006 , but ramelteon was not approved until 2005 , which limits the number of patients and the amount of data available for comparison with the other medication classes . it is possible that there may have been a time trend bias in the analysis . however , this would occur only if the risks for an accidental event had changed over the years for the other medications included in the analysis . also , if accidental events were more differentially reported prior to 2005 , the risk assessments may have been altered , but this seems unlikely . the small number of patients taking a melatonin receptor agonist ( compared with the other drug cohorts ) does make it difficult to draw definitive conclusions from this study . the small sample size may have contributed to the lack of an accidental event in the melatonin receptor agonist cohort . however , model convergence was used to adjust for the lack of an event , and the small sample size was accounted for in the statistical analyses . for these reasons , the melatonin receptor agonist cohort was used as a reference for comparison with the other medication cohorts , and no specific conclusions were drawn relating to the melatonin receptor agonist ( or between any of the cohorts ) . in the future , additional studies with a greater number of subjects receiving melatonin receptor agonists may be needed to determine how to most appropriately use hypnotics in older adults , both to treat insomnia successfully and to reduce the risks of accidental events . although more work is warranted to evaluate whether these findings can be supported in other patient populations , these results are nevertheless important . results of this study add to the literature on the risk of accidental events in patients who are taking insomnia medications . finally , although the sample size was small , the data in this retrospective claims analysis support findings from double - blind , placebo - controlled studies that indicate that selective melatonin receptor agonists lack the apparent drug - related or residual effects that can lead to accidental events , making the case for more selectively targeted treatments for insomnia.17,19,30,31 older adults taking a sedating antidepressant or any of the benzodiazepine receptor agonists appear to have a greater risk of an accidental event compared with a reference group taking a selective melatonin receptor agonist .

objective : this study examined the risk of accidental events in older adults prescribed a sedating antidepressant , long - acting benzodiazepine , short - acting benzodiazepine , and nonbenzodiazepine , relative to a reference group ( selective melatonin receptor agonist).methods : this was a retrospective cohort analysis of older adults ( 65 years ) with newly initiated pharmacological treatment of insomnia . data were collected from the thomson marketscan medicare supplemental and coordination of benefits databases ( january 1 , 2000 , through june 30 , 2006 ) . probit models were used to evaluate the probability of an accidental event.results:data were analyzed for 445,329 patients . patients taking a long - acting benzodiazepine ( 1.21 odds ratio [ or ] ) , short - acting benzodiazepine ( 1.16 or ) , or nonbenzodiazepine ( 1.12 or ) had a significantly higher probability of experiencing an accidental event during the first month following treatment initiation compared with patients taking the reference medication ( p < 0.05 for all ) . a significantly higher probability of experiencing an accidental event was also observed during the 3-month period following the initiation of treatment ( 1.62 long - acting benzodiazepine , 1.60 short - acting benzodiazepine , 1.48 nonbenzodiazepine , and 1.56 sedating antidepressant ; p < 0.05).conclusions : older adults taking an sad or any of the benzodiazepine receptor agonists appear to have a greater risk of an accidental event compared with a reference group taking an mr .

Introduction Patients and methods Study design Study population Measurements Statistics Results Demographics Probability of an accidental event Discussion Conclusions

PMC4847161

in the late 1990s , two - step etch and rinse and self - etch adhesives were introduced to the market . technique sensitivity has been reduced in self - etch adhesives by the use of non - rinse acidic primers . in the two - step self - etch adhesives , a self - etch primer and hydrophobic resin are applied in separate steps ; a mild self - etch primer is commonly used ( ph of approximately 2 ) . this primer is able to partially remove the smear layer and penetrate into the dentinal surface , resulting in less distinct resin tag formation ; thus , the hybrid layers produced are thinner than those in the etch and rinse systems . formation of a resin - reinforced hybrid layer has been generally accepted as the mechanism for improving bond strength of adhesives to dentin . higher bond strength and improved sealing ability with an increase in the adhesive layer thickness were explained by the improvement of stress distribution within the bonding interface and strain - induced relief of contraction stress of the composite resin , respectively . one - step self - etch adhesives include an etchant , primer and adhesive in one or two bottles and are marketed as all - in - one adhesives . in vitro microleakage studies indicated that self - etch adhesive systems at the dentinal margins were as effective as adhesives with a separate etching phase but the former group were less effective at the enamel margins [ 3,7 , 8 ] . mild acidity of the self - etch primers may be the reason for their weakness in enamel adhesion , while strong self - etchants can produce a more effective enamel etch than mild agents . high dentin bond strength for self - etch adhesives , comparable to that of etch and rinse adhesives , has also been reported [ 1012 ] . water storage and thermocycling are two common artificial aging techniques used to predict the durability and clinical performance of dental materials . significant decrease in bond strength has been reported even after short storage periods ( i.e. three months)[1215 ] . the current study was designed to evaluate the effect of water storage on microleakage and mtbs of two self - etch and two etch and rinse adhesives . a total of 128 extracted caries- and defect - free human third molars were selected and stored in distilled water containing sodium hypochlorite ( 10:1 ) at room temperature for no longer than three months after the extraction . a total of 32 teeth where sectioned approximately 5 mm below the cemento - enamel junction ( cej ) , at the furcation level , using number 2 round bur with high speed handpiece under cooling water . occlusal enamel was ground flat under running water with number 2 round bur and long cylindrical bur to provide a flat dentine surface . then , they were polished with number 3 sof - lex disc ( sof - lex , 3 m espe , st . paul , mn , usa ) at low - speed under cooling water for 15 seconds to create a uniform smear layer . composite core was built with incremental layers of z250 light - cure composite resin ( filtek z250 , 3 m espe st . after 24 hours of storage at room temperature , they were embedded in self - cure acrylic resin and were sectioned in buccolingual and mesiodistal directions with a water - cooled low - speed diamond saw ( m5-isomet diamond saw , buehler , esslingen , germany ) to create four to six sticks from each tooth . the cross - section of each stick was about 11 ( 0.2 ) mm . the specimens were stored in distilled water containing 0.5% chloramine t at 37c , to provide a wet environment preventing bacterial growth . each group was divided into two subgroups : one was tested at one day and the other was tested after six months of storage . the specimens were attached to the microtensile tester ( microtensile tester ; bisco , schaumburg , il , usa ) with cyanoacrylate glue ( mitreapel , beta chemical ind . & trade inc . the tensile load was applied at a crosshead speed of 1 mm / min until fracture occurred . then , the interface cross sections were measured by a digital micrometer ( mitutoyo corp . , class v cavities were prepared in 96 teeth ( 3 mm occlusogingivally , 4 mm mesiodistally and 2 mm depth on the buccal and lingual surfaces of each tooth ) . the gingival margin was placed 1.5 mm below the cej and the coronal margin was 1.5 mm above it . the cavities were prepared with a 008 diamond bur and high speed handpiece under copious water . the teeth were randomly assigned to four groups ( n=24 ) , and the adhesives were applied according to the manufactures instructions . before applying self - etch adhesives , the enamel margins were etched separately . then , cavities were restored with wedge - shaped light - cure composite increments ( filtekz250 , 3 m espe , st . restorations were finished using composite finishing burs and polished by sof - lex discs ( 3m - espe , st . the teeth were stored in 0.5% chloramine t solution ( chloramine t trihydrate , merck corp . , darmstadt , germany ) in distilled water and incubated ( shimifann , tehran , iran ) at 37c . microleakage assessment was performed for half the samples at one day and the other half was assessed after six months . for the six - month samples , the samples were first thermocycled for 1000 cycles in separate water baths at 5c and 55c with a dwell time of 30 seconds and three seconds of transfer time . the apical foramen and pits and fissures were sealed with composite resin and fissure sealants , respectively . then , two layers of nail varnish were applied to the entire tooth surface except for one millimeter margin around the restoration . after 24 hours of immersion in 0.5% fuchsin , the teeth were embedded in auto - polymerizing acrylic resin and sectioned longitudinally at the mid - buccal and midlingual surfaces using a water - cooled low speed diamond saw ( vafaei industrial factory , tehran , iran ) to obtain two sections from each tooth . each section ( 48 sections per group ) was examined under a stereomicroscope at 40 magnification ( carton optical industries , pathumthani , thailand ) . the degree of leakage was determined as follows : 0 = no dye penetration ; 1 = partial dye penetration along the occlusal or gingival wall ; 2 = dye penetration up to the full length of the cavity walls not including the axial walls ; 3 = dye penetration to the full length of the cavity walls including the axial walls . the effect of time and type of adhesive on bond strength were analyzed by two - way anova and since the interaction effect was significant at each time point , the comparison between the adhesive groups was made by one - way anova . the tamhane s post hoc test was used because the equality of variances was not assumed . for evaluating the differences between microleakage groups , the non - parametric kruskal - wallis test was applied and followed by a dunn s test . the differences in dye penetration between the occlusal and gingival margins in each group were analyzed by the wilcoxon signed rank test and the difference between the one day and six - month samples was analyzed by the mann whitney u test . all statistical analyses were performed by spss version 16 ( microsoft , il , usa ) ( p<0.05 ) . a total of 32 teeth where sectioned approximately 5 mm below the cemento - enamel junction ( cej ) , at the furcation level , using number 2 round bur with high speed handpiece under cooling water . occlusal enamel was ground flat under running water with number 2 round bur and long cylindrical bur to provide a flat dentine surface . then , they were polished with number 3 sof - lex disc ( sof - lex , 3 m espe , st . paul , mn , usa ) at low - speed under cooling water for 15 seconds to create a uniform smear layer . composite core was built with incremental layers of z250 light - cure composite resin ( filtek z250 , 3 m espe st . after 24 hours of storage at room temperature , they were embedded in self - cure acrylic resin and were sectioned in buccolingual and mesiodistal directions with a water - cooled low - speed diamond saw ( m5-isomet diamond saw , buehler , esslingen , germany ) to create four to six sticks from each tooth . the cross - section of each stick was about 11 ( 0.2 ) mm . the specimens were stored in distilled water containing 0.5% chloramine t at 37c , to provide a wet environment preventing bacterial growth . each group was divided into two subgroups : one was tested at one day and the other was tested after six months of storage . the specimens were attached to the microtensile tester ( microtensile tester ; bisco , schaumburg , il , usa ) with cyanoacrylate glue ( mitreapel , beta chemical ind . & trade inc . the tensile load was applied at a crosshead speed of 1 mm / min until fracture occurred . then , the interface cross sections were measured by a digital micrometer ( mitutoyo corp . , class v cavities were prepared in 96 teeth ( 3 mm occlusogingivally , 4 mm mesiodistally and 2 mm depth on the buccal and lingual surfaces of each tooth ) . the gingival margin was placed 1.5 mm below the cej and the coronal margin was 1.5 mm above it . the cavities were prepared with a 008 diamond bur and high speed handpiece under copious water . the teeth were randomly assigned to four groups ( n=24 ) , and the adhesives were applied according to the manufactures instructions . before applying self - etch adhesives , the enamel margins were etched separately . then , cavities were restored with wedge - shaped light - cure composite increments ( filtekz250 , 3 m espe , st . restorations were finished using composite finishing burs and polished by sof - lex discs ( 3m - espe , st . the teeth were stored in 0.5% chloramine t solution ( chloramine t trihydrate , merck corp . , darmstadt , germany ) in distilled water and incubated ( shimifann , tehran , iran ) at 37c . microleakage assessment was performed for half the samples at one day and the other half was assessed after six months . for the six - month samples , the samples were first thermocycled for 1000 cycles in separate water baths at 5c and 55c with a dwell time of 30 seconds and three seconds of transfer time . the apical foramen and pits and fissures were sealed with composite resin and fissure sealants , respectively . then , two layers of nail varnish were applied to the entire tooth surface except for one millimeter margin around the restoration . after 24 hours of immersion in 0.5% fuchsin , the teeth were embedded in auto - polymerizing acrylic resin and sectioned longitudinally at the mid - buccal and midlingual surfaces using a water - cooled low speed diamond saw ( vafaei industrial factory , tehran , iran ) to obtain two sections from each tooth . each section ( 48 sections per group ) was examined under a stereomicroscope at 40 magnification ( carton optical industries , pathumthani , thailand ) . the degree of leakage was determined as follows : 0 = no dye penetration ; 1 = partial dye penetration along the occlusal or gingival wall ; 2 = dye penetration up to the full length of the cavity walls not including the axial walls ; 3 = dye penetration to the full length of the cavity walls including the axial walls . the effect of time and type of adhesive on bond strength were analyzed by two - way anova and since the interaction effect was significant at each time point , the comparison between the adhesive groups was made by one - way anova . the tamhane s post hoc test was used because the equality of variances was not assumed . for evaluating the differences between microleakage groups , the non - parametric kruskal - wallis test was applied and followed by a dunn s test . the differences in dye penetration between the occlusal and gingival margins in each group were analyzed by the wilcoxon signed rank test and the difference between the one day and six - month samples was analyzed by the mann whitney u test . all statistical analyses were performed by spss version 16 ( microsoft , il , usa ) ( p<0.05 ) . single bond achieved the highest values at one day ( 27.423.67 mpa ) and six months ( 27.283.25 mpa ) . there was no significant difference between sbmp and cseb at one day ( p>0.980 ) . after six months of water storage , significant changes in bond strength values were observed in sbmp ( p<0.0350 ) and abse ( p<0.040 ) groups but not for cseb and sb groups . within the same column or row , identical letters or numbers indicate no differences ( p>0.05 ) . dye penetration scores for occlusal and gingival walls are presented in tables 3 and 4 . at one day , there was a significant difference between abse and other adhesive groups . at the occlusal margin and at the gingival margin , there were no statistically significant differences between groups ( p>0.05 ) . at six months , there was no statistically significant difference among groups at the occlusal margin ( p>0.05 ) , but at the gingival margin there was a statistically significant difference between self - etch adhesives ( cseb and abse ) and etch and rinse adhesives ( sbmp and sb ) ( p<0.001 ) . at the occlusal margin , significant differences were found between one day and six months groups except for sbmp ( p=0.054 ) . at the gingival margin , there was no statistically significant difference between one day and six months groups except for sb ( p<0.001 ) . one - day microleakage scores six - month microleakage scores the wilcoxon test indicated statistically significant differences between the occlusal and gingival scores of each group after one day and six months of storage ( p<0.001 ) , except for one day abse and six - month cseb ( p=0.4 and p=0.08 , respectively ) . however both of these mechanical properties are important clinically ; bond strength is required for restoration retention and microleakage must be minimized to prevent secondary caries and pulpal reactions and maintain marginal integrity . the mtbs test is a relatively new method for evaluating bond strength in dental research and has shown lower test variance compared to macrotensile test . aging by water storage and/or thermocycling are two common methods applied to simulate the bonding degradation overtime in the oral cavity . in the current study , among one day groups , abse showed the highest microleakage at the occlusal margin ; there was no significant difference at the gingival margin . several studies revealed more leakage in the occlusal margin of self - etch adhesives compared to etch and rinse adhesives ; however , no significant difference was reported for gingival margins [ 8,20 , 21 ] . high microleakage of self - etch adhesives may be due to incomplete etching of the surface by acidic monomers , allowing higher micro - leakage values than etch and rinse adhesives ( which use a separate phosphoric acid etchant ) . scanning electron microscopic ( sem ) studies have shown that applying phosphoric acid as an enamel etchant improves monomer penetration and the subsequent attachment of self - etch adhesives . in our study , however , abse did not cause a proper seal at the occlusal margin , which might be related to its formulation . since , two components of abse were mixed together immediately before use , water which is an indispensable component , creates permeable membranes , and hydrophobic resin layer formation will be compromised . after six months , sealing ability of self - etch groups at the occlusal margin was similar to that of etch and rinse groups . however , at the gingival margin , etch and rinse groups showed higher microleakage values . the depth of dentinal penetration is different between phosphoric acid and self - etch primers . in self - etch systems as the adhesive is applied , the dentinal surface is sealed causing a reduction in dentin permeability . by comparing the occlusal and gingival microleakage values , all groups leaked more at the gingival margin rather than the occlusal margin , as reported previously . the reason is related to the hydrated nature of dentin . in presence of water , creation of polymers with cross links will not occur which leads to weak polymerization of adhesive material ; however , this situation will not occur in enamel bonding . moreover , additional acid etching of enamel margins for self - etch groups may improve their sealing ability in occlusal margins except for the short - term results of abse group . however , cseb ( two - step self - etch adhesive ) had poorer short - term results than sb . generally , sb ( containing ethanol / water solvent ) functions well in laboratory studies [ 2729 ] . hosaka et al . demonstrated that ethanol in adhesive systems was responsible for higher bond strength values and durability overtime . ethanol is usually used to replace water in the collagen fibrils and is a better solvent for comonomers than water . it can also maintain the collagen fibrils in an expanded position after solvent evaporation to allow better resin infiltration . finally , a distinct surface layer of hydrophobic resin will be formed resulting in high bond strength values . after six months , the sbmp group showed a great reduction in bond strength , which is in agreement with the findings of shinohara et al . the cseb was the only self - etch adhesive with comparable bond strength durability to sb , which is in accordance with the results of armstrong et al , and implicates their high resistance against degradation . favorable durability of cseb has also been previously reported in several studies [ 25,34 , 35 ] . despite less distinct enamel etching patterns on sem micrographs in addition , the presence of 10-methacryloxydecy dihydrogen phosphate ( 10-mdp ) may also have contributed to the higher bonding durability . this functional monomer has a high chemical bonding potential to hydroxy - apatite and may cause a stable molecular adhesion . there are some reasons for suboptimal performance of one - step self - etch adhesive systems : ( i ) the more aggressive etching process , which may destabilize the collagen matrix , ( ii ) weak cohesive strength and ( iii ) low degree of polymerization . the applied method in this study for bond strength and microleakage evaluation had some limitations , such as lack of pulpal pressure upon the bonding procedure and aging , lack of mechanical loading during aging and absence of saliva . studies using fatigue tests and pulpal pressure simulation in the saliva media are recommended for better clinical generaliz - ability of results . under the conditions of this in vitro study , cseb had a relatively comparable performance to etch and rinse adhesive systems , regarding both microleakage and mtbs .

objectives : this study aimed to compare the durability of four adhesive systems by assessing their microtensile bond strength ( mtbs ) and microleakage during six months of water storage.materials and methods : a total of 128 human third molars were used . the adhesives tested were scotch bond multipurpose ( sbmp ) , single bond ( sb ) , clearfil - se bond ( cseb ) , and all - bond se ( abse ) . after sample preparation for mtbs testing , the microspecimens were subjected to microtensile tester after one day and six months of water storage . for microleakage evaluation , facial and lingual class v cavities were prepared and restored with composite . after thermocycling , microleakage was evaluated . bond strength values were subjected to one - way anova and tamhane s test , and the microleakage data were analyzed by the kruskal - wallis , dunn , mann whitney and wilcoxon tests ( p<0.05).results : single bond yielded the highest and abse yielded the lowest bond strength at one day and six months . short - term bond strength of sbmp and cseb was similar . after six months , a significant decrease in bond strength was observed in abse and sbmp groups . at one day , abse showed the highest microleakage at the occlusal margin ; however , at the gingival margin , there was no significant difference among groups . long - term microleakage of all groups at the occlusal margins was similar , whilst gingival margins of sbmp and sb showed significantly higher microleakage.conclusion:the highest mtbs and favorable sealability were obtained by clearfil se bond . water storage had no effect on microleakage of self - etch adhesives at the gingival margin or mtbs of cseb and sb .

INTRODUCTION MATERIALS AND METHODS Microtensile bond strength testing: Microleakage evaluation: Statistical analysis: RESULTS DISCUSSION CONCLUSION

PMC3923450

the united states food and drug administration and european medicines agency advise that , to improve safety in clinical development and postapproval , the potential for a new therapeutic agent to interact with established medications ( drug drug interaction , ddi ) should be assessed as early as possible during preclinical development . one major mechanism of ddi is the ability of chemical agents to regulate the expression of drug metabolism enzymes and transporters , often through the modulation of nuclear hormone receptor ( nhr ) activity , thereby altering the efficacy of both themselves and other compounds . it is valuable , therefore , to have a comprehensive understanding of the levels of expression of these protein factors and how they are modulated during therapy . in the liver , the primary site of drug metabolism , the majority of phase i ( modification ) reactions are carried out by cytochrome p450 ( cyp ) , with additional contributions made by alcohol dehydrogenase ( adh ) , aldehyde dehydrogenase ( aldh ) , aldo - keto reductase ( akr ) , epoxide hydrolase ( ephx ) , and flavin - containing monooxygenase ( fmo ) superfamilies . udp glucuronosyltransferases ( ugt ) and glutathione s - transferases ( gst ) enact most phase ii ( conjugation ) events . during laboratory study , if a broad expression profile of these dmes is required , dna microarray or high - density rt - pcr arrays are typically employed . while providing a practicable platform for this type of analysis a recent study by ohtsuki and colleagues demonstrated a poor correlation between protein and mrna levels for multiple cyp , ugt , and drug transporters , with only a handful of exceptions . in this study , the direct measurement of protein expression , as opposed to the measurement of mrna , correlated far better with enzymatic activity and is therefore a more appropriate readout for the evaluation of drug western blotting and other antibody - based approaches are the mainstay of protein expression analysis for dme , but our lab and others routinely struggle to interpret data due to the high degree of sequence homology of superfamily members , and hence cross - reactivity of antibody preparations . in order to circumvent this issue , recent developments have been made in stable isotope dilution mass spectrometry - based proteomics to simultaneously detect and quantify cyp and other drug metabolism related proteins . these studies utilize an absolute quantification technique ( aqua ) where known quantities of multiple synthetic stable isotope peptides for a protein / proteins of interest are spiked into proteolytic protein digests derived from liver samples , prior to lc - ms / ms analysis in a multiple reaction monitoring ( srm ) mode . unlabeled peptides co - elute , co - ionize , and are only differentiated by the difference in mass through lc - ms / ms analysis . using peak intensity ratios of the pairs of light and heavy isotopes , concentrations of analyte peptides a modified version of this procedure using stable isotope - labeled proteins expressed in and purified from escherichia coli as internal standards has also been developed which , post - translational modification and extraction variability excepted , accounts for efficiency of enzymatic digestion . but for comprehensive proteomic analysis , stable isotope labeling by amino acids in cell culture ( silac ) or in whole organisms ( in vivo silac , silam ) permits the simultaneous quantification of thousands of proteins with a much improved confidence due to the fact that both light and heavy analytes share near - identical chemical properties and environment . label - free shotgun proteomics is an alternative commonly used mass spectrometry based proteomics strategy . there are two mainstream label - free based lc - ms / ms approaches based on either spectral counting or ion intensity . the former compares the number of ms2 spectra assigned to a protein between samples , while the latter compares intensities of each precursor ion between samples . this approach aligns precursor ions between all lc - ms / ms runs using defined retention time and m / z windows . the signal intensities in each window for each sample are then integrated , normalized and compared between samples or groups . precursor ions showing significant differences or all precursors with ms2 spectra can then be identified . the success of this approach relies on tight control in sample processing and consistency in retention time in lc separation . in the current study , we harness the advantages of in vivo silac materials in conjunction with the label - free shotgun proteomics concept to quantify dmes in mouse liver , although the approach could be used for targeted quantification of any detectable protein or protein group of interest . peptides from dmes were identified in a metabolically labeled spike - in standard lysate and a complementary list of unlabeled peptides was constructed . this list of peptide pairs was stress - tested with a range of heavy to light input sample ratios and only the most reliable were retained . the final peptide list allowed us to quantify changes in expression of 51 dmes and was used to generate a constitutive androstane receptor ( car ) activation signature . 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene , 3,3,5,5-tetrachloro-1,4-bis(pyridyloxy)benzene ( tcpobop ) , corn oil , dl - dithiothreitol , and iodoacetamide were purchased from sigma ( dorset , uk ) . all mice were maintained under standard animal house conditions , with free access to food and water , and a 12 h light/12 h dark cycle . all animal work was carried out on male 8-week - old c57bl/6j mice in accordance with the animal scientific procedures act ( 1986 ) and after local ethical review . mice were administered either tcpobop ( a single intraperitoneal injection at 3 mg / kg ) in corn oil vehicle at 10 l / g body weight , or the same volume of corn oil vehicle alone . animals were sacrificed 7 days after dosing , and liver tissue excised and snap - frozen in liquid nitrogen for storage at 80 c . for lc - ms / ms , frozen liver tissue was thawed by addition of 9 volumes of sdt lysis buffer ( 4% sds , 0.1 m dtt , 100 mm tris - hcl ph7.6 ) then homogenized by rotor - stator ( 2 5 s at 20k revolutions ) . homogenate was heated to 95 c for 5 min , sonicated ( 2 5 s ) , and then centrifuged at 16 000 g for 10 min . supernatant ( protein sample for analysis ) was removed , aliquoted and stored at 80 c until use . protein samples ( total of 30 g / well ) were electrophoresed through a 12% bis - tris gel in mops running buffer supplemented with antioxidant ( all life technologies , paisley , uk ) alongside a spectra multicolour broad range protein ladder ( thermo fisher scientific , waltham , ma ) . gels were stained with coomassie blue , destained , and then rehydrated with milli - q water . gel regions containing proteins of interest , as described in the results section , were removed with a clean scalpel , sliced finely ( ca . 1 1 mm cubes ) , and added to 1.5 ml pcr - clean eppendorf tubes ( eppendorf , hamburg , germany ) . in - gel trypsin digest and peptide extraction was carried out according to the method of schevchenko and colleagues . peptide sample concentration was determined by nanodrop ( thermo fisher scientific ) and adjusted to 0.2 mg / ml in water containing 0.1% ( v / v ) trifluoroacetic acid . for western briefly , tissue was homogenized in 3 volumes of kcl buffer ( 1.15% w / v potassium chloride , 10 mm potassium phosphate , ph7.4 ) by rotor - stator ( 2 5 s at 20k revolutions ) followed by centrifugation at 11 000 g for 15 min . supernatant was ultracentrifuged at 100 000 g for 1 h and the resulting pellet resuspended in kcl buffer containing 0.25 m sucrose . protein concentration was adjusted to 1 mg / ml in lds sample buffer ( life technologies ) before electrophoresis through 10% acrylamide gels for 1 h at 200 v , followed by transfer onto nitrocellulose membranes ( 1 h at 100 v ) . our in - house panel of rabbit polyclonal antibodies for cyp detection has been summarized previously . fold changes were calculated from chemiluminescent signal intensity on a fujifilm las-3000 imager ( fujifilm , tokyo , japan ) . a nanoflow liquid chromatograph ( agilent 1200 , agilent , santa clara , ca ) with a ltq - orbitrap xl ( approximately 0.4 g total peptide was loaded onto a trap column at a flow rate of 10 l / min for 3 min and the flow was then reversed to an agilent zorbex nano c18 column ( 0.0075 mm i d ; 15 cm ; 3 m particle size ) . the peptides were resolved with a 3 h binary gradient at a flow rate of 300 nl / min as follows : 0% buffer b for 5 min followed by 230% buffer b for 140 min , 3090% buffer b for 15 min , 900% buffer b for 10 min , and 0% buffer b for 10 min . buffer a contained 2% acetonitrile and 0.1% formic acid in water , and buffer b contained 0.1% formic acid in acetonitrile . the column was periodically cleaned with a 2 l injection of buffer containing 50% acetonitrile and 0.1% formic acid in water . a proxeon nanospray source with a stainless steel emitter ( thermo fisher scientific ) was used to interface the agilent nanolc and ltq - orbitrap . the orbitrap was tuned using glu - fibrinogen b peptide . for the protein / peptide identification , a method that consisted of full scans between 330 and 1500 amu ( in orbitrap ) and data dependent ms / ms with top six precursor ions ( 2 to 4 charged ) in ltq was employed . orbitrap was operated in a profile mode at the resolution of 30 000 or 60 000 with a lock mass set at 445.1200 ( polycyclodimethylsiloxane ) , and ltq was operated in a centroid mode with isolation width = 1 ( m / z ) , normalized collision energy = 0.25 , and activation time = 30 ms . the max fill times for orbitrap and ltq were set at 500 and 50 ms , respectively . a dynamic exclusion of 30 s was used to maximize the acquisition of ms2 on peptides with lower intensity . for thr / sim analysis , a method that consisted of full scans between 330 and 1500 a.m.u ( in orbitrap ) and data dependent ms / ms scans with or without defined precursors was employed . a dynamic exclusion of 30 s and a threshold of 500 counts to trigger ms2 were also applied for ms / ms scans . nontargeted data dependent ms / ms was performed when there was no targeted precursor found in the ms scan . further details can be found in the method and tune files ( supporting information files s-1 and s-2 ) . protein and peptide database search was carried out using peaks version 6 ( bioinformatics solutions , waterloo , canada ) with an ipi - mouse database ( version 3.87 , european bioinformatics institute , hinxton , uk ) . the precursor mass tolerance was set at 7 ppm , and fragment ion mass tolerance set at 0.5 amu . the only permitted post - translational modifications were n - terminal acetylation and cysteine carbamidomethylation , while a maximum of two miscleavages were allowed . quantification of the predefined targeted peptides was carried out using sieve version 2.0 ( thermo fisher scientific ) using two seed files , one for each dme region of interest , containing retention time and m / z information . the precursor mass tolerance was set to 5 ppm , and the minimal intensity for alignment was set at 100 000 , with intensities derived from the first monoisotopic peak . data from sieve were exported to excel 2010 ( microsoft , redmond , wa ) for calculation of light to heavy peptide ratios . for protein quantification , light to heavy protein ratios were calculated within samples by summing average intensity values for all light peptides for each protein , then dividing by the corresponding heavy value . light to heavy protein ratios for technical replicates were averaged then biological replicates normalized to the average of control , before calculation of fold changes . for calculation of statistical significance , normalized values were log2-transformed and then analyzed by unpaired student s t test ( fdr = 0.5% ) using prism 6 ( graphpad , la jolla , ca ) ; * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . a workflow schematic of the thr / sim in vivo silac approach is shown in figure 1a . liver lysates were combined 1:1 with a liver lysate from metabolically labeled in vivo silac animals , which served as an internal standard for each experimental sample . gel electrophoresis was selected for sample preparation , as it allowed for fractionation and enrichment of dmes at the protein level . this selection was based on a pilot comparison of the sds - page in - gel digestion method to fasp . in the former , dmes were resolved by sds - page into two defined molecular weight regions which were excised for analysis ; dme region 1 ( 6640 kda ) was expected to contain adh , aldh , cyp , fmo , ugt , and ephx enzymes , while dme region 2 ( 4022 kda ) was expected to contain akr , gst , n - acetyltransferase ( nat ) , and sulfotransferase ( sult ) enzymes , based on calculated mw values ( figure 1b ) . protein in excised bands was reduced , alkylated , and digested with trypsin according to published protocols . a total of 64 dmes were identified by peaks analysis after the two - sample in - gel procedure , compared to 49 following the single - sample fasp procedure ( supporting information tables s-1s-3 ) . ( a ) metabolically labeled mouse liver tissue lysates were combined 1:1 with experimental sample lysates for sds - page . excised bands were processed by in - gel trypsin digestion for ls ms / ms analysis . heavy / light ratios from the first monoisotopic peaks were calculated for each peptide of interest in individual samples , with fold changes across samples calculated using the ratio / ratio value . ( b ) the gel bands excised , region 1 ( 6640 kda ) and region 2 ( 4022 kda ) , are predicted to contain the dmes of interest . similar to the principles of stable isotope dilution lc ms methods , the accuracy of measurements in thr / sim are highly dependent on the reliable detection of heavy stable isotope signals . it was therefore important to characterize the liver proteome of the spike - in reference lysate to determine which dmes could be monitored . as detailed above , sds - page and in - gel digestion of an in vivo silac liver sample permitted identification of a total of 64 dmes ( 2 adh , 7 akr , 11 aldh , 23 cyp , 1 ephx , 2 fmo , 9 gst , 0 nat , 2 sult , and 7 ugt ) by unique peptides ( fdr = 0.1% ) . in addition , due to the high degree of identity shared by certain enzymes , some were unidentifiable by unique peptides but were nonetheless retained as protein groups , for example , cyp2a4/2a5 . peptides without lysine were removed from the list , as were those with 10log p values < 17.3 ( fdr = 0.1% ) . heavy - only peptide list was converted to a list of silac peptide pairs by addition of the predicted counterpart light members . the utility of these peptide pairs for quantification was then stress - tested as follows . an unlabeled liver lysate was mixed with the in vivo silac liver lysate at ratios of 1:1 , 1:4 , and 1:16 in duplicate for processing and analysis by lc the ms data were analyzed in sieve using the silac peptide pair list as a seed file ( retention time window of 5 min ) . the average ratio value from technical replicates was then log4 transformed to evenly distribute the three data points , allowing calculation of an equally weighted r value . only those peptides showing strong linearity ( r > 0.9 ) were retained . the data output from sieve , along with calculations of r values , can be found in supporting information tables s-4 and s-5 . approximately 57% of identified peptides survived this filtration step ( supporting information figure s-1 ) . seed files containing m / z and retention time windows for each peptide can be found in supporting information tables s-6 ( dme region 1 ) and s-7 ( dme region 2 ) . an example of thr / sim in vivo silac is shown in figure 2 . three cyp1a2 peptides were used to define cyp1a2 levels , one of which eluted at 37.9 min and another at 94.7 min ( figure 2a ) . although some interfering signals were observed , these could be separated from the signals of interest by retention time gating and the matching of internal standard signals . for one of these peptides , nsiqditsalfk , an r value for linearity of 0.998 was observed ( figure 2b ) and the identities of both the light and heavy forms could be confirmed by their ms2 ( figure 2c and d ) . ( a ) using a 5 min retention time window ( gray boxes ) , nontarget signals could be effectively gated from nsiqditsalfk and fltnnnsaidk peptide pair quantification . ( b ) dilution linearity of the nsiqditsalfk peptide pair achieved r of 0.998 , while ms2 spectra were acquired for both ( c ) light and ( d ) heavy forms , confirming their identity . to compare the output of thr / sim in vivo silac to western blotting , we assessed the cyp induction profile in mice treated with a potent and specific inducer of the constitutive androstane receptor , tcpobop . animals ( n = 3 ) were administered the compound in corn oil , and , after 7 days , livers were harvested for analysis . western blotting for cyp indicated strong induction of multiple family members , compared to vehicle control ( figure 3a ) . depending on the origin of antisera , as well as results of previous studies ( not shown ) , the exact identity of individual enzymes may or may not be known . with our in - house panel of antibodies , up - regulation could be demonstrated for cyp1a1 ( 46.5-fold ) , cyp1a2 ( 5.1-fold ) , cyp2b10 ( 362.9-fold ) , and 3a11 ( 5.7-fold ) , while down - regulation could be demonstrated for cyp2e1 ( 0.8-fold ) , as these superfamily members are predictably and reproducibly identified . for other antisera , such as cyp2c and cyp2d , the precise identities of the proteins detected are unknown , so , although inductions are observed , these are not attributable to any family member in particular . for t - hr / sim - in vivo silac , data for individual animals were acquired and processed separately , and then average fold changes sd calculated at the last stage , to account for biological variability . cyp1a2 ( 3.5-fold ) , cyp2a4/5 ( 16.2-fold ) , cyp2c29 ( 15.7-fold ) , cyp2c37 ( 9.5-fold ) , cyp2d22 ( 2.5-fold ) , cyp3a13 ( 5.3-fold ) , and cyp8b1 ( 3.1-fold ) were significantly up - regulated ( figure 3b ) . although not statistically significant ( p = 0.08 ) , cyp2e1 levels were decreased ( 0.87-fold ) . due to the low abundance of cyp1a1 , cyp2b10 , and cyp3a11 in the spike - in standard , these enzymes could not be quantified by lc nevertheless , there was close agreement between methods of changes in cyp1a2 and cyp2e1 expression . moreover , western blotting suggested induction of unconfirmed cyp2c , cyp2d , and cyp3a family members , while lc ms / ms demonstrated induction of cyp2c29 , cyp2c37 , cyp2d22 , and cyp3a13 . cyp isoform in tcpobop - treated mice ( n = 3 ) , as compared to corn oil vehicle - treated control animals ( n = 3 ) , were measured by ( a ) western blot and ( b ) thr / sim - in vivo silac ( black bars , corn oil ; gray bars , tcpobop ) . following western blot of pooled samples , chemilluminescent signal was used to quantify fold changes in particular bands ( red boxes ) , with values presented alongside . note that , in the control , some protein levels were below the limit of detection by western blotting ( e.g. , cyp2b10 ) and therefore the fold changes calculated may not be accurate . for thr / sim - in vivo silac , the number of unique peptides used to calculate fold change is given ( gray text ) , and as samples for individual animals were analyzed separately , error bars are a reflection of biological variability , as well as technical error . to provide additional confidence that the assay was reliable , we extracted data for four proteins / protein families which are not expected to change significantly following tcpobop dosing : pro / albumin , calreticulin , -tubulin , and -tubulin . of the other quantifiable phase i enzymes , aldh1a1 ( 3.1-fold ) and fmo5 ( 3.1-fold ) were significantly up - regulated , while aldh2 ( 0.4-fold ) and aldh6a1 ( 0.5-fold ) were significantly down - regulated ( figure 4a ) . for phase ii , gsta3 ( 1.6-fold ) , gstm1 ( 7.5-fold ) , gstt3 ( 7.0-fold ) , sult3a1 ( 4.7-fold ) , ugt1a1/2 ( 2.9-fold ) , ugt2b34 ( 3.8-fold ) and ugt2b36 ( 1.8-fold ) were significantly up - regulated ( figure 4b ) . ugt2a3 ( 0.8-fold ) was the only enzyme for which significant down - regulation occurred . thr / sim - in vivo silac analysis of modulation of additional dmes by tcpobop . ( a ) other phase i and ( b ) phase ii enzymes were quantified . through the combination of in vivo silac and label - free approaches we have created a streamlined and simple targeted proteomics workflow permitting the quantitative analysis of 51 dmes in the mouse . this technique has utility in the investigation of pharmacodynamics , particularly when the potential for , or occurrence of , drug upstream , the use of metabolically labeled isotopic tissue as an internal standard reduces experimental variation derived from sample processing and ionization . this is especially important when error - prone methods such as in - gel digestion are used , and when the difference between experimental groups is relatively small . downstream , the strategy resembles that of stable isotope dilution lc - ms for quantification of small molecules . the narrow retention time and m / z windows reduce the influence of potential contaminant signals during data processing , while data analysis is simplified in comparison to more conventional silac procedures as only a predetermined list of proteins / peptides of interest is measured , permitting immediate and intuitive interpretation . one limitation of the thr / sim in vivo silac approach is that changes in expression can not be monitored in the dme for which heavy isotope labeled ms2 are not acquired . a potential solution to this problem would be to rerun a database search on the experimental ms files and , where ms2 for light peptides from nontargeted dme can be detected , manually interrogate the primary raw file , referencing the light signal to other heavy peptides eluted within the same time frame , or to total ion current as typically performed with label - free proteomics . caution must be reserved that such quantification may be subject to sources of peptide bias , such as differential recovery during sample processing and differential efficiency during ionization . although in the current study we believe high resolution sim mode is sufficient to monitor the levels of 51 dme proteins , mainly due to their higher relative abundance , the method can be easily adapted to an mrm mode where the precursor and fragment ion transitions are monitored . we have applied the thr / sim - in vivo silac workflow to the analysis of dme changes in response to the car activator , tcpobop . with an ec50 of approximately 100 nm , maximally effective dose of 3 mg / kg , and response duration of greater than 20 weeks in the mouse , it acts as a mitogen , nongenotoxic tumor - promoter and complete carcinogen . in the liver , in studies variously employing tcpobop and/or car knockout mice as tools to characterize the car - dependent gene battery , up - regulation of transcription of mrna has been demonstrated for cyp1a1 , 1a2 , 2a4 , 2a5 , 2b9 , 2b10 , 2b13 , 2c29 , 2c37 , 2c55 , 2c65 , 2f2 , and 3a11 , with decreases seen for 4a10 and 4a31 . at the protein level , widespread cyp induction has been demonstrated by western blot and in a postdigest o labeling study , where cyp1a2 , 2a4/5 , 2b10 , 2b20 , 2c29 , 2c37 , 2c38 , 3a11 , and 39a1 were shown to be up - regulated , with cyp2c40 , 2e1 , 3a41 , and 27a1 down - regulated . in the current study , we detected significant up - regulation of cyp1a2 , 2a4/2a5 , 2c29 , 2c37 , 2d22 , 3a13 , and 8b1 and nonsignificant down - regulation of cyp2e1 , broadly in agreement with the literature , although we did not see the previously reported changes in cyp2f2 . this could be due to post - transcriptional or post - translational regulation , or variation in genetic background . our data conflict with the report of cyp27a1 down - regulation , as we detected a nonsignificant increase . we did not observe any significant changes in adh or akr but , to our knowledge , the only previously reported car / tcpobop target within these superfamilies is akr1b7 , which we did not detect and were therefore unable to quantify . the only other reported car / tcpobop - regulated members of the phase i metabolism superfamilies under study are aldh1a1 , aldh1a7 , and fmo5 , which are up - regulated at the mrna level . our data agree with those for aldh1a1 and fmo5 , although we could not detect aldh1a7 protein . in previous reports , aldh1b1 , 2 , 6a1 , and 7a1 have shown a car / tcpobop - dependent down - regulation . our data agree completely with these observations , although only the more pronounced decreases in aldh2 and 6a1 achieve significance . for phase ii , at the mrna level , car / tcpobop can upregulate gsta1 , a2 , a4 , m1 , m2 , m3 , m4 , t1 and t3 , sult1a1 , 1e1 , 2a1 , 2a2 , 3a1 and 5a1 , ugt1a1 , 1a9 , 2b34 , 2b35 and 2b36 , and down - regulate ugt2a3 and 3a1/2 . in agreement with the literature , we observed significant induction of gsta3 , m1 , t3 and sult3a1 , and nonsignificant induction of gstm2 and t1 , and sult1a1 . for ugt , we detected induction of ugt1a1/2 , 2b34 , and 2b36 , with repression of 2a3 , all in agreement with previous ( mrna ) reports . our findings agree with the only report we are aware of regarding phase ii protein induction by car / tcpobop , that of ugt1a1 . therefore , as the vast majority of car targets have been established as such from mrna studies , it is noteworthy that , for many of these targets , we have provided the first evidence for their modulation at the protein level . moreover , despite the influence of post - transcriptional and post - translational regulation , we have demonstrated that the car activation signature is essentially conserved from mrna to protein . while we found that the thr / sim approach is satisfactory for the analysis of 51 dme proteins using a ltq - orbitrap , other recently developed ms2 based targeted quantitative proteomics approaches such as swath , ms , and parallel reaction monitoring ( prm ) may provide an alternative if a quadrupole - orbitrap or quadrupole - time - of - flight mass spectrometer is available . in particular , the prm approach has recently been shown to provide better sensitivity and specificity than sim . precursor ion pairs that are preisolated by a quadrupole , accumulated and fragmented in a c - trap . it is therefore anticipated that the number of dme proteins measured could be potentially improved using the prm approach . in conclusion , the application of thr / sim - in vivo silac for quantification of 51 dme presented here constitutes a comprehensive means of profiling altered capacity for drug metabolism in the mouse . an appreciation of this capacity is valuable when carrying out pharmacokinetic studies in this model organism , when up- or down - regulation of dme expression due to drug treatment or genetic manipulation has a bearing on results . moreover , this approach could provide an additional measure of the ddi potential of a candidate therapeutic compound during preclinical development . as modulation of dmes typically requires upstream activation of one or more nhr by direct binding to chemical agents , species differences in drug / receptor affinity mean that caution must be reserved in drawing conclusions from studies in model organisms . conceivably , thr / sim - in vivo silac could be used to measure ddi potential in nhr - humanized mice , such as the car / pxr double humanized line , constituting a more - accurately modeled in vivo approach and thereby reducing the gap between preclinical and clinical evaluation of ddi .

the modulation of drug metabolism enzyme ( dme ) expression by therapeutic agents is a central mechanism of drug drug interaction and should be assessed as early as possible in preclinical drug development . direct measurement of dme levels is typically achieved by western blotting , qpcr , or microarray , but these techniques have their limitations ; antibody cross - reactivity among highly homologous subfamilies creates ambiguity , while discordance between mrna and protein expression undermines observations . the aim of this study was to design a simple targeted workflow by combining in vivo silac and label - free proteomics approaches for quantification of dmes in mouse liver , facilitating a rapid and comprehensive evaluation of metabolic potential at the protein level . a total of 197 peptides , representing 51 phase i and phase ii dmes , were quantified by lc - ms / ms using targeted high resolution single ion monitoring ( thr / sim ) with a defined mass - to - charge and retention time window for each peptide . in a constitutive androstane receptor ( car ) activated mouse model , comparison of thr / sim - in vivo silac with western blotting for analysis of the expression of cytochromes p450 was favorable , with agreement in fold - change values between methods . the thr / sim - in vivo silac approach therefore permits the robust analysis of multiple dme in a single protein sample , with clear utility for the assessment of the drug drug interaction potential of candidate therapeutic compounds .

Introduction Materials and Methods Results Discussion

PMC5256076

it is my pleasure to review progress in numerical relativity based upon characteristic evolution . in the spirit of living reviews in relativity , we are now in an era in which einstein s equations can effectively be considered solved at the local level . several groups , as reported here and in other living reviews in relativity , have developed 3d codes which are stable and accurate in some sufficiently local setting . , there is no single code in existence today which purports to be capable of computing the waveform of gravitational radiation emanating from the inspiral and merger of two black holes , the premier problem in classical relativity . just as several coordinate patches are necessary to describe a spacetime with nontrivial topology , the most effective attack on the binary black hole problem may involve patching together pieces of spacetime handled by a combination of different codes and techniques . most of the effort in numerical relativity has centered about the cauchy { 3 + 1 } formalism , with the gravitational radiation extracted by perturbative methods based upon introducing an artificial schwarzschild background in the exterior region [ 1 , 4 , 2 , 3 , 181 , 180 , 156 ] . a different approach which is specifically tailored to study radiation is based upon the characteristic initial value problem . in the 1960 s , bondi [ 45 , 46 ] and it led to the first unambiguous description of gravitational radiation in a fully nonlinear context . it yields the standard linearized description of the plus and cross polarization modes of gravitational radiation in terms of the bondi news function n at future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the bondi news function is an invariantly defined complex radiation amplitude n = n + in , whose real and imaginary parts correspond to the time derivatives th and th of the plus and cross polarization modes of the strain h incident on a gravitational wave antenna . the major drawback of the characteristic approach arises from the formation of caustics in the light rays generating the null hypersurfaces . in the most ambitious scheme proposed at the theoretical level such caustics only a few structural stable caustics can arise in numerical evolution , and their geometrical properties are well enough understood to model their singular behavior numerically , although a computational implementation has not yet been attempted . in the typical setting for the characteristic initial value problem , the domain of dependence of a single nonsingular null hypersurface is empty . in order to obtain a nontrivial evolution problem , the null hypersurface must either be completed to a caustic - crossover region where it pinches off , or an additional boundary must be introduced . so far , the only caustics that have been successfully evolved numerically in general relativity are pure point caustics ( the complete null cone problem ) . when spherical symmetry is not present , it turns out that the stability conditions near the vertex of a light cone place a strong restriction on the allowed time step . point caustics in general relativity have been successfully handled this way for axisymmetric spacetimes , but the computational demands for 3d evolution would be prohibitive using current generation supercomputers . this is unfortunate because , away from the caustics , characteristic evolution offers myriad computational and geometrical advantages . as a result , at least in the near future , fully three - dimensional computational applications of characteristic evolution are likely to be restricted to some mixed form , in which data is prescribed on a non - singular but incomplete initial null hypersurface n and on a second boundary hypersurface b , which together with the initial null hypersurface determine a nontrivial domain of dependence . the hypersurface b may be either ( i ) null , ( ii ) timelike or ( iii ) spacelike , as schematically depicted in figure 1 . the first two possibilities give rise to ( i ) the double null problem and ( ii ) the nullcone - worldtube problem . it may be regarded as a cauchy initial boundary value problem where the outer boundary is null . an alternative interpretation is the cauchy - characteristic matching ( ccm ) problem , in which the cauchy and characteristic evolutions are matched transparently across a worldtube w , as indicated in figure 1 . figure 1the three applications of characteristic evolution with data given on an initial null hypersurface n and boundary b. the shaded regions indicate the corresponding domains of dependence . the three applications of characteristic evolution with data given on an initial null hypersurface n and boundary b. the shaded regions indicate the corresponding domains of dependence . in ccm , it is possible to choose the matching interface between the cauchy and characteristic regions to be a null hypersurface , but it is more practical to match across a timelike worldtube . ccm combines the advantages of characteristic evolution in treating the outer radiation zone in spherical coordinates which are naturally adapted to the topology of the worldtube with the advantages of cauchy evolution in treating the inner region in cartesian coordinates , where spherical coordinates would break down . in this review , we trace the development of characteristic algorithms from model 1d problems to a 2d axisymmetric code which computes the gravitational radiation from the oscillation and gravitational collapse of a relativistic star and to a 3d code designed to calculate the waveform emitted in the merger to ringdown phase of a binary black hole . and we trace the development of ccm from early feasibility studies to successful implementation in the linear regime and through current attempts to treat the binary black hole problem . florian siebel s thesis work , at the technische universitt mnchen , integrates an axisymmetric characteristic gravitational code with a high resolution shock capturing code for relativistic hydrodynamics . this coupled general relativistic code has been thoroughly tested and has yielded state - of - the - art results for the gravitational waves produced by the oscillation and collapse of a relativistic star ( see sections 5.1 and 5.2 ) . in yosef zlochower s thesis work , at the university of pittsburgh , the gravitational waves generated from the post - merger phase of a binary blacszk black hole is computed using a fully nonlinear three - dimensional characteristic code ( see section 3.8 ) . he shows how the characteristic code can be employed to investigate the nonlinear mode coupling in the response of a black hole to the infall of gravitational waves . a further notable achievement has been the successful application of ccm to the linearized matching problem between a 3d characteristic code and a 3d cauchy code based upon harmonic coordinates ( see section 4.7 ) . here the linearized cauchy code satisfies a well - posed initial - boundary value problem , which seems to be a critical missing ingredient in previous attempts at ccm in general relativity . the problem of computing the evolution of a self - gravitating object , such as a neutron star , in close orbit about a black hole is of clear importance to the new gravitational wave detectors . the interaction with the black hole could be strong enough to produce a drastic change in the emitted waves , say by tidally disrupting the star , so that a perturbative calculation would be inadequate . the understanding of such nonlinear phenomena requires well behaved numerical simulations of hydrodynamic systems satisfying einstein s equations . several numerical relativity codes for treating the problem of a neutron star near a black hole have been developed , as described in the living review in relativity on numerical hydrodynamics in general relativity by font . although most of these efforts concentrate on cauchy evolution , the characteristic approach has shown remarkable robustness in dealing with a single black hole or relativistic star . in this vein , state - of - the - art axisymmetric studies of the oscillation and gravitational collapse of relativistic stars have been achieved ( see section 5.2 ) and progress has been made in the 3d simulation of a body in close orbit about a schwarzschild black hole ( see sections 5.3 and 5.3.1 ) . in previous reviews , i tried to include material on the treatment of boundaries in the computational mathematics and fluid dynamics literature because of its relevance to the ccm problem . a separate living review in relativity on boundary conditions is certainly warranted and is presently under consideration . in view of this , i will not attempt to keep this subject up to date except for material of direct relevance to ccm , although i will for now retain the past material . animations and other material from these studies can be viewed at the web sites of the university of canberra , louisiana state university , pittsburgh university , and pittsburgh supercomputing center . characteristics have traditionally played an important role in the analysis of hyperbolic partial differential equations . however , the use of characteristic hypersurfaces to supply the foliation underlying an evolution scheme has been mainly restricted to relativity . this is perhaps natural because in curved spacetime there is no longer a preferred cauchy foliation provided by the euclidean 3-spaces allowed in galilean or special relativity . the method of shooting along characteristics is a standard technique in many areas of computational physics , but evolution based upon characteristic hypersurfaces is quite uniquely limited to relativity . bondi s initial use of null coordinates to describe radiation fields was followed by a rapid development of other null formalisms . these were distinguished either as metric based approaches , as developed for axisymmetry by bondi , metzner and van der burg and generalized to 3 dimensions by sachs , or as null tetrad approaches in which the bianchi identities appear as part of the system of equations , as developed by newman and penrose . at the outset , null formalisms were applied to construct asymptotic solutions at null infinity by means of 1/r expansions . soon afterward , penrose devised the conformal compactification of null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } ( scri ) , thereby reducing to geometry the asymptotic quantities describing the physical properties of the radiation zone , most notably the bondi mass and news function . the characteristic initial value problem rapidly became an important tool for the clarification of fundamental conceptual issues regarding gravitational radiation and its energy content . the initial focus on asymptotic solutions clarified the kinematic properties of radiation fields but could not supply the waveform from a specific source . it was soon realized that instead of carrying out a 1/r expansion , one could reformulate the approach in terms of the integration of ordinary differential equations along the characteristics ( null rays ) . the integration constants supplied on some inner boundary then determined the specific waveforms obtained at infinity . in the double - null initial value problem of sachs , the integration constants are supplied at the intersection of outgoing and ingoing null hypersurfaces . in the worldtube - nullcone formalism , the sources were represented by integration constants on a timelike worldtube . these early formalisms have gone through much subsequent revamping . some have been reformulated to fit the changing styles of modern differential geometry . rather than including a review of the extensive literature on characteristic formalisms in general relativity , i concentrate here on those approaches which have been implemented as computational evolution schemes . the existence and uniqueness of solutions to the associated boundary value problems , which has obvious relevance to the success of numerical simulations , is treated in a separate living review in relativity on theorems on existence and global dynamics for the einstein equations by rendall . the fundamental ingredient is a foliation by null hypersurfaces u = const . which are generated by a two - dimensional set of null rays , labeled by coordinates s , with a coordinate varying along the rays . in ( u , , x ) null coordinates , the main set of einstein equations take the schematic form 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_{,\lambda } } = { h_f}[f , g]$$\end{document } and 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{,u\lambda } } = { h_g}[f , g,{g_{,u}}].$$\end{document } here f represents a set of hypersurface variables , g a set of evolution variables , and hf and hg are nonlinear hypersurface operators , i.e. they operate locally on the values of f , g and g , u intrinsic to a single null hypersurface . in the bondi formalism , these hypersurface equations have a hierarchical structure in which the members of the set f can be integrated in turn in terms of the characteristic data for the evolution variables and prior members of the hierarchy . in addition to these main equations , there is a subset of four einstein equations which are satisfied by virtue of the bianchi identities , provided that they are satisfied on a hypersurface transverse to the characteristics . these equations have the physical interpretation as conservation laws . mathematically they are analogous to the constraint equations of the canonical formalism . but they are not necessarily elliptic , since they may be intrinsic to null or timelike hypersurfaces , rather than spacelike cauchy hypersurfaces . computational implementation of characteristic evolution may be based upon different versions of the formalism ( i.e. metric or tetrad ) and different versions of the initial value problem ( i.e. double null or worldtube - nullcone ) . however , most characteristic evolution codes share certain common advantages : the initial data is free . there are no elliptic constraints on the data , which eliminates the need for time consuming iterative constraint solvers and artificial boundary conditions . this flexibility and control in prescribing initial data has the trade - off of limited experience with prescribing physically realistic characteristic initial data.the coordinates are veryrigid , i.e. there is very little remaining gauge freedom.the constraints satisfy ordinary differential equations along the characteristics which force any constraint violation to fall off asymptotically as 1/r.no second time derivatives appear so that the number of basic variables is at most half the number for the corresponding version of the cauchy problem.the main einstein equations form a system of coupled ordinary differential equations with respect to the parameter varying along the characteristics . this allows construction of an evolution algorithm in terms of a simple march along the characteristics.in problems with isolated sources , the radiation zone can be compactified into a finite grid boundary with the metric rescaled by 1/r as an implementation of penrose s conformal method . because the penrose boundary is a null hypersurface , no extraneous outgoing radiation condition or other artificial boundary condition the analogous treatment in the cauchy problem requires the use of hyperboloidal spacelike hypersurfaces asymptoting to null infinity . for reviews of the hyperboloidal approach and its status in treating the associated three - dimensional computational problem , see [ 131 , 81].the grid domain is exactly the region in which waves propagate , which is ideally efficient for radiation studies . since each null hypersurface of the foliation extends to infinity , the radiation is calculated immediately ( in retarded time).in black hole spacetimes , a large redshift at null infinity relative to internal sources is an indication of the formation of an event horizon and can be used to limit the evolution to the exterior region of spacetime . while this can be disadvantageous for late time accuracy , it allows the possibility of identifying the event horizon on the fly , as opposed to cauchy evolution where the event horizon can only be located after the evolution has been completed . perhaps most important from a practical view , characteristic evolution codes have shown remarkably robust stability and were the first to carry out long term evolutions of moving black holes . there are no elliptic constraints on the data , which eliminates the need for time consuming iterative constraint solvers and artificial boundary conditions . this flexibility and control in prescribing initial data has the trade - off of limited experience with prescribing physically realistic characteristic initial data . the constraints satisfy ordinary differential equations along the characteristics which force any constraint violation to fall off asymptotically as 1/r . no second time derivatives appear so that the number of basic variables is at most half the number for the corresponding version of the cauchy problem . the main einstein equations form a system of coupled ordinary differential equations with respect to the parameter this allows construction of an evolution algorithm in terms of a simple march along the characteristics . in problems with isolated sources , the radiation zone can be compactified into a finite grid boundary with the metric rescaled by 1/r as an implementation of penrose s conformal method . because the penrose boundary is a null hypersurface , no extraneous outgoing radiation condition or other artificial boundary condition is required . the analogous treatment in the cauchy problem requires the use of hyperboloidal spacelike hypersurfaces asymptoting to null infinity . for reviews of the hyperboloidal approach and its status in treating the associated three - dimensional computational problem , the grid domain is exactly the region in which waves propagate , which is ideally efficient for radiation studies . since each null hypersurface of the foliation extends to infinity , the radiation is calculated immediately ( in retarded time ) . in black hole spacetimes , a large redshift at null infinity relative to internal sources is an indication of the formation of an event horizon and can be used to limit the evolution to the exterior region of spacetime . while this can be disadvantageous for late time accuracy , it allows the possibility of identifying the event horizon on the fly , as opposed to cauchy evolution where the event horizon can only be located after the evolution has been completed . characteristic schemes also share as a common disadvantage the necessity either to deal with caustics or to avoid them altogether . the scheme to tackle the caustics head on by including their development as part of the evolution is perhaps a great idea still ahead of its time but one that should not be forgotten . there are only a handful of structurally stable caustics , and they have well known algebraic properties . the structural stability of the singularities should in principle make this possible , and algorithms to evolve the elementary caustics have been proposed [ 69 , 202 ] . in the axisymmetric case , cusps and folds are the only structurally stable caustics , and they have already been identified in the horizon formation occurring in simulations of head - on collisions of black holes and in the temporarily toroidal horizons occurring in collapse of rotating matter [ 151 , 189 ] . in a generic binary black hole horizon , where axisymmetry is broken , there is a closed curve of cusps which bounds the two - dimensional region on the horizon where the black holes initially form and merge [ 144 , 133 ] . limited computer power , as well as the instabilities arising from non - hyperbolic formulations of einstein s equations , necessitated that the early code development in general relativity be restricted to spacetimes with symmetry . the techniques for relativistic fields which propagate on null characteristics are similar to the gravitational case . we postpone treatment of relativistic fluids , whose characteristics are timelike , until section 5 . it is often said that the solution of the general ordinary differential equation is essentially known , in light of the success of computational algorithms and present day computing power . but , in this spirit , it is fair to say that the general system of hyperbolic partial differential equations in one spatial dimension seems to be a solved problem in general relativity . codes have been successful in revealing important new phenomena underlying singularity formation in cosmology and in dealing with unstable spacetimes to discover critical phenomena . as described below one of the earliest characteristic evolution codes , constructed by corkill and stewart [ 69 , 201 ] , treated spacetimes with two killing vectors using a grid based upon double null coordinates , with the null hypersurfaces intersecting in the surfaces spanned by the killing vectors . they simulated colliding plane waves and evolved the khan - penrose collision of impulsive ( -function curvature ) plane waves to within a few numerical zones from the final singularity , with extremely close agreement with the analytic results . their simulations of collisions with more general waveforms , for which exact solutions are not known , provided input to the understanding of singularity formation which was unforeseen in the analytic treatments of this problem . many { 1 + 1}-dimensional characteristic codes have been developed for spherically symmetric systems . here initially the characteristic evolution of matter was restricted to simple cases , such as massless klein - gordon fields , which allowed simulation of gravitational collapse and radiation effects in the simple context of spherical symmetry . its application to hydrodynamics has made significant contributions to general relativistic astrophysics , as reviewed in section 5 . the synergy between analytic and computational approaches has already led to dramatic results in the massless klein - gordon case . on the analytic side , working in a characteristic initial value formulation based upon outgoing null cones , christodoulou made a penetrating study of the spherically symmetric problem [ 59 , 60 , 61 , 62 , 63 , 64 ] . in a suitable function space , he showed the existence of an open ball about minkowski space data whose evolution is a complete regular spacetime ; he showed that an evolution with a nonzero final bondi mass forms a black hole ; he proved a version of cosmic censorship for generic data ; and he established the existence of naked singularities for non - generic data . what this analytic tour - de - force did not reveal was the remarkable critical behavior in the transition to the black hole regime , which was discovered by choptuik [ 57 , 58 ] by computational simulation based upon cauchy evolution . this phenomenon has now been understood in terms of the methods of renormalization group theory and intermediate asymptotics , and has spawned a new subfield in general relativity , which is covered in the living review in relativity on critical phenomena in gravitational collapse by gundlach . the characteristic evolution algorithm for the spherically symmetric einstein - klein - gordon problem provides a simple illustration of the techniques used in the general case . it centers about the evolution scheme for the scalar field , which constitutes the only dynamical field . given the scalar field , all gravitational quantities can be determined by integration along the characteristics of the null foliation . this is a coupled problem , since the scalar wave equation involves the curved space metric . it illustrates how null algorithms lead to a hierarchy of equations which can be integrated along the characteristics to effectively decouple the hypersurface and dynamical variables . in a bondi coordinate system based upon outgoing null hypersurfaces u = const . and a surface area coordinate r , the metric is 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\beta}}du\left({{v \over r}du + 2dr } \right ) + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2}).$$\end{document } smoothness at r = 0 allows imposition of the coordinate conditions 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{v(u , r ) = r + { \mathcal o}({r^3 } ) } \\ { \beta ( u , r ) = { \mathcal o}({r^2 } ) . } \\ \end{array}$$\end{document } the field equations consist of the curved space wave equation = 0 for the scalar field and two hypersurface equations for the metric functions : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { , r } } = 2\pi r{({\phi _ { , r}})^2},$$\end{document } 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v_{,r } } = { e^{2\beta}}.$$\end{document } the wave equation can be expressed in the form 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\square^{(2)}}g - { \left({{v \over r } } \right)_{,r}}{{{e^{- 2\beta}}g } \over r } = 0,$$\end{document } where g = r and is the dalembertian associated with the two - dimensional submanifold spanned by the ingoing and outgoing null geodesics . initial null data for evolution consists of (u0 , r ) at initial retarded time u0 . because any two - dimensional geometry is conformally flat , the surface integral of g over a null parallelogram gives exactly the same result as in a flat 2-space , and leads to an integral identity upon which a simple evolution algorithm can be based . let the vertices of the null parallelogram be labeled by n , e , s , and w corresponding , respectively , to their relative locations ( north , east , south , and west ) in the 2-space , as shown in figure 2 . upon integration of equation ( 7 ) , curvature introduces an integral correction to the flat space null parallelogram relation between the values of g at the vertices : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_n } - { g_w } - { g_e } + { g_s } = - { 1 \over 2}\int\nolimits_\sigma { du\;dr } { \left({{v \over r } } \right)_{,r}}{g \over r}.$$\end{document } figure 2the null parallelogram . after computing the field at point n , the algorithm marches the computation to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } by shifting the corners by n n , e e , s e , w n. the null parallelogram . after computing the field at point n , the algorithm marches the computation to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } by shifting the corners by n n , e e , s e , w n. this identity , in one form or another , lies behind all of the null evolution algorithms that have been applied to this system . the prime distinction between the different algorithms is whether they are based upon double null coordinates or bondi coordinates as in equation ( 3 ) . when a double null coordinate system is adopted , the points n , e , s , and w can be located in each computational cell at grid points , so that evaluation of the left hand side of equation ( 8) requires no interpolation . as a result , in flat space , where the right hand side of equation ( 8) vanishes , it is possible to formulate an exact evolution algorithm . in curved space , of course , there is a truncation error arising from the approximation of the integral , e.g. , by evaluating the integrand at the center of . the identity ( 8) gives rise to the following explicit marching algorithm , indicated in figure 2 . let the null parallelogram lie at some fixed and and span adjacent retarded time levels u0 and uo + u . imagine for now that the points n , e , s , and w lie on the spatial grid , with rn rw = re rs = r . if g has been determined on the entire initial cone u0 , which contains the points e and s , and g has been determined radially outward from the origin to the point w on the next cone u0 + u , then equation ( 8) determines g at the next radial grid point n in terms of an integral over . the integrand can be approximated to second order , i.e. to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\delta r\delta u)$\end{document } , by evaluating it at the center of . to this same accuracy , the value of g at the center equals its average between the points e and w , at which g has already been determined . similarly , the value of ( v / r),r at the center of can be approximated to second order in terms of values of v at points where it can be determined by integrating the hypersurface equations ( 5 , 6 ) radially outward from r = 0 . after carrying out this procedure to evaluate g at the point n , the procedure can then be iterated to determine g at the next radially outward grid point on the u0 + u level , i.e. point n in figure 2 . upon completing this radial march to null infinity , in terms of a compactified radial coordinate such as x = r/(1 + r ) , the field g is then evaluated on the next null cone at u0 + 2u , beginning at the vertex where smoothness gives the startup condition that g(u , 0 ) = 0 . in the compactified bondi formalism , the vertices n , e , s , and w of the null parallelogram can not be chosen to lie exactly on the grid because , even in minkowski space , the velocity of light in terms of a compactified radial coordinate x is not constant . as a consequence , the fields g , , and v at the vertices of are approximated to second order accuracy by interpolating between grid points . however , cancellations arise between these four interpolations so that equation ( 8) is satisfied to fourth order accuracy . the net result is that the finite difference version of equation ( 8) steps g radially outward one zone with an error of fourth order in grid size , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({(\delta u)^2}{(\delta x)^2})$\end{document}. in addition , the smoothness conditions ( 4 ) can be incorporated into the startup for the numerical integrations for v and to insure no loss of accuracy in starting up the march at r = 0 . the resulting global error in g , after evolving a finite retarded time , is then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\delta u\delta x)$\end{document } , after compounding errors from 1/(ux ) number of zones . when implemented on a grid based upon the ( u , r ) coordinates , the stability of this algorithm is subject to a courant - friedrichs - lewy ( cfl ) condition requiring that the physical domain of dependence be contained in the numerical domain of dependence . in the spherically symmetric case , this condition requires that the ratio of the time step to radial step be limited by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(v / r)\delta u \leq 2\delta r$\end{document } , where r = [x/(1x ) ] . this condition can be built into the code using the value v / r = e , corresponding to the maximum of v / r at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the strongest restriction on the time step then arises just before the formation of a horizon , where v / r at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. this infinite redshift provides a mechanism for locating the true event horizon on the fly and restricting the evolution to the exterior spacetime . points near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } must be dropped in order to evolve across the horizon due to the lack of a nonsingular compactified version of future time infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${i^ + } $ \end{document}. the situation is quite different in a double null coordinate system , in which the vertices of the null parallelogram can be placed exactly on grid points so that the cfl condition is automatically satisfied . a characteristic code based upon double null coordinates was developed by goldwirth and piran in a study of cosmic censorship based upon the spherically symmetric gravitational collapse of a massless scalar field . their early study lacked the sensitivity of adaptive mesh refinement ( amr ) which later enabled choptuik to discover the critical phenomena appearing in this problem . subsequent work by marsa and choptuik combined the use of the null related ingoing eddington - finklestein coordinates with unruh s strategy of singularity excision to construct a 1d code that runs forever . later , garfinkle constructed an improved version of the goldwirth - piran double null code which was able to simulate critical phenomena without using adaptive mesh refinement . in this treatment , as the evolution proceeds on one outgoing null cone to the next , the grid points follow the ingoing null cones and must be dropped as they cross the origin at r = 0 . however , after half the grid points are lost they are then recycled at new positions midway between the remaining grid points . this technique is crucial for resolving the critical phenomena associated with an r 0 size horizon . an extension of the code was later used to verify that scalar field collapse in six dimensions continues to display critical phenomena . hamad and stewart also applied a double null code to study critical phenomena . in order to obtain the accuracy necessary to confirm choptuik s results they developed the first example of a characteristic grid with amr . they did this with both the standard berger and oliger algorithm and their own simplified version , with both versions giving indistinguishable results . their simulations of critical collapse of a massless scalar field agreed with choptuik s values for the universal parameters governing mass scaling and displayed the echoing associated with discrete self - similarity . hamad , horne , and stewart extended this study to the spherical collapse of an axion / dilaton system and found in this case that self - similarity was a continuous symmetry of the critical solution . brady , chambers , and goncalves used garfinkle s double null algorithm to investigate the effect of a massive scalar field on critical phenomena . the introduction of a mass term in the scalar wave equation introduces a scale to the problem , which suggests that the critical point behavior might differ from the massless case . they found that there are two different regimes depending on the ratio of the compton wavelength 1/m of the scalar mass to the radial size of the scalar pulse used to induce collapse . when m 1 , the critical solution is the one found by choptuik in the m = 0 case , corresponding to a type ii phase transition . however , when m 1 , the critical solution is an unstable soliton star ( see ) , corresponding to a type i phase transition where black hole formation turns on at a finite mass . a code based upon bondi coordinates , developed by husa and his collaborators , has been successfully applied to spherically symmetric critical collapse of a nonlinear -model coupled to gravity . critical phenomena can not be resolved on a static grid based upon the bondi r - coordinate . instead , the numerical techniques of garfinkle were adopted by using a dynamic grid following the ingoing null rays and by recycling radial grid points . they studied how coupling to gravity affects the critical behavior previously observed by bizo and others in the minkowski space version of the model . for a wide range of the coupling constant , they observe discrete self - similarity and typical mass scaling near the critical solution . the code is shown to be second order accurate and to give second order convergence for the value of the critical parameter . the first characteristic code in bondi coordinates for the self - gravitating scalar wave problem was constructed by gomez and winicour . they introduced a numerical compactification of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } for the purpose of studying effects of self - gravity on the scalar radiation , particularly in the high amplitude limit of the rescaling . as a , the red shift creates an effective boundary layer at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } which causes the bondi mass mb and the scalar field monopole moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q$\end{document } to be related by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_b } \sim \pi \vert q\vert/\sqrt 2$\end{document } , rather than the quadratic relation of the weak field limit . this could also be established analytically so that the high amplitude limit provided a check on the code s ability to handle strongly nonlinear fields . in the small amplitude case , this work incorrectly reported that the radiation tails from black hole formation had an exponential decay characteristic of quasinormal modes rather than the polynomial 1/t or 1/t falloff expected from price s work on perturbations of schwarzschild black holes . in hindsight , the error here was not having confidence to run the code sufficiently long to see the proper late time behavior . gundlach , price , and pullin [ 113 , 114 ] subsequently reexamined the issue of power law tails using a double null code similar to that developed by goldwirth and piran . their numerical simulations verified the existence of power law tails in the full nonlinear case , thus establishing consistency with analytic perturbative theory . they also found normal mode ringing at intermediate time , which provided reassuring consistency with perturbation theory and showed that there is a region of spacetime where the results of linearized theory are remarkably reliable even though highly nonlinear behavior is taking place elsewhere . these results have led to a methodology that has application beyond the confines of spherically symmetric problems , most notably in the the study of the radiation tail decay of a scalar field was subsequently extended by gmez , schmidt , and winicour using a characteristic code . they showed that the newman - penrose constant for the scalar field determines the exponent of the power law ( and not the static monopole moment as often stated ) . when this constant is non - zero , the tail decays as 1/t on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , as opposed to the 1/t decay for the vanishing case . ( they also found t log t corrections , in addition to the exponentially decaying contributions of the quasinormal modes . ) this code was also used to study the instability of a topological kink in the configuration of the scalar field . the kink instability provides the simplest example of the turning point instability [ 135 , 197 ] which underlies gravitational collapse of static equilibria . brady and smith have demonstrated that characteristic evolution is especially well adapted to explore properties of cauchy horizons . they examined the stability of the reissner - nordstrm cauchy horizon using an einstein - klein - gordon code based upon advanced bondi coordinates ( v , r ) ( where the hypersurfaces v = const are ingoing null hypersurfaces ) . they study the effect of a spherically symmetric scalar pulse on the spacetime structure as it propagates across the event horizon . their numerical methods are patterned after the work of goldwirth and piran , with modifications of the radial grid structure that allow deep penetration inside the black hole . in accord with expectations from analytic studies , they find that the pulse first induces a weak null singularity on the cauchy horizon , which then leads to a crushing spacelike singularity as r 0 . the null singularity is weak in the sense that an infalling observer experiences a finite tidal force , although the newman - penrose weyl component 2 diverges , a phenomenon known as mass inflation . these results confirm the earlier result of gnedin and gnedin that a central spacelike singularity would be created by the interaction of a charged black hole with a scalar field , in accord with a physical argument by penrose that a small perturbation undergoes an infinite redshift as it approaches the cauchy horizon . burko has confirmed and extended these results , using a code based upon double null coordinates which was developed with ori in a study of tail decay . he found that in the early stages the perturbation of the cauchy horizon is weak and in agreement with the behavior calculated by perturbation theory . brady , chambers , krivan , and laguna have found interesting effects of a non - zero cosmo - logical constant on tail decay by using a characteristic einstein - klein - gordon code to study the effect of a massless scalar pulse on schwarzschild - de sitter and reissner - nordstrom - de sitter spacetimes . first , by constructing a linearized scalar evolution code , they show that scalar test fields with 0 have exponentially decaying tails , in contrast to the standard power law tails for asymptotically flat spacetimes . rather than decaying , the monopole mode asymptotes at late time to a constant , which scales linearly with , in contrast to the standard no - hair result . this unusual behavior for the = 0 case was then independently confirmed with a nonlinear spherical characteristic code . using a combination of numerical and analytic techniques based upon null coordinates , hod and piran have made an extensive series of investigations of the spherically symmetric charged einstein - klein - gordon system dealing with the effect of charge on critical gravitational collapse and the late time tail decay of a charged scalar field on a reissner - nordstrm black hole [ 126 , 129 , 127 , 128 ] . these studies culminated in a full nonlinear investigation of horizon formation by the collapse of a charged massless scalar pulse . they track the formation of an apparent horizon which is followed by a weakly singular cauchy horizon which develops a strong spacelike singularity at r = 0 . this is in complete accord with prior perturbative results and nonlinear simulations involving a pre - existing black hole . oren and piran increased the late time accuracy of this study by incorporating an adaptive grid for the retarded time coordinate u , with a refinement criterion to maintain r / r = const . the accuracy of this scheme is confirmed through convergence tests as well as charge and constraint conservation . they were able to observe the physical mechanism which prohibits black hole formation with charge to mass ration q / m > 1 . electrostatic repulsion of the outer parts of the scalar pulse increases relative to the gravitational attraction and causes the outer portion of the charge to disperse to larger radii before the black hole is formed . inside the black hole , they confirm the formation of a weakly singular cauchy horizon which turns into a strong spacelike singularity , in accord with other studies . hod extended this combined numerical - analytical double null approach to investigate higher order corrections to the dominant power law tail , as well as corrections due to a general spherically symmetric scattering potential and due to a time dependent potential . he found ( log t)/t modifications to the leading order tail behavior for a schwarzschild black hole , in accord with earlier results of gmez et al . . these modifications fall off at a slow rate so that a very long numerical evolution ( t 3000 m ) is necessary to cleanly identify the leading order power law decay . the foregoing numerical - analytical work based upon characteristic evolution has contributed to a very comprehensive classical treatment of spherically symmetric gravitational collapse . sorkin and piran have investigated the question of quantum corrections due to pair creation on the gravitational collapse of a charged scalar field . for observers outside the black hole , several analytic studies have indicated that such pair - production can rapidly diminish the charge of the black hole . sorkin and piran apply the same double - null characteristic code used in studying the classical problem to evolve across the event horizon and observe the quantum effects on the cauchy horizon . the quantum electrodynamic effects are modeled in a rudimentary way by a nonlinear dielectric constant that limits the electric field to the critical value necessary for pair creation . the back - reaction of the pairs on the stress - energy and the electric current are ignored . they found that quantum effects leave the classical picture of the cauchy horizon qualitatively intact but that they shorten its lifetime by hastening the conversion of the weak null singularity into a strong spacelike singularity . the southampton group has constructed a { 1 + 1}-dimensional characteristic code for spacetimes with cylindrical symmetry [ 67 , 76 ] . the original motivation was to use it as the exterior characteristic code in a test case of ccm ( see section 4.4.1 for the application to matching ) . subsequently , sperhake , sjdin , and vickers [ 198 , 199 ] modified the code into a global characteristic version for the purpose of studying cosmic strings , represented by massive scalar and vector fields coupled to gravity . using a geroch decomposition with respect to the translational killing vector , they reduced the global problem to a { 2 + 1}-dimensional asymptotically flat spacetime , so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } can be compactified and included in the numerical grid . rather than the explicit scheme used in ccm , the new version employs an implicit , second order in space and time , crank - nicholson evolution scheme . the code showed long term stability and second order convergence in vacuum tests based upon exact weber - wheeler waves and xanthopoulos rotating solution , and in tests of wave scattering by a string . the results show damped ringing of the string after an incoming weber - wheeler pulse has excited it and then scattered to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the ringing frequencies are independent of the details of the pulse but are inversely proportional to the masses of the scalar and vector fields . the goal of computing waveforms from relativistic binaries , such as a neutron star spiraling into a black hole , requires more than a stable convergent code . it is a delicate task to extract a waveform in a spacetime in which there are multiple length scales : the size of the black hole , the size of the star , the wavelength of the radiation . it is commonly agreed that some form of mesh refinement is essential to attack this problem . mesh refinement was first applied in characteristic evolution to solve specific spherically symmetric problems regarding critical phenomena and singularity structure [ 90 , 118 , 51 ] . pretorius and lehner have presented a general approach for amr to a generic characteristic code . although the method is designed to treat 3d simulations , the implementation has so far been restricted to the einstein - klein - gordon system in spherical symmetry . the 3d approach is modeled after the berger and oliger amr algorithm for hyperbolic cauchy problems , which is reformulated in terms of null coordinates . the resulting characteristic amr algorithm can be applied to any unigrid characteristic code and is amenable to parallelization . they applied it to the problem of a massive klein - gordon field propagating outward from a black hole . the non - zero rest mass restricts the klein - gordon field from propagating to infinity . instead it diffuses into higher frequency components which pretorius and lehner show can be resolved using amr but not with a comparison unigrid code . one - dimensional characteristic codes enjoy a very special simplicity due to the two preferred sets ( ingoing and outgoing ) of characteristic null hypersurfaces . this eliminates a source of gauge freedom that otherwise exists in either two- or three - dimensional characteristic codes . however , the manner in which the characteristics of a hyperbolic system determine domains of dependence and lead to propagation equations for shock waves is the same as in the one - dimensional case . this makes it desirable for the purpose of numerical evolution to enforce propagation along characteristics as extensively as possible . in basing a cauchy algorithm upon shooting along characteristics , the infinity of characteristic rays ( technically , bicharacteristics ) at each point leads to an arbitrariness which , for a practical numerical scheme , makes it necessary either to average the propagation equations over the sphere of characteristic directions or to select out some preferred subset of propagation equations . the latter approach was successfully applied by butler to the cauchy evolution of two - dimensional fluid flow , but there seems to have been very little follow - up along these lines . the formal ideas behind the construction of two- or three - dimensional characteristic codes are similar , although there are various technical options for treating the angular coordinates which label the null rays . historically , most characteristic work graduated first from 1d to 2d because of the available computing power . the first characteristic code based upon the original bondi equations for a twist - free axisymmetric spacetime was constructed by isaacson , welling , and winicour at pittsburgh . the spacetime was foliated by a family of null cones , complete with point vertices at which regularity conditions were imposed . this allowed studies of the bondi mass and radiation flux on the initial null cone , but it could not be used as a practical evolution code because of instabilities . these instabilities came as a rude shock and led to a retreat to the simpler problem of axisymmetric scalar waves propagating in minkowski space , with the metric 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - d{u^2 } - 2du\;dr + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2})$$\end{document } in outgoing null cone coordinates . a null cone code for this problem was constructed using an algorithm based upon equation ( 8) , with the angular part of the flat space laplacian replacing the curvature terms in the integrand on the right hand side . this simple setting allowed one source of instability to be traced to a subtle violation of the cfl condition near the vertices of the cones . in terms of the grid spacing x , the cfl condition in this coordinate system takes the explicit form 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta u } \over { \delta r } } < - 1 + { [ { k^2 } + { ( k - 1)^2 } - 2k(k - 1)\cos \delta \theta ] ^{1/2}},$$\end{document } where the coefficient k , which is of order 1 , depends on the particular startup procedure adopted for the outward integration . far from the vertex , the condition ( 10 ) on the time step u is quantitatively similar to the cfl condition for a standard cauchy evolution algorithm in spherical coordinates . but condition ( 10 ) is strongest near the vertex of the cone where ( at the equator = /2 ) it implies that 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta u < k\delta r{(\delta \theta)^2}.$$\end{document } this is in contrast to the analogous requirement 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta u < k\;\delta r\;\delta \theta$$\end{document } for stable cauchy evolution near the origin of a spherical coordinate system . the extra power of is the price that must be paid near the vertex for the simplicity of a characteristic code . nevertheless , the enforcement of this condition allowed efficient global simulation of axisymmetric scalar waves . global studies of backscattering , radiative tail decay , and solitons were carried out for nonlinear axisymmetric waves , but three - dimensional simulations extending to the vertices of the cones were impractical at the time on existing machines . aware now of the subtleties of the cfl condition near the vertices , the pittsburgh group returned to the bondi problem , i.e. to evolve the bondi metric 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = \left({{v \over r}{e^{2\beta } } - { u^2}{r^2}{e^{2\gamma } } } \right)d{u^2 } + 2{e^{2\beta}}du\;dr + 2u{r^2}{e^{2\gamma}}du\;d\theta - { r^2}({e^{2\gamma}}d{\theta ^2 } + { e^{- 2\gamma}}{\sin ^2}\theta d{\phi ^2}),$$\end{document } by means of the three hypersurface equations 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { , r } } = { 1 \over 2}r{(\gamma , r)^2},$$\end{document } 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left [ { { r^4}{e^{2(\gamma - \beta)}}{u_{,r } } } \right]_{,r } } = 2{r^2}\left [ { { r^2}{{\left({{\beta \over { { r^2 } } } } \right)}_{,r\theta } } - { { { { ( { { \sin}^2}\theta \,\gamma)}_{,r\theta } } } \over { { { \sin}^2}\theta } } + 2{\gamma _ { , r}}{\gamma _ { , \theta } } } \right],$$\end{document } 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { v_{,r } } = - { 1 \over 4}{r^4}{e^{2(\gamma - \beta)}}{{({u_{,r}})}^2 } + { { { { ( { r^4}\sin \theta u)}_{,r\theta } } } \over { 2{r^2}\sin \theta}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { e^{2(\beta - \gamma)}}\left [ { 1 - { { { { ( \sin \theta \,{\beta _ { , \theta}})}_{,\theta } } } \over { \sin \theta } } + { \gamma _ { , \theta \theta } } + 3\cot \theta { \gamma _ { , \theta } } - { { ( { \beta _ { , \theta}})}^2 } - 2{\gamma _ { , \theta}}({\gamma _ { , \theta } } - { \beta _ { , \theta } } ) } \right ] } , \\ \end{array}$$\end{document } and the evolution equation 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{4r{{(r\gamma)}_{,ur}}{{\left\{{2r{\gamma _ { , r}}\;v - { r^2}\left [ { 2{\gamma _ { , \theta}}u + \sin \theta { { \left({{u \over { \sin \theta } } } \right)}_{,\theta } } } \right ] } \right\}}_{,r } } - 2{r^2}{{{{({\gamma _ { , r}}u\sin \theta)}_{,\theta } } } \over { \sin \theta } } } \\ { \quad \quad \quad \quad + { 1 \over 2}{r^4}{e^{2(\gamma - \beta)}}{{({u_{,r}})}^2 } + 2{e^{2(\beta - \gamma)}}\left [ { { { ( { \beta _ { , \theta}})}^2 } + \sin \theta { { \left({{{{\beta _ { , \theta } } } \over { \sin \theta } } } \right)}_{,\theta } } } \right ] . } \\ \end{array}$$\end{document } the beauty of the bondi equations is that they form a clean hierarchy . given on an initial null hypersurface , the equations can be integrated radially to determine , u , v , and ,u on the hypersurface ( in that order ) in terms of integration constants determined by boundary conditions , or smoothness if extended to the vertex of a null cone . the initial data is unconstrained except by smoothness conditions . because represents an axisymmetric spin-2 field , it must be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\sin ^2}\theta)$\end{document } near the poles of the spherical coordinates and must consist of l 2 spin-2 multipoles . in the computational implementation of this system by the pittsburgh group , the null hy - persurfaces were chosen to be complete null cones with nonsingular vertices , which ( for simplicity ) trace out a geodesic worldline r = 0 . the vertices of the cones were not the chief source of difficulty . a null parallelogram marching algorithm , similar to that used in the scalar case , gave rise to another instability that sprang up throughout the grid . in order to reveal the source of this instability , physical considerations suggested looking at the linearized version of the bondi equations , where they can be related to the wave equation . if this relationship were sufficiently simple , then the scalar wave algorithm could be used as a guide in stabilizing the evolution of . a scheme for relating to solutions of the wave equation had been formulated in the original paper by bondi , metzner , and van der burgh . however , in that scheme , the relationship of the scalar wave to was nonlocal in the angular directions and was not useful for the stability analysis . a local relationship between and solutions of the wave equation was found . this provided a test bed for the null evolution algorithm similar to the cauchy test bed provided by teukolsky waves . more critically , it allowed a simple von neumann linear stability analysis of the finite difference equations , which revealed that the evolution would be unstable if the metric quantity u was evaluated on the grid . for a stable algorithm , the grid points for u must be staggered between the grid points for , , and v. this unexpected feature emphasizes the value of linear stability analysis in formulating stable finite difference approximations . it led to an axisymmetric code [ 165 , 106 ] for the global bondi problem which ran stably , subject to a cfl condition , throughout the regime in which caustics and horizons did not form . stability in this regime was verified experimentally by running arbitrary initial data until it radiated away to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. also , new exact solutions as well as the linearized null solutions were used to perform extensive convergence tests that established second order accuracy . the code generated a large complement of highly accurate numerical solutions for the class of asymptotically flat , axisymmetric vacuum spacetimes , a class for which no analytic solutions are known . all results of numerical evolutions in this regime were consistent with the theorem of christodoulou and klainerman that weak initial data evolve asymptotically to minkowski space at late time . an additional global check on accuracy was performed using bondi s formula relating mass loss to the time integral of the square of the news function . the bondi mass loss formula is not one of the equations used in the evolution algorithm but follows from those equations as a consequence of a global integration of the bianchi identities . thus it not only furnishes a valuable tool for physical interpretation but it also provides a very important calibration of numerical accuracy and consistency . an interesting feature of the evolution arises in regard to compactification . by construction , the u - direction is timelike at the origin where it coincides with the worldline traced out by the vertices of the outgoing null cones . but even for weak fields , the u - direction generically becomes spacelike at large distances along an outgoing ray . geometrically , this reflects the property that i is itself a null hypersurface so that all internal directions are spacelike , except for the null generator . for a flat space time , the u - direction picked out at the origin leads to a null evolution direction at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } , but this direction becomes spacelike under a slight deviation from spherical symmetry . thus the evolution generically becomes superluminal near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document}. remarkably , this leads to no adverse numerical effects . this fortuitous property apparently arises from the natural way that causality is built into the marching algorithm so that no additional resort to numerical techniques , such as causal differencing , is necessary . stewart has implemented a characteristic evolution code which handles the bondi problem by a null tetrad , as opposed to metric , formalism . the geometrical algorithm underlying the evolution scheme , as outlined in [ 205 , 87 ] , is friedrich s conformal - null description of a compactified spacetime in terms of a first order system of partial differential equations . the variables include the metric , the connection , and the curvature , as in a newman - penrose formalism , but in addition the conformal factor ( necessary for compactification of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } ) and its gradient . without assuming any symmetry , there are more than 7 times as many variables as in a metric based null scheme , and the corresponding equations do not decompose into as clean a hierarchy . this disadvantage , compared to the metric approach , is balanced by several advantages : the equations form a symmetric hyperbolic system so that standard theorems can be used to establish that the system is well-posed.standard evolution algorithms can be invoked to ensure numerical stability.the extra variables associated with the curvature tensor are not completely excess baggage , since they supply essential physical information.the regularization necessary to treat \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } is built in as part of the formalism so that no special numerical regularization techniques are necessary as in the metric case . ( this last advantage is somewhat offset by the necessity of having to locate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } by tracking the zeroes of the conformal factor . ) the equations form a symmetric hyperbolic system so that standard theorems can be used to establish that the system is well - posed . the extra variables associated with the curvature tensor are not completely excess baggage , since they supply essential physical information . the regularization necessary to treat \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } is built in as part of the formalism so that no special numerical regularization techniques are necessary as in the metric case . ( this last advantage is somewhat offset by the necessity of having to locate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } by tracking the zeroes of the conformal factor . ) the code was intended to study gravitational waves from an axisymmetric star . since only the vacuum equations are evolved , the outgoing radiation from the star is represented by data ( 4 in newman - penrose notation ) on an ingoing null cone forming the inner boundary of the evolved domain . the inner boundary data is supplemented by schwarzschild data on the initial outgoing null cone , which models an initially quiescent state of the star . but by choosing a scenario in which a black hole is formed , it is possible to evolve the entire region exterior to the horizon . an obvious test bed is the schwarzschild spacetime for which a numerically satisfactory evolution was achieved ( although convergence tests were not reported ) . physically interesting results were obtained by choosing data corresponding to an outgoing quadrupole pulse of radiation . by increasing the initial amplitude of the data 4 , it was possible to evolve into a regime where the energy loss due to radiation was large enough to drive the total bondi mass negative . although such data is too grossly exaggerated to be consistent with an astrophysically realistic source , the formation of a negative mass was an impressive test of the robustness of the code . papadopoulos has carried out an illuminating study of mode mixing by computing the evolution of a pulse emanating outward from an initially schwarzschild white hole of mass m. the evolution proceeds along a family of ingoing null hypersurfaces with outer boundary at r = 60 m. the evolution is stopped before the pulse hits the outer boundary in order to avoid spurious effects from reflection and the radiation is inferred from data at r = 20 m. although gauge ambiguities arise in reading off the waveform at a finite radius , the work reveals interesting nonlinear effects : ( i ) modification of the light cone structure governing the principal part of the equations and hence the propagation of signals ; ( ii ) modulation of the schwarzschild potential by the introduction of an angular dependent mass aspect ; and ( iii ) quadratic and higher order terms in the evolution equations which couple the spherical harmonic modes . a compactified version of this study was later carried out with the 3d pitt code , which confirms these effects as well as new effects which are not present in the axisymmetric case ( see section 3.8 for details ) . the southampton group , as part of its goal of combining cauchy and characteristic evolution , has developed a code [ 74 , 75 , 172 ] which extends the bondi problem to full axisymmetry , as described by the general characteristic formalism of sachs . by dropping the requirement that the rotational killing vector be twist - free , they were able to include rotational effects , including radiation in the cross polarization mode ( only the plus mode is allowed by twist - free axisymmetry ) . the null equations and variables were recast into a suitably regularized form to allow compactification of null infinity . regularization at the vertices or caustics of the null hypersurfaces was not necessary , since they anticipated matching to an interior cauchy evolution across a finite worldtube . the code was designed to insure standard bondi coordinate conditions at infinity , so that the metric has the asymptotically minkowskian form corresponding to null - spherical coordinates . in order to achieve this , the hypersurface equation for the bondi metric variable must be integrated radially inward from infinity , where the integration constant is specified . this differs from the pittsburgh code in which all the equations are integrated radially outward , so that the coordinate conditions are determined at the inner boundary and the metric is asymptotically flat but not asymptotically minkowskian . the southampton scheme simplifies the formulae for the bondi news function and mass in terms of the metric . it is anticipated that the inward integration of causes no numerical problems because this is a gauge choice which does not propagate physical information . however , the code has not yet been subject to convergence and long term stability tests so that these issues can not be properly assessed at the present time . the matching of the southampton axisymmetric code to a cauchy interior is discussed in section 4.5 . numerical calculations of asymptotic quantities such as the bondi mass must pick off non - leading terms in an asymptotic expansion about infinity . this is similar to the experimental task of determining the mass of an object by measuring its far field . for example , in an asymptotically inertial frame ( called a standard bondi frame at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } ) , the mass aspect \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}(u,\theta , \phi)$\end{document } is picked off from the asymptotic expansion of bondi s metric quantity v ( see equation ( 16 ) ) of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v = r - 2{\mathcal m } + { \mathcal o}(1/r)$\end{document}. in gauges which incorporate some of the properties of an asymptotically inertial frame , such as the null quasi - spherical gauge in which the angular metric is conformal to the unit sphere metric , this can be a straightforward computational problem . however , the job can be more difficult if the gauge does not correspond to a standard bondi frame at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. one must then deal with an arbitrary coordinatization of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } which is determined by the details of the interior geometry . as a result , v has a more complicated asymptotic behavior , given in the axisymmetric case by 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{v - r = { { { r^2}{{(l\sin \theta)}_{,\theta } } } \over { \sin \theta } } + r{e^{2(h - k ) } } \times } \\ { \quad \quad \quad \left [ { \left({1 - { e^{- 2(h - k ) } } } \right ) + { { 2{{({h_{,\theta}}\sin \theta)}_{,\theta } } } \over { \sin \theta } } + { k_{,\theta \theta } } + 3{k_{,\theta}}\cot \theta + 4{{({h_{,\theta}})}^2 } - 4{h_{,\theta}}{k_{,\theta } } - 2{{({k_{,\theta}})}^2 } } \right ] } \\ { \quad \quad \quad - 2{e^{2h}}m + o({r^{- 1 } } ) , } \\ \end{array}$$\end{document } where l , h , and k are gauge dependent functions of ( u , ) which would vanish in a bondi frame [ 209 , 137 ] . the calculation of the bondi mass requires regularization of this expression by numerical techniques so that the coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } can be picked off . the task is now similar to the experimental determination of the mass of an object by using non - inertial instruments in a far zone which contains \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(1/r)$\end{document } radiation fields . it was accomplished in stewart s code by re - expressing the formula for the bondi mass in terms of the well - behaved fields of the conformal formalism . in the pittsburgh code , it was accomplished by re - expressing the bondi mass in terms of renormalized metric variables which regularize all calculations at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and make them second order accurate in grid size . the calculation of the bondi news function ( which provides the waveforms of both polarization modes ) is an easier numerical task than the bondi mass . it has also been implemented in both of these codes , thus allowing the important check of the bondi mass loss formula . an alternative approach to computing the bondi mass is to adopt a gauge which corresponds more closely to an inertial or bondi frame at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and simplifies the asymptotic limit . such a choice is the null quasi - spherical gauge in which the angular part of the metric is proportional to the unit sphere metric , and as a result the gauge term k vanishes in equation ( 18 ) . this gauge was adopted by bartnik and norton at canberra in their development of a 3d characteristic evolution code ( see section 3.5 for further discussion ) . it allowed accurate computation of the bondi mass as a limit as r of the hawking mass . mainstream astrophysics is couched in newtonian concepts , some of which have no well defined extension to general relativity . in order to provide a sound basis for relativistic astrophysics , it is crucial to develop general relativistic concepts which have well defined and useful newtonian limits . the results of characteristic codes show that the energy of a radiating system can be evaluated rigorously and accurately according to the rules for asymptotically flat spacetimes , while avoiding the deficiencies that plagued the pre - numerical era of relativity : ( i ) the use of coordinate dependent concepts such as gravitational energy - momentum pseudotensors ; ( ii ) a rather loose notion of asymptotic flatness , particularly for radiative spacetimes ; ( iii ) the appearance of divergent integrals ; and ( iv ) the use of approximation formalisms , such as weak field or slow motion expansions , whose errors have not been rigorously estimated . characteristic codes have extended the role of the bondi mass from that of a geometrical construct in the theory of isolated systems to that of a highly accurate computational tool . the bondi mass loss formula provides an important global check on the preservation of the bianchi identities . the numerical results demonstrate that computational approaches , rigorously based upon the geometrical definition of mass in general relativity , can be used to calculate radiation losses in highly nonlinear processes where perturbation calculations would not be meaningful . numerical calculation of the bondi mass has been used to explore both the newtonian and the strong field limits of general relativity . for a quasi - newtonian system of radiating dust , the numerical calculation joins smoothly on to a post - newtonian expansion of the energy in powers of 1/c , beginning with the newtonian mass and mechanical energy as the leading terms . this comparison with perturbation theory has been carried out to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(1/{c^7})$\end{document } , at which stage the computed bondi mass peels away from the post - newtonian expansion . it remains strictly positive , in contrast to the truncated post - newtonian behavior which leads to negative values . a subtle feature of the bondi mass stems from its role as one component of the total energy - momentum 4-vector , whose calculation requires identification of the translation subgroup of the bondi - metzner - sachs group . is tantamount to knowing the conformal transformation to a standard bondi frame in which the time slices of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } have unit sphere geometry . both stewart s code and the pittsburgh code adapt the coordinates to simplify the description of the interior sources . this results in a non - standard foliation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document}. the determination of the conformal factor which relates the 2-metric hab of a slice of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } to the unit sphere metric is an elliptic problem equivalent to solving the second order partial differential equation for the conformal transformation of gaussian curvature . in the axisymmetric case , the pde reduces to an ode with respect to the angle , which is straightforward to solve . stewart proposes an approach based upon the dyad decomposition 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{ab}}d{x^a}d{x^b } = { m_a}d{x^a}{\bar{m } _ b}d{x^b}.$$\end{document } the desired conformal transformation is obtained by first relating hab conformally to the flat metric of the complex plane . denoting the complex coordinate of the plane by , this relationship can be expressed as d = e ma dx . the conformal factor f can then be determined from the integrability condition 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{[{a^\partial}b]}}f = { \partial _ { [ { a^m}b]}}.$$\end{document } this is equivalent to the classic beltrami equation for finding isothermal coordinates . it would appear to be a more effective scheme than tackling the second order pde directly , but numerical implementation has not yet been carried out . there are now two independent 3d codes , one developed at canberra and the other at pittsburgh ( the pitt code ) , which have the capability to study gravitational waves in single black hole spacetimes , at a level still not mastered by cauchy codes . several years ago the pittsburgh group established robust stability and second order accuracy of a fully nonlinear 3d code which calculates waveforms at null infinity [ 42 , 31 ] and also tracks a dynamical black hole and excises its internal singularity from the computational grid [ 105 , 102 ] . the canberra group has implemented an independent nonlinear 3d code which accurately evolves the exterior region of a schwarzschild black hole . both codes pose data on an initial null hypersurface and on a worldtube boundary , and evolve the exterior spacetime out to a compactified version of null infinity , where the waveform is computed . however , there are essential differences in the underlying geometrical formalisms and numerical techniques used in the two codes and in their success in evolving generic black hole spacetimes . the pitt code uses a standard bondi - sachs null coordinate system , 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = -\left({{e^{2\beta}}{v \over r } - { r^2}{h_{ab}}{u^a}{u^b } } \right)d{u^2 } - 2{e^{2\beta}}du\;dr - 2{r^2}{h_{ab}}{u^b}du\;d{x^a } + { r^2}{h_{ab}}d{x^a}d{x^b},$$\end{document } where det(hab ) = det(qab ) for some standard choice qab of the unit sphere metric . this generalizes equation ( 13 ) to the three - dimensional case . the hypersurface equations derive from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_\mu}^v{\nabla _ v}u$\end{document } components of the einstein tensor . they take the explicit form 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { , r } } = { 1 \over { 16}}r\;{h^{ac}}{h^{bd}}{h_{ab , r}}{h_{cd , r}},$$\end{document } 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${({r^4}{e^{- 2\beta}}{h_{ab}}u_{,r}^b)_{,r } } = 2{r^4}{({r^ -}^2{\beta _ { , a}})_{,r } } - { r^2}{h^{bc}}{d_c}({h_{ab , r}})$$\end{document } 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{2{e^{- 2\beta}}{v_{,r } } = { \mathcal r } - 2{d^a}{d_a}\beta - 2({d^a}\beta){d_a}\beta + { r^ -}^2{e^{- 2\beta}}{d_a}\left({{{({r^4}{u^a})}_{,r } } } \right ) } \\{\quad \quad \quad \quad \ ; - { 1 \over 2}{r^4}{e^{- 4\beta}}{h_{ab}}u_{,r}^au_{,r}^b , } \\\end{array}$$\end{document } where da is the covariant derivative and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } the curvature scalar of the conformal 2-metric hab of the r = const . surfaces , and capital latin indices are raised and lowered with hab . given the null data hab on an outgoing null hypersurface , this hierarchy of equations can be integrated radially in order to determine , u and v on the hypersurface in terms of integration constants on an inner boundary . the evolution equations for the u - derivative of the null data derive from the trace - free part of the angular components of the einstein tensor , i.e. the components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h^{ab } } = 2m({a_{\bar m}}b)$\end{document}. they take the explicit form 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{{m^a}{m^b}\left({{{(r{h_{ab , u}})}_{,r } } - { 1 \over { 2r}}{{(rv{h_{ab , r}})}_{,r } } - { 2 \over r}{e^\beta}{d_a}{d_b}{e^\beta } + r{h_{ac}}{d_b}(u_{,r}^c ) } \right . } \\ { \quad \quad \quad - { { { r^3 } } \over 2}{e^{- 2\beta}}{h_{ac}}{h_{bd}}u_{,r}^cu_{,r}^d + 2{d_a}{u_b } + { r \over 2}{h_{ab , r}}{d_c}{u^c } } \\ { \quad \quad \quad + r{u^c}{d_c}({h_{ab , r } } ) + r{h_{ad , r}}{h^{cd}}\left . { ( { d_b}{u_c } - { d_c}{u_b } ) } \right ) = 0 . } \\ \end{array}$$\end{document } the canberra code employs a null quasi - spherical ( nqs ) gauge ( not to be confused with the quasi - spherical approximation in which quadratically aspherical terms are ignored ) . the nqs gauge takes advantage of the possibility of mapping the angular part of the bondi metric conformally onto a unit sphere metric , so that hab qab . the required transformation x y(u , r , x ) is in general dependent upon u and r so that the nqs angular coordinates y are not constant along the outgoing null rays , unlike the bondi - sachs angular coordinates . instead the coordinates y display the analogue of a shift on the null hypersurfaces u = const . the radiation content of the metric is contained in a shear vector describing this shift . this results in the description of the radiation in terms of a spin - weight 1 field , rather than the spin - weight 2 field associated with hab in the bondi - sachs formalism . in both the bondi sachs and nqs gauges , the independent gravitational data on a null hypersurface is the conformal part of its degenerate 3-metric . the bondi - sachs null data consists of hab , which determines the intrinsic conformal metric of the null hypersurface . in the nqs case , hab = qab and the shear vector comprises the only non - trivial part of the conformal 3-metric . both the bondi - sachs and nqs gauges can be arranged to coincide in the special case of shear - free robinson - trautman metrics [ 71 , 20 ] . the formulation of einstein s equations in the nqs gauge is presented in , and the associated gauge freedom arising from ( u , r ) dependent rotation and boosts of the unit sphere is discussed in . as in the pitt code , the main equations involve integrating a hierarchy of hypersurface equations along the radial null geodesics extending from the inner boundary to null infinity . in the nqs gauge the source terms for these radial ode s are rather simple when the unknowns are chosen to be the connection coefficients . however , as a price to pay for this simplicity , after the radial integrations are performed on each null hypersurface a first order elliptic equation must be solved on each r = const . the pitt code is an explicit second order finite difference evolution algorithm based upon retarded time steps on a uniform three - dimensional null coordinate grid . the straightforward numerical approach and the second order convergence of the finite difference equations has facilitated code development . most of these involve smoothing or filtering and have obvious advantage for removing short wavelength noise but would be unsuitable for modeling shocks . both codes require the ability to handle tensor fields and their derivatives on the sphere . spherical coordinates and spherical harmonics are natural analytic tools for the description of radiation , but their implementation in computational work requires dealing with the impossibility of smoothly covering the sphere with a single coordinate grid . polar coordinate singularities in axisymmetric systems can be regularized by standard tricks . in the absence of symmetry , these techniques do not generalize and would be especially prohibitive to develop for tensor fields . a crucial ingredient of the pitt code is the eth - module which incorporates a computational version of the newman - penrose eth - formalism . the eth - module covers the sphere with two overlapping stereographic coordinate grids ( north and south ) . it provides everywhere regular , second order accurate , finite difference expressions for tensor fields on the sphere and their covariant derivatives . the eth - calculus simplifies the underlying equations , avoids spurious coordinate singularities and allows accurate differentiation of tensor fields on the sphere in a computationally efficient and clean way . its main weakness is the numerical noise introduced by interpolations ( fourth order accurate ) between the north and south patches . for parabolic or elliptic equations on the sphere , the finite difference approach of the eth - calculus would be less efficient than a spectral approach , but no parabolic or elliptic equations appear in the bondi - sachs evolution scheme . the canberra code handles fields on the sphere by means of a 3-fold representation : ( i ) as discretized functions on a spherical grid uniformly spaced in standard ( , ) coordinates , ( ii ) as fast - fourier transforms with respect to ( , ) ( based upon a smooth map of the torus onto the sphere ) , and ( iii ) as a spectral decomposition of scalar , vector , and tensor fields in terms of spin - weighted spherical harmonics . the grid values are used in carrying out nonlinear algebraic operations ; the fourier representation is used to calculate ( , )-derivatives ; and the spherical harmonic representation is used to solve global problems , such as the solution of the first order elliptic equation for the reconstruction of the metric , whose unique solution requires pinning down the = 1 gauge freedom . the sizes of the grid and of the fourier and spherical harmonic representations are coordinated . in practice , the spherical harmonic expansion is carried out to 15th order in , but the resulting coefficients must then be projected into the 10 subspace in order to avoid inconsistencies between the spherical harmonic , grid , and fourier representations . the canberra code solves the null hypersurface equations by combining an 8th order runge - kutta integration with a convolution spline to interpolate field values . the radial grid points are dynamically positioned to approximate ingoing null geodesics , a technique originally due to goldwirth and piran to avoid the problems with a uniform r - grid near a horizon which arise from the degeneracy of an areal coordinate on a stationary horizon . the time evolution uses the method of lines with a fourth order runge - kutta integrator , which introduces further high frequency filtering . analytic stability analysis of the finite difference equations has been crucial in the development of a stable evolution algorithm , subject to the standard courant - friedrichs - lewy ( cfl ) condition for an explicit code . linear stability analysis on minkowski and schwarzschild backgrounds showed that certain field variables must be represented on the half - grid [ 106 , 42 ] . nonlinear stability analysis was essential in revealing and curing a mode coupling instability that was not present in the original axisymmetric version of the code [ 31 , 142 ] . this has led to a code whose stability persists even in the regime that the u - direction , along which the grid flows , becomes spacelike , such as outside the velocity of light cone in a rotating coordinate system . robust stability was established by imposing random initial data on the initial characteristic hypersurface and random constraint violating boundary data on an inner worldtube . ( robust stability was later adopted as one of the standardized tests for cauchy codes . ) the pitt code was the first 3d general relativistic code to pass this robust stability test . the use of random data is only possible in sufficiently weak cases where effective energy terms quadratic in the field gradients are not dominant . . runs for a time of 60,000 m were carried out for a moving , distorted schwarzschild black hole ( of mass m ) , with the marginally trapped surface at the inner boundary tracked and its interior excised from the computational grid [ 102 , 103 ] . this remains one of the longest simulations of a dynamic black hole carried out to date . furthermore , the scattering of a gravitational wave off a schwarzschild black hole was successfully carried out in the extreme nonlinear regime where the backscattered bondi news was as large as n = 400 ( in dimensionless geometric units ) , showing that the code can cope with the enormous power output nc / g 10 w in conventional units . this exceeds the power that would be produced if , in 1 second , the entire galaxy were converted to gravitational radiation . analytic stability analysis of the underlying finite difference equations is impractical because of the extensive mix of spectral techniques , higher order methods , and splines . although there is no clear - cut cfl limit on the code , stability tests show that there is a limit on the time step . the damping of high frequency modes due to the implicit filtering would be expected to suppress numerical instability , but the stability of the canberra code is nevertheless subject to two qualifications [ 22 , 23 , 24 ] : ( i ) at late times ( less than 100 m ) , the evolution terminates as it approaches an event horizon , apparently because of a breakdown of the nqs gauge condition , although an analysis of how and why this should occur has not yet been given . ( ii ) numerical instabilities arise from dynamic inner boundary conditions and restrict the inner boundary to a fixed schwarzschild horizon . second order accuracy has been verified in an extensive number of testbeds [ 42 , 31 , 102 , 228 , 229 ] , including new exact solutions specifically constructed in null coordinates for the purpose of convergence tests : linearized waves on a minkowski background in null cone coordinates.boost and rotation symmetric solutions .schwarzschild in rotating coordinates.polarization symmetry of nonlinear twist - free axisymmetric waveforms.robinson-trautman waveforms from perturbed schwarzschild black holes.nonlinear robinson - trautman waveforms utilizing an independently computed solution of the robinson - trautman equation.perturbations of a schwarzschild black hole utilizing an independent computed solution of the teukolsky equation . linearized waves on a minkowski background in null cone coordinates . nonlinear robinson - trautman waveforms utilizing an independently computed solution of the robinson - trautman equation . perturbations of a schwarzschild black hole utilizing an independent computed solution of the teukolsky equation . the complexity of the algorithm and nqs gauge makes it problematic to establish accuracy by direct means . exact solutions do not provide an effective convergence check , because the schwarzschild solution is trivial in the nqs gauge and other known solutions in this gauge require dynamic inner boundary conditions which destabilize the present version of the code . instead indirect tests by means of geometric consistency and partial convergence tests are used to calibrate accuracy . the consistency tests were based on the constraint equations , which are not enforced during null evolution except at the inner boundary . the balance between mass loss and radiation flux through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } is a global consequence of these constraints . no appreciable growth of the constraints was noticeable until within 5 m of the final breakdown of the code . in weak field tests where angular resolution does not dominate the error , partial convergence tests based upon varying the radial grid size verify the 8th order convergence in the shear expected from the runge - kutta integration and splines . when the radial source of error is small , reduced error with smaller time step can also be discerned . in practical runs , the major source of inaccuracy is the spherical harmonic resolution , which was restricted to 15 by hardware limitations . truncation of the spherical harmonic expansion has the effect of modifying the equations to a system for which the constraints are no longer satisfied . the pitt code was originally formulated in the second differential form of equations ( 22 , 23 , 24 , 25 ) , which in the spin - weighted version leads to an economical number of 2 real and 2 complex variables . subsequently , the variable 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_a } = { r^2}{e^{- 2\beta}}{h_{ab}}u_{,r}^b$$\end{document } was introduced to reduce equation ( 23 ) to two first order radial equations , which simplified the startup procedure at the initial boundary . although the resulting code has been verified to be stable and second order accurate , its application to increasingly difficult problems involving strong fields , and gradients have led to numerical errors that make important physical effects hard to measure . in particular , in initial attempts to simulate a white hole fission , gmez encountered an oscillatory error pattern in the angular directions near the time of fission . the origin of the problem was tracked to numerical error of an oscillatory nature introduced by terms in the hypersurface and evolution equations . gmez solution was to remove the offending second angular derivatives by introducing additional variables and reducing the system to first differential order in the angular directions . this suppressed the oscillatory mode and subsequently improved performance in the simulation of the white hole fission problem ( see section 3.7.2 ) . this success opens the issue of whether a completely first differential order code might perform even better , as has been proposed by gmez and frittelli . they gave a first order quasi - linear formulation of the bondi system which , at the analytic level , obeys a standard uniqueness and existence theorem ( extending previous work for the linearized case ) ; and they point out , at the numerical level , that a first order code could also benefit from the applicability of standard numerical techniques . the part of the code in which the operator introduced the oscillatory error mode was not identified in , i.e. whether it originated in the inner boundary treatment or in the interpolations between stereographic patches where second derivatives might be troublesome . there are other possible ways to remove the oscillatory angular modes , such as adding angular dissipation or using more accurate methods of patching the sphere . the economy of variables in the original bondi scheme should not be abandoned without further tests and investigation . a natural physical application of a characteristic evolution code is the nonlinear version of the classic problem of scattering off a schwarzschild black hole , first solved perturbatively by price . here the inner worldtube for the characteristic initial value problem consists of the ingoing branch of the r = 2 m hypersurface ( the past horizon ) , where schwarzschild data are prescribed . the nonlinear problem of a gravitational wave scattering off a schwarzschild black hole is then posed in terms of data on an outgoing null cone which describe an incoming pulse with compact support . part of the energy of this pulse falls into the black hole and part is backscattered to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. this problem has been investigated using both the pitt and canberra codes . the pittsburgh group studied the backscattered waveform ( described by the bondi news function ) as a function of incoming pulse amplitude . the computational eth - module smoothly handled the complicated time dependent transformation between the non - inertial computational frame at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and the inertial ( bondi ) frame necessary to obtain the standard plus and cross polarization modes . in the perturbative regime , the news corresponds to the backscattering of the incoming pulse off the effective schwarzschild potential . when the energy of the pulse is no larger than the central schwarzschild mass , the backscattered waveform still depends roughly linearly on the amplitude of the incoming pulse . its amplitude is greater than that predicted by linear scaling and its shape drastically changes and exhibits extra oscillations . in this very high amplitude case , the mass of the system is completely dominated by the incoming pulse , which essentially backscatters off itself in a nonlinear way . the canberra code was used to study the change in bondi mass due to the radiation . the hawking mass mh(u , r ) was calculated as a function of radius and retarded time , with the bondi mass mb(u ) then obtained by taking the limit r . the limit had good numerical behavior . for a strong initial pulse with = 4 angular dependence , in a run from u = 0 to u = 70 ( in units where the interior schwarzschild mass is 1 ) , the bondi mass dropped from 1.8 to 1.00002 , showing that almost half of the initial energy of the system was backscattered and that a surprisingly negligible amount of energy fell into the black hole . a possible explanation is that the truncation of the spherical harmonic expansion cuts off wavelengths small enough to effectively penetrate the horizon . the bondi mass decreased monotonically in time , as necessary theoretically , but its rate of change exhibited an interesting pulsing behavior whose time scale could not be obviously explained in terms of quasinormal oscillations . the bondi mass loss formula was confirmed with relative error of less than 10 . this is impressive accuracy considering the potential sources of numerical error introduced by taking the limit of the hawking mass with limited resolution . the code was also used to study the appearance of logarithmic terms in the asymptotic expansion of the weyl tensor . in addition , the canberra group studied the effect of the initial pulse amplitude on the waveform of the backscattered radiation , but did not extend their study to the very high amplitude regime in which qualitatively interesting nonlinear effects occur . the pitt code has also been implemented to evolve along an advanced time foliation by ingoing null cones , with data given on a worldtube at their outer boundary and on the initial ingoing null cone . the code was used to evolve a black hole in the region interior to the worldtube by implementing a horizon finder to locate the marginally trapped surface ( mts ) on the ingoing cones and excising its singular interior . the code tracks the motion of the mts and measures its area during the evolution . data at the outer worldtube was induced from a schwarzschild or kerr spacetime but the worldtube was allowed to move relative to the stationary trajectories ; i.e. with respect to the grid the worldtube is fixed but the black hole moves inside it . the initial null data consisted of a pulse of radiation which subsequently travels outward to the worldtube where it reflects back toward the black hole . the approach of the system to equilibrium was monitored by the area of the mts , which also equals its hawking mass . when the worldtube is stationary ( static or rotating in place ) , the distorted black hole inside evolved to equilibrium with the boundary . a boost or other motion of the worldtube with respect to the black hole did not affect this result . the marginally trapped surface always reached equilibrium with the outer boundary , confirming that the motion of the boundary was pure gauge . forever even when the worldtube wobbles with respect to the black hole to produce artificial periodic time dependence . an initially distorted , wobbling black hole was evolved for a time of 60 , 000 m , longer by orders of magnitude than permitted by the stability of other existing 3d black hole codes at the time . this exceptional performance opens a promising new approach to handle the inner boundary condition for cauchy evolution of black holes by the matching methods reviewed in section 4 . note that setting the pulse to zero is equivalent to prescribing shear free data on the initial null cone . combined with schwarzschild boundary data on the outer world tube however , the evolution of such shear free null data combined with kerr boundary data would have an initial transient phase before settling down to a kerr black hole . this is because the twist of the shear - free kerr null congruence implies that kerr data specified on a null hypersurface are not generally shear free . the event horizon is an exception but kerr null data on other null hypersurfaces have not been cast in explicit analytic form . ( curiously , kerr data on a null hypersurface with a conical type singularity do take a simple analytic form , although unsuitable for numerical evolution . ) using some intermediate analytic results of israel and pretorius , venter and bishop have recently constructed a numerical algorithm for transforming the kerr solution into bondi coordinates and in that way provide the necessary null data numerically . an important application of characteristic evolution is the calculation of the waveform emitted by binary black holes , which is possible during the very interesting nonlinear domain from merger to ringdown [ 144 , 224 ] . the evolution is carried out along a family of ingoing null hypersurfaces which intersect the horizon in topological spheres . it is restricted to the period following the merger , for otherwise the ingoing null hypersurfaces would intersect the horizon in disjoint pieces corresponding to the individual black holes . the evolution proceeds backward in time on an ingoing null foliation to determine the exterior spacetime in the post - merger era . it is an example of the characteristic initial value problem posed on an intersecting pair of null hypersurfaces [ 185 , 119 ] , for which existence theorems apply in some neighborhood of the initial null hypersurfaces [ 155 , 84 , 83 ] . here one of the null hypersurfaces is the event horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } of the binary black holes . the other is an ingoing null hypersurface j which intersects \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } in a topologically spherical surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}^ + } $ \end{document } approximating the equilibrium of the final kerr black hole , so that j approximates future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the required data for the analytic problem consists of the degenerate conformal null metrics of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } and j and the metric and extrinsic curvature of their intersection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}^ + } $ \end{document}. the conformal metric of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } is provided by the conformal horizon model for a binary black hole horizon [ 144 , 133 ] , which treats the horizon in stand - alone fashion as a three - dimensional manifold endowed with a degenerate metric ab and affine parameter t along its null rays . the metric is obtained from the conformal mapping \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\gamma _ { ab } } = { \omega ^2}{{\hat \gamma}_{ab}}$\end{document } of the intrinsic metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat \gamma}_{ab}}$\end{document } of a flat space null hypersurface emanating from a convex surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}_0}$\end{document } embedded at constant time in minkowski space . the horizon is identified with the null hypersurface formed by the inner branch of the boundary of the past of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}_0}$\end{document } , and its extension into the future . the flat space null hypersurface expands forever as its affine parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat t}$\end{document } ( minkowski time ) increases , but the conformal factor is chosen to stop the expansion so that the cross - sectional area of the black hole approaches a finite limit in the future . at the same time , the raychaudhuri equation ( which governs the growth of surface area ) forces a nonlinear relation between the affine parameters t and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat t}$\end{document}. this is what produces the nontrivial topology of the affine t - slices of the black hole horizon . the relative distortion between the affine parameters t and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat t}$\end{document } , brought about by curved space focusing , gives rise to the trousers shape of a binary black hole horizon . an embedding diagram of the horizon for an axisymmetric head - on collision , obtained by choosing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}_0}$\end{document } to be a prolate spheroid , is shown in figure 3 . the black hole event horizon associated with a triaxial ellipsoid reveals new features not seen in the degenerate case of the head - on collision , as depicted in figure 4 . if the degeneracy is slightly broken , the individual black holes form with spherical topology but as they approach , tidal distortion produces two sharp pincers on each black hole just prior to merger . at merger , the inner hole of the torus subsequently closes up to produce first a peanut shaped black hole and finally a spherical black hole . no violation of topological censorship occurs because the hole in the torus closes up superluminally . consequently , a causal curve passing through the torus at a given time can be slipped below the bottom of a trouser leg to yield a causal curve lying entirely outside the hole . in the degenerate axisymmetric limit , the pincers reduce to a point so that the individual holes have teardrop shape and they merge without a toroidal phase . figure 3 trousers shaped event horizon obtained by the conformal model figure 4upper left : tidal distortion of approaching black holes upper right : formation of sharp pincers just prior to merger . trousers shaped event horizon obtained by the conformal model upper left : tidal distortion of approaching black holes upper right : formation of sharp pincers just prior to merger . the conformal horizon model determines the data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}^ + } $ \end{document}. the remaining data necessary to evolve the exterior spacetime are given by the conformal geometry of j , which constitutes the outgoing radiation waveform . the determination of the merger - ringdown waveform proceeds in two stages . in the first stage , this outgoing waveform is set to zero and the spacetime is evolved backward in time to calculate the incoming radiation entering from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document}. ( this incoming radiation is eventually absorbed by the black hole . ) from a time reversed point of view , this evolution describes the outgoing waveform emitted in the fission of a white hole , with the physically correct initial condition of no ingoing radiation . preliminary calculations show that at late times the waveform is entirely quadrupolar ( = 2 ) but that a strong octopole mode ( = 4 ) exists just before fission . in the second stage of the calculation , this waveform could be used to generate the physically correct outgoing waveform for a black hole merger . the passage from the first stage to the second is the nonlinear equivalent of first determining an inhomogeneous solution to a linear problem and then adding the appropriate homogeneous solution to satisfy the boundary conditions . in this context , the first stage supplies an advanced solution and the second stage the homogeneous retarded minus advanced solution . when the evolution is carried out in the perturbative regime of a kerr or schwarzschild background , as in the close approximation , this superposition of solutions is simplified by the time reflection symmetry . the second stage has been carried out in the perturbative regime of the close approximation using a characteristic code which solves the teukolsky equation , as described in section 3.7 . more generally , beyond the perturbative regime , the merger - ringdown waveform must be obtained by a more complicated inverse scattering procedure , which has not yet been attempted . there is a complication in applying the pitt code to this double null evolution because a dynamic horizon does not lie precisely on r - grid points . as a result , the r - derivative of the null data , i.e. the ingoing shear of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h}$\end{document } , the ingoing shear is part of the free data specified at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}^ + } $ \end{document}. its value on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h}$\end{document } can be determined by integrating ( backward in time ) a sequence of propagation equations involving the horizon s twist and ingoing divergence . a horizon code which carries out these integrations has been tested to give accurate data even beyond the merger . the code has revealed new global properties of the head - on collision by studying a sequence of data for a family of colliding black holes which approaches a single schwarzschild black hole . the resulting perturbed schwarzschild horizon provides global insight into the close limit , in which the individual black holes have joined in the infinite past . a marginally anti - trapped surface divides the horizon into interior and exterior regions , analogous to the division of the schwarzschild horizon by the r = 2 m bifurcation sphere . in passing from the perturbative to the strongly nonlinear regime there is a rapid transition in which the individual black holes move into the exterior portion of the horizon . the data paves the way for the pitt code to calculate whether this dramatic time dependence of the horizon produces an equally dramatic waveform . see section 3.7.2 for first stage results . the nonlinear 3d pitt code has been calibrated in the regime of small perturbations of a schwarzschild spacetime [ 228 , 229 ] by measuring convergence with respect to independent solutions of the teukolsky equation . by decomposition into spherical harmonics , the teukolsky equation reduces the problem of a perturbation of a stationary black hole to a 1d problem in the ( t , r ) subspace perturbations for a component of the weyl tensor . campanelli , gmez , husa , winicour , and zlochower [ 56 , 134 ] have reformulated the teukolsky formalism as a double - null characteristic evolution algorithm . the evolution proceeds on a family of outgoing null hypersurfaces with an ingoing null hypersurface as inner boundary and with the outer boundary compactified at future null infinity . it applies to either the weyl component 0 or 4 , as classified in the newman - penrose formalism . the 0 component comprises constraint - free gravitational data on an outgoing null hypersurface and 4 comprises the corresponding data on an ingoing null hypersurface . in the study of perturbations of a schwarzschild black hole , 0 is prescribed on an outgoing null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } , representing an early retarded time approximating past null infinity , and 4 is prescribed on the inner white hole horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document}. the physical setup is described in figure 5 . the outgoing null hypersurfaces extend to future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } on a compactified numerical grid . consequently , there is no need for either an artificial outer boundary condition or an interior extraction worldtube . the outgoing radiation is computed in the coordinates of an observer in an inertial frame at infinity , thus avoiding any gauge ambiguity in the waveform . a star of mass m has undergone spherically symmetric collapse to form a black hole . the ingoing null worldtube \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } lies outside the collapsing matter . inside \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } ( but outside the matter ) there is a vacuum schwarzschild metric . outside of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , data for an ingoing pilse is specified on the initial outgoing null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document}. as the pilse propagates to the black hole event horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } , part of its energy is scattered to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the physical setup for the scattering problem . a star of mass m has undergone spherically symmetric collapse to form a black hole . the ingoing null worldtube \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } lies outside the collapsing matter . inside \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } ( but outside the matter ) there is a vacuum schwarzschild metric . outside of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , data for an ingoing pilse is specified on the initial outgoing null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document}. as the pilse propagates to the black hole event horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } , part of its energy is scattered to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the first calculations were carried out with nonzero data for 4 on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } and zero data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } ( so that no ingoing radiation entered the system ) . the resulting simulations were highly accurate and tracked the quasi - normal ringdown of a perturbation consisting of a compact pulse through 10 orders of magnitude and tracked the final power law decay through an additional 6 orders of magnitude . the measured exponent of the power law decay varied from 5.8 , at the beginning of the tail , to 5.9 near the end , in good agreement with the predicted value of 2 + 2 = 6 for a quadrupole wave . the accuracy of the perturbative solutions provide a virtual exact solution for carrying out convergence tests of the nonlinear pitt null code . in this way , the error in the bondi news function computed by the pitt code was calibrated for perturbative data consisting of either an outgoing pulse on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } or an ingoing pulse on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document}. for the outgoing pulse , clean second order convergence was confirmed until late times in the evolution , when small deviations from second order arise from accumulation of roundoff and truncation error . for the bondi news produced by the scattering of an ingoing pulse , clean second order convergence was again confirmed until late times when the pulse approached the r = 2 m black hole horizon . the late time error arises from loss of resolution of the pulse ( in the radial direction ) resulting from the properties of the compactified radial coordinate used in the code . the characteristic teukolsky code has been used to study radiation from axisymmetric white holes and black holes in the close approximation . the radiation from an axisymmetric fissioning white hole was computed using the weyl data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } supplied by the conformal horizon model described in section 3.6 , with the fission occurring along the axis of symmetry . the close approximation implies that the fission takes place far in the future , i.e. in the region of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } above the black hole horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document}. the data have a free parameter which controls the energy yielded by the white hole fission . the radiation waveform reveals an interesting dependence on the parameter . in the large limit , the waveform consists of a single pulse , followed by ringdown and tail decay . the amplitude of the pulse scales quadratically with and the width decreases with . as is reduced , the initial pulse broadens and develops more structure . in the small limit , the amplitude scales linearly with and the shape is independent of . since there was no incoming radiation , the above model gave the physically appropriate boundary conditions for a white hole fission ( in the close approximation ) . from a time reversed view point , the system corresponds to a black hole merger with no outgoing radiation at future null infinity , i.e. the analog of an advanced solution with only ingoing but no outgoing radiation . in the axisymmetric case studied , the merger corresponds to a head - on collision between two black holes . the physically appropriate boundary conditions for a black hole merger correspond to no ingoing radiation on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } and binary black hole data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document}. because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } are disjoint , the corresponding data can not be used directly to formulate a double null characteristic initial value problem . however , the ingoing radiation at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } supplied by the advanced solution for the black hole merger could be used as stage i of a two stage approach to determine the corresponding retarded solution . in stage ii , this ingoing radiation is used to generate the analogue of an advanced minus retarded solution . a pure retarded solution ( with no ingoing radiation but outgoing radiation at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } ) can then be constructed by superposition . the time reflection symmetry of the schwarzschild background is key to carrying out this construction . this two stage strategy has been carried out by husa , zlochower , gmez , and winicour . the superposition of the stage i and ii solutions removes the ingoing radiation from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } while modifying the close approximation perturbation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ + } $ \end{document } , essentially making it ring . the amplitude of the radiation waveform at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } has a linear dependence on the parameter , which in this black hole scenario governs the energy lost in the inelastic merger process . unlike the fission waveforms , there is very little -dependence in their shape and the amplitude continues to scale linearly even for large . it is not surprising that the retarded waveforms from a black hole merger differs markedly from the retarded waveforms from a white hole merger . the white hole process is directly visible at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } whereas the merger waveform results indirectly from the black holes through the preceding collapse of matter or gravitational energy that formed them . this explains why the fission waveform is more sensitive to the parameter which controls the shape and timescale of the horizon data . however , the weakness of the dependence of the merger waveform on is surprising and has potential importance for enabling the design of an efficient template for extracting a gravitational wave signal from noise . in the purely vacuum approach to the binary black hole problem , the stars which collapse to form the black holes are replaced by imploding gravitational waves . the imploding waves either emanate from a past singularity , in which case the time - reversed application of cosmic censorship implies the existence of an anti - trapped surface ; or they emanate from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document } , which complicates the issue of gravitational radiation content in the initial data and its effect on the outgoing waveform . these complications are avoided in the two stage approach adopted in the close approximation studies described in section 3.7.1 , where advanced and retarded solutions in a schwarzschild background can be rigorously identified and superimposed . computational experiments have been carried out to study the applicability of this approach in the nonlinear regime . from a time reversed viewpoint , the first stage is equivalent to the determination of the outgoing radiation from a fission of a white hole in the absence of ingoing radiation , i.e. the physically appropriate retarded waveform from a white hole fission . this fission problem can be formulated in terms of data on the white hole horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } and data representing the absence of ingoing radiation on a null hypersurface j which emanates from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } at an early time . the data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } is provided by the conformal horizon model for a fissioning white hole . this allows study of a range of models extending from the perturbative close approximation regime , in which the fission occurs inside a black hole event horizon , to the nonlinear regime of a fission visible from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the study concentrates on the axisymmetric spinless fission ( corresponding in the time reversed view to the head - on collision of non - spinning black holes ) . in the perturbative regime , the news function agrees with the close approximation waveforms . in the highly nonlinear regime , a bare fission was found to produce a dramatically sharp radiation pulse , which then undergoes a damped oscillation . because the fission is visible from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , it is a more efficient source of gravitational waves than a black hole merger and can produce a higher fractional mass loss . the pitt code has been used to model the nonlinear generation of waveforms by scattering off a schwarzschild black hole [ 228 , 229 ] . the physical setup is similar to the perturbative study in section 3.7 . a radially compact pulse is prescribed on an early time outgoing null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal j}^ -}$\end{document } and schwarzschild null data is given on the interior white hole horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^ -}$\end{document } , which is causally unaffected by the pulse . the input pulse is standardized to ( = 2 , m = 0 ) and ( = 2 , m = 2 ) quadrupole modes with amplitude a. the outgoing null hypersurfaces extend to future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } on a compactified numerical grid . the evolution code then provides the news function at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , in the coordinates of an observer in an inertial frame at infinity , thus avoiding any gauge ambiguity in the waveform . this provides a simple setting how the nonlinearities generated by high amplitudes affect the waveform . the study reveals several features of qualitative importance : the mode coupling amplitudes consistently scale as powers a of the input amplitude a corresponding to the nonlinear order of the terms in the evolution equations which produce the mode . this allows much economy in producing a waveform catalog : given the order n associated with a given mode generation , the response to any input amplitude a can be obtained from the response to a single reference amplitude.the frequency response has similar behavior but in a less consistent way . the dominant frequencies produced by mode coupling are in the approximate range of the quasinormal frequency of the input mode and the expected sums and diference frequencies generated by the order of nonlinearity.large phase shifts , ranging up 15% in a half cycle relative to the linearized waveform , are exhibited in the news function obtained by the superposition of all output modes , i.e. in the waveform of observational significance . these phase shifts , which are important for design of signal extraction templates , arise in an erratic way from superposing modes with different oscillation frequencies . this furnishes a strong argument for going beyond the linearized approximation in designing a waveform catalog for signal extraction.besides the nonlinear generation of harmonic modes absent in the initial data , there is also a stronger than linear generation of gravitational wave output . this provides a potential mechanism for enhancing the strength of the gravitational radiation produced during , say , the merger phase of a binary inspiral above the strength predicted in linearized theory.in the non - axisymmetric m = 2 case , there is also considerable generation of radiation in polarization states not present in the linearized approximation . in the simulations , input amplitudes in the range a = 0.1 to a = 0.36 lead to nonlinear generation of the polarization mode which is of the same order of magnitude as the mode ( which would be the sole polarization in the linearized regime ) . as a result , significant nonlinear amplification and phase shifting of the waveform would be observed by a gravitational wave detector , depending on its orientation . the mode coupling amplitudes consistently scale as powers a of the input amplitude a corresponding to the nonlinear order of the terms in the evolution equations which produce the mode . this allows much economy in producing a waveform catalog : given the order n associated with a given mode generation , the response to any input amplitude a can be obtained from the response to a single reference amplitude . the dominant frequencies produced by mode coupling are in the approximate range of the quasinormal frequency of the input mode and the expected sums and diference frequencies generated by the order of nonlinearity . large phase shifts , ranging up 15% in a half cycle relative to the linearized waveform , are exhibited in the news function obtained by the superposition of all output modes , i.e. in the waveform of observational significance . these phase shifts , which are important for design of signal extraction templates , arise in an erratic way from superposing modes with different oscillation frequencies . this furnishes a strong argument for going beyond the linearized approximation in designing a waveform catalog for signal extraction . besides the nonlinear generation of harmonic modes absent in the initial data , there is also a stronger than linear generation of gravitational wave output . this provides a potential mechanism for enhancing the strength of the gravitational radiation produced during , say , the merger phase of a binary inspiral above the strength predicted in linearized theory . in the non - axisymmetric m = 2 case , there is also considerable generation of radiation in polarization states not present in the linearized approximation . in the simulations , input amplitudes in the range a = 0.1 to a = 0.36 lead to nonlinear generation of the polarization mode which is of the same order of magnitude as the mode ( which would be the sole polarization in the linearized regime ) . as a result , significant nonlinear amplification and phase shifting of the waveform would be observed by a gravitational wave detector , depending on its orientation . these effects arise from the nonlinear modification of the schwarzschild geometry identified by papadopoulos in his prior work on axisymmetric mode coupling , reported in section 3.3.2 . although papadopoulos studied nonlinear mode generation produced by an outgoing pulse , as opposed to the case of an ingoing pulse studied in [ 228 , 229 ] , the same nonlinear factors were in play and gave rise to several common features . in both cases , analogs of features 1 , 2 , 3 , and 4 above are all apparent in papadopoulos s work . at the finite difference level , both codes respect the reflection symmetry inherent in einstein s equations and exhibit the corresponding selection rules arising from parity considerations . in the axisymmetric case considered by papadopoulos , this forbids the nonlinear generation of a mode from a mode , as described in feature 5 above . the evolution along ingoing null hypersurfaces in the axisymmetric work of papadopoulos has complementary numerical features with the evolution along outgoing null hypersurfaces in the 3d work . the grid based upon ingoing null hypersurfaces avoids the difficulty in resolving effects close to r = 2 m encountered with the grid based upon outgoing null hypersurfaces . the outgoing code would require amr in order to resolve the quasinormal ringdown for as many cycles as achieved by papadopoulos . however , the outgoing code avoids the late time caustic formation noted in papadopoulos work , as well as the complications of gauge ambiguity and backscattering introduced by a finite outer boundary . one attractive option would be to combine the best features of these approaches by matching an interior evolution based upon ingoing null hypersurfaces to an exterior evolution based upon outgoing null hypersurfaces , as implemented in for spherically symmetric einstein - klein - gordon waves . the waveform of relevance to gravitational wave astronomy is the superposition of modes with different frequency compositions and angular dependence . although this waveform results from a complicated nonlinear processing of the input signal , which varies with choice of observation angle , the response of the individual modes to an input signal of arbitrary amplitude can be obtained by scaling the response to an input of standard reference amplitude . the einstein - klein - gordon ( ekg ) system can be used to simulate many interesting physical phenomena . in 1d , characteristic ekg codes have been used to simulate critical phenomena and the perturbation of black holes ( see section 3.1 ) , and a cauchy ekg code has been used to study boson star dynamics . ( the characteristic approach has not yet been applied to the problem of stable 1d boson stars . ) extending these codes to 3d would open up a new range of possibilities , e.g. , the possibility to study radiation from a boson star orbiting a black hole . a first step in that direction has been achieved with the construction of a 3d characteristic code by incorporating a massless scalar field into the pitt code . since the scalar and gravitational evolution equations have the same basic form , the same evolution algorithm could be utilized . it was applied to the fully nonlinear simulation of an asymmetric pulse of ingoing scalar radiation propagating toward a schwarzschild black hole . the resulting scalar radiation and gravitational news backscattered to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } was computed . the amplitudes of the scalar and gravitational radiation modes exhibited the expected power law scaling with respect to the initial pulse amplitude . in addition , the computed ringdown frequencies agreed with the results from perturbative quasinormal mode calculations . its one disadvantage is the existence of either a caustic , where neighboring characteristics focus , or a milder version consisting of a crossover between two distinct characteristics . the vertex of a light cone is a highly symmetric caustic which already strongly limits the time step for characteristic evolution because of the cfl condition ( 11 ) . it does not appear possible for a single characteristic coordinate system to cover the entire exterior region of a binary black hole spacetime without developing very complicated caustics and crossovers . this limits the waveform determined by a purely characteristic evolution to the post merger period . ccm is a way to avoid such limitations by combining the strong points of characteristic and cauchy evolution into a global evolution . one of the prime goals of computational relativity is the simulation of the inspiral and merger of binary black holes . given the appropriate worldtube data for a binary system in its interior , characteristic evolution can supply the exterior spacetime and the radiated waveform . the potential advantages of ccm over traditional boundary conditions are accurate waveform and polarization state at infinity , computational efficiency for radiation problems in terms of both the grid domain and the computational algorithm , elimination of an artificial outer boundary condition on the cauchy problem , which eliminates contamination from back - reflection and clarifies the global initial value problem , anda global picture of the spacetime exterior to the horizon . accurate waveform and polarization state at infinity , computational efficiency for radiation problems in terms of both the grid domain and the computational algorithm , elimination of an artificial outer boundary condition on the cauchy problem , which eliminates contamination from back - reflection and clarifies the global initial value problem , and a global picture of the spacetime exterior to the horizon . these advantages have been realized in model tests , but ccm has not yet been achieved in fully nonlinear three - dimensional general relativity . the early attempts to implement ccm in general relativity involved the arnowitt - deser - misner ( adm ) formulation for the cauchy evolution . the difficulties were later traced to a pathology in the way boundary conditions have traditionally been applied in adm codes . instabilities introduced at boundaries have emerged as a major problem common to all adm code development . a linearized study [ 206 , 207 ] of adm evolution - boundary algorithms with prescribed values of lapse and shift shows the following : on analytic grounds , adm boundary algorithms which supply values for all components of the metric ( or extrinsic curvature ) are inconsistent.a consistent boundary algorithm only allows free specification of the transverse - traceless components of the metric with respect to the boundary.using such a boundary algorithm , linearized adm evolution can be carried out in a bounded domain for thousands of crossing times with no sign of exponential growing instability . on analytic grounds , adm boundary algorithms which supply values for all components of the metric ( or extrinsic curvature ) are inconsistent . a consistent boundary algorithm only allows free specification of the transverse - traceless components of the metric with respect to the boundary . using such a boundary algorithm , linearized adm evolution can be carried out in a bounded domain for thousands of crossing times with no sign of exponential growing instability . the evolution satisfied the original criterion for robust stability : that there be no exponential growth when the initial cauchy data and free boundary data are prescribed as random numbers ( in the linearized regime ) . these results gave some initial optimism that ccm might be possible with an adm code if the boundary condition was properly treated . however , it was subsequently shown that adm is only weakly hyperbolic so that in the linear regime there are instabilities which grow as a power law in time . in the nonlinear regime , it is symptomatic of weakly hyperbolic systems that such secular instabilities become exponential . this is necessarily so because interpolations continually introduce short wavelength noise into the neighborhood of the boundary . it was demonstrated some time ago that the pitt characteristic code has a robustly stable boundary ( see section 3.5.3 ) , but robustness of the cauchy boundary has only recently been studied . boundary conditions are both the most important and the most difficult part of a theoretical treatment of most physical systems . usually , that s where all the physics is . and , in computational approaches , that s usually where all the agony is . even with an analytic form of the correct physical boundary condition in hand , there are seemingly infinitely more unstable numerical implementations than stable ones . in general , a stable problem places more boundary requirements on the finite difference equations than on the corresponding partial differential equations . furthermore , the methods of linear stability analysis are often more unwieldy to apply to the boundary than to the interior evolution algorithm . the von neumann stability analysis of the interior algorithm linearizes the equations , while assuming a uniform infinite grid , and checks that the discrete fourier modes do not grow exponentially . the mode e , with k real , is not included in the von neumann analysis for periodic boundary conditions . however , for the half plane problem with a boundary to the right on the x - axis , one can legitimately prescribe such a mode as initial data as long as k > 0 so that it has finite energy . thus the stability of such boundary modes must be checked . in the case of an additional boundary to the left , the godunov - ryabenkii theory gives as necessary conditions for stability the separate von neumann stability of the interior and the stability of the allowed boundary modes . the correct physical formulation of any asymptotically flat cauchy problem also involves asymptotic conditions at infinity . these conditions must ensure not only that the total energy and the energy loss by radiation are both finite , but they must also ensure the proper 1/r asymptotic falloff of the radiation fields . however , when treating radiative systems computationally , an outer boundary is often established artificially at some large but finite distance in the wave zone , i.e. many wavelengths from the source . imposing an appropriate radiation boundary condition at a finite distance is a difficult task even in the case of a simple radiative system evolving on a fixed geometric background . nowhere is the boundary problem more acute than in the computation of gravitational radiation produced by black holes . the numerical study of a black hole spacetime by means of a pure cauchy evolution involves inner as well as outer grid boundaries . the inner boundary is necessary to avoid the topological complications and singularities introduced by the black holes . for multiple black holes , unruh suggested the commonly accepted strategy for cauchy evolution of black holes ( see ) . an inner boundary located at ( or near ) an apparent horizon ccm has a natural application to this problem . in the cauchy treatment of such a system , the outer grid boundary is located at some finite distance , normally many wavelengths from the source . attempts to use compactified cauchy hypersurfaces which extend to spatial infinity have failed because the phase of short wavelength radiation varies rapidly in spatial directions . when the system is nonlinear and not amenable to an exact solution , a finite outer boundary condition must necessarily introduce spurious physical effects into a cauchy evolution . the domain of dependence of the initial cauchy data in the region spanned by the computational grid would shrink in time along ingoing characteristics unless data on a worldtube traced out by the outer grid boundary is included as part of the problem . in order to maintain a causally sensible evolution , this worldtube data must correctly substitute for the missing cauchy data which would have been supplied if the cauchy hypersurface had extended to infinity . in a scattering problem , this missing exterior cauchy data might , for instance , correspond to an incoming pulse initially outside the outer boundary . in a problem where the initial radiation fields are confined to a compact region inside the boundary , these missing cauchy data are easy to characterize when dealing with a constraint free field , such as a scalar field where the appropriate cauchy data outside the boundary would be ,t = 0 . however , the determination of cauchy data for general relativity is a global elliptic constraint problem so that there is no well defined scheme to confine it to a compact region . furthermore , even if the data for a given problem were known on a complete initial hypersurface extending to infinity , it would be a formidable nonlinear evolution problem to correctly assign the associated boundary data on a finite outer boundary . another important issue arising in general relativity is whether the boundary condition preserves the constraints . it is typical of hyperbolic reductions of the einstein equations that the hamiltonian and momentum constraints propagate in a domain of dependence dictated by the light rays . unless the boundary conditions on the outer world tube enforce these constraints , they will be violated outside the domain of dependence of the initial cauchy hypersurface . this issue of a constraint - preserving initial boundary value problem has only recently been addressed . the first fully nonlinear treatment of a well - posed constraint preserving formulation of the einstein initial - boundary value problem ( ibvp ) has only recently been given by friedrich and nagy . their treatment is based upon a frame formulation in which the evolution variables are the tetrad , connection coefficients , and weyl curvature . although this system has not yet been implemented computationally , it has spurred the investigation of simpler treatments of einstein equations which give rise to a constraint preserving ibvp under various restrictions [ 55 , 208 , 54 , 88 , 112 ] . it is common practice in computational physics to impose some artificial boundary condition ( abc ) , such as an outgoing radiation condition , in an attempt to approximate the proper data for the exterior region . this abc may cause partial reflection of an outgoing wave back into the system [ 146 , 138 , 121 , 178 ] , which contaminates the accuracy of the interior evolution and the calculation of the radiated waveform . furthermore , nonlinear waves intrinsically backscatter , which makes it incorrect to try to entirely eliminate incoming radiation from the outer region . the resulting error is of an analytic origin , essentially independent of computational discretization . in general , a systematic reduction of this error can only be achieved by moving the computational boundary to larger and larger radii . a traditional abc for the wave equation is the sommerfeld condition . for a 3d scalar field this takes the form gt + gr = 0 , where g = r. this condition is exact only for a linear wave with spherically symmetric data and boundary . in that case , the exact solution is g = f1(t r ) + f2(t + r ) and the sommerfeld condition eliminates the incoming wave f2 . much work has been done on formulating boundary conditions , both exact and approximate , for linear problems in situations that are not spherically symmetric and in which the sommerfeld condition would be inaccurate . these boundary conditions are given various names in the literature , e.g. , absorbing or non - reflecting . see the articles [ 93 , 178 , 216 , 182 , 37 ] for general discussions . local abc s have been extensively applied to linear problems with varying success [ 146 , 78 , 28 , 215 , 121 , 44 , 139 ] . some of these conditions are local approximations to exact integral representations of the solution in the exterior of the computational domain , while others are based on approximating the dispersion relation of the so - called one - way wave equations [ 146 , 215 ] . higdon showed that this last approach is essentially equivalent to specifying a finite number of angles of incidence for which the abcperfect transmission . local abc s have also been derived for the linear wave equation by considering the asymptotic behavior of outgoing solutions , which generalizes the sommerfeld outgoing radiation condition . although this type of abc is relatively simple to implement and has a low computational cost , the final accuracy is often limited because the assumptions made about the behavior of the waves are rarely met in practice [ 93 , 216 ] . the disadvantages of local abc s have led some workers to consider exact nonlocal boundary conditions based on integral representations of the infinite domain problem [ 214 , 93 , 216 ] . even for problems where the green s function is known and easily computed , such approaches were initially dismissed as impractical ; however , the rapid increase in computer power has made it possible to implement exact nonlocal abc s for the linear wave equation and maxwell s equations in 3d [ 70 , 110 ] . if properly implemented , this kind of method can yield numerical solutions which converge to the exact infinite domain problem in the continuum limit , while keeping the artificial boundary at a fixed distance . however , due to nonlocality , the computational cost per time step usually grows at a higher power with grid size ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({n^4})$\end{document } per time step in three dimensions ) than in a local approach [ 93 , 70 , 216 ] . the problem is normally treated by linearizing the region between the outer boundary and infinity , using either local or nonlocal linear abc s [ 216 , 182 ] . the neglect of the nonlinear terms in this region introduces an unavoidable error at the analytic level . this is a subtle global problem because the correct boundary data must correspond to the continuity of fields and their normal derivatives when extended across the boundary into the linearized exterior . this is a clear requirement for any consistent boundary algorithm , since discontinuities in the field or its derivatives would otherwise act as spurious sheet source on the boundary , which contaminates both the interior and the exterior evolutions . but the fields and their normal derivatives constitute an overdetermined set of data for the boundary problem . so it is necessary to solve a global linearized problem , not just an exterior one , in order to find the proper data . the designation exact abc is given to an abc for a nonlinear system whose only error is due to linearization of the exterior . an exact abc requires the use of global techniques , such as the difference potential method , to eliminate back reflection at the boundary . to date thompson generalized a previous nonlinear abc of hedstrom to treat 1d and 2d problems in gas dynamics . these boundary conditions performed poorly in some situations because of their difficulty in adequately modeling the field outside the computational domain [ 212 , 93 ] . hagstrom and hariharan have overcome these difficulties in 1d gas dynamics by a clever use of riemann invariants . they proposed a heuristic generalization of their local abc to 3d , but this approach has not yet been validated . in order to reduce the level of approximation at the analytic level , an artificial boundary for a nonlinear problem must be placed sufficiently far from the strong - field region . there seems to be no numerical method which converges ( as the discretization is refined ) to the infinite domain exact solution of a strongly nonlinear wave problem in multi - dimensions , while keeping the artificial boundary fixed . ccm is a strategy that eliminates this nonlinear source of error . in the simplest version of ccm , cauchy and characteristic evolution algorithms are pasted together in the neighborhood of a worldtube to form a global evolution algorithm . the characteristic algorithm provides an outer boundary condition for the interior cauchy evolution , while the cauchy algorithm supplies an inner boundary condition for the characteristic evolution . the characteristic evolution is based upon the outgoing null hypersurfaces emanating from these slices , with the evolution proceeding from one hypersurface to the next by the outward radial march described earlier . there is no need to truncate spacetime at a finite distance from the sources , since compactification of the radial null coordinate used in the characteristic evolution makes it possible to cover the infinite space with a finite computational grid . in this way although characteristic evolution has limitations in the interior region where caustics develop , it proves to be both accurate and computationally efficient in the treatment of exterior regions . in its simplest form , ccm evolves a mixed spacelike - null initial value problem in which cauchy data is given in a spacelike region bounded by a spherical boundary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } and characteristic data is given on a null hypersurface emanating from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document}. the general idea is not entirely new . an early mathematical investigation combining space - like and characteristic hypersurfaces appears in the work of duff . the three chief ingredients for computational implementation are : ( i ) a cauchy evolution module , ( ii ) a characteristic evolution module and , ( iii ) a module for matching the cauchy and characteristic regions across their interface . the interface is the timelike worldtube which is traced out by the flow of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } along the worldlines of the cauchy evolution , as determined by the choice of lapse and shift . matching provides the exchange of data across the worldtube to allow evolution without any further boundary conditions , as would be necessary in either a purely cauchy or purely characteristic evolution . the most important application of ccm is anticipated to be the binary black hole problem . the 3d cauchy codes being developed to solve this problem employ a single cartesian coordinate patch , a stategy adopted in to avoid coordinate singularites . a thoroughly tested and robust 3d characteristic code is now in place , ready to match to the boundary of this cauchy patch . development of a stable implementation of ccm represents the major step necessary to provide a global evolution code for the binary black hole problem . from a cursory view , the application of ccm to this problem might seem routine , tantamount to translating into finite difference form the textbook construction of an atlas consisting of overlapping coordinate patches . in practice the underlying geometrical algorithm consists of the following main submodules : the boundary module which sets the grid structures . this defines masks identifying which points in the cauchy grid are to be evolved by the cauchy module and which points are to be interpolated from the characteristic grid , and vice versa . the reference structures for constructing the mask is the inner characteristic boundary , which in cartesian coordinates is the euclidean spherical worldtube x + y + z = r , and the outer cauchy boundary . the choice of lapse and shift for the cauchy evolution governs the dynamical and geometrical properties of the matching worldtube.the extraction module whose input is cauchy grid data in the neighborhood of the worldtube and whose output is the inner boundary data for the exterior characteristic evolution . this module numerically implements the transformation from cartesian { 3 + 1 } coordinates to spherical null coordinates . the algorithm makes no perturbative assumptions and is based upon interpolations of the cauchy data to a set of prescribed grid points on the worldtube . the metric information is then used to solve for the null geodesics normal to the slices of the worldtube . this provides the jacobian for the transformation to null coordinates in the neighborhood of the worldtube . the characteristic evolution module is then used to propagate the data from the worldtube to null infinity , where the waveform is calculated.the injection module which completes the interface by using the exterior characteristic evolution to supply the outer boundary data for the cauchy evolution . this is the inverse of the extraction procedure but must be implemented outside the worldtube to allow for overlap between cauchy and characteristic domains . the overlap region can be constructed either to have a fixed physical size or to shrink to zero in the continuum limit . in the latter case , the inverse jacobian describing the transformation from null to cauchy coordinates can be obtained to prescribed accuracy in terms of an affine parameter expansion along the null geodesics emanating from the worldtube . but the numerical stability of the scheme is not guaranteed . the boundary module which sets the grid structures . this defines masks identifying which points in the cauchy grid are to be evolved by the cauchy module and which points are to be interpolated from the characteristic grid , and vice versa . the reference structures for constructing the mask is the inner characteristic boundary , which in cartesian coordinates is the euclidean spherical worldtube x + y + z = r , and the outer cauchy boundary . the choice of lapse and shift for the cauchy evolution governs the dynamical and geometrical properties of the matching worldtube . the extraction module whose input is cauchy grid data in the neighborhood of the worldtube and whose output is the inner boundary data for the exterior characteristic evolution . this module numerically implements the transformation from cartesian { 3 + 1 } coordinates to spherical null coordinates . the algorithm makes no perturbative assumptions and is based upon interpolations of the cauchy data to a set of prescribed grid points on the worldtube . the metric information is then used to solve for the null geodesics normal to the slices of the worldtube . this provides the jacobian for the transformation to null coordinates in the neighborhood of the worldtube . the characteristic evolution module is then used to propagate the data from the worldtube to null infinity , where the waveform is calculated . the injection module which completes the interface by using the exterior characteristic evolution to supply the outer boundary data for the cauchy evolution . this is the inverse of the extraction procedure but must be implemented outside the worldtube to allow for overlap between cauchy and characteristic domains . the overlap region can be constructed either to have a fixed physical size or to shrink to zero in the continuum limit . in the latter case , the inverse jacobian describing the transformation from null to cauchy coordinates can be obtained to prescribed accuracy in terms of an affine parameter expansion along the null geodesics emanating from the worldtube . the above strategy provides a model of how cauchy and characteristic codes can be pieced together as modules to form a global evolution code . the full advantage of ccm lies in the numerical treatment of nonlinear systems where its error converges to zero in the continuum limit of infinite grid resolution [ 32 , 33 , 66 ] . for high accuracy , ccm is also by far the most efficient method . for small target error , it has been shown that the relative amount of computation required for ccm ( accm ) compared to that required for a pure cauchy calculation ( ac ) goes to zero , accm / ac o as o [ 42 , 39 ] . an important factor here is the use of a compactified characteristic evolution , so that the whole spacetime is represented on a finite grid . from a numerical point of view this means that the only error made in a calculation of the radiation waveform at infinity is the controlled error due to the finite discretization . accuracy of a cauchy algorithm which uses an abc requires a large grid domain in order to avoid error from nonlinear effects in its exterior . the computational demands of ccm are small because the interface problem involves one less dimension than the evolution problem . because characteristic evolution algorithms are more efficient than cauchy algorithms , the efficiency can be further enhanced by making the matching radius as small as possible consistent with the avoidance of caustics . at present , the computational strategy of ccm is exclusively the tool of general relativists who are used to dealing with novel coordinate systems . only recently [ 66 , 67 , 76 , 36 , 208 ] has its practicability been carefully explored . research on this topic has been stimulated by the requirements of the binary black hole grand challenge alliance , where ccm was one of the strategies pursued to provide boundary conditions and determine the radiation waveform . but i anticipate that its use will eventually spread throughout computational physics because of its inherent advantages in dealing with hyperbolic systems , particularly in three - dimensional problems where efficiency is desired . a detailed study of the stability and accuracy of ccm for linear and nonlinear wave equations has been presented in , illustrating its potential for a wide range of problems . in numerous analytic applications outside of general relativity , matching techniques have successfully cured pathologies in perturbative expansions . matching is a strategy for obtaining a global solution by patching together solutions obtained using different coordinate systems for different regions . by adopting each coordinate system to a length scale appropriate to its domain , a globally convergent perturbation expansion is sometimes possible in cases where a single coordinate system would fail . in general relativity , burke showed that matching could be used to eliminate some of the divergences arising in perturbative calculations of gravitational radiation . kates and kegles further showed that use of an exterior null coordinate system in the matching scheme could eliminate problems in the perturbative treatment of a scalar radiation field on a schwarzschild background . the schwarzschild light cones have drastically different asymptotic behavior from the artificial minkowski light cones used in perturbative expansions based upon a flat space green function . use of the minkowski light cones leads to nonuniformities in the expansion of the radiation fields which are eliminated by the use of true null coordinates in the exterior . kates , anderson , kegles , and madonna extended this work to the fully general relativistic case and reached the same conclusion . anderson later applied this approach to the slow motion approximation of a binary system and obtained a derivation of the radiation reaction effect on the orbital period which avoided some objections to earlier approaches . the use of the true light cones was also essential in formulating as a mathematical theorem that the bondi news function satisfies the einstein quadrupole formula to leading order in a newtonian limit . although questions of mathematical consistency still remain in the perturbative treatment of gravitational radiation , it is clear that the use of characteristic methods pushes these problems to a higher perturbative order . one of the first applications of characteristic matching was a hybrid numerical - analytical treatment by anderson and hobill of the test problem of nonlinear 1d scalar waves [ 7 , 8 , 9 ] . they matched an inner numerical solution to a far field solution which was obtained by a perturbation expansion . a key ingredient is that the far field is solved in retarded null coordinates ( u , r ) . because the transformation from null coordinates ( u , r ) to cauchy coordinates ( t , r ) is known analytically for this problem , the matching between the null and cauchy solutions is quite simple . causality was enforced by requiring that the system be stationary prior to some fixed time . this eliminates extraneous incoming radiation in a physically correct way in a system which is stationary prior to a fixed time but it is nontrivial to generalize , say , to the problem of radiation from an orbiting binary . later , a global , characteristic , numerical study of the self - gravitating version of this problem , by gmez and winicour , confirmed that the use of the true null cones is essential in getting the correct radiated waveform . for quasi - periodic radiation , the phase of the waveform is particular sensitive to the truncation of the outer region at a finite boundary . although a perturbative estimate would indicate an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(m / r)$\end{document } error , this error accumulates over many cycles to produce an error of order in the phase . anderson and hobill proposed that their method be extended to general relativity by matching a numerical solution to an analytic 1/r expansion in null coordinates . most perturbative - numerical matching schemes that have been implemented in general relativity have been based upon perturbations of a schwarzschild background using the standard schwarzschild time slicing [ 1 , 4 , 2 , 3 , 181 , 180 , 156 ] . it would be interesting to compare results with an analytic - numeric matching scheme based upon the true null cones . although the full proposal by anderson and hobill has not been carried out , characteristic techniques have been used [ 149 , 56 , 134 ] to study the radiation content of numerical solutions by treating the far field as a perturbation of a schwarzschild spacetime . most metric based treatments of gravitational radiation based upon perturbations of schwarzschild solve the underlying regge - wheeler and zerilli equations using traditional spacelike cauchy hypersurfaces . at one level , these approaches extract the radiation from a numerical solution in a region with outer boundary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal b}$\end{document } by using data on an inner worldtube \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } to construct the perturbative solution . ambiguities are avoided by use of gauge invariant perturbation quantities . for this to work , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } must not only be located in the far field but , because of the lack of proper outer boundary data , it is necessary that the boundary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal b}$\end{document } be sufficiently far outside \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } so that the extracted radiation is uncontaminated by back - reflection for some significant window of time . this extraction strategy has also been carried out using characteristic evolution in the exterior of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } instead of a perturbative solution , i.e. cauchy - characteristic extraction . babiuc , szilgyi , hawke , and zlochower have recently carried out a test comparison of the two methods . the contamination of the extracted radiation by back - reflection can only be eliminated by matching to an exterior solution which injects the physically appropriate boundary data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document}. cauchy - perturbative matching [ 181 , 180 ] has been implemented using the same modular structure described for ccm in section 4.2 . at present , perturbative matching and ccm share the common problem of long term stability of the outer cauchy boundary in 3d applications . these model problems provided a controlled environment for the development of ccm , in which either exact solutions or independent numerical solutions were known . the following studies showed that ccm worked like a charm in a variety of 1d applications , i.e. the matched evolutions were essentially transparent to the presence of the interface . the southampton group chose cylindrically symmetric systems as their model problem for developing matching techniques . in preliminary work , they showed how ccm could be consistently carried out for a scalar wave evolving in minkowski spacetime but expressed in a nontrivial cylindrical coordinate system . although the problem involves only one spatial dimension , there are two independent modes of polarization . the cauchy metric was treated in the jordan - ehlers - kompaneets canonical form , using coordinates ( t , r , , z ) adapted to the ( , z ) cylindrical symmetry . the advantage here is that u = t r is then a null coordinate which can be used for the characteristic evolution . they successfully recast the equations in a suitably regularized form for the compactification of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } in terms of the coordinate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y = \sqrt { 1/r}$\end{document}. the simple analytic relationship between cauchy coordinates ( t , r ) and characteristic coordinates ( u , y ) facilitated the translation between cauchy and characteristic variables on the matching worldtube , given by r = const . it is notable that they report no problems with instability , which have arisen in other attempts at unconstrained leapfrog evolution in general relativity . the characteristic evolution also used a leapfrog scheme for the evolution between retarded time levels u , while numerically integrating the hypersurface equations outward along the characteristics . the matching interface was located at points common to both the cauchy and characteristic grids . in order to update these points by cauchy evolution , these values were obtained by interpolation from characteristic grid points ( lying on three levels of null hypersurfaces in order to ensure second order accuracy ) . similarly , the boundary data for starting up the characteristic integration was obtained by interpolation from cauchy grid values inside the worldtube . the matching code was first tested using exact weber - wheeler cylindrical waves , which come in from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document } , pass through the symmetry axis and expand out to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the numerical errors were oscillatory with low growth rate , and second order convergence was confirmed . comparisons of ccm were made with cauchy evolutions using a standard outgoing radiation boundary condition . at high amplitudes the standard condition developed a large error very quickly and was competitive only for weak waves with a large outer boundary . in contrast , the matching code performed well even with a small matching radius . some interesting simulations were presented in which an outgoing wave in one polarization mode collided with an incoming wave in the other mode , a problem studied earlier by pure cauchy evolution . the code was next tested using exact cylindrically symmetric solutions , due to piran , safier , and katz , which contain both degrees of freedom . these solutions are singular at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } so that the code had to be suitably modified . the convergence rate of the numerical solution starts of as second order but diminishes to first order after long time evolution . this performance could perhaps be improved by incorporating subsequent improvements in the characteristic code made by sperhake , sjdin , and vickers ( see section 3.1 ) . a joint collaboration between groups at pennsylvania state university and the university of pittsburgh applied ccm to the ekg system with spherical symmetry . initial data were specified on the union of a spacelike hypersurface and a null hypersurface . the evolution used a 3-level cauchy scheme in the interior and a 2-level characteristic evolution in the compactified exterior . a constrained cauchy evolution was adopted because of its earlier success in accurately simulating scalar wave collapse . characteristic evolution was based upon the null parallelogram algorithm ( 8) . the matching between the cauchy and characteristic foliations was achieved by imposing continuity conditions on the metric , extrinsic curvature and scalar field variables , ensuring smoothness of fields and their derivatives across the matching interface . the extensive analytical and numerical studies of this system in recent years aided the development of ccm in this nontrivial geometrical setting by providing basic knowledge of the expected physical and geometrical behavior , in the absence of exact solutions . the ccm code accurately handled wave propagation and black hole formation for all values of m / r at the matching radius , with no symptoms of instability or back - reflection . second order accuracy was established by checking energy conservation . in further developmental work on the ekg model , the pittsburgh group used ccm to formulate a new treatment of the inner cauchy boundary for a black hole spacetime . in the conventional approach , the inner boundary of the cauchy evolution is located at an apparent horizon , which must lie inside ( or on ) the event horizon , so that truncation of the interior spacetime at the apparent horizon can not causally affect the gravitational waves radiated to infinity . however , instabilities reported in some early attempts at the conventional approach motivated an alternative treatment . in the ccm strategy , illustrated in figure 6 , the interior black hole region is evolved using an ingoing null algorithm whose inner boundary is a marginally trapped surface , and whose outer boundary lies outside the black hole and forms the inner boundary of a region evolved by the cauchy algorithm . in turn , the outer boundary of the cauchy region is handled by matching to an outgoing null evolution extending to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. data are passed between the inner characteristic and central cauchy regions using a ccm procedure similar to that already described for an outer cauchy boundary . the main diference is that , whereas the outer cauchy boundary data is induced from the bondi metric on an outgoing null hypersurface , the inner cauchy boundary is now obtained from an ingoing null hypersurface which enters the event horizon and terminates at a marginally trapped surface . a cauchy evolution , with data at t0 is matched across worldtubes r0 and r1 to an ingoing null evolution , with data at v0 , and an outgoing null evolution , with data at u0 . the ingoing null evolution extends to an inner trapped boundary q , and the outgoing null evolution extends to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. black hole excision by matching . a cauchy evolution , with data at t0 is matched across worldtubes r0 and r1 to an ingoing null evolution , with data at v0 , and an outgoing null evolution , with data at u0 . the ingoing null evolution extends to an inner trapped boundary q , and the outgoing null evolution extends to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the translation from an outgoing to an incoming null evolution algorithm can be easily carried out . the substitution + i/2 in the 3d version of the bondi metric ( 3 ) provides a simple formal recipe for switching from an outgoing to an ingoing null formalism . in order to ensure that trapped surfaces exist on the ingoing null hypersurfaces , such data can be obtained from initial cauchy data for a black hole . however , rather than extending the cauchy hypersurface inward to an apparent horizon , it was truncated sufficiently far outside the apparent horizon to avoid computational problems with the cauchy evolution . the initial cauchy data were then extended into the black hole interior as initial null data until a marginally trapped surface was reached . first , the inner matching surface must be chosen to be convex , in the sense that its outward null normals uniformly diverge and its inner null normals uniformly converge . ( this is trivial to satisfy in the spherically symmetric case . ) given any physically reasonable matter source , the focusing theorem guarantees that the null rays emanating inward from the matching sphere continue to converge until reaching a caustic . second , the initial null data must lead to a trapped surface before such a caustic is encountered . this is a relatively easy requirement to satisfy because the initial null data can be posed freely , without any elliptic or algebraic constraints other than continuity with the cauchy data . its performance showed that ccm provides as good a solution to the black hole excision problem in spherical symmetry as any previous treatment [ 186 , 187 , 150 , 11 ] . ccm is computationally more efficient than these pure cauchy approaches ( fewer variables ) and much easier to implement . depending upon the cauchy formalism adopted , achieving stability with a pure cauchy scheme in the region of an apparent horizon can be quite tricky , involving much trial and error in choosing finite difference schemes . there were no complications with stability of the null evolution at the marginally trapped surface . the cauchy evolution was carried out in ingoing eddington - finklestein ( ief ) coordinates . the initial cauchy data consisted of a schwarzschild black hole with an ingoing gaussian pulse of scalar radiation . since ief coordinates are based on ingoing null cones , it is possible to construct a simple transformation between the ief cauchy metric and the ingoing null metric . initially there was no scalar field present on either the ingoing or outgoing null patches . the initial values for the bondi variables and v were determined by matching to the cauchy data at the matching surfaces and integrating the hypersurface equations ( 5 , 6 ) . as the evolution proceeds , the scalar field passes into the black hole , and the marginally trapped surface ( mts ) grows outward . the mts is easily located in the spherically symmetric case by an algebraic equation . in order to excise the singular region , the grid points inside the marginally trapped surface the backscattered radiation propagated cleanly across the outer matching surface to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the strategy worked smoothly , and second order accuracy of the approach was established by comparing it to an independent numerical solution obtained using a second order accurate , purely cauchy code . as discussed in section 4.8 , this inside - outside application of ccm has potential application to the binary black hole problem . in a variant of this double ccm matching scheme , lehner has eliminated the middle cauchy region and constructed a 1d code matching the ingoing and outgoing characteristic evolutions directly across a single timelike worldtube . in this way , he is able to simulate the global problem of a scalar wave falling into a black hole by purely characteristic methods . the southampton ccm project is being carried out for spacetimes with ( twisting ) axial symmetry . the formal basis for the matching scheme was developed by dinverno and vickers [ 74 , 75 ] . similar to the pittsburgh 3d strategy ( see section 4.2 ) , matching is based upon an extraction module , which supplies boundary data for the exterior characteristic evolution , and an injection module , which supplies boundary data for the interior cauchy evolution . however , their use of spherical coordinates for the cauchy evolution ( as opposed to cartesian coordinates in the 3d strategy ) allows use of a matching worldtube r = rm which lies simultaneously on cauchy and characteristic gridpoints . this tremendously simplifies the necessary interpolations between the cauchy and characteristic evolutions , at the expense of dealing with the r = 0 coordinate singularity in the cauchy evolution . the characteristic code ( see section 3.3.3 ) is based upon a compactified bondi - sachs formalism . the use of a radial cauchy gauge , in which the cauchy coordinate r measures the surface area of spheres , simplifies the relation to the bondi - sachs coordinates . in the numerical scheme , the metric and its derivatives are passed between the cauchy and characteristic evolutions exactly at r = rm , thus eliminating the need of a matching interface encompassing a few grid zones , as in the 3d pittsburgh scheme . preliminary results in the development of the southampton ccm code are described by pollney in his thesis . the cauchy code is based upon the axisymmetric adm code of stark and piran and reproduces their vacuum results for a short time period , after which an instability at the origin becomes manifest . the characteristic code has been tested to reproduce accurately the schwarzschild and boost - rotation symmetric solutions , with more thorough tests of stability and accuracy still to be carried out . ccm has been successfully implemented in the fully 3d problem of nonlinear scalar waves evolving in a flat spacetime [ 37 , 36 ] . this study demonstrated the feasibility of matching between cartesian cauchy coordinates and spherical null coordinates , the setup required to apply ccm to the binary black hole problem . unlike spherically or cylindrically symmetric examples of matching , the cauchy and characteristic patches do not share a common coordinate which can be used to define the matching interface . this introduces a major complication into the matching procedure , resulting in extensive use of inter - grid interpolation . the accompanying short wavelength numerical noise presents a new challenge in obtaining a stable algorithm . the nonlinear waves were modeled by the equation 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c^{- 2}}{\partial _ { tt}}\phi = { \nabla ^2}\phi + f(\phi ) + s(t , x , y , z),$$\end{document } with self - coupling f( ) and external source s. the initial cauchy data (t0 , x , y , z ) and t(t0 , x , y , z ) are assigned in a spatial region bounded by a spherical matching surface of radius rm . the characteristic initial value problem ( 27 ) is expressed in standard spherical coordinates ( r , , ) and retarded time u = t r + rm : 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2{\partial _ { ur}}g = { \partial _ { rr}}g - { { { l^2}g } \over { { r^2 } } } + r(f + s),$$\end{document } where g = r and l is the angular momentum operator 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${l^2}g = - { { { \partial _ \theta}(\sin \theta \,{\partial _ ^2 g } \over { { { \sin}^2}\,\theta}}.$$\end{document } the initial null data consist of g(r , , , u0 ) on the outgoing characteristic cone u0 = t0 emanating at the initial cauchy time from the matching worldtube at r = rm . ccm was implemented so that , in the continuum limit , and its normal derivatives would be continuous across the matching interface . the use of a cartesian discretization in the interior and a spherical discretization in the exterior complicated the treatment of the interface . in particular , the stability of the matching algorithm required careful attention to the details of the inter - grid matching . nevertheless , there was a reasonably broad range of discretization parameters for which ccm was stable . one was based upon two overlapping stereographic grid patches and the other upon a multiquadric approximation using a quasi - regular triangulation of the sphere . also , two separate tactics were used to implement matching , one based upon straightforward interpolations and the other upon maintaining continuity of derivatives in the outward null direction ( a generalization of the sommerfeld condition ) . the solutions were second order accurate and the richardson extrapolation technique could be used to accelerate convergence . as expected , the nonlocal abc s yielded convergent results only in linear problems , and convergence was not observed for local abc s , whose restrictive assumptions were violated in all of the numerical experiments . the computational cost of ccm was much lower than that of current nonlocal boundary conditions . in strongly nonlinear problems , ccm appears to be the only available method which is able to produce numerical solutions which converge to the exact solution with a fixed boundary . although the individual pieces of the ccm module have been calibrated to give a second order accurate interface between cauchy and characteristic evolution modules in 3d general relativity , its stability has not yet been established . however , a stable version of ccm for linearized gravitational theory has recently been demonstrated . the cauchy evolution is carried out using a harmonic formulation for which the reduced equations have a well - posed initial - boundary problem . previous attempts at ccm were plagued by boundary induced instabilities of the cauchy code . although stable behavior of the cauchy boundary is only a necessary and not a sufficient condition for ccm , the tests with the linearized harmonic code matched to a linearized characteristic code were successful . the harmonic conditions consist of wave equations which can be used to propagate the gauge as four scalar waves using characteristic evolution . this allows the extraction world tube to be placed at a finite distance from the injection world tube without introducing a gauge ambiguity . furthermore , the harmonic gauge conditions are the only constraints on the cauchy formalism so that gauge propagation also insures constraint propagation . this allows the cauchy data to be supplied in numerically benign sommerfeld form , without introducing constraint violation . using random initial data , robust stability of the ccm algorithm figure 7 shows a sequence of profiles of the metric component \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\gamma ^{xy } } = \sqrt { - g } { g^{xy}}$\end{document } as a linearized wave propagates cleanly through the spherical injection boundary and passes to the characteristic grid , where it is propagated to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. figure 7sequence of slices of the metric component , evolved with the linear matched cauchy - characteristic code . in the last snapshot , sequence of slices of the metric component , evolved with the linear matched cauchy - characteristic code . in the last snapshot , it is clear that the three - dimensional inspiral and coalescence of black holes challenges the limits of present computational know - how . ccm offers a new approach for excising an interior trapped region which might provide the enhanced flexibility required to solve this problem . in a binary system , there are major computational advantages in posing the cauchy evolution in a frame which is co - rotating with the orbiting black holes . such a description seems necessary in order to keep the numerical grid from being intrinsically twisted . in this co - orbiting description , the cauchy evolution requires an inner boundary condition inside the black holes and also an outer boundary condition on a worldtube outside of which the grid rotation is likely to be superluminal . thus , successful implementation of ccm would solve the exterior boundary problem for this co - orbiting description . ccm also has the potential to handle the two black holes inside the cauchy region . as described earlier with respect to figure 6 , an ingoing characteristic code can evolve a moving black hole with long term stability [ 105 , 102 ] . this means that ccm might also be able to provide the inner boundary condition for cauchy evolution once stable matching has been accomplished . in this approach , the interior boundary of the cauchy evolution is located outside the apparent horizon and matched to a characteristic evolution based upon ingoing null cones . the inner boundary for the characteristic evolution is a trapped or marginally trapped surface , whose interior is excised from the evolution . in addition to restricting the cauchy evolution to the region outside the black holes , this strategy offers several other advantages . although finding a marginally trapped surface on the ingoing null hypersurfaces remains an elliptic problem , there is a natural radial coordinate system ( r , , ) to facilitate its solution . motion of the black hole through the grid reduces to a one - dimensional radial problem , leaving the angular grid intact and thus reducing the computational complexity of excising the inner singular region . ( the angular coordinates can even rotate relative to the cauchy coordinates in order to accommodate spinning black holes . ) the chief danger in this approach is that a caustic might be encountered on the ingoing null hypersurface before entering the trapped region . this is a gauge problem whose solution lies in choosing the right location and geometry of the surface across which the cauchy and characteristic evolutions are matched . there is a great deal of flexibility here because the characteristic initial data can be posed without constraints . this global strategy is tailor - made to treat two black holes in the co - orbiting gauge , as illustrated in figure 8 . two disjoint characteristic evolutions based upon ingoing null cones are matched across worldtubes to a central cauchy region . the interior boundaries of each of these interior characteristic regions border a trapped surface . at the outer boundary of the cauchy region , a matched characteristic evolution based upon outgoing null hypersurfaces propagates the radiation to infinity . the cauchy evolution is matched across two inner worldtubes 1 and 2 to two ingoing null evolutions whose inner boundaries excise the individual black holes . the outer cauchy boundary is matched across the worldtube to an outgoing null evolution extending to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. ccm for binary black holes , portrayed in a co - rotating frame . the cauchy evolution is matched across two inner worldtubes 1 and 2 to two ingoing null evolutions whose inner boundaries excise the individual black holes . the outer cauchy boundary is matched across the worldtube to an outgoing null evolution extending to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. present characteristic and cauchy codes can handle the individual pieces of this problem . their unification appears to offer the best chance for simulating the inspiral and merger of two black holes . the individual pieces of the fully nonlinear ccm module , as outlined in section 4.2 , have been implemented and tested for accuracy . the one missing ingredient is long term stability in the nonlinear gravitational case , which would make future applications very exciting . although the bondi - sachs evolution algorithm can easily be extended to include matter , the advantage of a light cone approach for treating fluids is not as apparent as for a massless field whose physical characteristics lie on the light cone . however , results from recent studies of relativistic stars and of fluid sources moving in the vicinity of a black hole indicate that this approach can provide accurate simulations of astrophysical relevance such as supernova collapse to a black hole , mass accretion , and the production of gravitational waves . miller and mota performed the first simulations of spherically symmetric gravitational collapse using a null foliation . baumgarte , shapiro and teukolsky subsequently used a null slicing to study supernovae and the collapse of neutron stars to form black holes . the use of a null slicing allowed them to evolve the exterior spacetime while avoiding the region of singularity formation . the group at the universidad de oriente in venezuela applied characteristic methods to study the self - similar collapse of spherical matter and charge distributions [ 14 , 18 , 15 ] . the assumption of self - similarity reduces the problem to a system of ode s , subject to boundary conditions determined by matching to an exterior reissner - nordstrm - vaidya solution . heat flow in the internal fluid is balanced at the surface by the vaidya radiation . their simulations illustrate how a nonzero total charge can halt gravitational collapse and produce a final stable equilibrium . it is interesting that the pressure vanishes in the final equilibrium state so that hydrostatic support is completely supplied by coulomb repulsion . font and papadopoulos have given a state - of - the - art treatment of relativistic fluids which is applicable to either spacelike or null foliations . their approach is based upon a high - resolution shock - capturing ( hrsc ) version of relativistic hydrodynamics in flux conservative form , which was developed by the valencia group ( for a review see ) . in the hrsc scheme , the hydrodynamic equations are written in flux conservative , hyperbolic form . in each computational cell , the system of equations is diagonalized to determine the characteristic fields and velocities , and the local riemann problem is solved to obtain a solution consistent with physical discontinuities . this allows a finite differencing scheme along the characteristics of the fluid that preserves the conserved physical quantities and leads to a stable and accurate treatment of shocks . because the general relativistic system of hydrodynamical equations is formulated in covariant form , it can equally well be applied to spacelike or null foliations of the spacetime . the null formulation gave remarkable performance in the standard riemann shock tube test carried out in a minkowski background . the code was successfully implemented first in the case of spherical symmetry , using a version of the bondi - sachs formalism adapted to describe gravity coupled to matter with a worldtube boundary . they verified second order convergence in curved space tests based upon tolman - oppenheimer - volkoff equilibrium solutions for spherical fluids . in the dynamic self - gravitating case , simulations of spherical accretion of a fluid onto a black hole were stable and free of numerical problems . accretion was successfully carried out in the regime where the mass of the black hole doubled . subsequently the code was used to study how accretion modulates both the decay rates and oscillation frequencies of the quasi - normal modes of the interior black hole . the characteristic hydrodynamic approach of font and papadopoulos was first applied to spherical symmetric problems of astrophysical interest . linke , font , janka , mller , and papadopoulos simulated the spherical collapse of supermassive stars , using an equation of state that included the effects due to radiation , electron - positron pair formation , and neutrino emission . they were able to follow the collapse from the onset of instability to black hole formation . the simulations showed that collapse of a star with mass greater than 5 10 solar masses does not produce enough radiation to account for the gamma ray bursts observed at cosmological redshifts . next , siebel , font , and papadopoulos studied the interaction of a massless scalar field with a neutron star by means of the coupled klein - gordon - einstein - hydrodynamic equations . they analyzed the nonlinear scattering of a compact ingoing scalar pulse incident on a spherical neutron star in an initial equilibrium state obeying the null version of the tolman - oppenheimer - volkoff equations . depending upon the initial mass and radius of the star , the scalar field either excites radial pulsation modes or triggers collapse to a black hole . the transfer of scalar energy to the star was found to increase with the compactness of the star . the approach included a compactification of null infinity , where the scalar radiation was computed . the scalar waveform showed quasi - normal oscillations before settling down to a late time power law decay in good agreement with the t dependence predicted by linear theory . global energy balance between the star s relativistic mass and the scalar energy radiated to infinity was confirmed . the approach initiated by font and papadopoulos has been applied in axisymmetry to pioneering studies of gravitational waves from relativistic stars . the gravitational field is treated by the original bondi formalism using the axisymmetric code developed by papadopoulos in his thesis [ 165 , 106 ] . because of the twist - free property of the axisymmetry in the original bondi formalism , the fluid motion can not have a rotational component about the axis of symmetry , i.e. the fluid velocity is constrained to the ( r , ) plane . in his thesis work , siebel extensively tested the combined hydrodynamic - gravity code in the nonlinear , relativistic regime and demonstrated that it accurately and stably maintained the equilibrium of a neutron star . as a first application of the code , siebel , font , mller , and papadopoulos studied axisymmetric pulsations of neutron stars , which were initiated by perturbing the density and -component of velocity of a spherically symmetric equilibrium configuration . the frequencies measured for the radial and non - radial oscillation modes of the star were found to be in good agreement with the results from linearized perturbation studies . the bondi news function was computed and its amplitude found to be in rough agreement with the value given by the einstein quadrupole formula . both computations involve numerical subtleties : the computation of the news involves large terms which partially cancel to give a small result , and the quadrupole formula requires computing three time derivatives of the fluid variables . these sources of computational error , coupled with ambiguity in the radiation content in the initial data , prevented any definitive conclusions . a hybrid equation of state was used to mimic stiffening at collapse to nuclear densities and shock heating during the bounce . the initial equilibrium state of the core was modeled by a polytrope with index = 4/3 . collapse was initiated by reducing the polytropic index to 1.3 . in order to break spherical symmetry , small perturbations were introduced into the -component of the fluid velocity . during the collapse phase , the central density increased by 5 orders of magnitude . at this stage the inner core bounced at supra - nuclear densities , producing an expanding shock wave which heated the outer layers . the collapse phase was well approximated by spherical symmetry but non - spherical oscillations were generated by the bounce . after the bounce , the bondi news function went through an oscillatory build up and then decayed in an = 2 quadrupole mode . however , a comparison with the results predicted by the einstein quadrupole formula no longer gave the decent agreement found in the case of neutron star pulsations . this discrepancy was speculated to be due to the relativistic velocities of 0.2c reached in the core collapse as opposed to 10c for the pulsations . however , gauge effects and numerical errors also make important contributions which cloud any definitive interpretation . this is the first study of gravitational wave production by the gravitational collapse of a relativistic star carried out with a characteristic code . it is clearly a remarkable piece of work which offers up a whole new approach to the study of gravitational waves from astrophysical sources . the pitt code has been coupled with a rudimentary matter source to carry out three - dimensional characteristic simulations of a relativistic star orbiting a black hole . a naive numerical treatment of the einstein - hydrodynamic system for a perfect fluid was incorporated into the code , but a more accurate hrsc hydrodynamic algorithm has not yet been implemented . the fully nonlinear matter - gravity null code was tested for stability and accuracy to verify that nothing breaks down as long as the fluid remains well behaved , e.g. , hydrodynamic shocks do not form . the code was used to simulate a localized blob of matter falling into a black hole , verifying that the motion of the center of the blob approximates a geodesic and determining the waveform of the emitted gravitational radiation at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. this simulation was a prototype of a neutron star orbiting a black hole , although it would be unrealistic to expect that this naive fluid code would reliably evolve a compact star for several orbits as it spiraled into a black hole . a 3d hrsc characteristic hydrodynamic code the code was applied to the problem of determining realistic initial data for a star in circular orbit about a black hole . in either a cauchy or characteristic approach to this initial data problem , a serious source of physical ambiguity is the presence of spurious gravitational radiation in the gravitational data . because the characteristic approach is based upon a retarded time foliation , the resulting spurious outgoing waves can be computed by carrying out a short time evolution . two very different methods were used to prescribe initial gravitational null data : a newtonian correspondence method , which guarantees that the einstein quadrupole formula is satisfied in the newtonian limit , andsetting the shear of the initial null hypersurface to zero . a newtonian correspondence method , which guarantees that the einstein quadrupole formula is satisfied in the newtonian limit , and setting the shear of the initial null hypersurface to zero . method 1 has only approximate validity in the relativistic regime of a star in close orbit about a black hole while method 2 completely ignores the gravitational lensing effect of the star . it was found that , independently of the choice of initial gravitational data , the spurious waves quickly radiate away , and that the system relaxes to a quasi - equilibrium state with an approximate helical symmetry corresponding to the circular orbit of the star . the results provide justification of recent approaches for initializing the cauchy problem which are based on imposing an initial helical symmetry , as well as providing a relaxation scheme for obtaining realistic characteristic data . one attractive way to avoid the computational expense of hydrodynamics in treating a star orbiting a massive black hole is to treat the star as a particle . this has been attempted using the pitt code to model a star of mass m orbiting a black hole of much larger mass , say 1000 m . the particle was described by the perfect fluid energy - momentum tensor of a rigid newtonian polytrope in spherical equilibrium of a fixed size in its local proper rest frame , with its center following a geodesic . the validity of the model requires that the radius of the polytrope be large enough so that the assumption of newtonian equilibrium is valid but small enough so that the assumption of rigidity is consistent with the tidal forces produced by the black hole . characteristic initial gravitational data for a double null initial value problem were taken to be schwarzschild data for the black hole . the evolution equations for the particle were arranged to take computational advantage of the energy and angular momentum conservation laws which would hold in the test body approximation . the evolution was robust and could track the particle for two orbits as it spiraled into the black hole . unfortunately , the computed rate of inspiral was much too large to be physically realistic : the energy loss was 10 greater than the value expected from perturbation theory . this discrepancy might have a physical origin , due to the choice of initial gravitational data that ignores the particle or due to a breakdown of the rigidity assumption , or a numerical origin due to improper resolution of the particle . the bondi news function , which represents the gravitational radiation field at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , is computed by post - processing the output data at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } for the primary evolution variables . this is a delicate numerical procedure involving large terms which partially cancel to give a small result . it is somewhat analogous to the experimental task of isolating a transverse radiation field from the longitudinal fields representing the total mass , while in a very non - inertial laboratory . in the original algorithm , the procedure was carried out in the coordinate system of the code by giving a geometric procedure for computing the news . this approach has been tested to be second order convergent in a wide number of testbeds [ 31 , 228 , 229 ] . alternatively , a coordinate transformation may be carried out to inertial bondi coordinates ( as originally formulated by bondi ) , in which the news calculation is quite clean . this approach was recently implemented in and shown to be second order convergent in robinson - trautman and schwarzschild testbeds . a direct comparison of the two approaches was not made , although it is clear both face the same delicate numerical problem of extracting a small radiation field in the presence of large gauge effects in the primary output data . the procedure is further complicated by sources of numerical error , such as the breakdown of the bondi surface area coordinate r near a stationary event horizon , strong nonlinearities and the gauge effects they produce near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , andsharp gradients . the breakdown of the bondi surface area coordinate r near a stationary event horizon , strong nonlinearities and the gauge effects they produce near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } , and these sources of error can be further aggravated by the introduction of matter fields , as encountered in trying to make definitive comparisons between the bondi news and the einstein quadrupole formula in the axisymmetric studies of supernova collapse described in section 5.2 . in the three - dimensional characteristic simulations of a star orbiting a black hole [ 41 , 38 ] , the lack of resolution introduced by a localized star makes an accurate calculation of the news highly problematical . there exists no good testbed for validating the news calculation in the presence of a fluid source . a perturbation analysis in bondi coordinates of the oscillations of an infinitesimal fluid shell in a schwarzschild background might prove useful for testing constraint propagation in the presence of a fluid . however , the underlying fourier mode decomposition requires the gravitational field to be periodic so that the solution can not be used to test computation of mass loss or radiation reaction effects .

i review the development of numerical evolution codes for general relativity based upon the characteristic initial value problem . progress is traced from the early stage of 1d feasibility studies to 2d axisymmetric codes that accurately simulate the oscillations and gravitational collapse of relativistic stars and to current 3d codes that provide pieces of a binary black hole spacetime . a prime application of characteristic evolution is to compute waveforms via cauchy - characteristic matching , which is also reviewed .

Introduction The Characteristic Initial Value Problem Prototype Characteristic Evolution Codes Cauchy-Characteristic Matching Numerical Hydrodynamics on Null Cones

PMC3199644

louis ix had a particular goal in mind when he founded the hospice des quinze - vingts in the mid - thirteenth century . mark otool has drawn attention to the close relationship between the king s own experience of chronic illness and his perception of the blind.17 he maintained a firm belief that those who suffered from any affliction including blindness were worthy of charity if they endured their afflictions with patient devotion and humility like he had.18 the quinze - vingts those who gave money to musicians there , and to those quinze - vingts beggars on the street , recognised that they were activating its spirit through sound . blind members and their sighted carers literally gave their voices to the quinze - vingts community when they entered . voice engaged in prayer , music and street - begging on behalf of the collective institution . quinze - vingts members were the focus of a significant number of bequests from the upper echelons of the bourgeois community of paris for the foundation of memorial masses.19 when the memorial mass was sung in the hospice chapel , the musicians from the community transported the soul of the benefactor to salvation through the sound of their blind - afflicted selves . the affective vocal quality of the blind musician s performance was directly replicated in the behaviour of the institutional space . this was primarily ritualistic , involving a specific initiation process , communal attendance at mass , bell ringing , and a particular moral code . quinze - vingts remained distinct from other religious houses through its particular sense of autonomy . quinze - vingts fought off other blind houses ( six - vingts , for example , a house for 120 blind people founded in 1292 by renaut barbou the elder ) for the most powerful right to sound the blind voice.20 quinze - vingts members saw themselves as retaining the uppermost privilege amongst other blind institutions to harness their own mark otool has explained : rather than marginalize the blind residents , this community was designed to care for them and even showcase their religiosity.21 this was reflected in the unique administrative structure of the institution allowing the blind , supported by the sighted , an important say in the running of the site . during the late seventeenth and early eighteenth centuries , the potent voicing of the collective quinze - vingts community remained an important feature of the urban landscape.22 the presence of quinze - vingts on the street still remained explicitly connected to the spirituality of blindness . in 1744 , for example , a group of quinze - vingts celebrated the king s return from the german war front . lord restore our sight that we may see the king , for just one moment , so that we no longer complain to you , that we have lost it : you will not be able to find , lord , our requests strange;when those least able to hear have the pleasureof hearing the praises . encoded within the song are all the characteristics of the spiritualised quinze - vingts self. blindness is viewed as a way of praising the divine kingdom . these praises are deliberately bold because they need to be heard by all ( even those least able to hear ) . quinze - vingts blindness was still expressed in the form of a spiritualised speech - act . yet , quinze - vingts musicians had also become closely associated with the more secular world of the burlesque . quinze - vingts musicians could be seen and heard on the pont neuf , at the caf des aveugles and , as we have seen , at saint ovid s fair.24 these appearances were influenced by the mediaeval tradition of the french blind farce.25 in this genre the singing blind beggar ( played by a sighted man ) was belittled through the appearance of a sighted guide who reveals the blind man to be morally unworthy of help . it is possible , however , to interpret the performance at the saint ovid s fair as somewhat more nuanced in meaning . by masquerading as singing blind beggars ( dressed up and led by their instruments ) , the quinze - vingts blind were recalling the genre whilst also maintaining their traditional style of performance . from the audiences point of view , the instrumental group resembles the farcical combination of passive blind beggars ( the blind playing the sighted playing the blind ) led by a sighted guide ( the musical instrument ) . the musical instrument is the ideal bodily accompaniment because , as in the original farce , it speaks for the blind man . from the blind s perspective , however , the costumes and the instruments are used to act up their traditional role as blind - afflicted selves . double bodies , enhancing the traditional quinze - vingts quality of bodily affliction by morphing with the blind body.26 thus , the grotesque body of the carnivalesque was , from the blind person s point of view , not necessarily contradictory to the suffering blind bodily figure emblematic of the quinze - vingts institution . form , still resided deep within quinze - vingts bodies that continued to reinvigorate themselves on the burlesque stage . double body form was out of place in the new enlightenment world of idealistic lingual purity . it is possible that hay saw , not only the belittling of the blind , but a dislocated sound and sight in the performance at saint ovid . here the voice of the quinze - vingts musician had a particular potency which sometimes transcended the presence of the physical body to which it belonged . hay was not alone in his fear of the raucous voice . during the later part of the eighteenth century , it was identified as a serious problem in the urban environment . in his 1783 essay , tableau de paris , louis - sbastien mercier expressed concern at some of the sounds and sights of parisian street life.27 he explained that he was deeply embarrassed by the number of beggars in the city who spilled out onto the street during the day from their various institutional houses . he made a specific attack on the mendiants valides [ able - bodied beggars ] who he saw as exploitative of everyday people.28 this was obvious in their physical demeanour which he described as ghastly and frightening . they follow you in the streets slowly uttering the sacred name of god in an unnatural , sorrowful , and monotonous voice.29 mercier saw such beggars as duplicitous . if you followed the beggars into the cabarets at night you would see and hear their violent behaviour . the lame person has discarded his crutch , the blind man his patch , the hunchback his horsehair hump , the one - armed person takes the violin , the dumb signal a frenetic lack of restraint they boast about taxes removed from public sensitivity , violence that they inflict on compassionate and gullible souls . they pass on their secrets to each other ; they repeat their pitiful roles with disrespectful outbursts of laughter.30 mercier s negative characterisation of the mendiants valides is through a figure of vocal and bodily separation . the whining voice of the beggar appears to come from a source outside the beggar s human self.31 the bodies of the figures move about like dismembered limbs . the beggars resemble mechanistic puppets , figures of the freudian uncanny.32 there is something frightening and deathly entering the beggar s human selves . not only does the voice sound horrible but it takes on a violent life of its own through secret communication and revolting laughter . it is possible that quinze - vingts musicians were amongst those depicted in this performance . voices are no longer recognised as spiritually meaningful , but overbearing and threatening . to a certain extent , mercier attempted to silence this kind of voice through his own condemnation in the text . the lame person has discarded his crutch , the blind man his patch , the hunchback his horsehair hump , the one - armed person takes the violin , the dumb signal a frenetic lack of restraint they boast about taxes removed from public sensitivity , violence that they inflict on compassionate and gullible souls . they pass on their secrets to each other ; they repeat their pitiful roles with disrespectful outbursts of laughter.30 mercier s reading reflects the anti - clericalism of the immediate post - revolution.33 the violence of these characters is caused by god . what is heard in their chattering speech is the hideous voice of an unseen god who corrupts the human form . it is the violent voice , however , that is the pivotal subject of mercier s description . it allows him to heighten the emotional level of the text and to create a sense of the threatening ( the beggars ) and the threatened ( himself and the public ) . in hay s eyes and ears , the quinze - vingts musicians at saint ovid s fair might also have posed a threat . they presented a literal voice that was shrill and confronting , but also a figurative voice that was inherently problematic from a bourgeois political and social point of view . hay s response was to desire another voice in the form of harmonious concerts that might drown these musicians out . hay believed he could help all the blind in this aim and he demonstrated with his work at the institute for blind youth that his programmes could be a success . these were closely constructed according to his philanthropic belief in human happiness and the power of the sciences and the arts to transform people s lives . the blind poor could learn a respectable trade through appropriate training . yet , like mercier , he was also expressing a certain anxiety about public health common amongst the post - revolutionary bourgeoisie : this was that the voice of the blind must be silenced or soothed . it is probable that hay heard the ugly , confused tones of popular speech , the visceral cry of birth and death in the music . this was bad music producing sounds far too closely related to the uncivilised cycles of bodily transformation . in his educational manifesto he was later to write : it is probably a lack of principles that reduce some to rushing around the streets going from door to door shattering ears with an out - of - tune instrument or a raucous voice , trying to obtain small change often given to them in return for quiet.34 hay s interpretation of the quinze - vingts voice was that it represented a corrupted body . cured through the implementation of training programmes within the institution and carefully controlled public presentation . catherine kudlick has traced its period of development from the middle of the eighteenth century to the cholera outbreaks of the mid - nineteenth century.35 kudlick explains that ideas relating to public health were initially furnished in the enlightenment in an attempt to improve poor people s lives.36 the revolution brought with it the turbulence that would require these ideas to be put into practice . it also brought an increasing division between the rich ( the givers of health ) and the poor ( the recipients of health ) . disease in the urban environment had been attributed to the behaviour and existence of the poor since the seventeenth century . as foucault has famously demonstrated , confinement was considered an effective means for the suppression of both poverty and disease.37 by the time of the revolution , quinze - vingts was administered entirely separately from the official houses for the poor , the hpitaux gnraux . nevertheless , it was considered similarly poor and diseased by the authorities on the basis of its perceived antisocial behaviour.38 the enlightenment culture of signs was now embedded in the language of the law.39 those unable to conform to an acceptable code of behaviour were considered close to savages and treated as ill. much of this political viewpoint was initially expressed in concern over the religious nature of its character . at stake just prior to the revolution the then superior general , the grand almoner cardinal de rohan , had been accused by the parlements of serious financial corruption during the hospice s move to the rue de charenton . an enquiry had been ordered.40 further problems , however , emerged later in the behaviour of the blind internal administrators , the jurors . frres , to swear allegiance to the institution in the manner of a religious brotherhood . the question constantly posed by authorities was : is there good order and decency within the hospital?41 such a question undermined the independent voice of the jurors . on 5 november 1790 the institution was officially placed under the protection of the national assembly [ assemble nationale ] and the jurors continued meeting unofficially . petition aside , quinze - vingts , now situated in one of the poorest quartiers of paris , was viewed by the state as an important participant in the new post - revolutionary culture of bienfaisance.42 like many of the parish - based institutions for poor - relief , quinze - vingts was transferred to the paris commune s municipal commission [ commission municipale ] in 1791 , which employed state commissioners to liaise with local clerical administrators to instil civic pride . this move sparked protest amongst quinze - vingts members , who resented the gaze of any specific administrator , religious or secular , appointed without their permission.43 during this time , the paris department had also received a complaint from a blind couple , m. gauthier and mme lidelle , who had been excluded from the institution by the members themselves during the turbulent period of institutional relocation.44 the jurors had expelled at least four members for marrying without their authorisation . this move went unreported during the affair , but the newly established committee of public assistance [ comit des secours publics ] was subsequently informed of the expulsions . in 1793 , the committee recommended suppression of the institution.45 of most concern was the perceived inappropriate behaviour of the jurors . it was the jurors who had created the need for the original enquiry , the committee complained , by expelling members who had rights to accommodation within the institution s grounds . they were also in agreement with complaints made by the commune clerk that the institution was deeply undemocratic , privileging only a few blind people from paris.46 the report by the committee of public assistance included a scathing attack on the institution s the institution was a collective body of sick individuals : so many people of any age or sex , most destined to live in idleness , can only be mutually depraved thus piled up ( if i may use this expression ) in cramped and enclosed conditions . also , for some years now , there have been serious complaints concerning the licentious life led in this house . the disturbances had inevitably grown in recent times as a result of the spirit of independence , division and anarchy which was adopted there.47 such a statement recalled the bodily sounds and images of the mendiants valides as described by mercier . though the individual blind self remained connected to its collective quinze - vingts home , it now belonged to a diseased group of citizens who created an extreme level of aggressive noise through unregulated behaviour . by recognising the violent voice in the dynamics of the quinze - vingts space , the committee of public assistance also implied the need for the firm voice of authority . so many people of any age or sex , most destined to live in idleness , can only be mutually depraved thus piled up ( if i may use this expression ) in cramped and enclosed conditions . also , for some years now , there have been serious complaints concerning the licentious life led in this house . the disturbances had inevitably grown in recent times as a result of the spirit of independence , division and anarchy which was adopted there.47 yet suppression was avoided . there was one authoritarian voice amongst the post - revolutionary who cherished the voice of the quinze - vingts blind . the reasons given for closing the institution were not plausible , it explained.48 the hospice had special qualities that should be preserved : humanity , justice and the general interest call for the preservation of this precious hospital.49 the system of jurors , previously attacked for its insularity , was suddenly praised for its qualities of corporative self - regulation : they support each other and watch over each other ; if there is a disturbance they are alerted immediately as it is in everyone 's interest to bring it to an end .50 in response to the criticism that the institution was only for the privileged few , observers replied that , as well as accepting blind people from departments beyond the paris zone , the institution could act as a model for the establishment of other similar institutions throughout the country , as it had originally in the middle ages . one , a m. maynier , for example , had raised his voice for the protection of the unfortunate and oppressed blind.51 the notion of voice here implies sympathy and compassion . citizens should join their voices with those of quinze - vingts described later in the document as such political authorities recognised the blind voice in much the same manner as mediaeval listeners did . they acknowledged that the blind voice expressed at quinze - vingts played an important role in society and should be cared for . quinze - vingts members , in turn , situated themselves within this revolutionary call for human rights . in demanding their right to a voice , they referred not only to their original charter of spiritual blind privilege ; they also referred to revolutionary observations , enshrined in the declaration of the rights of man [ dclaration des droits de lhomme ] , acknowledging all men ( including les malheureux ) the right to a voice . they echoed the parliamentary observation that some citizens required humanitarian assistance and a sympathetic ear from those in political control . to raise one s voice was to stage a political act of empowerment , as conveyed in the meaning of la voix as a term of personal enfranchisement ( donner la voix means to vote. ) such defiant voices , however , commenced as soon as quinze - vingts was placed under enquiry in 1789 . the blind asserted their voices as soon as they began to be perceived as a threat . in 1790 , the quinze - vingts blind wrote a long letter to the national assembly complaining about their treatment.52 it was lodged by a lawyer and signed by twelve members of the jurors : franois monier , franois gonoro , jean prevt , louis godevier , franois hoyez , pierre de longue , nicolas chadrin , franois crevier , jean louis barre , pierre le bros , jean louis baudoin , and caizergue . for ten years this class of men whose fate and misfortune are so moving for thinking souls , could hope that it would always be comforted in its adversity.53 they requested that their original form of administration be restored and attacks on their members be immediately stopped . they asked that their special status , conferred on them by the declaration of a king [ dclaration du roi ] , should be given the direct protection of the national assembly . brothers [ frres aveugles ] wrote a second petition to the national assembly in 1792 , demanding to be placed under the administration of the departments as a whole , not under the scrutiny of the local municipality , emphasising that their institution was established for all the blind of the empire.54 as if to demonstrate their desired independence , they had their demands transcribed by a sighted member of their community , instead of printed , and deposited the document themselves rather than through a lawyer . they signed their declaration not with a list of names but with the imperative phrase et ferez justice. during the immediate post - revolutionary period , quinze - vingts members played crucial roles in maintaining their independence . in 1802 , they wrote to protest against the amalgamation with the institute for blind youth . perpetual celibacy , since they would have to abandon their wives and children for a life of repression.55 it offends your sensitivity , citoyen consul , that the blind are treated as old people who will end their days in the hospice , or like sick persons who are there for a short time to recover their health , and , which is even harder , like vagrants to be removed from society, insisting instead that the blind are fathers with families or may become so ; they are welcomed at any age at the quinze - vingts and enjoy all the freedoms of the other citizens.56 in this statement , members demanded , not only their right to unqualified support as unfortunates of the state , but the freedom to express themselves as members of a newly established democratic society . national assembly explaining that it was the only authority in which these unfortunates can place their confidence and their hope.57 ironically , they also signed off the petition with a reference to their original foundation of royal privilege : you will not suffer , citoyen consul , that the history book , already open to record your virtues and your good deeds contains these words in the year ix of the republic , the hospice des quinze - vingts founded by a king , protected by all , enriched by good house - keeping and by its legacy.58 in their demands , the post - revolutionary blind referred to all past political regimes , however diverse , who had supported their right to a voice . the quinze - vingts voice was perceived once again by state authorities as threatening immediately after it was celebrated as exemplary . after the restoration of the institution in 1793 , the authorities aimed to shut down the literal musical voice of the institution and its associated figurative voice . the independent ethos of the institution continued to be perceived as a persistent source of the problem . quinze - vingts members were forced to put up with administrators appointed externally.59 the authorities also implemented hygienic reform to drown out the institution s spirited vocalising of blind affliction . this would not only improve the literal voices of the blind , but also reform the problematic figurative voice of the institution . violent voices would be transformed into beautiful ones which would make the public healthy when they heard them . after the institution s re - establishment , any constructive dialogue between the institution and political authorities collapsed . political documents either do not acknowledge the quinze - vingts voice ( the politicians are simply not listening ) or attend to it by attempting to silence it . quinze - vingts members protested against this treatment and continued to vocalise their presence in the urban environment through music . the tense situation was summed up by the agent general [ agent gnral ] when he reported to the minister of the interior in 1801 : the blind should be visited and be involved with so that you can get to know them better ; they are capable of anything ; they are allowed to do all things and they think that their infirmity protects them from everything.60 the blind are defined as threatening through their ability to express an unlawful voice . only by experiencing this voice first hand , the agent general warned , will the authorities know just how dangerous it could be . the authorities saw the merger between quinze - vingts and the institute for blind youth as an ideal opportunity to reform the voice of the entire institution . it would no longer be a place of bodily deviance , they argued , but of utility and cure . to articulate the blind voice begging was outlawed in the immediate post - revolutionary period for the reason that the institution was hiding money unlawfully . yet laws against begging remained enforced on the basis that they were associated with violence . in 1795 , the minister of the interior wrote to the director that a group of blind from the institution had been seen in the countryside to beg alongside ill - intentioned people and this is disturbing the public peace.61 the blind were not to wander from the house on their own without permission , he explained , and must be subject to the repressive laws on begging to prevent such a situation occurring again . musical instrument playing was officially permitted but a blind musician could easily be accused of begging rather than performing and then prohibited . laws against begging were incorporated into the rules and regulations of the institution during the consulat and the empire . the implementation of a set of policing regulations in 1800 by the then minister of the interior , lucien bonaparte , combined both a ban on begging and the official appointment of a director or agent general , not only to oversee the finances but also to supervise the movements of members . the blind - afflicted body now required containment both inside and outside the institution . like members of the post - revolutionary national assembly , bonaparte considered the institution like a body out of balance . it was in a state of the greatest disorder which would be completely transformed through the creation of a good system of administration and the establishment of a gentle and humane regime but fairly strong in order to maintain order , subordination and manners.62 the regulations centred on issues of hygiene and mess . if members threw rubbish into the corridors or left excrement on the toilet floor they would be fined ; if they kept pigeons or rabbits , the animal would be confiscated and the inhabitant fined ; dog mess would incur a fine . other rules addressed the supposedly unruly behaviour of the children of members , who traditionally lived in the compound with their parents . if they were not taken to school , or played violently , threw stones or fireworks , the parent would lose their journal subscription . members were also forbidden to have visitors to stay overnight or for a meal without permission . such regulations were attempts to impose practices of health on an institution that was essentially considered unwell . in 1801 , the then minister of the interior , chaptal , attempted to integrate hay s institution entitled , the national institute for blind workers [ institut national des aveugles - travailleurs ] originally the institution of blind youth [ institut des jeunes aveugles ] with quinze - vingts . chaptal saw the institution as a workhouse.63 this was totally contrary to hay s vision of the intellectual blind self , who read , wrote and learned music . more than half of the blind youth s day was thus spent in the wool spinnery , with the exception of a few students admitted to the music class or the print works.64 hay was unhappy with the emphasis on manual labour . it is possible to argue , however , that hay s agenda was not so far from chaptal s as might at first be thought . hay was , of course , attempting reform in a much more sensitive and progressive way . he took account of the intellectual abilities of the blind rather than relegating them to the workhouse . our institution will offer help to all , either for study , or for the practice of their art,65 he explained in his 1784 manifesto . hay developed a system of imprinting musical characters , along with letters of the alphabet , so that the blind could read and memorise music held in the bibliothque de got.66 hay admired the learning of the old masters such as bach , as well as french composers , couperin and balbastre.67 hay s programme of reform belonged to a much broader culture of musical education designed to cultivate the blind person . the musical education of the young blind during the post - revolutionary period was profoundly influenced by the composer and music theorist jean - philippe rameau . rameau reduced some of the more complex acoustical theories of cartesian mathematics into a single palatable system for pedagogical use.68 the simplicity and geometry of rameau s system made it perfect for teaching , and it formed the basis of harmony and composition programmes of post - revolutionary institutions , the paris conservatoire and the young blind at the institute for blind youth and quinze - vingts.69 students could memorise the progressions , absorb them and practise them over a year or more when they entered the institution . only after this process of intensive learning and practice could they produce performances on their instrument of choice and compositions of real taste. after memorisation , progressions could be skilfully reversed to create a perfect arch form , and in turn , the invention of a complete piece of music . rameau s system catered especially for blind pupils , a point he makes plain in the full title of his 1761 teaching manual , code de musique pratique , ou mthodes pour apprendre la musique , mme des aveugles [ practical music code , or methods for teaching music , even to the blind ] . blind students could shape their fingers and tune their ears producing the best results , precisely because they could not see . the system can best be described as a kind of physical intuition based on strict harmonic practice . as rameau explained : using this system , the fingers acquire a knowledge which nourishes the ear of all the harmonic routes , while it presents the mind with a reliable example of all the rules in which it must be educated , with the result that judgment , the ear and the fingers of intelligence work together to quickly produce the perfection that is desirable in this genre street criers and popular instrumentalists left listeners cold , rameau explained , because their music was based on an overly primitive theory of harmonics.71 street melodies were unsupported by an appropriate harmonic system . using this system , the fingers acquire a knowledge which nourishes the ear of all the harmonic routes , while it presents the mind with a reliable example of all the rules in which it must be educated , with the result that judgment , the ear and the fingers of intelligence work together to quickly produce the perfection that is desirable in this genre .70 to educate musicians using such a system was to remedy the problem of the raucous blind voice . it would create a blind self who was harmonised through a systemised set of sonic and gestural signs . hay believed that young blind musicians , trained in his institution , would lead the way . but he would also have hoped that the musical programme would have influenced all at quinze - vingts after the amalgamation . he wanted to create a completely new voice for the institution that was in keeping with civilised musical programmes of the late eighteenth - century bourgeoisie . but it could not be easily implemented alongside the blind voice at quinze - vingts . in 1816 , the blind youth institute departed quinze - vingts to form a separate institution once again . hay s musical programme was developed by the new director of the institute for blind youth now the royal institute for blind youth [ institution royale des jeunes aveugles ] and continued to influence generations of young blind musicians.72 quinze - vingts musicians , however , continued to perform on the streets of paris . yet they were met with further voices of political opposition that were to increase in volume as the century progressed . during the restoration , , they wrote to the grand almoner [ grand aumnier ] , gustave - maximilien - juste , prince de cro , their current superior general [ suprieur gnral ] , for an official restitution of the jurors.73 the jurors had continued to meet throughout the empire and restoration , though externally appointed administrators had often intervened in the running of the institution . unhappy with the administrators appointed by the prince , they demanded the official restitution of the juror system in order to safeguard against what they perceived as unlawful external interference . it would be much more economically expedient , the blind explained , to hand all moneys directly to the newly restored jurors , the observing and protective voice,74 who would then distribute the moneys appropriately . from the 1830s , kudlick and weygand have noted that during the restoration , the catholic ultra - royalists who ran the quinze - vingts looked particularly unfavourably upon their student musicians or singers who performed in such immoral places [ as the caf of the blind ] at all hours.75 in the 1820s , the almoner prohibited them from going to the caf , a venue they had continued to use for musical performance after the revolution.76 by the 1830s , the authorities realised that if the blind insisted on performing at the caf , boundaries had to be established by those in political control . in 1831 , they reluctantly granted permission for a band to visit the caf on the condition that it conformed to a strict curfew . most of the blind being able to devote themselves only to the art of music , the administrator will decide the extent to which they must be permitted to carry out this activity,77 they declared . the restriction of blind voices during the 1820s and 30s coincides with an intense period of panic amongst the parisian bourgeoisie concerning disease such as cholera . kudlick has explained that this heightened anxiety came from a new shift in perception that : paris suffered from a kind of internal imbalance rather than from externally imposed conditions of geography and climate ; the city was plagued by forces of its own making . implicit in this view was a moral judgement : not only was the capital becoming sick , but the sickness was part of the very fabric of urban existence and resulted from human activity rather than mysterious natural forces.78 by focusing on the individual behaviour of the poor and the environment that surrounded them , the bourgeoisie were reassured that they might not catch the disease . now , all the sites outside the institution frequented by the quinze - vingts musicians were under threat . paris suffered from a kind of internal imbalance rather than from externally imposed conditions of geography and climate ; the city was plagued by forces of its own making . implicit in this view was a moral judgement : not only was the capital becoming sick , but the sickness was part of the very fabric of urban existence and resulted from human activity rather than mysterious natural forces.78 though musicians continued to perform on the streets , their movements were heavily scrutinised . the authorities forced members , in particular the musicians , to wear a military - style uniform when out and about.79 such a uniform performed a completely different function from the fleur - de - lis robe offered to members by louis ix at the institution s foundation . post - revolutionary uniforms were designed to muffle or mute it with an instrument of authority . this was followed by much tighter forms of restrictions on public movements enforced by the prfecture de police.80 in 1842 , a list of quinze - vingts beggars was drawn up under the title : the blind who engage in any activity on the public highway in paris either by playing various instruments , or singing , fortune - telling , or peddling objects with no value as a way of disguising their begging.81 alongside a list of the licenced of the twenty - three individuals , eleven were instrumental musicians ( seven clarinettists and four violinists ) , with the remaining ( including an additional set of two small groups ) selling matches or asking for alms near churches . alongside the implementation of this system was a set of laws designed to ban the movements of quinze - vingts musicians outright.82 travelling beyond paris was now out of the question following the implementation of a revised and convoluted new application procedure . begging on the streets of paris ( either individually or in groups , though the latter was a more serious offence ) could also be permanently thrown out of the hospice , or have their pension dramatically reduced if they did not have the appropriate permission . attempts to silence the blind voice became much worse during the period of the july revolution . the classification of quinze - vingts members as violent , poor and diseased was suddenly extended to include revolutionary.83 in 1847 , a group of hospice inhabitants was expelled from the institution , presumably for begging on the street.84 they found their way to the house of victor hugo who arranged alternative accommodation for them in a provincial parish . he complained vehemently to the quinze - vingts chaplain , jean prompsault , who had managed to save the quinze - vingts chapel from closure by the church authorities during the 1830s . prompsault responded : the blind for whom you are concerned have been treated with a severity which , in my opinion , would be difficult to justify . i can state without fear of contradiction , that they are the best educated and the most peaceful men in the establishment.85 prompsault and hugo were amongst the few public figures during the mid - century period to recognise the blind voice . as prompsault explained : i have just received a letter from him [ the minister of the interior ] which states that this measure was intended to restore order , discipline and subordination , that it was successful in its aim , that the government of the republic does not need to be persuaded to take an interest in the blind , and that i must not intervene in an act of internal administration.86 meanwhile , the director proposed an ambitious and expensive renovation plan for the quinze - vingts site.87 though the plan was never executed , major work began on the bastille railway station next to the site in 1856 . a brand new ocular clinic was built in 1870 , and the site quickly transformed into an institution primarily designed for surgical practice.88 the blind were temporarily removed from the site altogether in 1885 . i have just received a letter from him [ the minister of the interior ] which states that this measure was intended to restore order , discipline and subordination , that it was successful in its aim , that the government of the republic does not need to be persuaded to take an interest in the blind , and that i must not intervene in an act of internal administration.86 the mid - nineteenth - century period of quinze - vingts history was dominated by aggressive modification of the institution s architecture , resulting in complete transformation . the independence of the figurative blind voice was silenced through the remodelling of its mouthpiece ( the institution ) by authorities and knowledgeable experts into something quite new and different . surgeons were eager to apply new methods in ocular surgery during this period to quinze - vingts patients and others throughout the world . their laudable aims also coincided with an assault on the blind voice . just prior to the installation of the surgical clinic at quinze - vingts , the quinze - vingts blind were the subject of a vicious verbal attack by lonce guyot - montpayroux , an active enemy of the empire and soon - to - be member of gambetta s government of the third republic . without mentioning them by name , he decried a group of citizens , les peureux et les aveugles [ the fearful and the blind ] dominating the tenth arrondissement of paris.89 these citizens , he explained , were extremists , either reactionaries or revolutionaries , who refused to change or be cured through their weakness or stubbornness of character . they could be identified through the terrible sounds they created as they wandered around the countryside in groups of musical bands . get close and listen to them while observing how each person likes to attract particular attention : the trombone will raise the pitch , the unfortunate clarinet will do its best not to be drowned out by the noise , and in a short time it will be a cacophony which will make you wish you were deaf.90 behind the attack was a political message in france , the interior administration is thus ; when it thinks that an eye is being kept on it , it acts in the same way as the clarinet that i have spoken about91 but the characterisation of the blind voice as threatening was all too familiar . the blind voice was a serious social problem and should be silenced . aside from efforts to maintain the voice of the institution , the blind actively protested against legislation designed to curb their voices . they discarded the uniform designed to muffle their voices on the street , complaining so vehemently about the restriction that the director recommended the rule be dropped.92 when work began on the bastille railway station they complained that the noise from the outside streets was unbearable.93 their voices , by implication , would be drowned out . a group of musicians from the institution was granted permission to perform on the streets of paris at the same time.94 unwanted noise would , once again , be met with blind voices . amongst these was franois leclerc , who requested permission to perform his clarinet on the bridges of the capital.95 by articulating their voices , quinze - vingts members presented the authorities with a formidable challenge . their vocalic mode of utterance gave them a particularly powerful presence in the urban landscape . they presented themselves in the highly public domain of the urban parisian street promoting a bodily image and vocal language that resisted any form of intervention by experts . lithographs printed during the period demonstrate that quinze - vingts musicians continued to be a lively presence in the urban environment despite regulations designed to suppress their activities at the time ( see figure 2).96figure 2gottfried engelmann , quinze - vingts members managed to maintain the right to sound the blind voice in the post - revolutionary period , but they were met with a series of authoritarian voices designed to silence them . the most sympathetic of these was produced by valentin hay , who desired reform amongst the blind through attention to social and intellectual improvement . quinze - vingts members protested against laws designed to silence them by continuing to sound their voices . they ignored curfews and continued to insist that protection of the quinze - vingts voice should be upheld . it is crucial that we acknowledge the blind voice of the post - revolutionary period . utterance gave blind musicians a sense of vocal power that was attached to a highly embedded , culturally defined status . they took care to continue to sound the blind self because it mattered to them . such vocal contributions , however , were much more than simply random acts of self - interest on behalf of a small minority group . self that so many french thinkers and writers had dreamed of in the period prior to the revolution . patient had the right to speak up and engage in a dialogue with their political and medical carers . political authorities also used voices to assert authority . their unwillingness to listen to the blind voice patient required the blind voice because its parameters of care were , in many ways , far too prescriptively confined . to acknowledge the sounding citizen the citizen patient at the hospice des quinze - vingts had a clear and startling voice .

this essay explores new models of the citizen patient by attending to the post - revolutionary blind voice. voice , in both a literal and figurative sense , was central to the way in which members of the hospice des quinze - vingts , an institution for the blind and partially sighted , interacted with those in the community . musical voices had been used by members to collect alms and to project the particular spiritual principle of their institution since its foundation in the thirteenth century . at the time of the revolution , the quinze - vingts voice was understood by some political authorities as an exemplary call of humanity . yet many others perceived it as deeply threatening . after 1800 , productive dialogue between those in political control and quinze - vingts blind members broke down . authorities attempted to silence the voice of members through the control of blind musicians and institutional management . the quinze - vingts blind continued to reassert their voices until around 1850 , providing a powerful form of resistance to political control . the blind voice ultimately recognised the right of the citizen patient to dialogue with their political carers .

Hearing and Understanding the Blind Voice Human Rights: Resisting Voices Silencing the Quinze-Vingts Voice

PMC4241926

when this extra body heat is not adequately released from the body or balanced , an elevation in body temperature is inevitable . as heat accumulates sweating is critical in managing body thermal load via evaporative cooling , especially while exercising in a hot environment . sweating reduces body fluid volume , resulting in dehydration , which increases physiological strain and reduces physical capacity . these adverse effects can be greater as the fluid deficit increases over exercise intensities . to minimize the negative effects of dehydration , voluntary drinking to ensure a hydrated state prior to and during exercise is recommended . rehydration by drinking is beneficial for reducing thermal and cardiovascular strain , stabilizing metabolic function , and decreasing fatigue . thus , rehydrating to maintain body fluid volume is a concern when exercising in a hot environment . although rehydration during exercise is recommended , individual variations in volume consumption and retention exist . to date , several factors influencing the volume of fluid consumption and volume retention have been proposed , including the contents of carbohydrates and electrolytes within the fluid [ 7 - 10 ] , heat acclimation status , age , and gender . however , subjects in these previous studies were tested without experiencing both exercise stress and heat acclimation at once and/or were examined only in a laboratory setting . for example , voluntary fluid intake was measured during exercise and recovery , and the volume intake was not controlled . these subjects were encouraged to drink , but the volume was not manipulated . in real life , frequent drinking during exercise and heat acclimation may occur . no reported study has investigated the impact of systemic fluid consumption training during short - term exercise and heat acclimation with a prescribed volume intake . although maintaining an adequate hydration level is believed to minimize thermoregulatory instability and maximize circulatory and metabolic capability while exercising in a hot environment , it has not been clearly demonstrated whether an active habitual drinking practice during training could modify voluntary drinking during subsequent exercise in a hot environment . this study was designed to investigate the impact of habitual fluid drinking practices while exercising and to evaluate the impact of heat stress on subsequent drinking volume while exercising in a hot environment . it was hypothesized that an active drinking practice would elicit greater fluid consumption and sweat rate versus a non - active drinking condition when fluid intake was allowed ad libitum . subjects participated in two experiments with identical procedures but different drinking interventions : 1 ) active hydration ( ah ) and 2 ) passive hydration ( ph ) . each experiment consisted of three sequential phases : 1 ) pre - testing , 2 ) 10-day training period , and 3 ) post - testing . , subjects ran on a treadmill in a hot environment . to achieve the study goal , they were required to drink different volume during two conditions . subjects were required to drink 1.5 times their body weight loss while exercising during ah and 0.5 times their body weight loss during ph . the two experiments were separated by at least 3 weeks , and the experiments were conducted during the late fall and winter . based on medical history , they were considered free from any pulmonary , metabolic , and orthopedic complications and had no heat illnesses . full study documentation and verbal explanations about the procedures involved were provided to all subjects , and they signed informed consent forms . the subjects physical characteristics including age , height , weight , and body fat content were measured using the bioimpedance method ( inbody 520 ; biospace , korea ) . during at least three separate occasions prior to the testing , the subjects were familiarized with running on a treadmill . using the modified bruce protocol , the maximal oxygen uptake ( vo2max ) of the subjects was measured while they ran on a treadmill ( q65 , quinton instrument co. , bothell , wa , usa ) . they exercised until volitional exhaustion while breathing through a breath - by - breath gas analysis system ( k4b , cosmed srl , rome , italy ) . after completion of the vo2max test , they rested for at least 30 min or until they returned to their resting heart rate ( hr ) . then , they ran once again to identify the speed of the treadmill that matched the range of each individual s exercise intensity ( 55 - 60% of the vo2max ) . initially , the speed of treadmill was set to elicit ~50% of the vo2max prediction ; additionally , it was adjusted every 3 min to match the target oxygen uptake ( vo2 ) . when they could steadily match their target vo2 for at least 56 min , the treadmill belt speed was recorded . that speed was used during testing and training . at 3 days after the pre - screening session their naked body weight was measured using a scale ( 150a , cas , seoul , korea ) , and a rectal probe ( barnant co. , barrington , il , usa ) was inserted 15 cm beyond the anal sphincter . thermocouples ( 692 - 0230 ; barnant co. , barrington , il , usa ) for measuring the subjects skin temperature were attached to three sites ( chest , triceps , and thigh ) using surgical tape ( micropore ; 3 m , st . an electronic hr monitor ( s610 ; polar electro oy , finland ) was strapped around the subjects chests . once all the equipment was adjusted , they entered a climate controlled chamber , with the temperature maintained at 39 1 with a relative humidity of 65 3% , and the subjects rested in a seated position for 23.3 7.6 min . after they adjusted to the environment , a blood sample was taken from the antecubital vein using a disposable syringe . then , they were asked to stand on the treadmill and begin running for 90 min at their previously determined treadmill belt speed . they were allowed to drink ad libitum during the testing , and they occasionally breathed through a mask for respiratory gas analysis . when they completed their exercise , all the measuring equipment was detached from their bodies , and they were dried completely . after their urine samples were collected , their naked weight was taken . subjects walked outside the chamber and recovered for 3 h at ambient temperature ( 25 2 ) without any vigorous physical activity or food consumption . during recovery , their naked body weight , blood samples , and urine samples were collected during the 1 and 3 hours . beginning 2 days after pre - testing , subjects participated in a 10-day training period where they exercised in an environment and at an exercise intensity identical to that in pre - testing . during the 90-min exercise , each subject had his own bottle filled with an assigned volume of fluid prepared by the investigators . the fluid volume to be consumed during the training period was determined during pre - testing . subjects in ah were asked to consume a sports beverage ( gatorade thirst quencher ) at 150% of their body weight loss while subjects in ph consumed 50% of their body weight loss . one - third of the target fluid volume was provided every 30 min of training . when they did not consume the amount provided within the time period , the leftover was discarded . although the amount and time frame for beverage consumption was decided for each subject , they were encouraged to drink continuously . within 2 days after training period completion after the experiments were completed , subjects underwent a second screening session , and the vo2max was evaluated . from height and weight , the body mass index ( kgm ) and body surface area ( m ) were calculated . all body weights were measured with accuracy of 10 g. all urine samples were used for volume measurement and urine specific gravity ( usg ) using a digital refractometer ( ug ; atago , tokyo , japan ) . rectal temperature ( tre ) and each skin site temperature ( tsk ) were monitored continuously using a thermocouple scanner ( barnant co. , barrington , il , usa ) and were recorded at 5-min intervals . the mean tsk was calculated by appropriately weighing the individual s tsk , 0.43 ( chest tsk ) + 0.25 ( triceps tsk ) + 0.32 ( thigh tsk ) . the mean body temperature ( tb ) was calculated as 0.73 tre + 0.27 mean tsk . during the testing the target volume of fluid consumption during the training was determined during pre - testing of each experiment . for this , the body mass difference between the baseline and at the end of the exercise was calculated with a correction for fluid volume consumed and voided . from this value , the relative amount of the individual beverage volume was calculated , and the value was considered as sweat loss . during testing , the fluid volume consumed , volume voided , and weight changes were calculated to estimate the sweat rate ( sr ) , expressed in mlkgh . blood samples were collected using a 10-ml sterilized disposable plastic syringe and were immediately transferred to a clot activator tube ( green vac - tube ; green cross ms corp . the serum was separated using a centrifuge ( ha-1000 - 3 ; hanil science co. , incheon , korea ) for 7 min and was analyzed for total proteins using a blood chemistry analyzer ( ektachem dt60 ii ; johnson & johnson , new brunswick , nj , usa ) . serum osmolality was measured in triplicate by using the freeze - point depression osmometer ( micro - osmometer model 210 ; fiske associates , norwood , ma , usa ) , and the average value of the three reading was taken . during exercise in the testing , the rating of perceived exertion ( rpe ; 11-point scale , 010 ) , thirst sensation ( 11-point scale ; 0 : no sensation to 10 : extremely thirsty ) , and heat sensation ( 11-point scale ; 0 : no sensation to 10 : extremely hot ) were asked and evaluated at the 15 and 90 minutes of exercise . the hr and vo2max were analyzed at the 15 , 40 , 65 , and 90 minutes during the testing . all variables are expressed as means standard deviation . a comparison of values between the ah and ph was made using student s t - test . when comparing conditions ( ah vs. ph ) to the testing period ( pre- vs. post - testing ) , within - subjects factorial analysis of variance with repeated measures was employed . subjects participated in two experiments with identical procedures but different drinking interventions : 1 ) active hydration ( ah ) and 2 ) passive hydration ( ph ) . each experiment consisted of three sequential phases : 1 ) pre - testing , 2 ) 10-day training period , and 3 ) post - testing . , subjects ran on a treadmill in a hot environment . to achieve the study goal , they were required to drink different volume during two conditions . subjects were required to drink 1.5 times their body weight loss while exercising during ah and 0.5 times their body weight loss during ph . the two experiments were separated by at least 3 weeks , and the experiments were conducted during the late fall and winter . based on medical history , they were considered free from any pulmonary , metabolic , and orthopedic complications and had no heat illnesses . full study documentation and verbal explanations about the procedures involved were provided to all subjects , and they signed informed consent forms . the subjects physical characteristics including age , height , weight , and body fat content were measured using the bioimpedance method ( inbody 520 ; biospace , korea ) . during at least three separate occasions prior to the testing , the subjects were familiarized with running on a treadmill . using the modified bruce protocol , the maximal oxygen uptake ( vo2max ) of the subjects was measured while they ran on a treadmill ( q65 , quinton instrument co. , bothell , wa , usa ) . they exercised until volitional exhaustion while breathing through a breath - by - breath gas analysis system ( k4b , cosmed srl , rome , italy ) . after completion of the vo2max test , they rested for at least 30 min or until they returned to their resting heart rate ( hr ) . then , they ran once again to identify the speed of the treadmill that matched the range of each individual s exercise intensity ( 55 - 60% of the vo2max ) . initially , the speed of treadmill was set to elicit ~50% of the vo2max prediction ; additionally , it was adjusted every 3 min to match the target oxygen uptake ( vo2 ) . when they could steadily match their target vo2 for at least 56 min , the treadmill belt speed was recorded . that speed was used during testing and training . at 3 days after the pre - screening session , pre - testing was conducted . on the day of testing , subjects reported to the laboratory after a 10-h overnight fast . their naked body weight was measured using a scale ( 150a , cas , seoul , korea ) , and a rectal probe ( barnant co. , barrington , il , usa ) was inserted 15 cm beyond the anal sphincter . thermocouples ( 692 - 0230 ; barnant co. , barrington , il , usa ) for measuring the subjects skin temperature were attached to three sites ( chest , triceps , and thigh ) using surgical tape ( micropore ; 3 m , st . an electronic hr monitor ( s610 ; polar electro oy , finland ) was strapped around the subjects chests . once all the equipment was adjusted , they entered a climate controlled chamber , with the temperature maintained at 39 1 with a relative humidity of 65 3% , and the subjects rested in a seated position for 23.3 7.6 min . after they adjusted to the environment , a blood sample was taken from the antecubital vein using a disposable syringe . then , they were asked to stand on the treadmill and begin running for 90 min at their previously determined treadmill belt speed . they were allowed to drink ad libitum during the testing , and they occasionally breathed through a mask for respiratory gas analysis . when they completed their exercise , all the measuring equipment was detached from their bodies , and they were dried completely . after their urine samples were collected , their naked weight was taken . subjects walked outside the chamber and recovered for 3 h at ambient temperature ( 25 2 ) without any vigorous physical activity or food consumption . during recovery , their naked body weight , beginning 2 days after pre - testing , subjects participated in a 10-day training period where they exercised in an environment and at an exercise intensity identical to that in pre - testing . during the 90-min exercise , each subject had his own bottle filled with an assigned volume of fluid prepared by the investigators . the fluid volume to be consumed during the training period was determined during pre - testing . subjects in ah were asked to consume a sports beverage ( gatorade thirst quencher ) at 150% of their body weight loss while subjects in ph consumed 50% of their body weight loss . one - third of the target fluid volume was provided every 30 min of training . when they did not consume the amount provided within the time period , the leftover was discarded . although the amount and time frame for beverage consumption was decided for each subject , they were encouraged to drink continuously . within 2 days after training period completion after the experiments were completed , subjects underwent a second screening session , and the vo2max was evaluated . from height and weight , the body mass index ( kgm ) and body surface area ( m ) were calculated . all body weights were measured with accuracy of 10 g. all urine samples were used for volume measurement and urine specific gravity ( usg ) using a digital refractometer ( ug ; atago , tokyo , japan ) . rectal temperature ( tre ) and each skin site temperature ( tsk ) were monitored continuously using a thermocouple scanner ( barnant co. , barrington , il , usa ) and were recorded at 5-min intervals . the mean tsk was calculated by appropriately weighing the individual s tsk , 0.43 ( chest tsk ) + 0.25 ( triceps tsk ) + 0.32 ( thigh tsk ) . the mean body temperature ( tb ) was calculated as 0.73 tre + 0.27 mean tsk . during the testing , the target volume of fluid consumption during the training was determined during pre - testing of each experiment . for this , the body mass difference between the baseline and at the end of the exercise was calculated with a correction for fluid volume consumed and voided . from this value , the relative amount of the individual beverage volume was calculated , and the value was considered as sweat loss . during testing , the fluid volume consumed , volume voided , and weight changes were calculated to estimate the sweat rate ( sr ) , expressed in mlkgh . blood samples were collected using a 10-ml sterilized disposable plastic syringe and were immediately transferred to a clot activator tube ( green vac - tube ; green cross ms corp . , the serum was separated using a centrifuge ( ha-1000 - 3 ; hanil science co. , incheon , korea ) for 7 min and was analyzed for total proteins using a blood chemistry analyzer ( ektachem dt60 ii ; johnson & johnson , new brunswick , nj , usa ) . serum osmolality was measured in triplicate by using the freeze - point depression osmometer ( micro - osmometer model 210 ; fiske associates , norwood , ma , usa ) , and the average value of the three reading was taken . during exercise in the testing , the rating of perceived exertion ( rpe ; 11-point scale , 010 ) , thirst sensation ( 11-point scale ; 0 : no sensation to 10 : extremely thirsty ) , and heat sensation ( 11-point scale ; 0 : no sensation to 10 : extremely hot ) were asked and evaluated at the 15 and 90 minutes of exercise . the hr and vo2max were analyzed at the 15 , 40 , 65 , and 90 minutes during the testing . all variables are expressed as means standard deviation . a comparison of values between the ah and ph was made using student s t - test . when comparing conditions ( ah vs. ph ) to the testing period ( pre- vs. post - testing ) , within - subjects factorial analysis of variance with repeated measures was employed . all subjects completed all procedures required in the study . during testing and training , they ran at a speed of 8.0 0.5 kmh for a distance of 11.6 1.4 km . the calculated exercise intensity during testing and training was 51 3.9% of vo2max . while subjects were requested to drink the full - strength beverage , most subjects preferred half the strength of the original beverage . the subjects baseline physical conditions during pre - testing in the two conditions did not differ ( table 1 ) . body weight and body mass index did not change between pre- and post - testing during ah and ph ( p > 0.05 ) , but body fat content decreased in both conditions between pre- and post - testing ( p < 0.05 , student s t - test ) . the target amount of beverage intake in training during ah was approximately three times greater than that during ph ( table 2 ) . the average volume consumed in training during ah was short of the target amount , but it was greater than 100% of the subjects body weight loss . during ph , when the actual beverage consumption was normalized by body weight , their consumption during ah was approximately three times greater than during ph . while exercising in post - testing during ah , the beverage consumption was approximately two times greater than during pre - testing ( p < 0.05 , table 2 ) . however , the beverage consumption while exercising during ph was not statistically significantly different between pre- and post - testing . during recovery , the amount of beverage consumption was neither different between pre- and post - testing nor between the ah and ph conditions . the total amount of beverage consumption during testing was greater in post - testing than in pre - testing during ah ( p < 0.05 ) , but not during ph ( p > 0.05 ) . both sweat loss and sweat rate were greater in post - testing compared with pre - testing during the exercise and recovery period of ah ( p < 0.05 , table 3 ) . however , these variables were only greater in post - testing compared with pre - testing during the recovery period of ph ( p < 0.05 ) . the final tre during exercise in post - testing was reduced compared with that in pre - testing in both experiments , but it was not statistically significant ( fig . the magnitude of the tre elevation during exercise was 3.0 0.7 , 2.8 0.4 , 3.2 0.8 , and 2.8 0.8 in pre - testing during ah , post -testing during ah , pre - testing during ph , and post - testing during ph , respectively , and no statistically significant difference was noted . the tsk and tb were also not signi - ficantly different among testing and experiments . the hrs also decreased during exercise in post - testing compared with those in pre - testing in both experiments , but the difference was not statistically significant ( fig . the magnitude of reduction of the final hr from pre - testing to post - testing was 14.7 14.6 beatsmin during ah and 9.6 16.4 beatmin during ph ( p > 0.05 ) . the usg , serum osmolality , and total protein were not different at baseline or at 3 h of recovery among testing and experiments ( table 4 ) . no psychometric variable during exercise was different among testing and experiments ( table 5 ) . dehydration during exercise and heat induces performance deterioration , mainly due to thermoregulatory and cardiovascular strain . fluid intake helps restore body fluid and fluid volume regulatory function , thereby improving physical capacity [ 1 - 3 ] . however , voluntary drinking during exercise , aimed at restoring fluids and regulatory function , does not always guarantee meeting the adequate volume requirement . we hypothesized that habitual drinking practices while exercising in a hot environment may help increase voluntary volume intake during subsequent exercise and heat stress . to our knowledge , this is the first reported study examining the effects of drinking a prescribed volume during exercise with short - term heat acclimation on fluid volume regulation . the most significant finding of the present study was that active habitual drinking practice raised the volume of voluntary drinking as well as increased the sweat rate while exercising in a hot environment . nevertheless , no thermoregulatory or circulatory benefit from the active drinking practice compared with the non - active practice was supported statistically . in this study , subjects participated in 10 days of exercise training in a hot environment , and they ran for 90 min while being required to drink 50% ( during ph ) or 150% ( during ah ) of their body weight loss during exercise . during the 10-day program , subjects drank approximately 84% ( during ph ; 42% of their weight loss ) and 83% ( during ah ; 123% of their weight loss ) of their target volume . after the program , subjects were tested and allowed to drink ad libitum while exercising in a hot environment . the volume of voluntary drinking and the sweat rate increased significantly during ah while no change was observed during ph . in both conditions , the tre and hr decreased , but no significant difference between the conditions was noted . our findings suggest that short - term exercise training in a hot environment while drinking , generally , may help reduce thermoregulatory and circulatory strain . these responses were not affected by drinking volume during exercise training , at least under the conditions used in this study . however , an increased active volume drinking practice during training caused an increased voluntary drinking volume and sweat rate . some studies have reported that short - term heat exposure with exercise increases voluntary fluid intake . greenleaf et al . reported that voluntary water intake increased from 450 ml / h on day 1 of exercise and heat stress increased to about 1,000 mlh on days 58 . in their study , subjects were free from a target drinking volume , which was used in the present study . also reported an increased daily fluid intake while exercising in a warm environment . in the present study , subjects drank about 570 mlh voluntarily in pretesting and 1,061 mlh in post - testing during ah . by comparing the volume consumed , the active drinking practice seemed to be no more beneficial over a non - target voluntary drinking volume . although 150% of weight loss was the target volume during ah , most subjects could not meet the requirement . despite the fact that thirst sensation increased by exercise - induced dehydration , driven by blood osmolality and angiotensin ii , this may not be sufficient to promote voluntary fluid intake to match water loss . studies have shown that voluntary drinking replaced , at most , 30 - 70% of water loss during exercise even when fluid was readily available [ 9 , 18 - 20 ] . the increase in volume loading due to exercising in a hot environment may stimulate the cardiopulmonary baroreceptor , resulting in suppression of the drinking desire . in his review , noakes noted that a high rate of fluid intake ( > 1 lh ) can be difficult during running and may lead to feelings of abdominal discomfort possibly due to the accumulation of unabsorbed fluid in the intestines . he also reported that most do not ingest fluid equal to the amount of fluid loss ; subsequently , he proposed that drinking practices during training may reduce the severity of these symptoms . the present study supported this notion because the increased active volume drinking practice resulted in an increased volume of voluntary drinking . as mentioned above , the exercise - heat stress may have reduced cardiopulmonary baroreceptor sensitivity leading to increased fluid intake . one notable observation was that subjects were reluctant to drink the full - strength sports drink while running in the heat . numerous studies have shown that carbohydrate - electrolyte solutions are more effective than plain water for stimulating voluntary fluid intake , and promoting rapid intestinal absorption [ 25 - 27 ] , although it is not always consistent in some populations . although a solution containing about 6% of carbohydrates is considered a sport drink , this concentration seemed to be too strong for the voluntary drinking conditions in the present study s subjects . a high concentration of a carbohydrate solution has been shown to decrease the rate of gastric emptying and absorption . additionally , drinking the full - strength beverage might have been uncomfortable for the present study s subjects and possibly reduced gastric emptying . osterberg et al . reported that volume retention between beverages containing 36% carbohydrate was not different . their usual drinking solution during exercise was plain water , instead of a full strength sports drink . although active drinking practices induced a higher drinking volume and sweat rate while exercising in a hot environment , it was not advantageous over passive drinking for thermo - regulatory or circulatory adjustments or psychometric responses . the general recommendation to prevent dehydration during exercise and heat stress is to prevent excessive dehydration ( > 2% body water deficit ) and excessive disturbance of the electrolyte balance . one reason why we did not observe any difference between the two conditions may have been the severity of the dehydration . during ph , although full recovery from weight loss by exercise was not noticed , more than 40% of the subjects body weight loss was replaced by voluntary drinking . this volume consumption may contribute to physiological and psychometric response adjustments and may subsequently allow at least a minimum adjustment to prevent dehydration . studies on more prolonged exercise interventions and/or severe volume restrictions during exercise may be warranted . active drinking practice while exercising in a hot environment induced greater voluntary fluid intake and sweat loss . although they were required to drink 150% of their weight loss , they actually consumed 123% of their weight loss . there was no additional benefit from the increased volume intake in terms of thermoregulatory and circulatory adjustments or psychometric parameters , which may be due to the severity of body weight loss while exercising under the two conditions .

[ purpose]to examine the effects of active drinking practices on fluid consumption and sweat rate while exercising in a hot environment.[methods]nine men completed two experiments . each consisted of 3 phases : pre - testing ( pre ) , training period , and post - testing ( post ) . during testing , the subjects ran on a treadmill at a moderate intensity for 90 min at 39 1 followed by a 3-h recovery . they drank ad libitum . during training , they ran for 90 min for 7 days while either drinking actively ( ah , 150% of weight loss ) or passively ( ph , 50% of weight loss).[results]the actual volume consumed in training was three times greater during ah than during ph . in post during ah , the volume of drinking was two times greater than pre ( 1592 953 and 855 551 ml , respectively ; p < 0.05 ) . no difference in volume consumption during ph between pre and post was found . the sweat loss during exercise was greater in post ( 1377 956 ml ) than in pre ( 558 642 ml ) during ah ( p < 0.05 ) , but not during ph . rectal temperature and heart rate decreased after training . serum osmolality following exercise were not different than the baseline or between the conditions.[conclusion]active drinking practices while exercising in a hot environment induced greater voluntary fluid intake and sweat loss .

INTRODUCTION METHODS Study design Subjects Pre-screening session Pre- and post-testing Training period Post-screening session Measurements and calculations Statistical analyses RESULTS DISCUSSION CONCLUSIONS

PMC2875105

following ethical approval from the university college london ethics of non - national health service human research committee , 531 families with children aged 7 - 9 years from five london schools were invited to take part in the physical exercise and appetite in children study ( peaches ) . this is a longitudinal study of the associations between eating behaviours , physical activity and adiposity during childhood . parents were sent a questionnaire that included measures of attitudes to their child s weight and their feeding practices . there was a 60% return rate ( n=244 ) , with the majority of respondents being mothers ( n=213 ) . because mothers have been reported to feed differently to fathers ( blissett et al . , 2006 ) , analyses were restricted to mothers for homogeneity of the sample . parents were asked to indicate their highest level of education ( in the uk system ) using seven response options ( no qualifications , o - level / gcse / equivalent , ethnicity was reported using 11 response options based on 1991 uk census ( 1991 census , crown copyright , esrc purchase ) , but because of the diversity of the non - white groups , subdividing the sample into different ethnic sub - groups produced very small samples , and so ethnicity was dichotomised into maternal feeding practices were assessed with the child feeding questionnaire ( cfq ; birch et al . restriction ( e.g. i have to be sure that my child does not eat too many high fat foods ) , and monitoring ( e.g. how much do you keep track of the snack food ( e.g. crisps , cheesy crackers ) that your child eats? ) . disagree , slightly disagree , neutral , slightly agree , and agree for the restriction scale . mean scale scores were computed if at least 70% of the items were completed ( tabachnick and fidell , 1996 ) . each mother s perception of her child s weight was assessed using one item : how would you describe your child s weight at the moment , with response options of : very underweight, overweight and very overweight. concern about overweight was assessed using the item : how concerned are you about your child becoming or remaining overweight in the future , with response options on a 5-point scale : concerned , fairly concerned, very concerned. both items have been used with parents of children of a similar age ( grimmett et al . , 2008 ; carnell et al . , children were weighed and measured in a private space at school by trained researchers using standardised procedures . height was measured to the nearest millimetre using a freestanding stadiometer ( leicester height measure , seca , birmingham , uk ) , and weight ( kg ) to the nearest 0.01 kilogramme using the tanita tbf-300ma body composition analyser ( tanita corporation , japan ) . inter - rater reliability in a sub - sample of 30 children was high for weight ( r=1.0 ) and height ( r=0.99 ) . body mass index ( bmi : kg / m ) was calculated and converted into age- and sex - adjusted standard deviation scores ( bmi sd - score ) according to 1990 british reference data ( cole et al . , 1995 ) using the imsgrowth macro ( http://homepage.mac.com/tjcole ) . bmi sd - scores were grouped into four weight categories based on the international obesity taskforce ( iotf ) criteria for healthy - weight , overweight and obesity ( cole et al . , 2000 ) , and the recently proposed criteria for underweight ( thinness grades 1 , 2 or 3 ) ( cole et al . in addition , the healthy - weight group was subdivided into lower - healthy - weight ( 50th centile ) and higher - healthy - weight ( > 50th centile but not meeting criteria for overweight ) to create an additional sub - division of largest sub - group . because of small numbers in the obese category , obese and overweight were combined into an overweight / obese category . very overweight , so just three categories of perceived weight ( underweight , normal weight , overweight ) were used in all analyses . the patterning of each maternal feeding practice across weight groups was examined using trend analysis in spss one - way anova , both unadjusted and adjusted for demographic factors ( child sex and age , maternal ethnicity and education ) . because the weight distribution was similar to uk norms ( hse , 2006 , www.data.archive.co.uk ) , weighted rather than unweighted linear terms similarly , the patterning of each feeding practice across weight perception groups and weight concern groups was examined using trend analysis both unadjusted and adjusted for socio - demographic factors ( child sex and age , maternal ethnicity and education ) to determine the suitability for correlation analyses . pearson s bivariate correlations were run to test the first three ( of four ) steps in mediation analysis : ( a ) that the predictor ( bmi - sd - score ) is associated with the outcome ( feeding practices ) ( b ) that the predictor is associated with the mediators ( perceptions or concerns ) , and ( c ) that the mediator is associated with the outcome . to quantify the predictive value of bmi sd - score and perceptions and concerns on maternal feeding practices , and of bmi sd - score on perceptions and concerns , variables that were significantly correlated were entered into separate regression models . to test step four of the mediation analysis , child bmi sd - score ( the predictor variable ) must significantly predict maternal feeding style ( outcome variable ) in the absence of perceptions / concerns about child weight ( mediator variables ) and perceptions / concerns should be associated with both predictor and outcome variables . mediation is assumed if the association between the predictor and the outcome is significantly reduced by inclusion of the mediator in the regression model ( baron and kenny , 1986 ) . in this case , if the association between child adiposity and maternal feeding style is reduced when maternal weight perception or concern is added into the regression model , perceptions and concerns are implicated as mediators . as a stronger test of mediation , the preacher and hayes ( 2004 ) bootstrapping approach to the sobel test ( sobel , 1982 ) was used to demonstrate the significance of an indirect effect of adiposity on maternal feeding styles via perceptions and concerns . following ethical approval from the university college london ethics of non - national health service human research committee , 531 families with children aged 7 - 9 years from five london schools were invited to take part in the physical exercise and appetite in children study ( peaches ) . this is a longitudinal study of the associations between eating behaviours , physical activity and adiposity during childhood . parents were sent a questionnaire that included measures of attitudes to their child s weight and their feeding practices . there was a 60% return rate ( n=244 ) , with the majority of respondents being mothers ( n=213 ) . because mothers have been reported to feed differently to fathers ( blissett et al . , 2006 ) , analyses were restricted to mothers for homogeneity of the sample . parents were asked to indicate their highest level of education ( in the uk system ) using seven response options ( no qualifications , o - level / gcse / equivalent , vocational , a - level / equivalent , bachelor degree / equivalent , higher degree and other , please state ) . these were combined as ethnicity was reported using 11 response options based on 1991 uk census ( 1991 census , crown copyright , esrc purchase ) , but because of the diversity of the non - white groups , subdividing the sample into different ethnic sub - groups produced very small samples , and so ethnicity was dichotomised into maternal feeding practices were assessed with the child feeding questionnaire ( cfq ; birch et al . , 2001 ) , which has three subscales assessing i try to get him / her to eat anyway ) , restriction ( e.g. i have to be sure that my child does not eat too many high fat foods ) , and monitoring ( e.g. how much do you keep track of the snack food ( e.g. crisps , cheesy crackers ) that your child eats? ) . sometimes , often , and always for the pressure to eat and monitoring scales , and mean scale scores were computed if at least 70% of the items were completed ( tabachnick and fidell , 1996 ) . each mother s perception of her child s weight was assessed using one item : how would you describe your child s weight at the moment , with response options of : very underweight, overweight and very overweight. concern about overweight was assessed using the item : how concerned are you about your child becoming or remaining overweight in the future , with response options on a 5-point scale : very concerned. both items have been used with parents of children of a similar age ( grimmett et al . , 2008 ; children were weighed and measured in a private space at school by trained researchers using standardised procedures . height was measured to the nearest millimetre using a freestanding stadiometer ( leicester height measure , seca , birmingham , uk ) , and weight ( kg ) to the nearest 0.01 kilogramme using the tanita tbf-300ma body composition analyser ( tanita corporation , japan ) . inter - rater reliability in a sub - sample of 30 children was high for weight ( r=1.0 ) and height ( r=0.99 ) . parents were asked to indicate their highest level of education ( in the uk system ) using seven response options ( no qualifications , o - level / gcse / equivalent , vocational , a - level / equivalent , bachelor degree / equivalent , higher degree and other , please state ) . these were combined as ethnicity was reported using 11 response options based on 1991 uk census ( 1991 census , crown copyright , esrc purchase ) , but because of the diversity of the non - white groups , subdividing the sample into different ethnic sub - groups produced very small samples , and so ethnicity was dichotomised into maternal feeding practices were assessed with the child feeding questionnaire ( cfq ; birch et al . , 2001 ) , which has three subscales assessing i try to get him / her to eat anyway ) , restriction ( e.g. i have to be sure that my child does not eat too many high fat foods ) , and monitoring ( e.g. how much do you keep track of the snack food ( e.g. crisps , cheesy crackers ) that your child eats? ) disagree , slightly disagree , neutral , slightly agree , and agree for the restriction scale . mean scale scores were computed if at least 70% of the items were completed ( tabachnick and fidell , 1996 ) . each mother s perception of her child s weight was assessed using one item : how would you describe your child s weight at the moment , with response options of : very underweight, overweight and very overweight. concern about overweight was assessed using the item : how concerned are you about your child becoming or remaining overweight in the future , with response options on a 5-point scale : concerned , fairly concerned, very concerned. both items have been used with parents of children of a similar age ( grimmett et al . , 2008 ; carnell et al . , 2005 ) children were weighed and measured in a private space at school by trained researchers using standardised procedures . height was measured to the nearest millimetre using a freestanding stadiometer ( leicester height measure , seca , birmingham , uk ) , and weight ( kg ) to the nearest 0.01 kilogramme using the tanita tbf-300ma body composition analyser ( tanita corporation , japan ) . inter - rater reliability in a sub - sample of 30 children was high for weight ( r=1.0 ) and height ( r=0.99 ) . body mass index ( bmi : kg / m ) was calculated and converted into age- and sex - adjusted standard deviation scores ( bmi sd - score ) according to 1990 british reference data ( cole et al . , 1995 ) using the imsgrowth macro ( http://homepage.mac.com/tjcole ) . bmi sd - scores were grouped into four weight categories based on the international obesity taskforce ( iotf ) criteria for healthy - weight , overweight and obesity ( cole et al . , 2000 ) , and the recently proposed criteria for underweight ( thinness grades 1 , 2 or 3 ) ( cole et al . , 2007 ) . lower - healthy - weight ( 50th centile ) and higher - healthy - weight ( > 50th centile but not meeting criteria for overweight ) to create an additional sub - division of largest sub - group . because of small numbers in the obese category , obese and overweight were combined into an overweight / obese category . very overweight , so just three categories of perceived weight ( underweight , normal weight , overweight ) were used in all analyses . concerned , fairly concerned or very concerned about their child s weight , so these categories were combined to form a single category named the patterning of each maternal feeding practice across weight groups was examined using trend analysis in spss one - way anova , both unadjusted and adjusted for demographic factors ( child sex and age , maternal ethnicity and education ) . because the weight distribution was similar to uk norms ( hse , 2006 , www.data.archive.co.uk ) , weighted rather than unweighted linear terms similarly , the patterning of each feeding practice across weight perception groups and weight concern groups was examined using trend analysis both unadjusted and adjusted for socio - demographic factors ( child sex and age , maternal ethnicity and education ) to determine the suitability for correlation analyses . pearson s bivariate correlations were run to test the first three ( of four ) steps in mediation analysis : ( a ) that the predictor ( bmi - sd - score ) is associated with the outcome ( feeding practices ) ( b ) that the predictor is associated with the mediators ( perceptions or concerns ) , and ( c ) that the mediator is associated with the outcome . to quantify the predictive value of bmi sd - score and perceptions and concerns on maternal feeding practices , and of bmi sd - score on perceptions and concerns , variables that were significantly correlated were entered into separate regression models . to test step four of the mediation analysis , child bmi sd - score ( the predictor variable ) must significantly predict maternal feeding style ( outcome variable ) in the absence of perceptions / concerns about child weight ( mediator variables ) and perceptions / concerns should be associated with both predictor and outcome variables . mediation is assumed if the association between the predictor and the outcome is significantly reduced by inclusion of the mediator in the regression model ( baron and kenny , 1986 ) . in this case , if the association between child adiposity and maternal feeding style is reduced when maternal weight perception or concern is added into the regression model , perceptions and concerns are implicated as mediators . as a stronger test of mediation , the preacher and hayes ( 2004 ) bootstrapping approach to the sobel test ( sobel , 1982 ) was used to demonstrate the significance of an indirect effect of adiposity on maternal feeding styles via perceptions and concerns . maternal and child characteristics are shown in table 1 . as intended in the sampling procedure , there was high ethnic diversity with 38% of mothers classified as non - white ; compared with national statistics ( ons , 2005 , www.ons.gov.uk ) . there were no significant differences in children s characteristics betwee those whose parents responded or did not respond to the parental questionnaire . differences in bmi sd - scores between boys and girls approached significance ( t=(211)1.89 , p=.06 ) , with girls ( m=.07 , sd=1.29 ) being slightly thinner than boys ( m=.26 , sd=1.28 ) . prevalence of overweight and obesity ( 16% ) was lower than the general population ( 33% ) ( www.dh.gov.uk ) , which may be due to families with heavier children choosing not to participate in the study . linear trend analysis demonstrated a significant negative trend across child weight groups for pressure to eat in the adjusted ( f(3,190)=3.78 , p=.012 ) and unadjusted models ( f(1,208)=6.93 , p=.003 ) , and a significant positive trend across weight groups for restriction in the adjusted ( f(3,189)=2.95 , p=.034 ) and unadjusted models ( f(1,206)=6.25 , p=.01 ) . normal weight. only 41% of underweight children were perceived as underweight , and only 44% of overweight or obese children were perceived as overweight . for concern about overweight , 50% of mothers of children classified as overweight / obese were concerned or very concerned about their child staying or becoming overweight in the future . there was a significant linear trend for perceived weight across child weight groups in unadjusted ( f(1,209)=76.69 , p<.001 ) and adjusted ( f(3,188)=28.51 , p<.001 ) analyses , indicating that although mothers perception of their child s weight was often incorrect , relative perception was correct . concern about overweight showed a significant linear trend across child weight groups in the unadjusted ( f(1,209)=33.48 , p<.001 ) and adjusted models ( f(3,202)=17.49 , p<.001 ) . mean cfq subscale scores across categories of perceived weight and concern for overweight are illustrated in table 3 . there was a significant linear trend for pressure to eat across perceived weight categories in the adjusted ( f(2,188)=4.79 , p= .009 ) and unadjusted models ( f(1,208)=11.15 , p=.001 ) , with lower use of pressure for children who were perceived to be more overweight . restriction across categories of concern about overweight in both unadjusted ( f(1,206)=21.24 , p<.001 ) and adjusted models ( f(2,188)=9.60 , p<.001 ) , with restriction increasing among mothers who were more concerned about overweight . table 4 illustrates the bivariate correlations between bmi sd - score , perceptions , concerns and maternal feeding practices . bmi sd - score , perception of child weight and maternal pressure to eat were intercorrelated , as was bmi sd - score , concern and restriction ; fulfilling the criteria to test mediation and were taken forward into two separate mediation analyses . monitoring was not included , because it was not associated with bmi sd - score or mediators . linear regression analysis showed that bmi sd - score significantly predicted pressure to eat ( =0.23 , p=.001 ) , restriction ( =.16 , p=.02 ) , concern about weight ( =.39 , p<.001 ) , and perception of weight ( =.56 , p<.001 ) . perception of weight significantly predicted pressure to eat ( =.023 , p=.001 ) , and concern about overweight significantly predicted following baron and kenny s criteria ( baron and kenny , 1986 ) , mediation analysis was appropriate for pressure to eat and restriction was the only feeding practice significantly associated with both child weight group and maternal concern about overweight , and pressure was the only feeding practice associated with both child weight group and perception of child weight . table 5 presents results of change in associations between bmi sd - score , and maternal feeding practices upon the addition of maternal perceptions or concerns . when concern for child overweight was added into the regression model , the association between child bmi sd - score and restriction became non - significant ( =.04 , p=.44 ) , but concern remained a significant predictor ( =.30 , p<.001 ) , with the full model explaining 9.9% of the variance . using bootstrapping , the sobel test confirmed the significance of an indirect effect of the association between child adiposity and restriction ( z=3.29 , p=.001 ) , indicating that maternal concern about child overweight mediates the relationship between the child s actual weight and parental use of restrictive feeding . when perception of child weight was added to the model to predict pressure to eat , the effect was reduced , but the association between child bmi sd - score and pressure remained significant ( =.17 , p=.04 ) with the full model explaining 7.1% of the variance . linear trend analysis demonstrated a significant negative trend across child weight groups for pressure to eat in the adjusted ( f(3,190)=3.78 , p=.012 ) and unadjusted models ( f(1,208)=6.93 , p=.003 ) , and a significant positive trend across weight groups for restriction in the adjusted ( f(3,189)=2.95 , p=.034 ) and unadjusted models ( f(1,206)=6.25 , p=.01 ) . normal weight. only 41% of underweight children were perceived as underweight , and only 44% of overweight or obese children were perceived as overweight . for concern about overweight , 50% of mothers of children classified as overweight / obese were concerned or very concerned about their child staying or becoming overweight in the future . there was a significant linear trend for perceived weight across child weight groups in unadjusted ( f(1,209)=76.69 , p<.001 ) and adjusted ( f(3,188)=28.51 , p<.001 ) analyses , indicating that although mothers perception of their child s weight was often incorrect , relative perception was correct . concern about overweight showed a significant linear trend across child weight groups in the unadjusted ( f(1,209)=33.48 , p<.001 ) and adjusted models ( f(3,202)=17.49 , p<.001 ) . mean cfq subscale scores across categories of perceived weight and concern for overweight are illustrated in table 3 . there was a significant linear trend for pressure to eat across perceived weight categories in the adjusted ( f(2,188)=4.79 , p= .009 ) and unadjusted models ( f(1,208)=11.15 , p=.001 ) , with lower use of pressure for children who were perceived to be more overweight . no significant trends were found for restriction or monitoring across perceived weight groups . there was a significant linear trend for restriction across categories of concern about overweight in both unadjusted ( f(1,206)=21.24 , p<.001 ) and adjusted models ( f(2,188)=9.60 , p<.001 ) , with restriction increasing among mothers who were more concerned about overweight . table 4 illustrates the bivariate correlations between bmi sd - score , perceptions , concerns and maternal feeding practices . bmi sd - score , perception of child weight and maternal pressure to eat were intercorrelated , as was bmi sd - score , concern and restriction ; fulfilling the criteria to test mediation and were taken forward into two separate mediation analyses . monitoring was not included , because it was not associated with bmi sd - score or mediators . linear regression analysis showed that bmi sd - score significantly predicted pressure to eat ( =0.23 , p=.001 ) , restriction ( =.16 , p=.02 ) , concern about weight ( =.39 , p<.001 ) , and perception of weight ( =.56 , p<.001 ) . perception of weight significantly predicted pressure to eat ( =.023 , p=.001 ) , and concern about overweight significantly predicted following baron and kenny s criteria ( baron and kenny , 1986 ) , mediation analysis was appropriate for pressure to eat and restriction was the only feeding practice significantly associated with both child weight group and maternal concern about overweight , and pressure was the only feeding practice associated with both child weight group and perception of child weight . table 5 presents results of change in associations between bmi sd - score , and maternal feeding practices upon the addition of maternal perceptions or concerns . when concern for child overweight was added into the regression model , the association between child bmi sd - score and restriction became non - significant ( =.04 , p=.44 ) , but concern remained a significant predictor ( =.30 , p<.001 ) , with the full model explaining 9.9% of the variance . using bootstrapping , the sobel test confirmed the significance of an indirect effect of the association between child adiposity and restriction ( z=3.29 , p=.001 ) , indicating that maternal concern about child overweight mediates the relationship between the child s actual weight and parental use of restrictive feeding . when perception of child weight was added to the model to predict pressure to eat , the effect was reduced , but the association between child bmi sd - score and pressure remained significant ( =.17 , p=.04 ) with the full model explaining 7.1% of the variance . this study demonstrated that mothers concern about their child s weight significantly mediated the association between their use of restrictive feeding practices and their child s adiposity . this supports our hypothesis that restriction is more likely to be a response to maternal concern about overweight than a cause of weight gain . , 2006 ; carnell and wardle , 2007 ; spruijt - metz et al , 2002 ) , child adiposity was inversely associated with the pressure to eat feeding style , but neither maternal perception of the child s weight , nor their concern about their child being overweight in the future mediated this effect . that maternal concern about child overweight mediates the association between child adiposity and restriction suggests a plausible causal relationship : mothers who are not concerned about their child staying or becoming overweight in the future see no need to restrict their child s intake of unhealthy foods , while concerned mothers limit their child s intake in an attempt to control weight gain . restriction may therefore be a result of maternal concern rather than a cause of weight gain . the cross - sectional nature of our study means causality is only implicated , not confirmed , but some support comes from a longitudinal study of 7 - 14 yr old african- and white - american children in which maternal concern about child weight was associated with lower increase in fat mass 3 years later ( spruijt - metz et al . , 2006 ) . if maternal concern results in greater control in child feeding , this may be protective against childhood weight gain . perceptions of child weight did not fully mediate the association between child adiposity and pressure to eat , but there was little variation in mother s perception of their child s weight with most mothers perceiving their child as normal - weight . however , concern for overweight also did not mediate the association between child adiposity and pressure to eat . previous research in a sample of mothers and their infants ( n=634 ) showed that maternal concern about their child being underweight was associated with pressure to eat ( baughcum et al . , 2001 ) ; suggesting that mothers who perceive their child as underweight use more pressuring strategies . a similar trend across perceived weight groups was found in the present study . a more sensitive measure of concern for underweight might help test whether pressure to eat is , like restriction , also a child - responsive parental feeding practice , rather than accepting the more unlikely hypothesis that pressure to eat is protective against weight gain . however , pressure is more likely to be related to diet quality generally than weight specifically because it is often exerted to ensure that healthy foods are consumed . mothers who are worried about the quality of their child s diet might also be expected to exert more pressure , but perceptions of diet quality were not assessed in this study . one limitation of this study is that the measurement of maternal concerns was limited to a single question asking about both current and future weight concerns . it is therefore not possible to know whether the concern was about either current or future overweight or a combination of the two . furthermore , maternal concerns are likely to extend beyond weight to include the child s eating and activity patterns . studies assessing the influence of such concerns on controlling feeding practices and their future consequences on children s weight are needed . there was limited variance in the measure of maternal perceptions and concerns , making it necessary to combine response options into three categories , and thereby lose detail . another limitation is the cross - sectional nature of this study , precluding causal inference . in these analyses , the child s body size ( bmi sd - score ) was treated as the independent variable rather than outcome , but the alternative model in which concern causes restriction and thereby weight gain can not be ruled out . other possible interpretations , such as overlap of the concern and restriction categories , or that another untapped variable is relevant to how parents feed their children , may account for the findings . importantly , the current study has identified a significant mediating factor in the relationship between child adiposity and parental feeding practices proving the need for further research . because peaches is a longitudinal study , future work will be able to assess longitudinal associations between child weight , maternal concern and maternal feeding practices , allowing more explicit testing of causal relationships . another weakness is that the sample was lean relative to the wider population ; which we believe to be due to heavier children electing not to participate . however , a greater spread across the weight trajectory should strengthen rather than weaken the associations observed here . the socio - economic and ethnic diversity of the sample is a strength , allowing greater generalisability of the findings . our results indicate that attributing overweight to excessive maternal control in feeding , in particular to mothers use of restriction , is unjustified . mothers appear to adopt specific restrictive feeding strategies in response to their concerns about their child s weight . instead of blaming parents for influencing childhood weight through detrimental feeding practices , future work should explore maternal concerns and beliefs about feeding in more detail , and identify the type and degree of control that is effective for optimal diet and growth .

objectiveto determine whether controlling parental feeding practices are associated with children s adiposity and test the hypothesis that any associations are mediated by maternal perception of their child s weight.methodchildren aged 7 - 9 yrs ( n=405 ) were weighed and measured at school as part of the physical exercise and appetite in children study ( peaches ) . adiposity was indexed with bmi sd - scores . the child feeding questionnaire ( cfq ) was completed by 53% of mothers of participating children ( n=213 ) . mothers reported whether they thought their child was overweight , normal weight or underweight , and rated their concern about future overweight on a 5-point scale.resultshigher child adiposity was associated with lower pressure to eat and higher restriction scores . restriction increased linearly with maternal concern about overweight , and maternal concern about overweight fully mediated the association between child adiposity and restriction . use of pressure increased as mothers perceived their child to be thinner , but perceived weight did not mediate the association between child weight status and maternal pressure to eat . monitoring was not associated with child adiposity , maternal perception of weight or concern about overweight.conclusionrestriction appears to be a consequence of mothers concern about their child becoming overweight rather than a cause of children s weight gain . pressure may be a more complex response that is influenced by the desire to encourage consumption of healthy foods as well as ensure adequate energy intake and appropriate weight gain .

Methods Participants Measures Maternal socio-demographic information Maternal feeding practices Maternal perception of weight and concern about overweight Childrens anthropometry Data treatment and statistical analysis Results Trend analysis: Maternal feeding practices across child weight groups Trend analysis: Maternal perceptions and concerns across child weight groups Trend analysis: Maternal feeding practices across child weight perception and concern groups Mediation analysis Discussion

PMC4893568

epidemiological studies suggest childbearing is an important contributor to development of obesity in many women [ 17 ] . weight retention associated with pregnancy is estimated to be between 0.5 and 3.8 kg at 2.5 years postpartum [ 1 , 2 , 410 ] . according to several long - term us studies , including the national maternal and infant health survey and the first national health and nutrition examination survey ( nhanes ) , average weight gain associated with one live birth is approximately 1.6 to 1.7 kg above age - associated weight gain . higher than average permanent weight retention , however , is experienced by many women who become overweight or obese following the birth of a single child . for example , according to nhanes , having one live birth increases the risk of becoming moderately overweight ( body mass index , bmi > 27.3 kg / m ) by 60% and becoming obese ( bmi > 30.0 weight loss at 6 months postpartum has emerged as a key predictor of long - term weight retention [ 1 , 4 ] , highlighting the importance of early postpartum weight reduction . in one long - term study of 795 women , those who lost all pregnancy - associated weight gain by 6 months postpartum were 2.4 kg heavier eight to ten years later , whereas those who retained weight were 8.3 kg heavier . even though breastfeeding often emerges as a protective factor against weight retention [ 4 , 12 , 13 ] , in fact , butte and hopkinson observed that the effect of lactation on postpartum weight reduction is highly variable and may even contribute to net weight gain in some mothers . although socioeconomic , environmental , and lifestyle factors , including number of children , maternal age , and exercise habits , are important determinants of weight retention following pregnancy , it is possible that hormonal changes that occur as a result of pregnancy or lactation may also be at play . recent evidence has brought attention to the gut hormones as key contributors to the regulation of food intake and energy balance [ 1619 ] . these hormones include the stomach - derived orexigenic peptide ghrelin and the intestinal - derived anorexigenic peptides glucagon - like peptide-1 ( glp-1 ) and peptide yy ( pyy ) , which serve as signals to the appetite - regulating centers of the brain . it is not known , however , whether ghrelin , pyy , or glp-1 ( or other modulators of the brain - gut axis ) is altered in the postpartum state , particularly during lactation . a few human studies have measured circulating ghrelin in postpartum women [ 2024 ] ; however , it remains unclear if and how these hormones fluctuate across the first postpartum year , both during fasting and in response to a meal , and whether circulating concentrations influence postpartum weight retention . the primary purpose of this study is to determine whether the appetite - regulating hormones ghrelin , pyy , and glp-1 are altered during lactation ( both fasting and in response to a meal ) and to determine whether circulating concentrations of these hormones , along with insulin and/or leptin , are associated with body weight retention in the year following childbirth . a secondary purpose is to explore whether these hormones are acutely associated with appetite and food intake in an ad libitum meal . based on the foregoing , it is plausible to hypothesize that fasting or meal - induced concentrations of these hormones are altered during lactation and are associated with body weight retention . in particular , variations of one or all of these hormones could help explain the highly variable differences in body weight and body composition noted in lactating women . in agreement with this premise , cummings et al . proposed that ghrelin is a thrifty gene product that evolved to help animals consume and store fat well , thereby increasing survival during times of reduced food availability or famine . it could be argued that lactation is a time when thrifty metabolism , acting to preserve maternal adiposity , might offer a survival advantage for both mother and offspring . alternately , elevated ghrelin and/or lowered pyy or glp-1 during fasting may serve as maternal signals to increase food intake to support the energy demands of lactation . twenty - four healthy , primiparous , postpartum , exclusively breastfeeding women and 20 never - pregnant controls were recruited from november 2009 through april 2013 and followed for one year . women were recruited during late pregnancy and early postpartum using recruitment flyers distributed to clinics , physician offices , lactation consultants , and area hospitals . to qualify , participants had to be at least 18 yr of age , nonsmokers in good general health , and with a hemoglobin and thyroid stimulating hormone ( tsh ) concentration within the normal range ( hemoglobin = 12.018.0 g / dl ; tsh = 0.355.50 u / ml ) . participants were excluded if they had renal , hepatic , endocrine , gastrointestinal , pulmonary , cardiac , or hematologic disease , including elevated blood pressure ( > 140/90 mm hg ) ; showed signs of depression , anxiety , disordered eating , alcoholism , or other psychological or substance abuse issues ; or used prescription or over - the - counter medications / herbal preparations that could influence metabolism . lactating participants were excluded if they had complications of pregnancy ( e.g. , gestational diabetes or pregnancy - induced hypertension ) , had a multiple birth , or either were not currently exclusively breastfeeding or did not intend to breastfeed for at least a year . women were included regardless of delivery method as long as they had no activity restrictions at 4 - 5 wk postpartum . the never - pregnant controls were of an age and bmi range that allowed matching to the postpartum lactating group at baseline ( 4 - 5 weeks postpartum ) for age , bmi , and race / ethnicity ; this was accomplished by recruiting groups of control women following the baseline visits of groups of postpartum participants . never - pregnant control women were excluded if they planned on becoming pregnant or relocating from the area within the year following their baseline visit . volunteers were fully informed of possible risks during all procedures before providing written informed consent . lactating women were studied 46 weeks after delivery and at 6 and 12 months postpartum ( figure 1 ) . control women were initially evaluated and followed up at 5 and 11 months from their baseline date . at the initial visit , body weight , body composition , and resting metabolic rate ( rmr ) were measured and a meal test was performed to measure the hormonal ( ghrelin , pyy , glp-1 , and insulin ) responses to a standardized breakfast meal and ad libitum lunch . these same measures excluding the rmr and the meal testing were performed at follow - up ( figure 1 ) . blood was drawn after a 12-hour overnight fast for analysis of ghrelin , pyy , glp-1 , insulin , and leptin . test days were scheduled during the follicular phase of the menstrual cycle for controls and lactating women who had resumed menstruation ; serum progesterone concentrations were measured in all women to confirm status ( i.e. , serum progesterone < 2.0 ng / ml ) . body composition was assessed by dual energy x - ray absorptiometry ( lunar prodigy , ge healthcare , fairfield , ct ) and waist circumference was measured at the level of the umbilicus . food intake was controlled for 24 hours prior to baseline testing by providing a controlled diet which consisted of commercially available foods / beverages . energy content of the diet was estimated using the equation of redman et al . based on body weight , age , and sex with an estimate of 1900 kj ( 454 kcal ) added for lactating women . participants were asked to consume all food / beverages provided and to return empty wrappers and any unconsumed food / beverages . two snack bars ( 28.4 g , ~418 kj ; clif bar & company , berkeley , ca ) were given as optional snacks above calculated energy requirements if the participant felt in need of additional energy . participants were allowed plain water ad libitum but were asked not to consume other beverage or food not provided by the researchers . participants were also asked to refrain from moderate - to - strenuous exercise for 24 hours before each test day . on the test day morning rmr and fasting respiratory quotient ( rq ) were measured over a 30 min period by indirect calorimetry using a commercially available metabolic cart ( parvomedics trueone 2400 , sandy , utah ) , calibrated before each test with standardized gases . immediately following the rmr , an intravenous catheter was inserted into a forearm or hand vein and connected to a sterile saline solution ( 0.9% sodium chloride ) that was slowly infused ( ~30 cc / hour ) to keep the catheter patent . after the baseline blood draw , participants were provided with a breakfast smoothie ( 15% protein , 60% carbohydrate , and 25% fat ) that they were to consume in 15 minutes . the energy content of the test meal was individualized to provide 20% of each subject 's estimated total energy expenditure ( ~16752500 kj depending on weight , age , and lactation status ) . blood samples were drawn 30 , 60 , 90 , 120 , and 150 minutes after test meal ingestion and 60 minutes following the ad libitum lunch meal ( see below ) . ad libitum food intake was used as a marker of appetite ( commonly defined as the desire to eat ) and was assessed following the 150-minute blood draw . ad libitum intake was determined individually in a room with the following food / beverages , in gram - weight portions , arranged on the table : ice water , cooked pasta ( 160 g dry ) , marinara sauce ( 140 g ) , alfredo sauce ( 140 g ) , parmesan cheese , meat balls ( 3 ) , hard - boiled eggs ( 2 ) , whole wheat bread ( 2 slices ) , white bread ( 2 slices ) , fresh apples and oranges , milk , fruited - yogurt ( 3 , individual containers ) , and individual portions of margarine , assorted - jellies , honey , and peanut butter . subjects were told the meal was provided to allow baseline ratings for hunger and satiety on a full stomach and were instructed to eat as much as desired within 20 minutes . subjects were not allowed to read or study during the meal or carry bags / backpacks or coats into the room . the amount of food and fluid consumed was determined by weighing food remaining in the buffet as well as unconsumed food on the subjects ' plates . protein , fat , carbohydrate , and total energy were calculated using a food analysis program ( nutribase 8 professional edition v.8.3.6 ) based on the usda national nutrient database . hunger and satiety were assessed at baseline and at 30 , 60 , 90 , 120 , and 150 minutes after test meal ingestion ( in accordance with blood draws ) and at 20 and 60 minutes following the ad libitum meal using standard visual analogue scales ( vas ) . ( 2 ) how satisfied do you feel ? ( 3 ) how full do you feel ? the vas was 100 mm in length and was anchored at each end by words describing the extremes of the appetite being measured . blood samples were collected , placed on ice , and cold centrifuged ( 28c ) to prevent protein degradation . for the gut peptides , 150 l of aprotinin and 40 l of dpp - iv were added to 4 ml edta - containing tubes immediately after collection for analysis of pyy and glp-1 concentrations . blood for analysis of ghrelin and its specific active or acylated form ( ghrelinacyl ) was collected into prechilled tubes containing 100 l of 200 mm aebsf in addition to edta . following separation of plasma , 200 l of 1 n hydrochloric acid was added for each 1 ml of plasma for ghrelin analysis only . leptin , total ghrelin , ghrelinacyl , pyy , and glp-1 were analyzed by ria using commercially available kits ( millipore corp . , insulin analysis was also completed using a commercially available kit ( siemens diagnostics , tarrytown , ny ) . fasting blood drawn at baseline was analyzed for leptin , progesterone , and prolactin as specified by the kit manufacturer ( progesterone , siemens diagnostics ; prolactin , cayman chemical company , ann arbor , mi ) . intra - assay cvs are as follows : leptin 8.1% , insulin 12.1% , ghrelin 4.4% , ghrelinacyl 11.8% , pyy 10.1% , glp-1 6.5% , and prolactin 2.3% . interassay cv for assays analyzed in multiple batches was as follows : leptin 16.4% , ghrelin 9.7% , ghrelinacyl 23.5% , pyy 10.5% , and glp-1 17.9% . during the 6- and 12-month follow - up visits , participants reported to the laboratory between 7:00 and 9:00 am following a 12 h fast . height , weight , and body composition were measured and blood was obtained for analysis of fasting concentrations of leptin , insulin , ghrelinacyl , total ghrelin , pyy , glp-1 , and progesterone . information on prepregnancy body weight , weight gain during pregnancy , infant 's birth weight , and lactation status was collected from lactating women , and information on menstrual status was collected from all women via questionnaire and logs . participants were provided logs to document lactation status and menstrual function which were initially provided at baseline and returned at the 6- and 12-month visits . 15 to 20 subjects per group were initially selected based on power / sample size calculations . specifically , sample size calculations were performed using fasting and 60 min postprandial ghrelin from our previous study which were the only data available at study initiation . these calculations determined that a sample size of 10 to 26 subjects would be required to detect ( with 8099% power ) a difference between the postpartum lactating and nonpregnant control groups at either time point based on average concentrations in the lactating and control groups and a common standard deviation . sample size calculations performed for the association between fasting ghrelin and fasting pyy and the change in body fat over the one - year follow - up period suggested 14 to 29 postpartum subjects would be required to detect ( with 8099% power ) an association between fasting postpartum ghrelin and change in body fat if the true change in body fat mass is 0.007 kg per pg / ml change in fasting ghrelin ; the standard deviation of fasting ghrelin is 39 pg / ml and the standard deviation of body fat change is 2.0 kg . at baseline , independent sample t - tests were used to test for differences in body weight / composition , food intake , and fasting hormone concentrations between lactating and control women . changes in hormone concentrations and appetite ratings in response to the standardized meal were analyzed using anova to test for a group ( lactating , control ) by time interaction or main effects for time or group . data collected in response to the ad libitum lunch meal were not included in these analyses . area under the curve ( auc ) was calculated using the trapezoidal method corrected for baseline values ( prism version 6.05 for windows , graphpad software , inc . , la jolla , ca ) for appetite ratings and hormones that changed with meal ingestion including insulin , total ghrelin , ghrelinacyl , pyy , glp-1 , and the appetite ratings . for hormones and appetite ratings that were initially decreased after meal , including total ghrelin , ghrelinacyl hunger , and desire to eat , both aucs below ( negative ) and above ( positive ) the effect of group for the aucs was determined using independent sample t - tests . separate anova models were used to test for appetite and hormone responses to the ad libitum lunch . for the longitudinal analysis , changes in body weight , body composition , and the fasting hormones over the 12-month period were analyzed by repeated measures anova to test for a group by time ( baseline , 6 months , and 12 months ) interaction or main effects of time or group and by comparing the deltas of key variables ( i.e. , delta body weight from baseline to 6 and 12 months ) using independent sample t - tests . weight retention was analyzed similarly using delta body weight from reported prepregnancy weight to weight at 6 and 12 months . standard correlation and anova procedures were used to compare differences between women who lost within 2 kg of prepregnancy weight versus those who retained weight . physical and anthropometric characteristics of the postpartum lactating and never - pregnant control women are summarized in table 1 . of the 24 enrolled , 8 were overweight prior to pregnancy ( bmi > 24.9 kg / m ) and 16 were normal weight ( bmi = 18.524.5 kg / m ) . lactating women gained 16.1 4.1 kg during pregnancy , based on reported prepregnancy weight , and were studied at 5.0 0.7 weeks postpartum . the never - pregnant controls were studied on day 5.3 3.4 of their menstrual cycle and reported being weight stable for at least the past year . lactating and control women were matched at baseline for age , height , weight , bmi , and body composition ( table 1 ) . lactating women , however , had larger waist and hip circumferences than control women ( p < 0.001 ) . all women had progesterone concentrations < 2.0 ng / ml during baseline study , indicative anovulation or follicular phase of the menstrual cycle . baseline hip and waist circumferences are missing for one lactating subject and resting metabolism data are missing for two lactating and three control participants due to equipment failure or scheduling conflicts . complete blood and hormonal concentration data were also not available for one lactating woman at baseline due to problems with catheter insertion during the meal test . in addition , occasional samples are missing for two lactating and two control women for ghrelin and ghrelinacyl , one lactating and one control participant for pyy , and two controls and five lactating for glp-1 due to problems with indwelling catheter clotting at specific time points . missing blood data that occurred at 60 , 90 , or 120 min were filled in by using the average of previous and subsequent points . missing blood data at baseline , 30 min ( after meal ) , or 150 min ( immediately before ad libitum meal ) however was considered missing ; participants with missing data at these time points were not included in the repeated measures analyses and auc analysis . energy and macronutrient intakes of the controlled diet averaged 9514 2192 kj ( 2274 524 kcal ; 16.1 1.3% protein , 23.8 3.4% fat , and 62.6 5.2% carbohydrate ) in the lactating and 9799 1494 kj ( 2342 357 kcal ; 15.3 1.1% protein , 23.4 2.5% fat , and 64.3 4.0% carbohydrate ) in the control groups ( p > 0.05 ) . the rmr and resting rq of lactating and control women are summarized in table 1 . no differences were detected between groups ( p > 0.05 ) even after adjusting for differences in fat - free mass , fat mass , and age . tsh was within normal limits for all women but was significantly lower in lactating compared to control women ( p = 0.05 ) . prolactin concentration was also higher in lactating compared to control women ( p = 0.001 ) . fasting insulin , leptin , ghrelin , ghrelinacyl , pyy , and glp-1 concentrations , however , did not differ ( p > 0.05 ) between groups . as summarized in table 3 , fasting leptin and insulin concentrations were associated with total body weight and body adiposity but not lean mass , whereas fasting ghrelin tended to correlate negatively with body fat . there were no associations between fasting concentrations of ghrelinacyl , pyy , and glp-1 and body mass , fat mass , or fat - free mass . the standardized breakfast meal provided 2087 452 kj ( 499 108 kcal ; 14.3 1.1% protein , 22.9 0.8% fat , 58.9 0.5% carbohydrate , and 2.8 0.7 grams of fiber ) for lactating women and 1720 134 kj for control women ( 411 32 kcal ; 14.2 0.4% protein , 22.9 0.9% fat , 58.9 0.8% carbohydrate , and 2.3 0.4 grams of fiber ) . the insulin , ghrelinacyl , pyy , and glp-1 responses to the standardized breakfast meal are shown in figure 2 and auc responses for all hormones presented in table 2 . ghrelin ( not shown ) and ghrelinacyl did not change significantly in response to meal ingestion ( p > 0.05 ) . the concentration of ghrelinacyl was visibly higher in lactating versus control women after meal but there was only a trend for a group effect ( p = 0.13 at 150 minutes ) . in contrast , insulin , pyy , and glp-1 increased significantly ( p < 0.001 ) following the standardized meal and fell over the next 150 minutes . these meal - induced responses , however , were not different between lactating and control women ( p > 0.05 , group time ) . peak insulin responses ( highest insulin concentration minus fasting insulin ) were also not different between groups and averaged 19.6 18.7 in the controls and 14.0 13.3 u / ml ( p = 0.27 ) . ratings of hunger and desire to eat decreased after the standardized breakfast meal and then increased over the 150 minutes postprandially in both groups ( p < 0.05 , time effect ; figure 3 ) . ratings of satiety and fullness demonstrated the opposite pattern . despite small visual differences in appetite ratings between the lactating and control women between 60 and 120 min , no significant group differences were found ( p > 0.10 , group time and group effect ) . the auc for the four appetite ratings was also not different between groups ( p > 0.10 ) . the lactating women consumed more energy ( p < 0.05 ) ( 3678 1146 kj ; 879 274 kcal ) , protein ( 36.9 10.8 grams ) , and carbohydrate ( 134.7 47.6 grams ) than the controls ( 2971 816 kj ; 710 195 kcal , 29.8 10.0 g protein , and 108.6 36.4 g carbohydrate ) but the macronutrient composition and fiber content were similar ( p > 0.05 ) between lactating ( 16.9 2.2% protein , 24.6 6.9% fat , 61.5 8.4% carbohydrate , and 9.4 3.5 g fiber ) and control groups ( 16.6 3.0% protein , 25.6 7.2% fat , 61.0 9.6% carbohydrate , and 7.6 2.4 g fiber ) . the ad libitum meal resulted in increased ( p < 0.05 ) serum concentrations of pyy , glp-1 , and insulin at 60 min postprandial compared to before initiation of the ad libitum meal ( i.e. , 150 minutes ) ( figure 2 ) . hunger and desire to eat were decreased greater than those of the standardized breakfast and satiety and fullness were increased ( figure 3 ) . ghrelin and ghrelinacyl , however , did not change significantly over time ( p > 0.05 for time ) following consumption of the ad libitum lunch but plasma concentrations of ghrelinacyl were greater ( p = 0.04 ) at 60 minutes postprandially in lactating compared to control women ( figure 2 ) . energy intake was not predicted by concentrations of appetite - regulating hormones or hunger or satiety ratings prior to the ad libitum meal or by hormone response to the previous standardized meal . however , higher serum concentrations of ghrelinacyl prior to the meal ( at 150 min ) and a higher ghrelinacyl response ( auc ) were associated with selection of more carbohydrate ( p < 0.05 , r = 0.33 , and r = 0.39 , resp . ) and less protein ( p < 0.05 , r = 0.035 , and r = 0.46 ) as a percent of total energy in the ad libitum lunch meal . a higher pyy auc was correlated with higher absolute carbohydrate intake ( r = 0.33 ; p < 0.05 ) . of the initial 24 lactating and 20 control women , 6 lactating and 5 controls were unable to complete the longitudinal measurements because of relocation , time constraints , or undisclosed reasons . of the 18 lactating women , 9 continued to breastfeed for the duration of the study and 9 stopped lactating between baseline and the 6-month ( n = 4 ) and 12-month follow - up ( n = 5 ) . the anthropometric characteristics of these 18 lactating and 15 controls are shown in table 4 . anthropometric characteristics of the women lost to attrition did not differ ( p > 0.05 , data not shown ) from those completing the study . during the follow - up period , lactating women lost 5.3 2.2 kg of body mass whereas the body weight of control women remained stable ( p = 0.02 ; group time interaction , figure 4 ) . lactating women also experienced a reduction of fat mass , body fat percentage , and waist circumference ( p = 0.011 , p = 0.007 , and p = 0.027 , resp . lean mass remained unchanged ( p = 0.947 ) in both groups . the majority of loss in waist circumference , weight , and body fat occurred during the first six months postpartum ( figure 4 ; table 2 ) . at 12 months postpartum , 15 of the lactating women were within 2 kg of their prepregnancy weight while the remaining three were 6.0 to 17.5 kg heavier than prepregnancy weight . change in total body mass and fat mass did not differ at either time point in the lactating women who continued to lactate at 12 months ( n = 9 ) compared to those who discontinued lactation ( n = 9 ) ( p > 0.05 ) . despite decreases in total body weight and adiposity in lactating women , fasting concentrations of leptin , ghrelin , ghrelinacyl , and glp-1 remained relatively stable across the first postpartum year ( table 4 ) . fasting concentrations of insulin and pyy increased slightly in both lactating and control women ( p < 0.05 for time ) . absolute change in body mass and body fat for lactating women at either 6 or 12 months was not explained by concentrations of the appetite - regulating hormones at baseline either during fasting or in response to the standardized breakfast meal ( auc ) . comparisons between women who returned to within 2 kg of their prepregnancy weight ( n = 15 ) and those that had retained their pregnancy - associated weight gain ( n = 3 ) suggested that those retaining weight had gained more during pregnancy ( 22.0 5.7 versus 15.0 3.4 kg , p < 0.05 ) despite similar self - reported weight and bmi ( 63.2 8.9 kg , 25.2 3.0 kg / m versus 64.6 8.5 kg , 22.6 9.5 the weight retainers also had higher fasting insulin during baseline testing ( 1.4 0.8 versus 0.0 0.0 ) and greater insulin ( total auc , 1299 542 versus 446 362 , p < 0.05 ) and ghrelin rebound responses ( i.e. , higher response after the initial postmeal fall ; positive auc , 17155 27648 versus 2893 3033 , p < 0.05 ) after the standardized breakfast meal . failure to lose weight following pregnancy is an important and identifiable risk factor for long - term obesity [ 1 , 4 ] and alterations in circulating fasting and meal - induced appetite regulators during lactation were considered as a possible mechanism of postpartum weight retention . we determined that fasting concentrations of these hormones and their postprandial pattern following standardized intake were not different between lactating and never - pregnant control women matched for age , body mass , and body composition . the only notable difference was the tendency for higher postprandial ghrelinacyl in the lactating women ( figure 2 ) that was higher after the ad libitum lunch despite consumption of 24% more energy than control women . circulating concentrations of appetite - regulating hormones were also not predictive of postpartum weight loss ; however , higher postprandial ghrelin and insulin responses were observed in women who did not lose pregnancy - associated body mass by the end of the first postpartum year . this suggests that a greater response of the orexigenic hormone ghrelin may work against postpartum weight loss by either promoting increased appetite / food intake and/or preventing body fat loss . to our knowledge , this is the first study to comprehensively evaluate differences in fasting and meal - induced responses of the gut peptides ghrelin , pyy , and glp-1 along with insulin and the adipose - derived hormone leptin . these hormones act in concert serving as signals between the gut , adipose tissue , and brain and are involved in appetite , glucose homeostasis , and body weight regulation [ 1619 ] . although the actions of these hormones are not completely elucidated , ghrelin is thought to enhance food intake through interactions with neuropeptide y ( npy ) and agouti - related protein- ( agrp- ) expressing neurons of the hypothalamic arcuate nucleus whereas pyy , glp-1 , and leptin work via dampening these neurons [ 16 , 18 ] . the role of these hormones in obesity pathogenesis is not yet delineated but peripheral infusion of ghrelin is shown to promote food intake in humans and decrease fat oxidation and promote adiposity in animal models , whereas infusion of pyy and glp dampens appetite and food intake in humans [ 3436 ] . our results suggest that differences in these hormones do not , however , predict intake in an ad libitum meal . curiously , higher serum concentrations of ghrelinacyl prior to the ad libitum meal and a higher ghrelinacyl auc were associated with selection of more carbohydrate and less protein . somewhat in contrast to the current results , our previous study found lactating and nonlactating postpartum women had lower fasting ghrelin concentrations compared to never - pregnant controls . our earlier study , however , measured only total ghrelin ( not ghrelinacyl ) and pyy at 60 min postprandially and was complicated by the higher body mass and adiposity in the postpartum women compared to leaner controls . the current study , which carefully matched lactating and control groups at baseline for body mass and body composition , also did not find differences in postprandial hormone concentrations apart from the consistently higher postprandial ghrelinacyl in lactating women that was statistically different after lunch . given the potential role of ghrelin , pyy , and glp-1 in appetite regulation and energy balance , we had expected identifying a different fasting postprandial pattern in lactating women reflecting their increased energy demands . the higher ghrelinacyl concentration in lactating women 60 min after consumption of the ad libitum lunch , despite consumption of 24% more energy , is the only notable finding and may suggest reduced suppression of hunger even after a full meal . this could promote shorter frequency of food intake initiation between meals . on the other hand , similar to our generally null findings , serum concentrations of ghrelin and pyy are also unaffected by lactation in rats and pigs [ 3739 ] . however , higher circulating ghrelin concentration in lactating dairy cows is predictive of greater pasture intake during pasture grazing . another purpose of the current study was to evaluate whether varying concentrations or patterns of appetite - regulating hormones predict weight retention over the first year postpartum . this could help to explain the highly variable differences in body mass and body composition changes observed after childbirth and during lactation . butte and hopkinson speculated over fifteen years ago that body composition changes during lactation are in response to a sequence of complex neuroendocrine and biochemical stimuli , many of which were unknown . although we did not find that fasting or meal - induced changes in gut peptides predict absolute body mass change , a greater ghrelin rebound after meal was observed in the three women who retained postpartum weight at the end of the first postpartum year . a higher fasting insulin and greater insulinemic response was further noted and is likely due to the higher adiposity of these women . altered ghrelin may work to promote food intake and/or dampen fat oxidation preventing spontaneous body weight loss . women who increase body mass excessively during the first pregnancy or retained weight after delivery also have a higher risk in subsequent pregnancies of overweight and obesity . although well controlled , the current study was limited by sample size , use of a liquid versus a solid standardized meal , and the known general variability of both circulating concentrations of gut peptides among participants and the intra / intervariability of the assays . even though our sample size of 24 lactating and 20 never - pregnant controls is relatively large given recruitment feasibility and well within the a priori target sample , it may have limited our ability to detect differences for various peptides particularly ghrelinacyl and glp-1and subjective appetite ratings . a larger and more diverse sample may have provided more statistical power to detect differences in the groups . longitudinal data was likely limited by sampling from a healthy university community as well as the approximate 25 percent attrition and may have contributed to the small number of weight retainers at one year ( 3 versus 15 ) . our observation that weight retainers had higher ghrelin rebound responses ( i.e. , higher response after the initial postmeal fall ) , higher fasting insulin , and greater insulin meal after the standardized breakfast meal should be followed up in future studies . a final possible limitation was our decision to match controls based on body mass in early versus late postpartum . this may have masked true differences in the early postpartum period when lactating women are expected to be heavier than never - pregnant women . results of the current study are not in support of the hypothesis that appetite - regulating gut peptides and leptin are altered during lactation and associated with postpartum body weight retention . the potential for differences in the ghrelinacyl and glp-1 responses to a meal during lactation should be further evaluated in future larger - scale studies . similarly , our observation that postpartum weight loss may be countered by alterations in postprandial ghrelinacyl at early postpartum deserves further exploration in a larger , more diverse sample of women . understanding how these and other appetite- and body weight - regulating hormones function during this particularly obesogenic stage of the women 's lifecycle may be helpful in furthering the overall understanding of obesity pathogenesis .

to determine whether fasting and meal - induced appetite - regulating hormones are altered during lactation and associated with body weight retention after childbearing , we studied 24 exclusively breastfeeding women ( bmi = 25.2 3.6 kg / m2 ) at 4 - 5 weeks postpartum and 20 never - pregnant controls ( bmi = 24.0 3.1 kg / m2 ) . ghrelin , pyy , glp-1 , and appetite ratings were measured before / and 150 minutes after a standardized breakfast and 60 minutes after an ad libitum lunch . body weight / composition were measured at 6 and 12 months . fasting and area under - the - curve responses for appetite - regulating hormones did not differ between lactating and control groups ; ghrelinacyl , however , tended to track higher after the standardized breakfast in lactating women and was higher ( p < 0.05 ) after the ad libitum lunch despite a 24% higher energy intake ( p < 0.05 ) . by 12 months , lactating women lost 5.3 2.2 kg ( n = 18 ) , whereas control women ( n = 15 ) remained weight stable ( p = 0.019 ) ; fifteen of the lactating women returned to within 2.0 kg of prepregnancy weight but three retained > 6.0 kg . the retainers had greater ( p < 0.05 ) postmeal ghrelin rebound responses following breakfast . overall these studies do not support the hypothesis that appetite - regulating hormones are altered during lactation and associated with postpartum weight retention . altered ghrelin responses , however , deserve further exploration .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

PMC5330804

brain - machine interfaces ( bmis ) impose arbitrary associations between neural activity patterns and prosthetic actions . a neuroprosthetic skill is considered successfully acquired when this association is learned and the prosthesis can be controlled in a goal - directed manner . the underlying principle is that neural activity , reinforced via operant conditioning , can be volitionally generated . operant conditioning of cortical neurons has been successfully achieved in monkeys ( engelhard et al . , 2013 , fetz , 1969 , fetz and baker , 1973 , hwang et al . , 2013 , moritz et al . , 2008 , 2014 ) , rodents ( arduin et al . , 2013 , clancy et al . , 2014 , gage et al . , 2005 , 2012 ) , and humans ( cerf et al . , 2010 ) by translating their activity into auditory or visual feedback signals and reinforcing desired patterns with reward . because this paradigm specifies which neurons control an action , it circumvents the neural complexity associated with natural behavior . its use goes , therefore , beyond bmi applications ; it can serve as a research tool that facilitates dissecting the involved plasticity mechanisms ( sadtler et al . , 2014 ) . a closed - loop implementation in which the feedback takes the form of direct cortical activation ( i.e. , an artificial sensory channel ) would offer the advantage of having full control of the neurons directly involved in the association to be learned . implementing such an artificial position sense ( lebedev et al . , 2011 ) in a bmi setting could not only offer more flexible and faster feedback mappings , but , more importantly , substitute the position sense of a neuroprosthetic device when natural sensation is lost . , 2013 ) ; bias ( salzman et al . , 1990 ) , mimic ( oconnor et al . 2013 ) , or even augment ( thomson et al . , 2013 ) natural perception ; and cue goal - directed movements ( fitzsimmons et al . it can also be optimally integrated with natural sensory cues during real reaching movements ( dadarlat et al . , 2015 ) . however , in conjunction with volitional neural control , cortical stimulation has only been able to provide cues for choosing ( odoherty et al . , 2009 ) or for discriminating ( odoherty et al . , 2011 ) between virtual targets , while natural sensory feedback was used to guide learning and the actual execution . the feasibility of artificial sensations to not only cue , but to actually instruct in real time the generation of conditioned neural patterns , remains currently unexplored . this would mean that the brain can create an association between an action and its real - time sensory consequence when both are restricted to activities of different sets of cortical neurons . stable activity patterns of local populations of motor cortex neurons have been found to emerge during motor learning ( peters et al . , 2014 ) , and highly interconnected cortical ensembles ( harris and mrsic - flogel , 2013 ) might be responsible for the emergence of such population dynamics where individual neurons matter little . operant conditioning of a single motor cortex neuron might thus be expected to entrain its ensemble during learning and just be a participant of a changing population code . recent findings , however , suggest that learning might be confined to the conditioned neurons rather than involving a cortical ensemble ( arduin et al . , 2013 , clancy et al . , 2014 ) conclusive evidence of such learning specificity requires conditioning single neurons and simultaneous unambiguous tracking of a large number of neighboring non - conditioned neurons . in the present study , we therefore address the following questions : can mice learn to use a fabricated feedback channel to control single - neuron activity ? how do responses of the conditioned and neighboring cortical neurons change with learning ? for this purpose , we developed an all - optical bmi , a system for simultaneous wide - field two - photon population imaging of neurons expressing genetically encoded calcium ( ca ) indicators in primary motor cortex ( m1 ) and simultaneous activation of neurons expressing optogenetic actuators in primary somatosensory cortex ( s1 ) . operant conditioning of a single m1 neuron was performed by reading out its activity in real time , transforming it into a rate code of optogenetic stimulation pulses in s1 and reinforcing above - threshold activations with reward . we tested the necessity of the optogenetic feedback signal for identifying the reinforced activations and for learning to produce them more often over time . we then analyzed learning - related changes observed in conditioned neurons in comparison to those in neighboring , longitudinally tracked , non - conditioned neurons . our results unveil basic properties of l2/3 processing , which may also characterize cortical activity during , but not be delineable with , natural behaviors . to test if artificial sensory feedback can guide operant conditioning of cortical neurons , we first developed a novel two - photon imaging system designed for simultaneous optogenetic stimulation of cortical areas in head - fixed mice ( figures 1a1d ; see star methods ) . mice expressed the genetically encoded ca indicator gcamp6f in forelimb m1 and the optogenetic actuator channelrhodposin-2 ( chr2 ) in the corresponding somatosensory representation ( figure 1a ) . we used a cre - dependent reporter mouse line ( ai32 ) to ensure a stable level of chr2 expression over time . to monitor the effect of the optogenetic stimulation , we measured the local field potential in the contralateral s1 with electrocorticograms ( ecogs ; figure 1b ) . experimental animals were either classified as chr2 mice ( n = 8) or control mice ( n = 11 ) depending on whether ecog responses were detectable upon optogenetic stimulation ( figures 1c , s1a , and s1b , available online ) . the absence of optogenetically driven responses in control mice was due to insufficient or lack of expression of chr2 ( see star methods ) . two - photon images of large populations of individual l2/3 neurons ( 461 164 , mean sd ) were acquired at 30hz and simultaneously streamed to a dedicated computer for real - time processing ( figure 1d ) . to condition the activity of a single neuron , we extracted its fluorescence changes from the image and used it as a proxy for its neuronal activity . the fluorescence transients were transformed directly into a rate code dictating the frequency of the optogenetic stimulation pulses targeting s1 . a water reward was delivered whenever the conditioned neuron s activity and , by extension , the rate of optogenetic stimulation crossed a chosen threshold , thereby reinforcing high activation levels ( figure 1e ) . we trained the chr2 and control mice in the operant conditioning task for at least 15 consecutive daily sessions , for 3040 min per session ( figure s2a ; see star methods for details ) . potential visual cues from the optogenetic stimulation were effectively masked with blue light flashes ( figure s1c ) . learning was defined as significant increases in threshold crossing rate ( tcr ) of the conditioned neuron s ( cn ) ca response between session start and end ( two - tailed paired t test , p < 0.05 ) . according to this definition , chr2 mice learned on average within single sessions ( figure 2a ) , with six out of eight individuals showing significantly increased tcrs . a significant increase in tcr compared to session start was already observed in the second time bin ( p < 10 ) , suggesting that operant control of the cn ensued after 4.9 1.64 min ( mean sd ) of conditioning . control mice , which underwent identical experimental procedures and were rewarded at similar rates as chr2 mice ( figure s2b ) , did not learn on average , with only one out of eleven showing an increase in the tcr ( figure 2b ) . baseline tcr at the start of a session did not differ between mice that learned and the ones that did not learn ( non - paired t test , p = 0.15 ; figure s2c ) . 0.05 ) became more frequent across successive days of training in chr2 mice only ( figure 2c ) . after more than 10 consecutive days of conditioning , the probability of observing learning in a given chr2 animal reached 40% . during these learning sessions , the number of threshold crossings increased by 0.66 0.08 ( mean sem ) every minute . movement artifacts in the two - photon images and activation of the cn by the optogenetic stimulation via direct inputs from s1 were negligible and did not contribute to threshold crossings ( figures s2d learning was also not accompanied by more frequent contralateral forepaw movements ( figure s2h ) . in previous studies , operant conditioning of cortical neurons in the absence of sensory feedback ( reward only ) was found to be either not possible ( koralek et al . , 2012 ) or only successful in experienced animals previously trained with visual feedback ( fetz , 1969 ) or extensively exposed to reward - based learning ( hira et al . , 2014 ) . our results indicate that in naive animals , providing feedback by chr2-based cortical stimulation can expedite operant conditioning of cortical neurons when compared to reward reinforcement only . therefore , the brain is able to form associations between the activity of a single neuron and proportional feedback stimulation in s1 within single behavioral sessions . given that learning occurs surprisingly fast , have the mice truly formed an association between activity increases of the cn and the reward outcome ? is optogenetic feedback a necessary cue for maintaining this association after the initial learning has occurred ? contrary to normal task conditions where rewards were automatically delivered , these questions were addressed with a condition , during which mice had to initiate licks upon threshold crossings to receive the reward . this condition therefore tested if the mice can behaviorally detect instances of threshold crossings ( i.e. , the actions that lead to reward ) and was introduced for brief intervals of time at the end of the last five experimental sessions ( figure s2a ) . to learn and maintain an association between above - threshold activation and the reward outcome , mice could also rely on internal predictions related to the generation of the neuron s activity ( i.e. , analogous to efference copies ) . to assess the relative contribution of the involved cues , we uncoupled the optogenetic feedback from the cn s activity in feedback removal and playback conditions . in the normal feedback condition , detection probability , defined as the proportion of threshold crossings successfully detected by subsequent licking , was close to unity ( figures 2d and 2e ) . when feedback was transiently removed , detection probability significantly dropped ( p < 10 , kruskal - wallis test ) and became no different than would be expected by chance ( figure s4 ; see star methods for details ) . these results reveal that the association has indeed been learned , that the optogenetic stimulus remains necessary , and that potential internal cues alone are not sufficient for detecting correct performance . to assess if optogenetic stimulation alone was sufficient for identifying the rewarded actions , we also introduced a playback condition , during which the stimulation pattern was transiently decoupled from the cn s real - time activity and controlled by a recording from a previous day ( figures 2d and 2e ) . during playback , because rewarded actions were not generated anymore by the animal , their identification could only be based on the s1 stimulation pattern . detection probability during playback was not different compared to normal feedback periods ( p = 0.33 ) , demonstrating the importance optogenetic stimulation acquired during learning ( figures 2d and 2e ) . to exclude reliance on any other sensory cues , such as visual percepts evoked by the blue light pulses , we also delivered playback stimuli to a control area not expressing chr2 ( figure 1a ) . under these conditions , detection ability was significantly compromised ( p < 10 ) and indistinguishable from either the stimulus removal condition ( p = 0.84 ) or chance occurrence ( figure s4 ) . these results indicate that the artificial sensory signal is not only needed for learning to rapidly occur , but also to behaviorally identify correct performance during the task and remain the sole necessary cue for this purpose thereafter . ca events might occur more frequently over time and thus the rate of those that cross threshold also increases . alternatively , the ca events might become larger and thereby cross threshold more often . to examine this , we looked at activity changes of the cns in all sessions with significant learning ( n = 33 ) . within - session increases in tcr were guided by increases in the rate of ca events ( i.e. , occurrence of spike bursts ) , as well as increases in their individual amplitudes ( i.e. , number of spikes in a burst ) ( figures 3a and 3b ) . while both changes in ca event rate and amplitude contributed to tcr increases , event amplitude changes accounted significantly more on average for the variability in tcr during a session ( general linear model ; figure 3b , inset ) . furthermore , if either increase of the amplitude or rate of the conditioned events still persists , it would indicate that this activity change might be the result of learning - related plasticity . we found that upon feedback removal event rate significantly dropped , leading to a decreased number of threshold crossings , yet the event amplitude remained elevated ( figure 3b ) . the persistence of the elevated amplitude might therefore be an indication of lasting plasticity changes in the system . the immediate decrease in the rate of events , on the other hand , indicates that feedback remains necessary for maintaining the learned behavior and that the observed increases in cn s activity are not signs of irreversible ca accumulation or related to the acquisition of a habit . we next asked if these learning - related changes are specific to the cn or if they can also be found in simultaneously imaged neighboring neurons . we found that 6.4% ( quartiles , 2.3% and 10.6% ) of neighboring neurons had significant within - session linear increases in either event rate or amplitude and were therefore labeled as increasing neurons ( ins ) these increases , however , did not match the characteristics of the learning - related activity changes of the cns ( figures 3c and s5 ) . whereas the rate changes of ins were similar to those of cns ( two - sided paired t test , p = 0.51 , t(26 ) = 0.661 ) , the mean changes in event amplitude , which are mostly responsible for learning , were statistically different ( p < 0.001 , t(26 ) = 4.257 ) . furthermore , the onset of ca events in ins lagged both rewarded ca events of the cn and reward onsets ( figure 3d ) . thus , activity increases observed in non - conditioned neurons probably reflect the behavioral consequences ( e.g. , reward anticipation , collection , or consumption ) rather than causes of above - threshold activations of the cn . it follows that learning - related changes are restricted to the cn and do not involve the collective activation of a local population of m1 neurons . because the feedback signal represents a pseudo - random stimulation pattern for all neurons other than the cn , the observed neuronal specificity suggests that it is the activity - locked feedback signal , and not just the s1 optogenetic stimulation per se , that is important for learning . the neuronal specificity further suggests that the conditioned activity was not primarily related to other variables such as forelimb movements that would most likely result in driving all forelimb - related neurons in concert . in spite of an absence of learning - related activity increases in neighboring neurons , it is still possible that the cn is driven by activity in the local network . indeed , we found that a small fraction of neighboring neurons ( 2.5% ; quartiles , 0% and 8.2% ) exhibited ca events that preceded those of the cn more often than would be expected by chance ( p < 0.01 , hypergeometric probability ) ( figure 3e ) and could therefore potentially constitute pre - synaptic inputs . these events occurred sparsely ( 30% 10% , mean sd of threshold crossings ) , and in only 16% of these leading neurons , an ideal observer could discriminate whether a conditioned ca event would cross threshold or not ( p < 0.05 , permutation test , receiver operating characteristic [ roc ] analysis ; see star methods for details ) . leading neurons , but not the ins , were situated in closer spatial proximity to the cn than a size - matched random neuronal sample ( figures 3e and 3f ) . small spatially organized functional clusters of neurons might therefore exist in l2/3 of m1 , as previously suggested ( hira et al . these putative local pre - synaptic drivers might actually be more numerous , yet be individually weak and therefore below the detection threshold of ca imaging used in this study . therefore , even if the animals learned to control a single neuron instead of an m1 subpopulation , the nearby neurons might still play an active role in providing part of the drive to the cn . long - term tracking of individual l2/3 neurons has revealed that their population representation might be stable , but their individual responsiveness and/or functional tunings are flexible day to day in motor ( huber et al . , 2012 , peters et al . , 2014 ) , as well as sensory ( unpublished data ) cortices . similarly , consistent cross - day learning of multi - unit neuroprosthetic control has been found to require either daily retraining or iteratively adapting the decoder to the day - to - day variability of neuronal activity ( orsborn et al . , 2014 , taylor et al . , 2002 ) . redundant representations of a motor or sensory function across many neurons might allow such flexibility and reflect a necessary adaptability of neural responses . however , operant conditioning of a single m1 neuron attributes a functional role to that neuron only . it might therefore be expected that its responses would become more stable and stereotyped over time . we analyzed the stability of the cns across multiple days of learning . despite their exclusive status , cns were readily found inactive at the start of a new session and often remained silent for several consecutive days ( figures 4a and s6 ) . this prevented further conditioning with the same neuron and forced us to choose a new one . the decision to switch conditioning to a new neuron was based on a baseline recording prior to session start ( see star methods for details ) , whereas the data in figure 4a depict the neuron s average activity during the entire session . the distribution of day - to - day changes in the overall ca event rate of the cns was not different from that of the non - conditioned neurons ( figure 4b ; two - sample kolmogorov - smirnov goodness - of - fit test , p = 0.15 ) . conditioned l2/3 neurons seem therefore not to be immune to flexibility , even when they acquire an exclusive functional role , which further highlights the robustness of this seemingly intrinsic property of l2/3 representations . taking together the remarkable speed and specificity with which the conditioned neuron adapts to the imposed constraints , our results suggest the existence of functionally and spatially specific plasticity mechanisms able to tune the activity of an l2/3 neuron on very short timescales . to determine if more complex mappings can also be rapidly learned with artificial feedback , we trained mice to modulate the activity of three neurons simultaneously . chr2 mice previously trained on the single - neuron task were taught to co - activate two arbitrarily chosen m1 neurons , while silencing a third neuron to gain reward . the amplitude of the fluorescence signal of each neuron was transformed with a logistic function . the outputs of neurons 1 and 2 were summed and that of neuron 3 subtracted to obtain an ensemble signal ( figure 5a ) . the ensemble signal dictated the frequency of optogenetic stimulation pulses according to the same transfer function as in figure 1d , and above - threshold crossings triggered reward . the threshold level was chosen so that only simultaneous activations of neurons 1 and 2 , and near - baseline activity of neuron 3 , led to reward . any activation , no matter how strong , of either neuron 1 or 2 alone was not sufficient for threshold crossing , and any above - baseline activity of neuron 3 prevented it ( figure 5b ) . we found that chr2 mice can also learn this more complex mapping , as indicated by within - session linear increases in tcrs ( figure 5c ) . as imposed by the mapping rule , neurons 1 and 2 were co - activated at threshold crossings of the ensemble activity ( figure s7b ) . the probability of these co - activations might fortuitously increase during a session as the rate of ca events increases in the two neurons , or even in only one of them . both neurons indeed increased their overall activity throughout a session ( figure s7a ) . but to test whether mice also generated more co - activation per se , we analyzed if the number of co - activations per produced ca event changes during learning . we found that this ratio significantly increased between session start and end for both neuron 1 ( p = 0.003 , kruskal - wallis test ) and neuron 2 ( p = 0.03 ) , demonstrating that more frequent co - activation did not occur fortuitously . a comparison of each neuron s activity aligned on any ca event of the other , between the start and end of a session , confirms that they became more co - active during learning ( figure s7b ) . to illustrate a practical neuroprosthetic application of this mapping , we also translated the imposed rules into mechanical constraints governing the multi - joint displacement of a robotic arm ( figure 5a ; movie s1 ) . the activity of each neuron was transformed into a joint angle , rendering the ensemble activity proportional to the distance ( in joint angle coordinates ) from the arm s endpoint to a target area . the optogenetic stimulation thus provided feedback of the arm s position relative to the target . the increased number of threshold crossings was therefore akin to more frequent target hits ( figure 5c ) . although unbeknownst to the mouse , increases in the rate of successful displacements toward the target illustrate how multiple l2/3 neurons can be conditioned to execute goal - directed movements of a prosthetic device under the guidance of artificial sensory feedback . this study reveals that artificial sensory feedback about the current activity state of one or multiple volitionally modulated neurons can guide rapid learning during operant conditioning . we effectively fabricated an optical version of an artificial cortical communication channel ( jackson et al . , 2006 ) , which was used by mice to rapidly ( i.e. , in less than an hour ) learn to produce reinforced neural activity patterns in a goal - directed manner , similarly to using natural sensorimotor associations . schematic depictions of such a channel are readily included in idealized views of neuroprosthetic control where artificial sensory information from the prosthetic device is fed back directly to the brain ( bensmaia and miller , 2014 , kwok , 2013 ) . our experiments provide a seminal proof of concept of such a system using an adaptation - based approach ( bensmaia and miller , 2014 ) and illustrate that it could in principle be used to modulate multiple neurons in concert for executing goal - directed multi - joint prosthetic movements . a more biomimetic implementation ( bensmaia and miller , 2014 ) it would necessitate that multiple instances of the artificial channel be learned in parallel , each activating and providing proprioceptive feedback of a single joint . graded stimulation of the cortex has previously only been used to guide natural forelimb reaches ( dadarlat et al . , 2015 ) , albeit under the assistance of proprioception and a lengthy ( i.e. , several months ) learning procedure involving pairings with visual cues . in contrast , we demonstrate that both sensory and motor peripheries can be bypassed and that unassisted , arbitrary associations can be fashioned between activities of two different sets of cortical neurons ( not necessarily invoking direct anatomical pathways ) . a key component in our experiments was the wide - field imaging of a large number of cortical neurons and their longitudinal tracking during conditioning . it enabled us to peek into cortical mechanisms underlying volitional neural control and how the brain drives its own activity . conceptually , the reinforced activity of the conditioned neuron ( i.e. , the controlled variable ) is driven by a this might seem surprising given that cortical networks are locally highly interconnected ( harris and mrsic - flogel , 2013 ) . it is , however , consistent with the general sparseness of l2/3 neuronal firing ( petersen and crochet , 2013 ) where population activity is thus not expected to reflect a common motor function and might be less correlated compared to the output neurons of the deeper layers of cortex . previous studies actually report a similar confinement of learning - related activity to conditioned cortical neurons ( arduin et al . , 2013 , clancy et al . , 2014 ) . the original single - neuron conditioning experiments of fetz and baker ( fetz and baker , 1973 ) targeted deeper cortical layers ( probably layer 5 ) using microelectrodes to simultaneously record the conditioned and an adjacent neighboring neuron . their findings revealed that in about half of the recorded pairs , the responses were correlated during threshold crossings . we can therefore hypothesize that spatial specificity is more prominent in l2/3 and might be related to the necessary flexibility of this cortical layer for the purposes of learning new skills and adaptive mechanisms in general ( huber et al . it follows that the volitional controller driving l2/3 neurons most likely involves other , more distributed or distant cortical activations or subcortical pathways ( koralek et al . , 2012 ) . our results also demonstrate that when reward is contingent on the volitional activation of a single neuron , neural mechanisms can home in on that cell and ignore its immediate cortical neighbors . in a way , this mirrors a concept put forward by in vivo single - cell stimulation experiments ( brecht et al . , 2004 , houweling and brecht , 2008 ) . the latter suggests the existence of circuits downstream of the stimulated cortical neuron capable of reading out single - cell activity . our finding , on the other hand , suggests that activity in circuits upstream of the conditioned neuron can be routed to a single cortical cell . both emphasize the behavioral relevance of single - neuron activity , which might be more significant than previously thought . the closest direct precedent to our study is the work of clancy and colleagues ( clancy et al . , 2014 ) . they also used wide - field imaging to condition cortical neurons and were thus also able to unambiguously track activity of a local subpopulation of individual neurons during learning . instead of conditioning a single neuron , they conditioned ensembles of up to eleven neurons . the auditory feedback and reward delivery were dependent on the summed activity of the ensemble . additionally , they calibrated the feedback transform coefficients and reward thresholds daily based on baseline activity levels , whereas we kept them the same throughout the experiment for each conditioned neuron . their approach was hence more immune to day - to - day fluctuations in the activity of single neurons and probably explains why apart from observing within - session learning as we did , their mice also learned across days . similar coordination between non - conditioned neurons , those outside the ensembles , was not present and did not increase over time . such specificity of learning - related changes to conditioned neurons is confirmed in the present study , but our findings go further by demonstrating that this remains true even when conditioning is restricted to just a single neuron . clancy and colleagues also identified that some non - conditioned cells were activated around threshold crossings , which was more evident for those nearby than those distant from the conditioned ensemble . although a direct comparison is not possible because they do not report precise temporal relationships , these nearby cells might be similar to our spatially clustered leading neurons , which represent a putative pre - synaptic drive of operant activity in the local network . the finding that increased amplitudes of the ca events ( reflecting stronger bursts ) guided learning and persisted despite a performance drop after feedback removal led us to speculate that learning might in part be the result of neuronal plasticity , such as synaptic potentiation or more synchronous inputs to the conditioned neuron . future experiments could thus be aimed at identifying potentiated synapses and subsequently tracing the source of the related pre - synaptic input ( wickersham et al . , 2007 ) . since the volitional controller , again conceptually , uses the feedback signal to modify the controlled variable ( fetz , 2007 ) , the fact that we could artificially constrict the feedback to a set of cortical neurons might further facilitate its identification . a potential site of plasticity could be the cn s synapses receiving direct input from s1 . in the mouse vibrissal system , s1 projections directly target l2/3 neurons in m1 ( mao et al . , 2011 ) , and analogous the sequential activation of the cn and s1 afferents might have , by design , led to a hebbian - like spike timing - dependent plasticity ( markram et al . , 1997 ) and have been a contributing factor to producing more post - synaptic spikes over time . if that were the case , it is expected that s1 stimulation would evoke higher ca levels in the cn after , as compared to before , learning . however , we were not able to detect any significant fluorescence transients in the cn upon optogenetic activation of s1 , neither before nor after learning ( figure s3c ) . learning - related activity increases in the cn therefore do not seem to be mediated by direct s1-to - m1 projections , reinforcing the idea that more intricate pathways are involved . actually , because s1 stimulation does not lead , but follows , the cn s activation by a few milliseconds , it is more likely that the synapses in question were depressed and not potentiated ( jacob et al . , 2007 ) . indeed , stimulation of a cortical site proportional to real - time activity recorded at another site induces neural plasticity that is consistent with synaptic potentiation of pathways from the recording to the stimulation site , and not vice versa ( jackson et al . , 2006 ) . further work is therefore required to unequivocally identify the recruited pathways and the source of the input driving the conditioned neurons in m1 . in practical terms , this means that attaching an artificial position sense to neuroprosthetic actuators is feasible and can in principle be rapidly learned ( figure 5a ; movie s1 ) . these findings in rodents might also be applicable to humans and pave the way toward developing artificial feedback systems for patients , which could be implemented even with currently available electrode - based approaches . besides neuroprosthetic applications , our study also provides a novel behavioral tool for in vivo circuit dissection : an in cerebro learning paradigm that bypasses sensory and motor peripheries by imposing the learned action and its feedback directly in the cortex . this approach can potentially substitute for natural behavior and thereby facilitate dissecting neural circuits underlying sensorimotor learning . for instance , trans - synaptic tracing ( beier et al . , 2011 , wickersham et al . , 2007 ) can identify the presynaptic drivers of the conditioned neuron and the postsynaptic targets of the stimulated neurons , thus revealing how the feedback signal is routed to instruct volitional control and what ultimately drives the output . in addition , structural imaging and functional manipulations of the spines of conditioned neurons or the boutons of stimulated cells can provide insights into the synaptic plasticity mechanisms at play . finally , the accuracy of circuit dissection can be improved by producing a more specific feedback signal aimed at individual neurons with two - photon targeted activation and simultaneous imaging ( packer et al . , 2015 , reagent or resourcesourceidentifierantibodiesanti - chr2mfd diagnosticsclone 15e2biotinylated goat anti - mouse iggvector labscat # ba-9200 ; rrid : ab_2336171bacterial and virus strainsaav2.1-syn-gcamp6f.wpre.sv40university of pennsylvaniacat # cs0201aav2.1-hsyn-cre.wpre.hghuniversity of pennsylvaniacat # cs0342chemicals , peptides , and recombinant proteinssigma fast dab tabletsigma - aldrichcat # d4293 - 50setexperimental models : organisms / strainsmouse : ai32 : rosa - cag - lsl - chr2(h134r)-eyfp - wprejackson laboratoryrrid : imsr_jax:012569mouse : wild type : c57bl/6ncrlcharles rivercat # 027 further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact , daniel huber ( [email protected] ) . we used 8 to 12 weeks old ai32 transgenic homozygote male mice carrying a floxed chr2(h134r)-eyfp fusion gene inserted in the gt(rosa)26sor locus in a c57/bl6 background mouse strain ( madisen et al . , 2012 ) the animals were housed in an animal facility , maintained on a 12:12 light / dark cycle and were placed under a water restriction regime ( 1 ml / day ) ( median weight : 22.5 g , range : 19.2 g to 24.1 g ) . the animals did not undergo any previous surgery , drug administration or experiments and were housed in groups of maximum 5 animals per cage . all procedures were approved by the institutional animal care and use committee of the university of geneva and geneva veterinary offices . all surgeries were conducted under isoflurane anesthesia ( 1.5%2% ) and additional analgesic ( 0.1 mg / kg buprenorphine intramuscular ( i.m . ) ) , local anesthetic ( 50 - 100 l 1% lidocaine subcutaneous ( s.c . ) under the scalp ) and anti - inflammatory drugs ( 2.5 mg / kg dexamethasone i.m . and 5 mg / kg carprofen s.c . ) were administered as necessary . a custom made titanium head bar was implanted in the skull to allow for subsequent head fixation . a craniotomy was performed over the left frontal cortex and two viral injections ( 30 - 50 nl each ) were delivered ( 10 - 20 nl / min ) into the forepaw representation in s1 ( 0.75 mm anterior , 2.25 mm or 1.75 mm lateral to bregma ) and two in the forepaw m1 ( 1.75 mm anterior and either 2 mm or 1.5 lateral ) . in 8 mice , ( aav2.1-syn - cre , 1:100 for 2 mice and 1:1000 for 6 mice , 0.2% fastgreen in sterile saline , virus stock titer 1.46 10 genome copy per ml ( gc / ml ) ) . in m1 , ( aav2.1-syn - gcamp6f , 1:10 , 2.96 10 gc / ml ) was injected . the coordinates for the somatosensory representation of the forepaw were based on a series of intrinsic signal imaging sessions ( 7 additional ai32 mice ) . after virus injection , cortex was rinsed for 1 - 2 min with dexamethasone ( 0.03% ) . two hand - cut glass coverslips ( 150 m thick ) that matched the shape of the craniotomy were glued together with optical adhesive ( norland 61 ) . the cranial window was placed on top of the cortex , glued to the bone with cyanoacrylic glue and secured with dental cement . the correspondence between the s1 injection sites and the sensory forepaw representation was confirmed post - operatively with intrinsic signal imaging in each mouse . the m1 injection coordinates were calculated relative to the s1 coordinates and based on a database of cortico - cortical projections in somatic sensorimotor areas ( zingg et al . , 2014 ) . in addition , all mice were implanted with cortical surface electrodes ( teflon coated gold wires , au-3 t , science products , germany ) in the contralateral somatosensory ( 0.75 mm anterior , 2 mm lateral to bregma ) and visual cortices ( 3 mm posterior , 1 mm lateral to bregma ) . after a four day recovery period , mice were placed under a water restriction regime ( 1 ml / day ) . neural responses could be evoked by optogenetic stimulation of s1 in all 8 mice ( figures s1a and s1b ) . histological verification of chr2 expression revealed dense labeling of cell bodies and neuropil in l2/3 and l5 at the s1 site , whereas no neurons were retrogradely labeled in m1 ; only axonal projections of chr2 neurons from s1 could be observed ( figure s8a ) . 11 additional male mice that underwent identical surgery and injection procedures were used as controls as they did not exhibit ecog responses to the optogenetic stimulus ( figures s1a and s1b ) and showed no or only sparse expression of chr2:4animals were wild - type siblings not carrying the floxed chr2-eyfp gene.2ai32 mice were heterozygote siblings4ai32 homozygote mice received a highly diluted cre - virus injection in s1 ( 1:10000).2ai32 homozygote mice received a diluted cre - virus injection in s1 ( 1:1000 ) , but showed only very sparse expression of chr2 ( figure s8b ) . we explain the low chr2 expression levels and lack of evoked responses in these mice by the failed virus delivery during surgery . ai32 mice were heterozygote siblings ai32 homozygote mice received a highly diluted cre - virus injection in s1 ( 1:10000 ) . ai32 homozygote mice received a diluted cre - virus injection in s1 ( 1:1000 ) , but showed only very sparse expression of chr2 ( figure s8b ) . we explain the low chr2 expression levels and lack of evoked responses in these mice by the failed virus delivery during surgery . at the end of the experiments , mice were deeply anesthetized with pentobarbital , transcardially perfused with cold saline and paraformaldehyde 4% ( pfa ) and the brains were removed and stored overnight in pfa 4% . then , the brains were transferred to a sucrose solution ( 20% in pbs 0.1 m ) for at least 24 hr and sliced in 50 m coronal sections with a freezing microtome . tissue was stored in a solution of sodium azide ( 0.01% in pbs 0.1 m ) until histological processing . slices were incubated with a mouse primary monoclonal antibody anti - chr2 ( clone 15e2 , mfd diagnostics ) and with a biotinylated secondary antibody anti - mouse ( vector labs ) . the tissue was finally processed with diaminobenzidine ( sigma - aldrich ) for the colorimetric reaction . slices were mounted and imaged with a wide field scanner microscope ( olympus vs120 ) at 10x . mice were head - fixed and placed on a heated platform under light isoflurane anesthesia ( 0.75% ) . ten 1 s vibrotactile stimuli consisting of a 100 hz sinusoidal vibration were delivered to their forepaw with a 10 s inter stimulus interval . the cranial window was illuminated by a collimated red light led ( 630 nm ) and imaged at 10 fps with a 256 by 332 pixels resolution . the average difference image between the stimulation period and a 1.5 s baseline period was processed by a 50 by 50 pixels spatial averaging filter and subsequently smoothed by a 5 by 5 pixels gaussian low pass filter with 0.5 pixels standard deviation . imaging was performed with a custom built two - photon microscope ( mimms ; https://openwiki.janelia.org/wiki/display/public/home ) controlled by scanimage 4.2 ( scanimage.org ) using a 16x 0.8 na objective ( nikon ) and with excitation wavelength at 940 nm ( ultra ii , tunable ti : sapphire laser , coherent ) . 512 by 512 pixel images covering 626 by 665 m of cortex were acquired at 29.57 hz using bidirectional scanning with a resonant scanner system ( thorlabs ) . the power was modulated with pockels cell ( 350 - 80-la-02 , conoptics ) and calibrated with a photodiode ( thorlabs ) . optogenetic stimuli were generated with a 473 nm laser ( dhom - w473 - 200mw , ultralasers ) which was custom modified to be gated electronically using ttl pulses . the power was calibrated before each session to be 20mw at the stimulation site on the brain surface . the imaging and optogenetic beams were combined using a longpass dichroic mirror ( 700dcxxr , chroma ) between the scan lenses and the tube lens . the primary mirror for imaging was a custom polychroic ( chroma , zt470/561/nir - trans ) transmitting the infrared and blue light , while reflecting the green . before detection , the remaining ir light was filtered with a colored glass band pass filter ( bg39 , chroma ) , whereas the remaining blue light was removed with a short pass filter ( cg475 , chroma ) . images were continuously acquired using gated photo multiplier tubes ( gpmt ) ( h11706p-40 sel , hamamatsu ) and written in 16 bit format to disk in separate files ( 1110 frames / file ) . the start / end of file trigger was issued by a separate pc and later used for temporal alignment of ca traces with behavioral variables . the objective was aligned perpendicular to the imaging window for each mouse using a custom built laser - based alignment device . at the start of each session , the imaging field was manually located using reference images of previous recording days and slow drifts manually corrected for throughout the session . to correct for lateral movements , a custom matlab registration algorithm was used to align each image to a template taken as the average image of a 30 s resting baseline period recorded at the start of each session . the cross - correlation was computed between each image and the template by multiplying the two - dimensional discrete fourier transform of one with the complex conjugate of the fourier transform of the other and taking the inverse fourier transform of the product . the row and column location of the peak cross - correlation value was taken as the vertical and horizontal shift , respectively . 10% of each image was cropped at the boundaries for the purposes of this computation . semi - automatic custom matlab routines were used to draw regions of interests ( rois ) for individual neurons using the session average image . the ca - dependent fluorescence time series were extracted by averaging pixels within each roi . the time - varying baseline f0 of a fluorescence trace was computed as the average value in a 20 s moving time window by excluding values that surpass 20% of this average . at each time point , the updated baseline value f0 was subtracted from the raw fluorescence value f to yield f and compute the relative change to baseline as f / f0 . images of scanned brain tissue acquired with scanimage were streamed on the local network using the user datagram protocol ( udp ) . each end - of - frame signal triggered a custom coded matlab routine that sent , in succession , 32 equal - sized lines of the 512 by 512 pixel image ( i.e. , 16 packets / image ) over the network . the packets were read on the appropriate udp port on a separate pc , the images reassembled and the relative change with respect to baseline of the conditioned neuron(s ) computed by extracting the average pixel intensity in a defined region of interest comprising a single cell . the extracted activity was downsampled to 0.2 s by averaging blocks of 6 consecutively received images and changes relative to a preceding 20 s moving baseline window were computed ( f / f0 ) . f / f0 values exceeding 20% were excluded when updating the baseline value . a second custom written matlab instance running on the same pc transformed , at every time sample t , f / f0 into a frequency value f(t ) according to f(t)=aen(t)a where n(t ) is the f / f0 value at time t. the obtained frequency value was then binned into one of 17 bins ranging from 1 hz to 15.12 hz in quarter - octave increments and produced a rate signal for the optogenetic pulses . the pulse rate was updated whenever the binned f(t ) differed from the binned f(t-1 ) . linear changes in neural activity therefore resulted in exponential changes in the feedback signal ( figure 1d , inset ) . a third custom written matlab instance used the rate code to generate an analog output signal on a data acquisition board ( national instruments , austin , tx ) that produced the optogenetic feedback to the mouse . the same instance also triggered a 0.35 s auditory beep and a reward pulse that opened a valve to deliver a drop of water to the mouse whenever the signal crossed a threshold level . the water drop was sucked away with a peristatic pump ( minipulse 2 , gilson , middleton , wi ) right upon delivery , thereby requiring immediate consumption and instigating anticipatory licking . rewards were always delivered automatically upon threshold crossings except in the lick - triggered reward condition of the last 5 sessions ( see behavioral training section below ) . the threshold level was initially chosen for each neuron on a trial - and - error basis to yield a minimum of approximately two rewards per minute , based on a baseline recording period , and was kept fixed thereafter . that was binned in the maximum 15.12 hz bin and was set by adjusting the coefficient a. only threshold crossings triggered a reward and the 15 hz stimulation was turned off if a sustained activity above threshold continued in excess of 1 s. the optogenetic feedback was reinstated once such sustained activity came back below threshold . this was done to pair the maximum stimulation frequency ( 15 hz ) with activity threshold crossings only , as this is what we sought to reinforce . by turning the stimulus off we avoided optogenetically reinforcing high levels of activity sustained above threshold that did not lead to reward . the total median time delay between frame completion and feedback generation was uniformly distributed between 19.2 ms and 219.2 ms ( see table).median ( 5% and 95% quantiles ) time delays incurred by the different processing stagesframe transmission11.2 ( 9.2 , 13.0 ) mssignal extraction7.1 ( 6.8 , 10.1 ) msrate code computation0.84 ( 0.8 , 5.4 ) msfeedback generation0.052 ( 0.05 , 3.2 ) ms median ( 5% and 95% quantiles ) time delays incurred by the different processing stages the choice of the conditioned neurons was biased toward neurons in the middle of the imaged field of view . other selection criteria were a clear identifiable morphology and a minimal ca event rate of 2 events / minute ( assessed during initial baseline measurements lasting 2 to 3 min ) . once a neuron was chosen , it was used for conditioning on all subsequent sessions as long as it remained active ( produced more than 2 ca events / minute ) . if the criterion was not met , conditioning was switched to a new neuron meeting the criterion before the start of the session . the functional role ( relationship to movements ) of the chosen neurons was not assessed and therefore remained unknown . because image movement correction was not performed for the real - time data , we quantified the artifactual transitions in the conditioned f / f0 traces that were induced by lateral motions ( figures s2d the optogenetic feedback was guided by mirrors through the imaging objective and visually aligned on the chr2 expression site in forepaw s1 using a ccd camera and a reference image of the blood vessel map . the squared pulses were used to electrically switch the laser on and off creating as such a fast and silent shutter for the blue light stimulus . the same pulse signal elongated by 1 ms turned off the gpmt used for imaging during photostimulation periods . this resulted in at most one band of 92 lines of the 512 by 512 pixel images to be blacked out over a two frame period for the maximal 15 hz stimulation level . eventual blanking of the cn was detected online and the frame in question was ignored in subsequent processing stages . as a visual mask , a collimated blue light led ( 473 nm , roithner ) was turned on and the gpmt turned off during flyback periods of scanning ( 1.5 ms pulses at the 29.57 hz acquisition rate ) , thus not affecting the acquired images while providing a perceptually stable visual stimulus . this pulsated blue light illuminated the otherwise dark setup from session start to session end . the feedback signal was delivered continuously without any trial structure but was paused for 1 s at 37.5 s intervals to allow for logging acquired data to separate files . this end / start of file trigger was also used to periodically update the saved image files on the scanimage pc and later used for aligning extracted ca traces with behavioral variables . mice sat head - fixed in a tube ( 25 mm inner diameter ) . the forepaw contralateral to the imaged and stimulated cortical sites was resting on a hold bar to maintain balance while the ipsilateral forepaw was restrained inside the tube . the water reward was delivered through a spout placed at licking distance below the snout . the tube and the mouse were connected to a 5 v node and , when touched , the conductive hold bar and reward spout shunted the 5 v to a circuit that pulled a respective analog input signal to the data acquisition board to high . licks and forepaw rests were in this manner continuously acquired at a sampling rate of 1 khz . releases of the hold bar were used to assess if mice consistently used contralateral forepaw movements to solve the task ( figure s2h ) . we used 4 control mice to test the efficacy of a blue visual mask in suppressing visually evoked ecog responses by the optogenetic stimulus . with the mask turned off , visually evoked potentials were observed in response to the onset and offset of the blue laser pulse train , but were completely suppressed when the mask was turned on ( figures s1c and s1d ) . mice were trained in the operant conditioning task for 15 to 17 consecutive daily sessions . the experimental timeline of each session was divided into condition blocks ( figure s2a ) . each conditioning session began with a three minute period of playback where a neural activity recorded on a previous day controlled the optogenetic feedback and reward delivery , in otherwise identical experimental settings . the f / f0 trace of the conditioned neuron saved in a previous session was used for this purpose . instead of the f / f0 values extracted from the streamed images at each time sample in real - time , the optogenetic feedback and threshold crossings were determined by the f / f0 values of that saved trace . control was then switched to the conditioned neuron and after an average of 25 min of real - time conditioning , the optogenetic feedback was removed for 3 min and reinstated thereafter . lick - triggered rewards were introduced during the first 3 min of playback and 3 min before , during and after optogenetic feedback removal . instead of being automatically delivered , mice had to initiate licking in a 500 ms time window following threshold crossings to obtain the reward . because rewards were otherwise always automatically delivered , these brief introductions of the lick - triggered reward condition were not significant enough to incentivize mice to engage in continuous licking as is sometimes observed in such lick - triggered reward paradigms . additionally , each session ended with 6 min of playback under the lick - triggered reward condition . in the first 3 min , optogenetic stimulation was applied to a control non - chr2 site and moved back to the s1 chr2 site in the last 3 min of playback . the positions corresponding to the chr2 and control sites were located on the blood vessel map using a ccd camera and their image coordinates recorded . the optogenetic beam was then positioned with a pair of galvanometric mirrors on those same image coordinates ( using a reflective surface ) and the corresponding voltages applied to the two mirrors were recorded for each site . the beam was then moved between chr2 and control sites , during the experiment , by applying the appropriate voltage levels at relevant times . all transitions between different experimental conditions were not cued by experiment interruptions or additional sensory stimuli . in the three neuron conditioning experiments , each neuron s activity n was transformed by a logistic function according tof(n)=12[1+tanh(a(nc ) ) ] . the ensemble activity f(n1)+f(n2)f(n3 ) controlled the rate of optogenetic feedback pulses and reward delivery . a and c were set to 2.2 and 0.6 , respectively and the reward threshold to 1.75 . this mapping constrained neurons n1 and n2 to co - activate and n3 to remain silent to bring the ensemble activity above threshold and trigger a reward . more specifically , f / f0 values of n3 above 0.36 prevented threshold crossings irrespective of n1 and n2 activity . concomitant f / f0 values of n1 and n2 above 1.15 resulted in threshold crossings provided the activity of n3 remained at zero . no statistical methods were used to predetermine sample size and all trained animals were included in the analysis . two - tailed student s t test ( for samples with equal variances ) or welch s t test ( for samples with unequal variances ) equal variances were determined with the two - sample f - test and the normality assumption was tested with the kolmogorov - smirnov test . the efficacy of chr2 activation ( figures s1a and s1b ) was assessed by measuring differentially between the two contralateral cortical electrode responses evoked by a 0.5 s train of 5 ms optical pulses delivered at 15 hz . peak responses in a 20 ms time window following the onset of each 5 ms light pulse were compared across all stimulation trials ( n = 30 ) to peak responses to the same number of simulated light pulses in the 0.5 s baseline period preceding the stimulus train onset . animals with significant differences ( paired t test , p < 0.05 ) between true and simulated mean peak values were classified as chr2 mice and as control mice otherwise . no method of randomization was used and the investigator was not blind with respect to the classification of 5 control animals , but was blind for the other 6 and for all chr2 animals . learning curves were calculated for the conditioning period between the end of the initial playback and either the onset of feedback removal or onset of the lick - triggered reward condition that precedes feedback removal . threshold crossing rate ( tcr ) was calculated for each of 8 equally sized time bins in this window . learning sessions were defined as those with significant linear increases of tcr across the 8 bins ( linear regression , p < 0.05 ) . mice with significantly bigger tcr in the last compared to the first bin across all sessions were labeled as learners and as non - learners otherwise . tcr normalization was performed by offsetting individual session learning curves to the same tcr value in the first bin , taken as the mean of all first bin tcr values , and dividing the rate of each bin by it . chance levels of detection probability of threshold crossings assessed in the lick - triggered reward condition ( figure s4 ) were simulated separately for each mouse and each experimental condition as follows : 1 . we identified the experimental epoch corresponding to each individual data point included in the mean value calculations of figure 2e . we isolated licks that occur only during baseline stimulation ( i.e. , 1 hz ) and attributed them to spontaneous licking . we then simulated spontaneous licking for the whole epoch by randomly reproducing the isolated lick sequences , thus preserving their temporal statistics ( i.e. , the inter - lick - interval and lick duration ) . chance detection probability was then calculated using these simulated licks and the same neural activity as for the actual data . as such , this procedure yielded for each individual data point a corresponding chance level and allowed paired statistical comparisons . ca events were detected by taking the first derivative of the smoothed f / f0 trace ( savitzky - golay filter , second order polynomial , 15 data points ) and an event onset was defined when the z - scored trace crossed a value of 2 and event end when it decreased again below 0 . event amplitude was defined as the difference of the f / f0 values between event end and event onset time points . event rate and average event amplitude were calculated for the same 8 time bins as the learning curves . a general linear model with the threshold crossing rate ( tcr ) z score as the dependent and the z scores of the event rate ( er ) and event amplitude ( ea ) as the independent variables was fit to each of the 33 learning sessions as follows : tcr = berer+beaea . the relative contribution of er and ea changes to tcr increases was evaluated by comparing the distributions of the fitted ber and bea values expressed in terms of number of standard deviations across all learning sessions . event rate and event amplitude change factors correspond to the ratios between the last and first time bins of the two measures , respectively . temporal ca event probability ( figure s3a ) was computed by transforming each f / f0 trace into an event trace which was set to 1 between each event onset and end , and to 0 otherwise . the reward - triggered average of all event traces of a given neuron yielded the event probability . increasing neurons were defined , in a given learning session , as those having significant linear increases ( p < 0.05 , linear regression ) of either their eas or ers between the start and end of learning , evaluated with the 8 time - binned values . leading neurons were identified , in a given learning session , by computing the hypergeometric cumulative distribution function value for each non - cn according top=1i=0x(ki)(mkni)/(mn),where x is the number of instances in which the non - cn produced a ca event in a 1 s interval preceding the onset of the cn s threshold crossing ca event . k is the product of the number of time samples in the 1 s interval and the number of threshold crossings in a given session ( number of samples drawn ) , m is the product of the number of time samples in a 4 s interval preceding the cn s event onset and the number of threshold crossings ( size of the population ) and n is the number of instances in which non - cn events occurred in the 4 s interval ( number of items with the desired characteristic in the population ) . non - cns with p < 0.01 were defined as leading neurons ( i.e. , those that produce ca events that precede the cn s threshold crossing ca event more often than what would be expected by chance ) . a receiver operating characteristic ( roc ) analysis was carried out on the leading neurons to assess whether their activity can predict threshold crossings of the cn s ca trace . for each learning session , the cn s ca events were classified into rewarded ( those that crossed threshold , event type x ) and unrewarded ( those that did not cross threshold , event type y ) , and each leading neuron s f / f0 trace in a 1 s interval preceding the onset of these events was taken into account . a discrimination score ( dvx ) was computed for each leading neuron for the i event type x as the dot - product of the neuron s f / f0 trace ( xi ) and the mean trace across all type x events ( excluding the i event , x ) minus the dot - product of xi and the mean trace across all type y events ( y ) . the discrimination score dvy was analogously obtained for the i event type y and , thus , according todvx = xi(xjiy)dvy = yi(xyji ) . an roc curve was constructed by plotting , for each criterion value c varied across the range of dvx and dvy values , p(dvx > c ) ( i.e. , the fraction of dvx values exceeding the criterion ) against p(dvy > c ) ( i.e. , the fraction of dvy values exceeding the criterion ) . the area under the roc curve ( auc ) was computed using trapezoidal numerical integration ( trapz ( ) function in matlab ) and corresponds to the fraction of events correctly discriminated by an ideal observer using the obtained discrimination scores . a permutation test , consisting of shuffling the x and y event labels and calculating auc values with 1999 shuffled subsets , yielded a p value for each leading neuron , taken as the fraction of shuffled auc values that were more extreme than the auc corresponding to the non - shuffled data . leading neurons with p < 0.05 were deemed to correctly predict , above chance level , weather a cn s ca event would cross threshold . bootstrap hypothesis testing of a test statistic being different from zero was performed by taking at random , with replacement , n values from the total set of n measurements of a variable , 1999 times . the two - tailed bootstrap p value was then computed from the 1999 sample measurements of a test statistic x as follows : p=2min(1bj=1bi(xj0),1bj=1bi(xj>0)),where b = 1999 , xj is the j bootstrap sample of x and i ( ) is the indicator function , which is equal to 1 when its argument is true and 0 otherwise .

summaryneuronal motor commands , whether generating real or neuroprosthetic movements , are shaped by ongoing sensory feedback from the displacement being produced . here we asked if cortical stimulation could provide artificial feedback during operant conditioning of cortical neurons . simultaneous two - photon imaging and real - time optogenetic stimulation were used to train mice to activate a single neuron in motor cortex ( m1 ) , while continuous feedback of its activity level was provided by proportionally stimulating somatosensory cortex . this artificial signal was necessary to rapidly learn to increase the conditioned activity , detect correct performance , and maintain the learned behavior . population imaging in m1 revealed that learning - related activity changes are observed in the conditioned cell only , which highlights the functional potential of individual neurons in the neocortex . our findings demonstrate the capacity of animals to use an artificially induced cortical channel in a behaviorally relevant way and reveal the remarkable speed and specificity at which this can occur .

Introduction Results Discussion STARMethods Author Contributions

PMC3125794

small angle x - ray scattering ( saxs ) is a technique that allows the study of the structure and interactions of biological molecules in solution . it can be used to probe proteins , nucleic acids , and their complexes under a variety of conditions , from near physiological to highly denaturing , without the need to crystallize the sample and without the molecular weight limitations inherent in other methods such as nmr spectroscopy . the increasing availability of high - flux , third - generation synchrotron sources , improvements in detector technology and algorithmic developments for data analysis have made saxs a technique of choice for a range of biological applications ( 1 ) . the basic principle of saxs is to scatter x - ray photons elastically off molecules in solution , and to record the scattering intensity as a function of the scattering angle . the intensity profile of the buffer is subtracted from the profile of the macromolecule in the buffer , yielding an excess intensity profile , related to the excess electronic density of the molecule and its environment . the saxs profile provides information about the global structure and conformation of the studied molecule(s ) . several reviews on the physical principles and theory of saxs describe in detail how the scattering data can be analyzed and how different parameters can be fit and interpreted ( 26 ) . existing computational approaches for modeling a macromolecular structure based on its saxs profile can be separated into two classes : profile - to - model ( ab initio methods ) and model - to - profile approaches . the former aims at proposing coarse shapes represented by dummy beads that fit the experimental profile ( 1016 ) , while the latter aims at comparing the theoretical profile of a given atomic or coarse grained model to the experimental one ( 17,18 ) . the model - to - profile approach consists in computing the theoretical profile of a given atomic structure and providing a measure of the goodness - of - fit to the experimental profile . it is useful for many applications where one needs to decide whether the proposed model is in agreement with the experiment , and to make assumptions about why they differ , if they do . several tools have been designed for that purpose , following various methods ( 1924 ) . to our knowledge , the most accurate method to date for computing saxs profiles of a given macromolecule has been achieved by treating the solvent ( excluded and hydrating ) explicitly ( 24 ) . through this method , even higher resolution profiles resulting from wide angle x - ray scattering ( waxs ) experiments were also reasonably reproduced . all other methods rely on a continuum representation of the solvent - excluded volume , first proposed by fraser et al . ( 25 ) : the effective - atomic - scattering - form - factor method . the programs sassim ( 22 ) and fast - saxs ( 23 ) rely on configurational averaging over molecular dynamics simulations to treat the excess in electron density of the hydration layer relative to bulk solvent . the widely used program crysol ( 19 ) makes the assumption that the hydration shell surrounding the solute is a constant density layer of given thickness , while foxs ( 20 ) attributes an additional term to every effective - atomic - scattering - form - factor proportional to their solvent accessible area to account for the hydration layer 's contribution . aquasaxs aims at providing a new way to treat the solvation layer accurately without the need for extensive molecular dynamics simulations . to achieve this goal , aquasaxs takes advantage of recently developed methods that compute the solvent - distribution around a given solute on a 3d grid such as the poisson boltzmann langevin ( pbl ) formalism ( 26 ) ; pbl is implemented in aquasol that is freely available online as a web server ( 27 ) or as a software available upon request from the authors ( 28 ) . here , aquasaxs can also take advantage of other methods that compute the solvent - distribution surrounding a macromolecule . for example , outputs from 3d - rism ( three dimensional - reference interaction site model ) calculations can readily be used by aquasaxs . the 3d - rism theory has been implemented in the latest ambertools package ( 29 ) . in addition , aquasaxs provides a way for the user to check and/or tune the atomic types and the corresponding parameters that are used for computing the solute and the solvent - excluded - volume contribution to the saxs profile . in the following sections , we describe the method and interface of aquasaxs . in the infinite dilution limit , assuming that the macromolecule has one preferential conformation , the scattering intensity of a sample solution containing the macromolecule is proportional to the scattering intensity of a single macromolecule of scattering density ( r ) , surrounded by a solvent of average electron density . the scattering intensity of this macromolecule at a given value of the wavevector norm q is the spherical average of the scattering intensity on the sphere of radius q in reciprocal space ( equation 1 ) . ( 1 ) where a is the excess form factor of the system and are respectively the form factor of the solute in vacuo and of the solvent - excluded - volume . we perform the spherical averaging using the cubature formulae ( 30 ) . following the effective - atomic - scattering - form - factor method ( 25 ) , the solute and the solvent - excluded - volume 's form factors are computed as a sum over all n non - hydrogen atoms of the solute ( equations 2 and 3 ) . ( 2 ) ( 3 ) where fj is the atomic form factor in vacuo ( 19 ) , computed as a sum of a constant and four gaussians whose parameters depend on the atom type , is the solvent volume displaced by atom j. is an overall expansion factor , as defined in ( 19 ) , with c1 being the ratio of the adjusted and computed average atomic radii ( default value = 1.0 ) . programs such as aquasol ( 28 ) [ or 3d - rism / amber ( 29 ) ] compute solvent density maps around the solute , based on the physical interactions within the system . boltzmann formalism , where the solvent is no longer described as a continuum dielectric medium but rather as an assembly of self - orienting dipoles of variable density on a grid . it was shown that the resulting water distribution is in good agreement with experimental data and with the chemical nature of the atoms exposed to the solvent , both at the atomic and residue - level ( 26 ) . these maps are typically cubic grids of given size and resolution ( a ) , where each grid point r is associated to a given density value . basically , in such maps , one expects a density of 0 inside the solute , and 1 ( in units of bulk density ) in the bulk region of the solvent , i.e. far from the solute . at the boundary between the solute and bulk region , ( 4 ) the sum runs over all points with nonzero density . in practice , to reduce computation time , grid points with a density close to 1 ( i.e. typically within 1.10 ) are removed from the sum . on urate oxidase ( example mentioned below ) , allowing a tolerance of 1.10 slowed down the computation by a factor of three and did not affect the resulting profile : the same fitting parameters were found , and the goodness - of - fit ( cf equation 6 ) was similar ( 1.688 versus 1.691 ) . besides the solute and solvent , another possible contributor to the saxs profile is the ion atmosphere surrounding the solute . aquasol ( 28 ) computes the density maps of free cations and anions , and , in principle , these maps could be used to compute the excess form factors of ions . however , at physiological concentrations ( 200 mm nacl ) the ratio of the fugacities of ions and water is < 0.5% . at this stage , the contribution of ions was not implemented into aquasaxs , except in the form of explicitly bound and fixed ions . nevertheless , the presence of free ions can indirectly affect the solvent density in the hydration shell ( screening effect ) , so the user is prompted for the ionic strength of the solution . alternatively , the hydration shell 's form factor can be computed as in foxs ( 20 ) , following equation 5 . ( 5 ) where is the fraction of solvent accessible surface of the atom j ( 31 ) and is the water form factor . is a scale factor used to adjust the hydration shell 's contribution ( default value = 1.0 ) . the computed profile is fitted to a given experimental saxs profile ( with experimental error ) by minimizing the goodness - of - fit function with respect to three adjustable parameters : c1 , c2 and c ( equation 6 ) . ( 6 ) c1 and c2 values are scanned within a given range ( , and ) , in steps of 0.0055 , 0.014 and 0.04 , resp . , and for each pair , a linear - least - squares minimization is performed to adjust the scaling constant c. the pair leading to the minimal is kept to compute the returned profile . aquasaxs was successfully tested with all pdb ( 32 ) structures that have an experimental saxs profile in the open access bioisis database ( 33 ) , and gave results similar to crysol and foxs . the calculation scales linearly with the number of points at which the profile is sampled ( ) , as well as with the number of non - hydrogen atoms of the solute . it scales to the cube of the number of points per grid edge , although this expensive cost is attenuated by a preliminary compressing process discarding all points with an excess density close to zero . for the solvent - map solvation option , for maps with 65 grid points per edge ( about 2 grid size ) , the calculation typically takes from less than a minute to a few minutes for systems of a few thousands atoms to dozens of thousands of atoms ( with ) , which is a few times slower than crysol and foxs . for a waxs spectrum , . for the surface - accessible solvation option figure 1 shows the flowchart of a typical aquasaxs calculation . starting with a pdb file or a pqr file , the user is expected to make two decisions , namely the selection of the solvation method , and whether a comparison is made with an experimental saxs profile or not . a pdb or pqr file is mandatory for the calculation , and depending on the chosen method for solvation , a solvent map in cns format must be fed into the server , or the pdb file must be in pqr format . if one wants to fit a theoretical profile to an experimental one , the latter must be provided as a formatted separate file . after the calculation , the web browser is redirected to the result 's page , where the profiles and relative residuals are displayed , as well as a summary of the run . a pdb or pqr file is mandatory for the calculation , and depending on the chosen method for solvation , a solvent map in cns format must be fed into the server , or the pdb file must be in pqr format . if one wants to fit a theoretical profile to an experimental one , the latter must be provided as a formatted separate file . after the calculation , the web browser is redirected to the result 's page , where the profiles and relative residuals are displayed , as well as a summary of the run . there are three options for the solvation method : either a solvation map is provided as input , or a solvation map is computed using aquasol ( 28 ) , or a hydration layer is defined using the accessible surface of each atom , following the method introduced in foxs ( 20 ) . the solvent - map solvation option will account for the hydration layer following equation 4 . if a solvent map ( in cns format ) is provided by the user in input , it will be taken into account . if no solvent map is provided , aquasol ( 28 ) will be run in default mode prior to aquasaxs ; in that case , the pdb input file must be in pqr format ( 34 ) ( partial charges and atomic radius must be known for all atoms ) . the surface - accessible solvation option uses a form factor defined as in equation 5 . optionally , an ascii text file containing the experimental saxs profile can be given as input to aquasaxs . lines containing data must consist of at least two columns and an optional , but recommended , third one . the first column contains the values of the wavevector q. by default , aquasaxs defines q as in , where is the scattering angle and is the wavelength of the incident x - ray beam . if not provided , the experimental error will be assumed to be equal to the difference between the intensity in qn , and the average intensity at qn1 and qn+1 . if the corresponding atom is a hydrogen or belongs to a water molecule , it is discarded . otherwise , the residue and atom 's names are parsed among the standard pdb protein , nucleic acids and ligands library ( 32 ) . thirteen atom types are currently recognized : carbons with zero , one , two or three bound hydrogens , nitrogen with zero , one , two or three bound hydrogens , oxygen with zero or one bound hydrogen , sulfur with zero or one bound hydrogen , and phosphorus . once the atomic type of atom j has been recognized , its position rj is stored and it is assigned the corresponding form factor fj , excluded volume and radius . on the flowchart web - page , the user is given the possibility to check whether the atomic types of the residues in the provided pdb / pqr file can be recognized . if the user wants to define other atomic types than those listed above , two optional files can be given as input to the program : one listing the atom types of a given residue , the other listing the atomic parameters of new atom types . several other options / parameters can be set : the maximum q - value considered , the sampling resolution of the profile , the bulk average electron density ( in e. ) , the subset chains in the structure to be considered , as well as the values of c1 and c2 in the nonfitting mode . the computation is performed in real time and the browser is redirected to the result 's page when the calculation has finished . if an email address is provided , an email will be sent to the user . depending on the system 's size and server 's queue load , the typical running time ranges from less than a minute to a few minutes . the result 's page displays a plot of the computed profile ( see figure 2 ) , superimposed to the experimental profile , if provided , as well as the run logfile . links to three output files are displayed , to retrieve the logfile , the profile file , and a pdb file listing all the atoms that have been considered in the calculation . possibly , links toward the output files of aquasol ( 28 ) are displayed too ( computed solvent map and logfile ) . 65 points per edge , equally spaced by 2.2 define the cubic grid ( using a higher resolution map did not significantly improve the fit ) . the solute was immersed in an ion atmosphere of 0.1 m nacl , and the solute region was defined by its solvent - accessible surface ( with a probe radius of 1.4 ) . one of the profile displayed here ( in blue ) was output by aquasaxs after fitting , along with the fitting parameters : c1 = 1.021 and c2 = 1.022 . the goodness of fit is : = 1.69 . the computation took less than 5 minutes . the profiles fitted using foxs ( green ) and crysol ( orange ) are shown for comparison . their respective values for the goodness - of - fit is 2.46 and 1.53 . foxs used c1 = 1.09 and c2 = 2.9 as fitting parameters , while crysol used = 0.025 , ra = 1.560 and vol = 179 493 ( which corresponds to a volume 20% more important than the volume actually deduced from the average radius ra ) . additional parameters for crysol were the use of up to the 30th order of spherical harmonics , and 18th order for the fibonacci grid . in every case , the bulk density was set at 0.334 e..the figure in inset displays the goodness - of - fit computed by aquasaxs for the range of c1 and c2 scanned by the program . only values of between the minimum and 6 are shown , for clarity . 65 points per edge , equally spaced by 2.2 define the cubic grid ( using a higher resolution map did not significantly improve the fit ) . the solute was immersed in an ion atmosphere of 0.1 m nacl , and the solute region was defined by its solvent - accessible surface ( with a probe radius of 1.4 ) . one of the profile displayed here ( in blue ) was output by aquasaxs after fitting , along with the fitting parameters : c1 = 1.021 and c2 = 1.022 . the profiles fitted using foxs ( green ) and crysol ( orange ) are shown for comparison . their respective values for the goodness - of - fit is 2.46 and 1.53 . foxs used c1 = 1.09 and c2 = 2.9 as fitting parameters , while crysol used = 0.025 , ra = 1.560 and vol = 179 493 ( which corresponds to a volume 20% more important than the volume actually deduced from the average radius ra ) . additional parameters for crysol were the use of up to the 30th order of spherical harmonics , and 18th order for the fibonacci grid . in every case , the bulk density was set at 0.334 e..the figure in inset displays the goodness - of - fit computed by aquasaxs for the range of c1 and c2 scanned by the program . in addition to single conformation fitting of experimental saxs profiles , aquasaxs provides two methods to deal with multiple structure files . the first one , called sequential fit , provides a way to compute the saxs profile of a set of pdb / pqr files in a single run . that way , if an experimental profile is provided , the user can readily compare the relevance of provided models , without having to rerun the server several times . the second method , called ensemble fit , aims at plugging in all possible models and refining their population , and is directly inspired from ( 35 ) . another interesting way to take profit of this approach would be the following : starting from a given pdb structure , several models are built along a given deformation parameter ( e.g. a normal mode ) ; the best value for this deformation parameter would likely be detected by the refinement process . in practice , this method couples a mean - field optimization of the model 's populations with a simulated annealing protocol . let us consider an ensemble of m models , each model m being associated the scattered intensity . noting pm the associated probability ( or population ) of model m , the total scattered intensity ( in the limit of infinite dilution ) is then : ( 7 ) if one defines a free energy f of the form : ( 8) where is defined as in equation 6 , s is the entropy , and t is the temperature , then minimizing the free energy with respect to pm gives : ( 9 ) where is the inverse of , and is a normalization constant given by . the refinement starts with uniform values of the probabilities , which are updated at each cycle of the refinement until a self - consistent solution is obtained ; at each cycle the derivative are evaluated at the current solution , i.e. the current set of pm values . the temperature governs the contrast between the different populations , the contrast being higher as the temperature is lower . after convergence at a given temperature , the set of populations found is used as initial guess for a new mean - field refinement at lower temperature , until the final temperature is reached . results of both methods applied on synthetic data are presented in supplementary materials . we have described a program that allows structural biologists to compare their saxs data to the theoretical one for a model given as a pdb or pqr file . its major novelty resides in the possibility to better model the hydration layer through a physically sound representation of the solvent density map , combined with the use of the cubature method for spherical averaging . the user - friendly interface allows to modify ( or add new entries to ) the list of scatterers and their parameters . the possibility to fit the data with multiple models , either independently , or through population refinement has also been implemented . future developments will allow for the possibility to refine the coordinates of the model against the experimental data . in that case , care must be taken to use as few degrees of freedom as possible . one possibility is to restrict the deformation of the model along a small number of finally , the possibility to compute the theoretical anomalous saxs profile of a solute containing atoms with anomalous contribution ( e.g. bromide or cesium ions ) will be made available soon .

small angle x - ray scattering ( saxs ) techniques are becoming more and more useful for structural biologists and biochemists , thanks to better access to dedicated synchrotron beamlines , better detectors and the relative easiness of sample preparation . the ability to compute the theoretical saxs profile of a given structural model , and to compare this profile with the measured scattering intensity , yields crucial structural informations about the macromolecule under study and/or its complexes in solution . an important contribution to the profile , besides the macromolecule itself and its solvent - excluded volume , is the excess density due to the hydration layer . aquasaxs takes advantage of recently developed methods , such as aquasol , that give the equilibrium solvent density map around macromolecules , to compute an accurate saxs / waxs profile of a given structure and to compare it to the experimental one . here , we describe the interface architecture and capabilities of the aquasaxs web server ( http://lorentz.dynstr.pasteur.fr/aquasaxs.php ) .

INTRODUCTION AQUASAXS METHOD PERFORMANCE OF AQUASAXS AQUASAXS WEB SERVER ADDITIONAL FEATURES CONCLUSION SUPPLEMENTARY DATA FUNDING

PMC4495497

over the previous two decades , nephron - sparing surgery ( nss ) has become the preferred surgical treatment for t1a and t1b renal masses . the minimally invasive approach of laparoscopic partial nephrectomy ( lpn ) combines the benefits of laparoscopic surgery with the benefits of open nss , resulting in decreased post - operative pain and length of stay in lpn with equivalent oncologic outcomes and post - operative renal function . however , lpn is a technically challenging procedure with a steep learning curve and a higher post - operative complication rate . laparoscopic robot - assisted partial nephrectomy ( lrpn ) is less challenging than lpn because the robot facilitates precise tumor excision and renal reconstruction with increased degrees of freedom and tremor elimination . thus , lrpn is increasingly being utilized by surgeons for minimally invasive nss ( nationwide inpatient sample , unpublished data 2007 - 2012 ) . the goal of our project was to assess the learning curve for lrpn by examining the outcomes of warm ischemia time ( wit ) , estimated blood loss , complication rates and operating time . upon defining the learning curve using wit , we investigated the impact of tumor complexity on the learning curve by matching for renal nephrometry scores before and after the learning curve . furthermore , we analyzed the learning curve with the perspective of our contemporary urologic goals concerning maximum wits . between october 2007 and april 2014 , a single surgeon experienced in lpn completed 171 consecutive lrpns at an academic medical center for t1a and t1b localized renal masses . the surgeon was fellowship trained in endourology with 10 years of post - fellowship clinical experience . characteristics and perioperative outcomes for these patients were recorded in a prospective database , approved for use by the institutional review board . initially , reconstruction of the tumor resection site was performed with a running base suture layer followed by a second cortical layer of interrupted sutures . starting in february 2010 , running sutures were used for both the base and the cortical layer . in 14 of 44 ( 31.8% ) procedures completed between july 2013 and april 2014 , this goal was revised to 20 min within 10 months following the june 2010 publication of results associating damage to renal function with ischemia times over 20 min . complications were reported using the clavien dindo classification system with a cut - off of 30 days post - operation for noting post - operative complications . additionally , we noted the procedures with intra- or post - operative incidences of urine leaks and bleeding . notably , there were no fatal complications or urine leaks ( confirmed post - operatively by examining drain creatinine levels ) . change in renal function was determined using the percent difference in pre- and post - operative glomerular filtration rate ( gfr ) . gfr was calculated using the modification of diet in renal disease 2 ( mdrd ) equation . pre - operative gfr was calculated using the most recent creatinine value within 4 weeks preceding the surgery , while the post - operative ( nadir ) gfr was calculated using the highest creatinine value over the course of the patient 's hospital stay . the post - operative follow - up gfr was calculated using the most recent creatinine value between 3 weeks and 18 months following the operation . to define the learning curve , the surgical outcomes of estimated blood loss , operative time , wit and complications were evaluated . wit was examined on a month - wise basis ; surgeries with no warm ischemia were not included in this analysis . the learning curve was defined to be overcome at the first month of surgeries to consistently have a wit of 20 min or less ( our current goal ) . following determination of the learning curve , surgeries were matched by renal nephrometry score to account for tumor complexity between cases . after blinding for wit , surgeries were stratified first by nephrometry score and then by operation date . the cases were matched 1:1 before and after the learning curve ; surgeries that could not be matched were removed . statistical analyses were conducted with spss version 20 ( ibm , armonk , ny , usa ) and stata 13.1 ( stata corp . the mean values of characteristics and outcomes were compared using student 's t - test . in comparing complications between two groups ( first 30 to last 30 ; pre- and post - learning curve ) , a chi - squared analysis was used . to determine predictors of wit , a univariate linear regression was used to compare continuous and categorical variables with wit . a multivariable linear regression was then used to analyze variables found to be independent predictors of wit on univariate analysis ; all variables with a p value of 0.05 or less were included in the multivariable model . between october 2007 and april 2014 , a single surgeon experienced in lpn completed 171 consecutive lrpns at an academic medical center for t1a and t1b localized renal masses . the surgeon was fellowship trained in endourology with 10 years of post - fellowship clinical experience . characteristics and perioperative outcomes for these patients were recorded in a prospective database , approved for use by the institutional review board . initially , reconstruction of the tumor resection site was performed with a running base suture layer followed by a second cortical layer of interrupted sutures . starting in february 2010 , running sutures were used for both the base and the cortical layer . in 14 of 44 ( 31.8% ) procedures completed between july 2013 and april 2014 , this goal was revised to 20 min within 10 months following the june 2010 publication of results associating damage to renal function with ischemia times over 20 min . complications were reported using the clavien dindo classification system with a cut - off of 30 days post - operation for noting post - operative complications . additionally , we noted the procedures with intra- or post - operative incidences of urine leaks and bleeding . notably , there were no fatal complications or urine leaks ( confirmed post - operatively by examining drain creatinine levels ) . change in renal function was determined using the percent difference in pre- and post - operative glomerular filtration rate ( gfr ) . gfr was calculated using the modification of diet in renal disease 2 ( mdrd ) equation . pre - operative gfr was calculated using the most recent creatinine value within 4 weeks preceding the surgery , while the post - operative ( nadir ) gfr was calculated using the highest creatinine value over the course of the patient 's hospital stay . the post - operative follow - up gfr was calculated using the most recent creatinine value between 3 weeks and 18 months following the operation . to define the learning curve , the surgical outcomes of estimated blood loss , operative time , wit and complications were evaluated . wit was examined on a month - wise basis ; surgeries with no warm ischemia were not included in this analysis . the learning curve was defined to be overcome at the first month of surgeries to consistently have a wit of 20 min or less ( our current goal ) . following determination of the learning curve , surgeries were matched by renal nephrometry score to account for tumor complexity between cases . after blinding for wit , surgeries were stratified first by nephrometry score and then by operation date . the cases were matched 1:1 before and after the learning curve ; surgeries that could not be matched were removed . statistical analyses were conducted with spss version 20 ( ibm , armonk , ny , usa ) and stata 13.1 ( stata corp . the mean values of characteristics and outcomes were compared using student 's t - test . in comparing complications between two groups ( first 30 to last 30 ; pre- and post - learning curve ) , , a univariate linear regression was used to compare continuous and categorical variables with wit . a multivariable linear regression was then used to analyze variables found to be independent predictors of wit on univariate analysis ; all variables with a p value of 0.05 or less were included in the multivariable model . the characteristics and outcomes of the lrpn patients were examined [ table 1 ] . comparing the first 30 with the 30 most recent operations , there was a marked increase in mean tumor diameter ( 2.43.4 cm , p = 0.02 ) and mean tumor complexity ( nephrometry score : 5.97.0 , p = 0.02 ) . despite this increase in complexity , a significant decrease in wit ( median : 23.015.2 min , p < 0.01 ) was observed . all of the remaining characteristics and outcomes remained unchanged or statistically insignificant between the first 30 and the 30 most recent lrpns , including mean operating room time ( 206.8202.9 min , p = 78 ) , mean estimated blood loss ( ebl ) ( 172.7171.2 ml , p > 0.9 ) , post - operative bleeding ( 02 , p = 0.16 ) , clavien grade 3 complications ( 02 , p = 0.16 ) , clavien grade 4 complications ( 10 , p = 0.32 ) , mean body mass index ( bmi ) ( 31.731.4 , p = 0.87 ) and mean age ( 56.259.1 , p = 0.38 ) . there were no complications with a clavien grade of 5 in this study [ table 1 ] . characteristics and outcomes of the total and the first 30 and the last 30 patients in this study next , surgical outcomes as a function of operative date were compared . wit was determined to be the most informative dependent variable ; there were no significant changes in other outcomes , including complication rate , blood loss and total operation time . thus , wit was used in defining the learning curve ; using the contemporary goal of maintaining wit at 20 min or less , the learning curve was determined to be overcome after 58 lrpns [ figure 1 ] . two patients were converted to radical nephrectomy in the first 58 cases due to the central or hilar position of the tumor . one patient was converted to open surgery in the first five cases and one case to pure laparoscopic partial in the first 40 cases . scatter plot of warm ischemia time ( wit ) versus operative date for the laparoscopic robot - assisted partial nephrectomies . the graph is a line indicating a wit of 20 min matching based on nephrometry score before and after the learning curve created two groups of 52 patients . most patient characteristics and outcomes remained the same pre and post - learning curve [ table 2 ] . notably , nephrometry score remained the same as it was the matching parameter while wit exhibited a significant decrease . figure 2 shows the comparison between wit and operative date updated for matched nephrometry scores between the pre- and post - learning curve groups . comparison of mean outcomes and characteristics between lrpn patients before and after the learning curve using matched data scatter plot of warm ischemia time versus operative date for the laparoscopic robot - assisted partial nephrectomies matched by nephrometry score before and after the learning curve . surgeries performed after the learning curve are indicated in the shaded box above upon examining the characteristics and outcomes that were continuous variables , a univariate analysis revealed that nephrometry score ( p < 0.001 ) , tumor diameter ( p comparing categorical variables , only the year of surgery was found to be a predictor of wit ( p < 0.001 ) . finally , a multivariable linear regression confirmed that nephrometry score ( = 1.2 min , p < 0.001 ) , tumor diameter ( = 1.8 min , p < 0.001 ) , cortical renorrhaphy ( = 5.3 min , p = 0.004 ) and year of surgery ( = -1.9 min , p < 0.001 ) were significant predictors of wit . univariable and multivariable linear regression analysis for predictors of warm ischemia time comparing wit with operative date , the surgeries were stratified by the surgeon 's goal concerning maximum wit during the time of operation [ figure 3 ] . between 2007 and april 2011 , the mean wit was 25.4 min , while the mean wit between april 2011 and june 2013 was 19.1 min . for the lrpns performed between july 2013 and april 2014 , cortical renorrhaphy was omitted in 14 ( 31.8% ) patients ; the mean wit during this time was 15.7 min . a single - factor anova found the differences in wits between these groups to be statistically significant ( p < 0.01 ) . scatter plot of warm ischemia time ( wit ) versus operative date for the laparoscopic robot - assisted partial nephrectomies . surgeries are stratified by goal for maximum wit ( 30 min , 20 min , 20 min with omission of cortical renorrhaphy ) the characteristics and outcomes of the lrpn patients were examined [ table 1 ] . comparing the first 30 with the 30 most recent operations , there was a marked increase in mean tumor diameter ( 2.43.4 cm , p = 0.02 ) and mean tumor complexity ( nephrometry score : 5.97.0 , p = 0.02 ) . despite this increase in complexity , a significant decrease in wit ( median : 23.015.2 min , p < 0.01 ) was observed . all of the remaining characteristics and outcomes remained unchanged or statistically insignificant between the first 30 and the 30 most recent lrpns , including mean operating room time ( 206.8202.9 min , p = 78 ) , mean estimated blood loss ( ebl ) ( 172.7171.2 ml , p > 0.9 ) , post - operative bleeding ( 02 , p = 0.16 ) , clavien grade 3 complications ( 02 , p = 0.16 ) , clavien grade 4 complications ( 10 , p = 0.32 ) , mean body mass index ( bmi ) ( 31.731.4 , p = 0.87 ) and mean age ( 56.259.1 , p = 0.38 ) . there were no complications with a clavien grade of 5 in this study [ table 1 ] . characteristics and outcomes of the total and the first 30 and the last 30 patients in this study next , surgical outcomes as a function of operative date were compared . wit was determined to be the most informative dependent variable ; there were no significant changes in other outcomes , including complication rate , blood loss and total operation time . thus , wit was used in defining the learning curve ; using the contemporary goal of maintaining wit at 20 min or less , the learning curve was determined to be overcome after 58 lrpns [ figure 1 ] . two patients were converted to radical nephrectomy in the first 58 cases due to the central or hilar position of the tumor . one patient was converted to open surgery in the first five cases and one case to pure laparoscopic partial in the first 40 cases . scatter plot of warm ischemia time ( wit ) versus operative date for the laparoscopic robot - assisted partial nephrectomies . matching based on nephrometry score before and after the learning curve created two groups of 52 patients . most patient characteristics and outcomes remained the same pre and post - learning curve [ table 2 ] . notably , nephrometry score remained the same as it was the matching parameter while wit exhibited a significant decrease . figure 2 shows the comparison between wit and operative date updated for matched nephrometry scores between the pre- and post - learning curve groups . comparison of mean outcomes and characteristics between lrpn patients before and after the learning curve using matched data scatter plot of warm ischemia time versus operative date for the laparoscopic robot - assisted partial nephrectomies matched by nephrometry score before and after the learning curve . upon examining the characteristics and outcomes that were continuous variables , a univariate analysis revealed that nephrometry score ( p < 0.001 ) , tumor diameter ( p < 0.001 ) and renorrhaphy status ( p < 0.001 ) were predictors of wit [ table 3 ] . comparing categorical variables , only the year of surgery finally , a multivariable linear regression confirmed that nephrometry score ( = 1.2 min , p < 0.001 ) , tumor diameter ( = 1.8 min , p < 0.001 ) , cortical renorrhaphy ( = 5.3 min , p = 0.004 ) and year of surgery ( = -1.9 min , p < 0.001 ) were significant predictors of wit . comparing wit with operative date , the surgeries were stratified by the surgeon 's goal concerning maximum wit during the time of operation [ figure 3 ] . between 2007 and april 2011 , the mean wit was 25.4 min , while the mean wit between april 2011 and june 2013 was 19.1 min . for the lrpns performed between july 2013 and april 2014 , cortical renorrhaphy was omitted in 14 ( 31.8% ) patients ; the mean wit during this time was 15.7 min . a single - factor anova found the differences in wits between these groups to be statistically significant ( p < 0.01 ) . scatter plot of warm ischemia time ( wit ) versus operative date for the laparoscopic robot - assisted partial nephrectomies . surgeries are stratified by goal for maximum wit ( 30 min , 20 min , 20 min with omission of cortical renorrhaphy ) in our analysis of a single surgeon 's lrpns , we found that wit was the only outcome that consistently changed between the first 30 and the last 30 patients . there were no urine leaks or complications with a clavien score of 5 , while differences in the number of clavien grade 3 and 4 complications and post - operative bleeds remained statistically insignificant . thus , wit was examined for a learning curve and using the most recent goal of 20 min was found to be 58 cases . all conversions to radical ( 2 ) , open ( 1 ) or pure laparoscopic partial ( 1 ) were seen in the first 58 cases . finally , we matched between pre- and post - learning curve lrpns based on renal nephrometry score . even after matching , we noticed a significant decrease in wit , although the other median patient characteristics and surgical outcomes were not statistically different . finally , we found four predictors of wit : nephrometry score , tumor diameter , cortical renorrhaphy and the year the lrpn was performed . although the effect of wit on post - operative renal function is controversial , a wit of up to 20 min has been our goal since 2011 because of its association with minimal damage to renal function . we defined the learning curve as the number of cases needed to consistently achieve this goal ; however , it is notable that prior to 2011 , our goal was to limit wit to 30 min . our goal was revised in june 2010 based on literature supporting a maximum wit of 30 min . the changing nature of our goals , reflected in figure 3 , made it difficult for us to examine a learning curve . between october 2007 and june 2010 , of 29 cases , only three had a wit greater than 30 min , leading us to conclude that the lrpn learning curve for an established laparoscopic surgeon is minimal . within 10 months of the june 2010 publication , we had adopted and were consistently achieving our new goal of 20 min [ figure 3 ] . the omission of cortical renorrhaphy resulted in a decrease in wit from the second half of 2013 onwards [ figure 3 ] . no urine leaks or bleeding complications were seen when the cortical renorrhaphy was omitted ( n = 14 ) . by definition , this method utilizes early unclamping , wherein the renal artery is unclamped following the completion of the base layer of sutures at the reconstruction site . previously , the adoption of early unclamping has resulted in decreased wit . however , unlike previously described methods of early unclamping , our method omits cortical renorrhaphy the second , outer layer of sutures that is otherwise completed following unclamping . we hypothesize that omitting cortical renorrhaphy will improve renal function loss and improve visualization of the base layer to improve access to arterial bleeders . in fact , the % gfr loss improved ( -8.1% vs. -2.7% ) during the final 30 cases where cortical renorrhaphy was selectively omitted , although this did not reach statistical significance . interestingly , when looking at the matched cohort [ table 2 ] , despite a significant decline in ischemia time there was no improvement in renal function supporting the principle that resection and reconstruction are important drivers of renal function after partial nephrectomy . a recent matched study showed both improved volume loss ( % volume loss : 9 cm vs. 17 cm , p = 0.003 ) and renal function decline ( risk of 10% gfr loss : 13% vs 47% , p = 0.03 ) in a group where cortical renorrhaphy was omitted compared with a group with cortical renorrhaphy during partial nephrectomy . recent studies have also found minimal learning curves for experienced laparoscopic surgeons adopting lrpn . however , there are some notable differences between our study and the others . first , in comparison with our 171 patients , the four other studies examined between 38 and 50 patients in determining their respective learning curves . benway et al . found learning curves for the cumulative number of cases needed to reach the absolute minimum operating time ( 19 cases ) and wit ( 26 cases ) . haseebuddin et al . defined the curve as the number of cases needed to reach the point of minimal variation in operating room time ( 16 cases ) and in wit ( 26 cases ) . ellison et al . calculated their curve as the number of cases needed to reach a plateau in wit and ebl ( 33 lrpns for both ) , but found no significant decrease in operating room times as the cumulative lrpn number increased . determined the curve as the number of lrpns needed to reach the average operating time and average wit of the 18 most recent lpns ( five lrpns for both variables ) . upon examining our complication rate , we found that we experienced a seamless transition in adopting robotic - assisted partial nephrectomy . although gill and colleagues observed a significant complication rate during the adoption of pure lpn , our complication rate was very minimal . however , comparisons to this study are limited as we did not include pure laparoscopic cases in the current study . our results compare favorably with those of recent studies that also examined the complication rate for robot - assisted partial nephrectomy . we attempted to minimize selection bias by including all patients who underwent lrpn , although we excluded procedures with no wit for the purpose of examining the learning curve . second , not all of the procedures were conducted with the same method for reconstruction . additionally , we used only one outcome measure , wit , for determining the learning curve . finally , our analysis included the lrpns of only a single surgeon who was experienced in laparoscopic surgery . because wit is an easy variable to track , setting an ideal limit for wit provided the surgeon a clear goal to strive toward and based on this association , we hypothesize that creating useful metrics for surgeon feedback can result in changes of surgeon behavior and improved outcomes . we believe that our experience supports the use of evidence - based medicine in urologic practice . for a surgeon who is experienced in laparoscopic partial nephrectomies , adoption of robotic assistance can be performed with a very low complication rate . the ischemia time decreased while no significant changes in blood loss , operating time or complications were seen . the decrease in ischemia time was not felt to be related to a learning curve , but rather to the adoption of stricter goals in 2011 , early vascular unclamping and the omission of cortical renorrhaphy from 2013 onwards . nephrometry score , tumor diameter , cortical renorrhaphy and year of surgery were found to be predictors of wit on adjusted analysis .

introduction : the learning curve for robotic partial nephrectomy was investigated for an experienced laparoscopic surgeon and factors associated with warm ischemia time ( wit ) were assessed.materials and methods : between 2007 and 2014 , one surgeon completed 171 procedures . operative time , blood loss , complications and ischemia time were examined to determine the learning curve . the learning curve was defined as the number of procedures needed to reach the targeted goal for wit , which most recently was 20 min . statistical analyses including multivariable regression analysis and matching were performed.results:comparing the first 30 to the last 30 patients , mean ischemia time ( 23.015.2 min , p < 0.01 ) decreased while tumor size ( 2.43.4 cm , p = 0.02 ) and nephrometry score ( 5.97.0 , p = 0.02 ) increased . body mass index ( p = 0.87 ) , age ( p = 0.38 ) , complication rate ( p = 0.16 ) , operating time ( p = 0.78 ) and estimated blood loss ( p = 0.98 ) did not change . decreases in ischemia time corresponded with revised goals in 2011 and early vascular unclamping with the omission of cortical renorrhaphy in selected patients . a multivariable analysis found nephrometry score , tumor diameter , cortical renorrhaphy and year of surgery to be significant predictors of wit.conclusions:adoption of robotic assistance for a surgeon experienced with laparoscopic surgery was associated with low complication rates even during the initial cases of robot - assisted partial nephrectomy . ischemia time decreased while no significant changes in blood loss , operating time or complications were seen . the largest decrease in ischemia time was associated with adopting evidence - based goals and new techniques , and was not felt to be related to a learning curve .

INTRODUCTION MATERIALS AND METHODS Patient selection and surgical technique Complications and renal function Determination of learning curves and matching Statistical analyses RESULTS Patient characteristics and surgical outcomes Determining the learning curve Investigation and matching for tumor complexity of the patients Examining Predictors of WIT Comparison of ischemia time with contemporary goals DISCUSSION CONCLUSIONS

PMC4799570

a considerable proportion of patients with depression previously experienced a certain extent of stressful life events ( sles ) before the onset of depression . previous studies reported that sles can cause a decrease in gray matter volume in some key brain regions , such as the anterior cingulate , hippocampus , and parahippocampal gyrus . as is widely known , alterations in gray matter volume are commonly found in depressed patients . for example , kim et al . found that the gray matter volume was decreased in the bilateral caudate nucleus and the thalamus in patients with major depression . another previous study found that to some extent , the pattern of structural abnormalities observed in the brains of depressed patients is similar to the pattern of abnormalities in patients with posttraumatic stress disorder , a disorder caused by extreme stress . collectively , the findings of these studies led to the hypothesis that alteration in gray matter caused by stress - related factors may be the neurobiological basis of subsequent depression . for example , some epidemiological studies reviewed by paykel found that not all depression patients experience obvious sles before the onset of depressive symptoms . these findings suggest that structural alterations in the brains of patients with major depression are intrinsic and independent of whether a patient had experienced an sle . unfortunately , to the best of our knowledge , no studies have explored whether brain structural differences exist between depression patients with and without prior sles . thus , the relationships among stress factors , brain structure , and depressive episodes remain unclear . based on the results of previous epidemiological studies , we postulated that the experience of sles before the onset of depression likely causes brain structural alterations , and these alterations are associated with the subsequent onset of depression . in addition , such alterations are probably different from brain structural alterations found in depression patients without sle experiences . the aim of this pilot study was to compare the gray matter volume between two groups of drug - naive female patients who had or had not experienced sles prior to the onset of major depression by high - field magnetic resonance imaging ( mri ) and to provide a foundation for large sample follow - up studies to investigate the associations among stress factors , brain structural alterations , and the onset of depression . this study was approved by the ethics committee of affiliated hospital of jining medical university in shandong province , china . written informed consent was obtained from all participants and their legal guardians when applicable . both inpatients and outpatients were treated in the department of psychiatry of our hospital between february 2011 and february 2013 . due to a previous finding that male depression patients are more likely to be alcohol or nicotine abusers , both of which could confound the mri results , only female patients were enrolled in this study . all participants were diagnosed by two senior psychiatrists according to the first depressive episode criteria of icd-10 . none of the participants had ever been treated with anti - depressants , mood stabilizers , or antipsychotics . patients were excluded from the study if they met any of the following criteria : ( 1 ) history of disturbance of consciousness for more than 5 min , ( 2 ) diagnosis of neurological disease , ( 3 ) depression accompanied by serious psychotic symptoms or substance abuse , ( 4 ) presence of physical disease not suitable for an mri scanning , ( 5 ) pregnant or breastfeeding , ( 6 ) affected by claustrophobia or other disorders that could affect mri scanning , and ( 7 ) history of alcohol consumption or nicotine use . the 24-item hamilton depression scale was used to evaluate the severity of the depressive symptoms . the life event scale was used to evaluate life events that occurred within 6 months as well as the severity of stress experienced within 6 months before the emergence of the first episode of major depression . mri data were collected using siemens 3.0 t magetom trio , a tim system ( siemens , germany ) . the scanning parameters were as follows : ( 1 ) for t1-weighted images ( t1-wi ) sequences : repetition time ( tr ) = 350 ms , echo time ( te ) = 2.5 ms , slice thickness = 5.5 mm , gap = 1.1 m , matrix = 320 320 , and field of view ( fov ) = 230 mm 230 mm . ( 2 ) for t2-wi sequences : tr = 6000 ms , te = 93 ms , slice thickness = 5.5 mm , gap = 1.1 m , matrix = 320 320 , and fov = 230 mm 230 mm . a 3dt1 sequence was obtained if no organic disease was found in the regular scans and tr / te = 1900 ms/9.5 ms , fov = 250 mm 250 mm , matrix = 128 128 , slice thickness = 0.9 mm , and interval = 0.45 cm . spm8 ( the fil methods group , uk ) and vbm8 ( structural brain mapping group , germany ) software programs were used in both preprocessing and processing for scanned images . the raw digital imaging and communication in medicine mri data were converted to nifti format using spm8 running on matlab r2010b ( the mathworks inc . , brain volume normalizing , bias correcting , and segmentation into gray matter , white matter , and cerebrospinal fluid were performed using vbm8 toolbox . vbm8 toolbox is based on an optimized voxel - based morphometry protocol that helps increase the signal to noise ratio . the total volumes of gray matter , white matter , and cerebrospinal fluid were assessed by calculating the resulting tissue probabilities . total brain volume was defined as the sum of the gray matter and white matter volumes . the volume images were smoothed using an isotropic gaussian kernel ( full width at half maximum = 8 mm ) . continuous and categorical variables were compared between groups using independent samples t - tests and chi - square analysis . a p value of < 0.05 was considered statistically significant . this study was approved by the ethics committee of affiliated hospital of jining medical university in shandong province , china . written informed consent was obtained from all participants and their legal guardians when applicable . both inpatients and outpatients were treated in the department of psychiatry of our hospital between february 2011 and february 2013 . due to a previous finding that male depression patients are more likely to be alcohol or nicotine abusers , both of which could confound the mri results , only female patients were enrolled in this study . all participants were diagnosed by two senior psychiatrists according to the first depressive episode criteria of icd-10 . none of the participants had ever been treated with anti - depressants , mood stabilizers , or antipsychotics . patients were excluded from the study if they met any of the following criteria : ( 1 ) history of disturbance of consciousness for more than 5 min , ( 2 ) diagnosis of neurological disease , ( 3 ) depression accompanied by serious psychotic symptoms or substance abuse , ( 4 ) presence of physical disease not suitable for an mri scanning , ( 5 ) pregnant or breastfeeding , ( 6 ) affected by claustrophobia or other disorders that could affect mri scanning , and ( 7 ) history of alcohol consumption or nicotine use . the 24-item hamilton depression scale was used to evaluate the severity of the depressive symptoms . the life event scale was used to evaluate life events that occurred within 6 months as well as the severity of stress experienced within 6 months before the emergence of the first episode of major depression . mri data were collected using siemens 3.0 t magetom trio , a tim system ( siemens , germany ) . the scanning parameters were as follows : ( 1 ) for t1-weighted images ( t1-wi ) sequences : repetition time ( tr ) = 350 ms , echo time ( te ) = 2.5 ms , slice thickness = 5.5 mm , gap = 1.1 m , matrix = 320 320 , and field of view ( fov ) = 230 mm 230 mm . ( 2 ) for t2-wi sequences : tr = 6000 ms , te = 93 ms , slice thickness = 5.5 mm , gap = 1.1 m , matrix = 320 320 , and fov = 230 mm 230 mm . a 3dt1 sequence was obtained if no organic disease was found in the regular scans and tr / te = 1900 ms/9.5 ms , fov = 250 mm 250 mm , matrix = 128 128 , slice thickness = 0.9 mm , and interval = 0.45 cm . spm8 ( the fil methods group , uk ) and vbm8 ( structural brain mapping group , germany ) software programs were used in both preprocessing and processing for scanned images . the raw digital imaging and communication in medicine mri data were converted to nifti format using spm8 running on matlab r2010b ( the mathworks inc . , brain volume normalizing , bias correcting , and segmentation into gray matter , white matter , and cerebrospinal fluid were performed using vbm8 toolbox . vbm8 toolbox is based on an optimized voxel - based morphometry protocol that helps increase the signal to noise ratio . the total volumes of gray matter , white matter , and cerebrospinal fluid were assessed by calculating the resulting tissue probabilities . total brain volume was defined as the sum of the gray matter and white matter volumes . the volume images were smoothed using an isotropic gaussian kernel ( full width at half maximum = 8 mm ) . continuous and categorical variables were compared between groups using independent samples t - tests and chi - square analysis . a p value of < 0.05 was considered statistically significant . in total , 43 women participated in the study ( 23 patients in the sle group and 20 patients in the non - sle group ) . no significant differences in age , education level , or the duration and severity of the depressive symptoms were observed between the two groups . the levels of sle stimulation were significantly higher in sle group than in non - sle group [ table 1 ] . all patients who reported sles noted that they experienced negative events , such as divorce or unemployment . life events scale comparison between sle and non - sle groups data are shown as mean standard deviation . compared with that in non - sle group , the gray matter volume in sle group was less in the bilateral temporal lobe , right occipital lobe , and right limbic lobe [ table 2 and figure 1 ] . however , these differences in gray matter volume were no longer statistically significant after the application of false discovery rate ( fdr ) correction . mni coordinate of the decreased gray matter volume regions in the sle group compared to that in the non - sle group without false discovery rate correction sle : stressful life event . regions of decreased gray matter volume displayed by xjview ( without false discovery rate correction ) . blue color indicates decreased brain volume , and the number to the left of each image represents the layer location . a color scale for the t value in total , 43 women participated in the study ( 23 patients in the sle group and 20 patients in the non - sle group ) . no significant differences in age , education level , or the duration and severity of the depressive symptoms were observed between the two groups . the levels of sle stimulation were significantly higher in sle group than in non - sle group [ table 1 ] . all patients who reported sles noted that they experienced negative events , such as divorce or unemployment . life events scale comparison between sle and non - sle groups data are shown as mean standard deviation . compared with that in non - sle group , the gray matter volume in sle group was less in the bilateral temporal lobe , right occipital lobe , and right limbic lobe [ table 2 and figure 1 ] . however , these differences in gray matter volume were no longer statistically significant after the application of false discovery rate ( fdr ) correction . mni coordinate of the decreased gray matter volume regions in the sle group compared to that in the non - sle group without false discovery rate correction sle : stressful life event . regions of decreased gray matter volume displayed by xjview ( without false discovery rate correction ) . blue color indicates decreased brain volume , and the number to the left of each image represents the layer location . a color scale for the t value to our knowledge , the present study is the first mri study to compare gray matter volumes in female drug - naive depression patients who did or did not experience sles before the onset of depression . in patients with sles , the gray matter volume was less in the temporal lobe , right occipital lobe , and right limbic lobe in comparison to that in the non - sle patients . however , these differences were no longer statistically significant after fdr correction . these results indicated that stressful experiences before the onset of depression did not lead to changes in brain gray matter volume . this finding challenges our primary hypothesis that patients who experience sles before the onset of major depression likely exhibit specific alterations in brain gray matter that may be related to both previous sles and subsequent onset of depression . at first glance , previous studies have reported that there may be no changes in gray matter in the first episode depression patients . for example , guo et al . observed abnormalities only in local brain functional activities but not in brain gray matter volume in drug - naive first episode depression patients compared to healthy controls . their results suggest that there may be no gray matter changes within the early stage of the onset of depression and support the results of our present study to some extent . in addition , kim et al . reported that a change in gray matter volume is influenced by the brain - derived neurotrophic factor val66met single nucleotide polymorphism and related to the patients resilience to sles . thus , genetic factors may help explain , to some extent , the inconsistency between our results and the findings of other studies although further studies are needed to provide experimental evidence for this theory . in addition , several previous studies have suggested a direct association between stress and brain structural alterations in patients with major depression . qiao et al . reported a positive association between sensitivity to individual life events and the regional gray matter volume in the ventrolateral prefrontal cortex , and kronmller et al . found a significant negative association between life events and hippocampal volumes in patients after the first episode of depression . in addition , zhang et al . reported that gray matter volume was reduced in the left precentral gyrus and right fusiform gyrus in patients with depressive cognitive tendencies compared to that in healthy controls . of note , these previous studies drew their conclusions from comparisons of characteristics between patients and healthy controls rather than between patients with different histories of sles . also , the results of the previous studies are inconsistent and even contradictory in some cases . in the present study , we compared observations in two categories of depression patients with different histories of sles , and this difference in the patient samples may be one factor explaining the discrepancy between our results and the results of these previous studies . our study has some limitations and longitudinal clinical studies with large and genetically varied patient samples are needed to overcome these limitations and effectively elucidate the definitive relationship between sles and the subsequent onset of depression . first , this study included menopausal women who are reported to be prone to depression , which may affect the results . second , clinical studies should be designed with collection of mri and genetic data from the following four groups with sufficient numbers of cases in each : ( 1 ) individuals who experienced sles but did not develop depression , ( 2 ) patients who experienced sles and subsequently developed depression , ( 3 ) patients who experienced a first episode of major depression without prior experience of sles , and ( 4 ) healthy controls without sles . although such a study will be extremely challenging and costly , it will offer the ability to elucidate the definitive relationship between stress and the onset of depression and provide important information for the prevention of depression . in conclusion , although the results of the present study suggests that after fdr correction , there are no significant differences in brain gray matter volume between patients who have previously experienced sles and those who have not , this study still provides useful information for further exploration of the definitive role of stress in the onset of depression . this work was supported by grants from the china postdoctoral science foundation funded project ( no . 2012m520585 ) , the development of medical science and technology project of shandong province ( no . 2011hz011 ) , the natural science foundation of shandong province ( no . zr2011hm023 ) and the science and technology fund of tianjin health bureau ( no . this work was supported by grants from the china postdoctoral science foundation funded project ( no . 2012m520585 ) , the development of medical science and technology project of shandong province ( no .

background : this study aimed to observe the differences in brain gray matter volume in drug - naive female patients after the first episode of major depression with and without stressful life events ( sles ) before the onset of depression.methods:forty-three drug - naive female patients voluntarily participated in the present study after the first major depressive episode . the life event scale was used to evaluate the severity of the impact of sles during 6 months before the onset of the major depressive episode . high - field magnetic resonance imaging ( mri ) scans were obtained , and the vbm and spm8 software process were used to process and analyze the mri.results:compared to that in patients without sles , the volume of brain gray matter was lower in the bilateral temporal lobe , right occipital lobe , and right limbic lobe in the sle group . however , the gray matter volume did not differ significantly between the two groups after the application of false discovery rate ( fdr ) correction.conclusions:although the results of the present study suggest the absence of significant differences in brain gray matter volume between female drug - naive patients after the first episode of major depression with and without sles after fdr correction , the study provides useful information for exploring the definitive role of stress in the onset of depression .

I M Patients Clinical assessments Magnetic resonance imaging sequence and imaging parameters Magnetic resonance imaging data analysis Statistical analysis R Demographic characteristics Comparison of gray matter volume D Financial support and sponsorship Conflicts of interest

PMC3883326

the aim of endodontic treatment is to clean , disinfect and shape the root canals with the purpose of enabling easy and appropriate filling , thus restoring the function of the tooth . for this purpose , removing the agents that cause irritation of the pulp and periapical tissue through biomechanical preparation is needed . all these stages are particularly important , but the sum of all the stages is the key to successful endodontic treatment . the final stage of endodontic treatment consists in filling the root canal system completely and compactly with materials that have physical - chemical properties , which are capable of securing hermetic sealing , making marginal leakage difficult , preventing re - infection and creating a favorable biological environment in order to enable periapical tissue repair . in several cases , after root canal filling , the tooth is not permanently restored in the same session , jeopardizing the recently filled root canal due to exposure . the same could be said about conventional endodontic therapy , where temporary restorative materials are extensive used to perform root canal sealing between sessions . therefore , the placement of a temporary restoration with adequate properties is indispensable to prevent marginal microleakage . thus , the aim of the present study was to assess coronal marginal leakage of different types of temporary restorative materials used in root canals after endodontic treatment . the null hypothesis tested was that there would be no difference in the performance of the different materials . a total of 88 single - rooted human teeth from the tooth bank were used , after previous approval from the research ethics committee of the institution . the teeth were submitted to the pulp chamber access procedure using spherical diamond bur no . 3 ( kg soresen , barueri , sp , brazil ) coupled at high - speed ms 400 ( dabi atlante , ribeiro preto , sp , brazil ) and finishing was performed with a no . the biomechanical preparation was performed using the crown - down technique and the apical stop was made with k - type file # 40 ( dentsply - maillefer , ballaigues , switzerland ) . at each change of the instrument , the canals were irrigated with 1 ml of 2.0% sodium hypochlorite solution ( biodynmica , ibipor , pr , brazil ) using a disposable syringe and navitip needle ( ultradent , south jordan , usa ) . after , the canals were dried with absorbent paper points and filled using the lateral condensation technique with endofill sealer ( dentsply , petrpolis , rj , brazil ) . next , the teeth were randomly separated into four groups ( n = 22 ) and stored in individual receptacles . the temporary sealing materials tested on each group were as follows : group i : clip f temporary sealing material ( voco , cuxhaven , germany)group ii : bioplic temporary sealing material ( biodinmica , ibipor , sp , brazil)group iii : vitremer temporary sealing material ( 3 m espe , sumar , sp , brazil)group iv : ketak n100 temporary sealing material ( 3 m espe , sumar , sp , brazil ) . group i : clip f temporary sealing material ( voco , cuxhaven , germany ) group ii : bioplic temporary sealing material ( biodinmica , ibipor , sp , brazil ) group iii : vitremer temporary sealing material ( 3 m espe , sumar , sp , brazil ) group iv : ketak n100 temporary sealing material ( 3 m espe , sumar , sp , brazil ) . to perform coronal sealing , 4 mm of the filling material were removed below the root canal entrance using a no . 5 gattes - gliden bur ( dentsply - maillefer ) , but leaving enough space to place the temporary sealing materials that would fill the entire pulp chamber . after performing coronal sealing , the root surfaces were made impermeable with cyanoacrylate ester ( scotch - bond , 3 m espe , sumar , sp , brazil ) to prevent dye penetration along the root surface . next , the specimens were immersed in indian ink ( acrilex , so paulo , sp , brazil ) for 30 and 60 days , 10 specimens for each time interval . after the immersion period , the specimens were washed under running water for 1 h , dried , and the cyanoacrylate was removed from the root surfaces with the aid of a no . after decalcification , the roots were washed under running water and placed into alcohol ascendant scale ( 70% , 80% , 92% and 100% ) for 1 h in each scale , followed by immersion into methyl salicylate for the clarification process . the positive control group differed from the other specimens because it did not receive the temporary sealing material . on the other hand , after diaphanization , the specimens were placed on a glass slide and taken to the optical microscope all 03 ( alliance , campinas , sp , brazil ) at 12 magnification to assess the depth of coronal leakage . the values of coronal marginal leakage were measured in millimeters from the root canal entrance up to the most longitudinal point of dye penetration . the data of all measurements were submitted to the kruskal - wallis statistical test , at a 5% of level significance , using the software graphpad prism 4.0 ( graphpad software , la jolla , ca , eua ) . the values obtained for the coronal marginal leakage using different sealing materials may be seen in table 1 . irrespective of the time intervals , bioplic presented the highest mean values of leakage and vitremer the lowest , however , with no statistically significant difference when compared to each other and to the other materials ( p > 0.05 ) [ figure 1 ] . mean values ( mm ) and standard deviations of coronal marginal leakage linear coronal leakage measurement under optical microscope . ( a ) test specimen from vitremer group ( lowest microleakage mean values ) . ( b ) test specimen from negative control group ( no dye penetration -arrow ) ( left ) and test specimen from bioplic group ( right ) in the present study , coronal marginal leakage of different types of temporary sealing materials used in root canals after endodontic treatment was assessed . based on the results obtained , it could be affirmed that the tested hypothesis was accepted , since there was no significant difference in the performance of the different materials . coronal microleakage between tooth surface and temporary restorative materials can be assessed using different methods , being the dye penetration test the most used . temporary restorative materials are used in the pulp chamber access to seal the root canal system between visits in endodontic office , until a permanent restoration is performed . such material should effectively seal the root canal entrance from microorganism 's contamination , be dimensionally stable in the presence of moist and have adequate mechanical properties . moreover , some treatments require temporary restorations for longer periods than others . in such cases , due to the facility of removal between sessions and lower cost , studies demonstrated that in the absence of an adequate coronal temporary seal , contamination of root canal system could occur in less than 3 days . several studies have shown that these materials are incapable of preventing coronal marginal leakage , leading to root canal contamination and inducing the appearance of periapical lesions . as found in the scientific literature , the present study demonstrated that the materials tested were incapable of preventing coronal marginal microleakage in any of the samples . although some studies have found statistical significant differences in the performance of temporary sealing materials against coronal marginal leakage , other studies have not and are in agreement with the present study . the materials that presented the highest microleakage mean values , clip f and bioplic , have a resinous matrix - based composed of bis - gma , silicon dioxide , groups of dimethacrylates and organic filler particles . according to the manufacturer , polymerization shrinkage is minimal and it does not influence sealing , however , in addition to possible minimal shrinkage , they do not present any type of dentin adhesion that could avoid marginal leakage . on the other hand , the materials that presented the lowest mean values of marginal leakage , vitremer and ketac n100 , are glass ionomer - based cements . the use of glass ionomer cements as temporary restorative materials in endodontics has been investigated for years and studies report favorable results . glass ionomer - based cements have a chemical bonding capacity due to the adhesion between the carboxylic groups of the polyacids and the calcium ions existent in enamel , dentin and cementum . therefore , it was probably due to this fact that they were less prone to marginal leakage . moreover , srikumar et al . in a recent study , immersed teeth in 1% indian ink dye after restoration with several temporary restorative materials and submitted them to bleaching and thermo cycling for 7 days . the results were demonstrated that zinc phosphate and zinc oxide eugenol - based cements were affected by the bleaching agent action and thermo cycling procedure . however , minimal leakage values were observed for hydrophilic pre - mixed temporary restorative materials under the same conditions . the hydrophilic characteristics of both materials ( pre - mixed temporary restorative material and glass ionomer - based cements ) could explain the favorable results observed in the different studies . although there is no a temporary restorative material able to completely avoid contamination for a long period , these materials are capable of minimizing such contamination during and after endodontic treatment . furthermore , none of the materials tested in the present study was capable of preventing marginal leakage within the 30 and 60 days period in any of the specimens . it was observed that coronal marginal leakage progresses over the course of time , however , with no significant differences in the performance of materials after 30 and 60 days . this fact allow us to conclude that , irrespective of the period of time used , temporary sealing materials must not be kept in the root canals for a long period of time due to the risk of contamination . despite the limitations of this in vitro study , it could be stated that the glass ionomer - based cements presented the lowest microleakage values . however , none of the tested materials was able to prevent microleakage for a long period of time .

aim : the aim of this study is to assess the coronal marginal leakage of three temporary restorative materials used for root canal sealing after endodontic treatment.materials and methods : a total of 88 single - rooted teeth were submitted to biomechanical preparation and filled by lateral condensation technique . after obturation process , the teeth were randomly separated into four groups , being two teeth of each group used as positive and negative control . temporary sealing was performed as follows : gi - clip f ( voco ) ; gii - bioplic ( biodinmica ) ; giii - vitremer ( 3 m espe ) and giv - ketak n100 ( 3 m espe ) . next , the specimens were immersed into indian ink for 30 and 60- days , being 10 specimens for each time interval and then submitted to diaphanization to verify the amount of coronal leakage using a measuring microscope.results:leakage mean values within the 30-day period were as follows : vitremer ( 0.3 mm ) , ketak n100 and clip f ( 0.6 mm ) and bioplic ( 1.7 mm ) . within the 60-day period , leakage means were 1.1 mm , 1.5 mm , 2.2 mm and 2.6 mm , respectively.conclusions:none of the materials was capable of preventing marginal leakage within the 30- and 60-day period . in both time intervals , bioplic presented the highest mean of leakage and vitremer the lowest .

Introduction Materials and Methods Results Discussion Conclusion

PMC5426407

turnaround time for any pathological laboratory is very important which depends on the preparation and diagnosis of the pathological lesions . the rapidity advantages the clinician to treat acutely ill patients and influence the work practice of the pathologist.1 with the advent of modernization , tissue processing is modified from the point of tissue removal to embedding for instant histopathological diagnosis by various techniques or methods . after the surgical removal , the tissue undergoes preparatory protocol for the preparation of sections , which usually involves impregnation with a suitable supporting medium . the stages of tissue processing include fixation , dehydration , clearing , impregnation , and embedding for designated durations of time to ensure completion of the procedure.2 , 3 the reproducibility and relatively low expense attached to the most commonly employed methods continue to recommend it as a valuable tool after nearly 100 years of existence . however , with the demand for faster or early reporting , newer techniques such as rapid manual and microwave processing are getting introduced . each of them is unique with their own set of advantages and disadvantages.2 - 4 the conventional tissue processing is reliable and cost - effective , but time consumption , reagent toxicity , and delay in providing diagnosis are the major disadvantages . the rapid manual tissue processing has major disadvantages such as the use of noxious chemicals and greater degree of tissue distortion and shrinkage which led to exploration for new short - processing schedules . the microwave tissue processing eliminates the use of noxious chemicals , causes lesser distortion of tissue , and has shorter processing time , but the cost involved in instrumentation is very high.4 , 5 microwaves were invented by spencer in 1945 , which work on the principle of producing heat by oscillating or exciting polar molecules . the microwave irradiation forces dipolar molecules of proteins to rotate through 180 at the rate of 2.45 billion cycles per second.3,6 these excited molecules due to kinetics cause collision with adjacent molecules resulting in transfer of rotational energy . this friction causes production of heat within the material itself , leading to the accelerated diffusion of processing fluids ; hence , faster processing is possible.7 the advantages associated with microwave processing led to the production of commercially available microwaves , specifically designed for tissue processing ; however , the cost involved in these is very high.1,8 domestic microwaves are readily available , affordable and had been used for tissue processing with good results earlier by some authors . thus , the aim of the present study was to compare and analyze the efficacy of three tissue processing techniques . in the present study , sixty specimens were selected randomly from the department of oral pathology and microbiology , genesis institute of dental sciences and research , ferozepur , punjab , india . the soft - tissue specimens fixed in 10% neutral - buffered formalin for 24 h were included in the current study . hard tissues specimens such as cartilage , bone , and tooth were not included in the study . the gross features of the specimen were recorded and the tissues were cut into three pieces of same size ( approximately 2 cm 2 cm ) to be processed by three methods . the size of the tissues was recorded before processing and after impregnation with the help of a standard ruler to calculate shrinkage of the tissues . the tissue sections obtained after processing were subsequently stained with hematoxylin and eosin ( h and e ) . the stained slides in each group processed by three techniques were randomly numbered for a blind study and circulated among four observers referred as o1-o4 . the observers graded each parameter by following specific criteria as given in tables 1 and 2 . grading criteria for assessment of cytoplasmic and nuclear details grading criteria for assessment of color intensity the microwave oven was calibrated as the microwave energy is non - uniform within the chamber . thus , hot and cold spots were detected in the chamber with the use of thermal paper sheet , instead of the use of extra water load as suggested by various authors . the cold spot provided the most consistent results every time . the technique was self - standardized by trial and error method in the domestic microwave ( lg model no . the microwave was operated at the maximum output power of 40% ( approximately 360 w ) with rotating tray and ring removed . the cut piece from a fixed tissue sample was placed in a plastic tissue cassette and washed with the running tap water so that tissue was free of formalin . the tissue was irradiated in 200 ml of 100% methanol ( qualikems , vadodara , gujarat , india ) and 200 ml of 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) for dehydration at cold spot for 2 cycles of 10 min each , respectively , in the microwave . after dehydration , tissue was impregnated in 200 ml of molten paraffin wax ( qualikems , vadodara , gujarat , india ) for 2 cycles at the cold spot of 10 min each and was embedded in paraffin wax ( qualikems , vadodara , gujarat , india ) . microwave used for microwave - assisted tissue processing the cut piece from a fixed tissue sample was placed in a metal tissue cassette and washed with the running tap water so that tissue should be free of formalin . the tissue was dehydrated in 70% isopropyl alcohol ( one change ) , 90% isopropyl alcohol ( one change ) , and 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) , three changes of 1 h each , respectively . after dehydration , tissue was cleared in two changes of xylene of 1 h each . finally , tissue was impregnated in two changes of molten paraffin wax ( qualikems , vadodara , gujarat , india ) for 1 h each and was embedded . the cut piece from a fixed tissue sample was washed with the running tap water so that tissue should be free of formalin ; after that , tissue was wrapped in a filter paper and dehydrated in 95% isopropyl alcohol , 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) for 20 min on a stir plate . the dehydrated tissue was cleared in xylene ( qualikems , vadodara , gujarat , india ) for 20 min on a stir plate . tissue was impregnated in two changes of molten paraffin wax ( qualikems , vadodara , gujarat , india ) of 1 h each and was then embedded . the processed tissues by respective techniques were stained as per the protocol ( table 3 ) . protocol for staining the values obtained from different observers after assessment of sections processed by the three techniques were subjected to statistical analysis by kruskal one - way analysis of variance ( anova ) was used for comparing mean shrinkage in tissues processed by the three tissue processing techniques . the microwave oven was calibrated as the microwave energy is non - uniform within the chamber . thus , hot and cold spots were detected in the chamber with the use of thermal paper sheet , instead of the use of extra water load as suggested by various authors . the technique was self - standardized by trial and error method in the domestic microwave ( lg model no . the microwave was operated at the maximum output power of 40% ( approximately 360 w ) with rotating tray and ring removed . the cut piece from a fixed tissue sample was placed in a plastic tissue cassette and washed with the running tap water so that tissue was free of formalin . the tissue was irradiated in 200 ml of 100% methanol ( qualikems , vadodara , gujarat , india ) and 200 ml of 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) for dehydration at cold spot for 2 cycles of 10 min each , respectively , in the microwave . after dehydration , tissue was impregnated in 200 ml of molten paraffin wax ( qualikems , vadodara , gujarat , india ) for 2 cycles at the cold spot of 10 min each and was embedded in paraffin wax ( qualikems , vadodara , gujarat , india ) . the cut piece from a fixed tissue sample was placed in a metal tissue cassette and washed with the running tap water so that tissue should be free of formalin . the tissue was dehydrated in 70% isopropyl alcohol ( one change ) , 90% isopropyl alcohol ( one change ) , and 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) , three changes of 1 h each , respectively . after dehydration , tissue was cleared in two changes of xylene of 1 h each . finally , tissue was impregnated in two changes of molten paraffin wax ( qualikems , vadodara , gujarat , india ) for 1 h each and was embedded . the cut piece from a fixed tissue sample was washed with the running tap water so that tissue should be free of formalin ; after that , tissue was wrapped in a filter paper and dehydrated in 95% isopropyl alcohol , 100% isopropyl alcohol ( qualikems , vadodara , gujarat , india ) for 20 min on a stir plate . the dehydrated tissue was cleared in xylene ( qualikems , vadodara , gujarat , india ) for 20 min on a stir plate . tissue was impregnated in two changes of molten paraffin wax ( qualikems , vadodara , gujarat , india ) of 1 h each and was then embedded . the processed tissues by respective techniques were stained as per the protocol ( table 3 ) . protocol for staining the values obtained from different observers after assessment of sections processed by the three techniques were subjected to statistical analysis by kruskal one - way analysis of variance ( anova ) was used for comparing mean shrinkage in tissues processed by the three tissue processing techniques . all observers were assumed to be reliable as the cronbach s reliability test was statistically significant ( table 4 ) . reliability test was carried out among observers to evaluate the alpha value the histopathological evaluation of the tissues ( tables 5 - 7 ) revealed that the nuclear - cytoplasmic contrast was good and cellular outline was distinct in tissues processed by microwave - assisted technique , followed by conventional processing and rapid manual processing techniques . the secretory products can be easily appreciated , and the red blood cells and inflammatory cells were intact ( figures 2 - 4 ) . histopathological evaluation for cellular morphology histopathological evaluation for nuclear morphology histopathological evaluation for staining characteristics squamous cell carcinoma processed by conventional method ( a and b ) , microwave method ( c and d ) , and rapid manual method ( e and f ) ( h and e , 10 , 40 ) fibrous histiocytoma processed by conventional method ( a and b ) , microwave method ( c and d ) , and rapid manual method ( e and f ) ( h and e , 10 , 40 ) submandibular salivary gland processed by conventional method ( a and b ) , microwave method ( c and d ) , and rapid manual method ( e and f ) ( h and e , 10 , 40 ) the color intensity of the tissues ( tables 5 - 7 ) revealed that the microwave sections were crisper and there was a good contrast between the h and e - stained areas . one - way anova revealed a significant value on comparison of the tissue shrinkage processed by the three techniques ( table 8) . the dimensions of the tissues were recorded before dehydration and before paraffin embedding with the ruler , respectively . the mean percentage of shrinkage in rapid manual technique was significantly higher as compared to the other two techniques , whereas statistically non - significant value was obtained by comparing conventional and microwave method of tissue processing . microwaves are electromagnetic waves9 which cause oscillation and excitation of polar molecules which are usually dipolar molecules of proteins in tissues . the excited molecules cause collision with adjacent molecules due to kinetics producing friction and cause production of heat within the material itself . the heat produced enhances the rate of diffusion of fluids to permeate into the tissues . this has resulted in a substantial reduction in the basic steps of histoprocessing , thereby reducing turnaround time and providing the same day diagnosis.6 , 10 the applications of microwaves are extensive which includes tissue fixation , stabilization of large specimens , tissue processing for light and electron microscopy , and histochemical and immunohistochemical staining . microwave tissue processing technique was introduced by boon and kok in 1985 , but the potential application of microwave energy was first recognized by mayers in 1970 , who successfully fixed tissue with a microwave generator.11 boon et al.12 reported that it was possible to produce a significant acceleration of tissue processing using microwave radiation . the first processor described was able to complete the processing in 30 - 120 min , thus reducing the processing time from 24 h to just 1 - 2 h providing early reporting and easier patient management.13 thus , the aim of the present study was to compare the cytoplasmic and nuclear details as well as staining characteristics of tissue sections processed by conventional , rapid manual , and microwave techniques . the noxious chemicals used in conventional tissue processing were replaced in microwave tissue processing . in the microwave processing in contrast to conventional tissue processing , isopropyl alcohol was replaced by methanol as dehydrating agent and xylene by isopropyl alcohol as intermediate agent . the reagent selection agreed with babu et al.,14 who also used methanol , isopropyl alcohol , and molten paraffin wax for microwave tissue processing . microwave radiation when enters the chamber , gets reflected by chamber walls until these get absorbed by the material placed inside the chamber . however , the spreading is not even throughout the chamber , leading to formation of hot and cold spot zones.16 - 18 hence , hot and cold spots should be detected to achieve consistent results . microwave processing was self - standardized by trial and error method , in which the hot and cold spots were detected using a damp thermal paper.16 all the procedures in the microwave were carried out in the cold spot zones as suggested by sharp and paperiello,19 bernard,20 rangell and keller21 in their respective studies . microwavable plastic tissue cassettes were used for microwave tissue processing which are cheap and reusable as metallic utensils are contraindicated in the microwave because the electric fields of the waves produced by microwave magnetron are completely reflected at the same frequency by metals which can lead to sparking.22 in the present study , the staining protocol for microwave was followed as given by babu et al.,14 which included the stains used to be accelerated in the microwave . domestic microwave oven has been used for acceleration of the various stains without the production of any deleterious effects on staining which also included h and e stain.23 , 24 in our study , the three pieces of tissue processed by three techniques sectioned by a soft - tissue microtome and stained as per their respective protocols were evaluated ( figures 5 - 7 ) . we adopted the criterion for evaluation of tissue sections given by kango and deshmukh.25 comparison between conventional , microwave , and rapid manual tissue processing for cytoplasmic details comparison between conventional , microwave , and rapid manual tissue processing for nuclear details staining characteristics of conventional , microwave , and rapid manual tissue processing the overall quality of the tissue sections processed by microwave and manual methods was comparable . the microwave processed sections had the same or similar cytoplasmic and nuclear details ( figures 2 - 4 ) with good erythrocyte integrity and lymphocytic appearance than the manual methods . we also observed that the stroma in some cases was slightly more condensed focally in microwave processed tissue sections , which is similar to the findings reported by boon et al.12 this leads to the erroneous categorization of these cases as indistinct in studies by kango and deshmukh.25 since our criterion was adopted from the above - mentioned study , we also placed focal condensation of stroma as indistinct . in contrast , kok et al.11 refuted the importance of focal condensation of stroma in diagnostic pathology . the color intensity of the tissues graded by four observers revealed that the microwave sections were crisper and there was a good contrast between the hematoxyphilic and eosinophilic areas ( figures 2 - 4 ) . the microwave processed tissues showed an increased reaction to h and e. the sections stained were slightly more eosinophilic as compared to the manual techniques . similar findings were reported by hopwood et al.,26 boon et al.,12 leong and price,27 and panja et al.4 hopwood et al.26 suggested that this eosinophilia could be easily corrected by altering the stain composition or staining time in eosin . in contrast , leong and price27 observed that eosinophilia of the cytoplasm was advantageous as it produced good nuclear - cytoplasmic contrast and enhancement of the cellular features . the dysplastic features , i.e. hyperchromatism , pleomorphism of tumor cells and mitotic figures , were easily appreciable in the microwave processed tissue sections of malignancy . there was also an easy appreciation of the giant cells in the tissues of central giant cell granuloma and tubercular lymphadenitis processed by microwave processing technique . rapid processing of histopathologic material is becoming increasingly desirable for intraoperative consultations and timely diagnosis . we found positive impact on turnaround time in microwave method as the time taken for block preparation from fixed tissue was 1h as compared to conventional method ( 9 h ) and rapid manual method ( 3 h ) . as assessed in our study , the effects of the three methods of histoprocessing on cytoplasmic and nuclear details of epithelial , fibrous , and glandular tissue showed no statistically significant variation . the microwave - assisted tissue processing method reproducibly yields similar histologic quality to that provided by conventional processing . it has many advantages including feasibility , safety , and elimination of noxious chemicals that might be used for improvement in the practice of the histopathology laboratory , permitting the preparation of diagnostic material within a day . domestic microwaves are easily available and cost - effective but have certain notable disadvantages such as uneven heating and inability to record and maintain temperature within the chamber . further exploration in the field is required for the development of cost - effective microwave histoprocessors for histopathology , which provides similar histologic material for rapid processing and same - day reporting .

objectives : tissue processing for years is carried out by the conventional method , which is a time - consuming technique resulting in 1-day delay in diagnosis . however , in this area of modernization and managed care , rapid diagnosis is increasingly desirable to fulfill the needs of clinicians . the objective of the present study was to compare and determine the positive impact on turnaround times of different tissue processing methods by comparing the color intensity , cytoplasmic details , and nuclear details of the tissues processed by three methods.methods:a total of sixty biopsied tissues were grossed and cut into three equal parts . one part was processed by conventional method , second by rapid manual , and third by microwave - assisted method . the slides obtained after processing were circulated among four observers for evaluation . sections processed by the three techniques were subjected to statistical analysis by kruskal wallis test . cronbach s alpha reliability test was applied to assess the reliability among observers . one - way analysis of variance ( anova ) was used for comparing mean shrinkage before and after processing.results:all observers were assumed to be reliable as the cronbach s reliability test was statistically significant . the results were statistically non - significant as observed by kruskal wallis test . one - way anova revealed a significant value on comparison of the tissue shrinkage processed by the three techniques . the histological evaluation of the tissues revealed that the nuclear - cytoplasmic contrast was good in tissues processed by microwave , followed by conventional and rapid manual processing techniques . the color intensity of the tissues processed by microwave was crisper , and there was a good contrast between the hematoxylin and eosin - stained areas as compared to manual methods.conclusion:the overall quality of tissues from all the three methods was similar . it was not feasible to distinguish between the three techniques by observing the tissue sections . microwave - assisted tissue processing has reduced the time from sample reception to diagnosis , thus enabling the same - day processing and diagnosis .

Introduction Methods Microwave tissue processing Conventional tissue processing Rapid manual tissue processing Results Discussion Conclusion

PMC3038574

contemporary medical practice is anchored in advanced engineering technology mostly provided by the developed world . most developing countries like nigeria rely on over 90% of imported medical technology for the running of her healthcare delivery systems as has been noted in the literature . this in itself poses a great limitation in the efficiency of the health systems in many developing countries because essential equipment are often insufficient , poorly managed , or difficult to replace . thus in the face of a shaky world economy and lack of adequate funding for the continued importation of these technologies , the health care system is bound to fail with a consequent high mortality and morbidity [ 2 , 3 ] . a typical nigerian tertiary hospital would require a minimum of 20 functional incubators to adequately handle its high admission delivery resulting from high population of people within its catchment zones [ 4 , 5 ] . reported that the neonatal centres of most of the nigeria 's premier tertiary hospitals were without an adequate number of functional incubators to handle their numerous referral cases . the hospitals were rather littered with carcases of old and obsolete models of incubators that had long been phased out by the manufacturers and hence without available spare parts for maintenance . the problem was not limited to obsolete incubators as the functionality of more current models was also compromised due to inefficient supply chain of spare parts . many of these hospitals resorted to make - shift hot boxes and poorly serviced obsolete incubators that often led to uncontrollable heating , overheating some babies to death . the very old and crude method of hot water bottles in cot returned as a regular practice in place of functional incubators [ 5 , 7 , 8 ] . this led to diverse kinds of disasters , exposed nurses to different degrees of burns and some newborns died from spilling hot water [ 9 , 10 ] . thermal distress became a major player for high neonatal mortality exacerbated by a rate of 62% point - of - admission hypothermia as reported by ogunlesi et al . . clinicians and nurses hence practiced with unprecedented frustration [ 5 , 11 , 12 ] . it has been shown that effective maintenance of thermoneutrality and appropriate humidification in neonates can enhance survival rates , while neonatal hypothermia is a major cause of rapid death in newborns [ 1318 ] . however , it was impossible to equip the neonatal centres with adequate number of functional incubators due to poor funding and because the country depended on 100% importation of these at unaffordable costs . services were epileptic in the few places where any of these incubators existed at all due to complete lack of system maintenance that could only be rendered by the manufacturers ' foreign technicians . it became obvious that nigeria needed some kind of locally manageable engineering approach to save the teaming population of her neonates for which an estimated 284,000 die each year or 778 per day . to this end , amadi et al . developed the recycled incubator technique this paper presents how recycled incubators may have contributed to reverse a deteriorating neonatal delivery system in nigeria . this is a multicentre followup on a previous study by amadi et al . that developed the application of recycled incubator technique ( rit ) that was used to revamp various obsolete incubators in nigerian tertiary hospitals . fourteen of these hospitals , chosen on the basis of availability of a neonatal unit and the need to cover all the geographical regions of the country 's tropical climate , were involved in this study , ( see figure 1 ) . prior to the introduction of the recycled incubators , various ideas as contributed from initial interviews and on - the - spot assessments were collated to generate effective solutions for the provision of adequate functional incubators at affordable costs to re - equip the hospitals . the systems so - recycled were used to completely re - equip the hospitals or supplement the relatively expensive modern incubators that were in short supply . in rit application , the canopies , access windows and doors , window blinds , filtration systems , and other peripheral assemblies were refashioned in simple designs that were locally producible . temperature and humidity processing and air reconditioning assemblies , oxygen concentration assemblies , and safety devices were reorganised to fit the existing compartments of the particular model . these were cost - competitively chosen and procured based on their output effects that were capable of substituting the requirement of various assembly components of the incubator model . process reconfiguration , additional inter - connectivity , and assembling interfaces for all basic internal assemblies were designed , tailored to every specific model and adjusted to suit outputs from the procured components . clinical trials and performance assessments of initial 11 units of the produced rit incubators were rigorously conducted for six months at the neonatal units of the university of nigeria , enugu and jos university teaching hospitals . based on initial satisfactory performance of the rit , it was introduced to other teaching hospitals to either re - equip or supplement the few modern ones in use . these include uith ilorin , luth lagos , upth portharcourt , and fmc owerri . currently over 100 units of the systems are being used in more than 14 tertiary hospital centres , covering all geopolitical regions of nigeria ( figure 1 ) . the six - year followup performance assessment was conducted based on analyses of three indicators , ( 1 ) impact of rit systems on overall neonatal mortality , ( 2 ) rit impact on nursing practice , ( 3 ) technical evaluation of rit functional parameters . the pre - rit and post - rit neonatal survivals and mortalities were directly assessed from the record books of 3 centres . this involved the assessment of at least 12 months of neonatal care before and after the introduction of rit systems in each centre . this was rigorously done as each neonatal case was carefully examined to isolate admissions that involved hypothermic complications and required a possible intervention of an incubator at some stage . most centres opted out of this retrospective study segment due to shortfalls in record book content such that cases of interest could not be effectively isolated . pre - rit record examination extended only as far back as the number of available corresponding months of post - rit records in the participated centres for a fair comparison . figures noted in this exercise included ( 1 ) total overall number of babies admitted during the period , ( 2 ) total number of individual incubator - dependent neonates ( idns ) admitted , ( 3 ) total number of these idns that were successfully discharged , ( 4 ) total number of those discharged by patient carers ' choice or referred to other centres , and ( 5 ) total number of deaths . these were applied to quantify the relative fraction the surviving neonates constituted based on the total number that was fully nursed ( admission to discharge ) at the centre . poor performance of centres due to lack of functional equipment often leads to the failure of carers in choosing such centres for their patients . therefore the relative overall number of babies admitted at the centres was applied as a contributing measure of carers ' confidence . this application particularly ensured that increase or decrease in admission was not due to a growing / dwindling catchment population or change in payment structure that increased / decreased access to care during this period . availability of functional incubators may also have a contributory role for a faster weight gain and hence earlier successful discharge . seventy - eight nurses in 9 hospital centres , mean ( sd ) of 8.7 ( 2.8 ) , participated in a questionnaire - based assessment of the impact of rit in neonatal nursing in their centres . stringent measures were applied to ensure that only qualifying nurses from each centre assessed their practice experiences . hospital nursing management in nigeria often impose compulsory rotation of staff around the hospital wards with the exception of very few . therefore to qualify as a questionnaire respondent , a nursing staff must have continuously worked in the neonatal centre for at least 12 months leading to rit introduction and at least 12 months immediately following this . four factors required were : to be assessed in the questionnaire according to the private opinion of each respondent . how has rit assisted ( 1 ) your neonatal survival confidence , ( 2 ) actual neonatal survival , ( 3 ) your overall nursing enthusiasm , and ( 4 ) your desire to use more rit incubators in your practice . they were asked to normalise their pre - rit practice experience of these factors to 1 and hence score these against post - rit experience in a scale of 0 to 10 ( from very poor to very good ) ; score 0 signifying poorer post - rit experience , score 1 signifying no perceived practice difference , and this was conducted on 31 incubators that had completed a minimum of six years of continuous service in 6 tertiary hospitals across nigeria . forty - two clinical attendants from the hospitals independently provided performance scores for rit systems they use . these were on the following parameters : ( a ) set point accuracy , ( b ) maintainability ( defined as ease of availability of system 's spare parts and maintenance engineers in nigeria ) , ( c ) ease of hood accessibility , ( d ) heating transient response , ( e ) sensitivity and response to erratic power supply , ( f ) humidification , ( g ) system aesthetic appreciation , ( h ) operation inherent noise , ( i ) response to high ambient temperatures , and ( j ) cost of system procurement . the scores were such that the ideal desirable level of an index was scored 10 and what was considered a very poor level was scored 0 . overall satisfaction of the performance of rit systems was chosen between very dissatisfied , dissatisfied , satisfied , and very satisfied . there was no comparative performance assessment against the non - rit incubators as most of the centres did not have functional non - rit incubators with up to 6 years of continuous service . these were either reported to have been out of use for lack of spare parts or effective maintenance personnel . however , the relative presence of functional non - rit incubators was noted in all centres and applied to compute the average rit contribution towards the survival of nigeria 's incubator - dependent neonates . seventy - eight of an expected 86 questionnaires ( 91% ) were fully completed and returned by the participating nurses . the returned questionnaires indicated that on average , nurses reported an overwhelming increase in their confidence and for their overall nursing enthusiasm with an average ranking of over 8 on the scale of 0 to 10 with 1 signifying no perceived practice difference ( figure 2 ) . actual retrospective measure of impact through patient files shows an average decrease in mortality by 22% ( figure 3 ) from 254/000 to 198/000 . all of the clinicians and nurses reported overall improvement in practice and better success rates in neonatal survival . eight - eight percent of these believe that their perceived higher success rate was mainly due to the incubation boost from rit systems as most of the other clinical factors did not change significantly . four of the providers ( 9.1% ) however think that other factors have equal contribution to the improved success rates , including recently introduced use of apnoea monitors in their respective centres . figure 4 shows a summary of the average scores for the various indices of rit system assessment by the users . on overall performance satisfaction , 37 out of 42 ( 88.1% ) of users were very satisfied and the rest were satisfied . figure 5 shows three informative bars describing availability of functional incubators in each centre ; these are ( 1 ) the total number of functional incubators at the introduction of rit systems ( total at start ) , ( 2 ) the total number of functional incubators current at the time of this study ( total presently ) , and finally ( 3 ) the total number of those current incubators that are powered by rit . at the present , only the university of portharcourt ( upth ) and lagos university ( luth ) teaching hospitals have at least 15 units of functional systems each . for the 14 hospitals listed , none of the initial non - rit incubators that were met in functional condition at the introduction of rit systems in the 6 centres was found functional during this study . the world health organisation ( who ) states that only six countries , including nigeria , jointly contribute 50% of annual global deaths of children of less than five years of age . this figure is largely a reflection of the terrifically high neonatal mortality within the country . our collaborative experience shows that some of the neonatal deaths in the referral hospitals occur as a result of inefficient maintenance of neonatal thermo - neutrality as effective practice would require . this study agrees with others and shows that the blame is partly due to lack of adequate supply of functional incubators and other essential equipment needed in the hospitals [ 5 , 21 , 22 ] . it is arguably correct that facility deliveries and kangaroo warming practices ( direct skin to skin contact with the mother ) during transport are factors that could help decrease the high point - of - admission hypothermia . however , cultural and demographic situations in nigeria , for example , make these factors only partially effective as poverty , long travel distances and ineffective motorized transport still work together to cause many of the babies to arrive at facilities weak or hypothermic . the prevalence of these societal factors means that availability of an adequate number of functional incubators can not be compromised . we set out to apply the idea of recycled incubator technique ( rit ) to supplement the existing incubators in the neonatal centres of fourteen participating tertiary hospitals in nigeria . the generic - components approach adopted in rit incubators allowed the technique its robust ability to easily accept a switchover at any time to another generic , more readily available and cost - effective unit of a component . this also enables rit incubators to remain operational irrespective of an economic downturn affecting the producers of any of its functional components . its operational design , tailored to suit the common traditional constraints of environmental , power , and social factors of low - income countries makes this a user - friendly piece of equipment . jos university teaching hospital ( juth ) applied the initial 10 systems in 2003 , and these have remained the only functional systems in use in the hospital until this current study . the 6-year followup assessment of system performance in all the participating hospitals shows that the system is reliable with a high user - satisfaction rating . this may have , in no small measure , contributed to the reversal of a once dwindling practice enthusiasm and high neonatal mortality in these hospitals . the hospitals listed in this study constitute most of the premier nigerian hospitals and hence reflect the national situation . this suggests that rit might have provided an unprecedented contribution towards the saving of neonates in nigeria . its usage is also well represented across the country and constitutes over 74% of all the functional incubators in the study hospitals , hence significantly contributing to the resolution of a seemingly national problem . evidently , from the report of ademola oyedeji et al . in 1983 to that of ogunlesi et al . in 2008 , nigerian hospitals have many incubator carcasses but fewer than three functional units for application at one time [ 5 , 6 , 12 ] . rit has however changed this trend in the participating hospitals with many of these consistently operating with over 10 functional incubators . these include the lagos university teaching hospital that currently operates with up to 24 functional incubators . direct measurements of neonatal mortality ( figure 3 ) appear to , in part , agree with the perception of the nurses ( figure 2 ) . however , it is understood that careful parametric isolation needed to have been initially defined and prospectively measured during intervention to be able to ascertain the exact incubator contributions towards individual survival . we therefore can only report that these findings correlate with and contributed to a positive clinical impact of the rit systems . furthermore , functional incubators may have also helped to enhance carers ' confidence and motivated increased care - seeking behaviour of patients as evidenced by the increased number of admissions . in the present 6-year followup study , rit systems were found to comprise three - quarters of functional incubators in 14 nigerian centres . system performance was rated high ; provider satisfaction and confidence were much improved ; neonatal mortality coincidentally declined . rit may be a way forward for the rest of the public hospitals in low - income countries especially as high neonatal mortality in these may , in part , be due to the short supply of functional incubators . the success of rit demonstrates that locally manageable and appropriate technological solutions could apply to improve nigeria 's rating in infant and neonatal mortality . rit could be a sustainable application in low - income countries if used incubators from developed countries were donated for recycling . this study may also act as a challenge and motivation for engineers from low income countries to get involved in supplementing imported technologies to make the healthcare delivery system of their countries more functional and affordable . h. o. amadi conceptualised and designed the rit application ; he also coordinated and contributed at all levels of the present work , from study design to article submission . h. o. amadi , j. c. azubuike , u. s. etawo , u. r. offiong , c. ezeaka , o. eyinade , g. n. adimora , a. osibogun , n. ibeziako , e. o. iroha , a. i. dutse , c. o. chukwu , e. e. okpere , m. b. kawuwa , a. u. el - nafaty , s. a. kuranga , and o. a. mokuolu all contributed equally to this work on initial installations in their respective hospitals , system monitoring , system optimization , study reviews and manuscript readiness .

nigeria has a record of high newborn mortality as an estimated 778 babies die daily , accounting for a ratio of 48 deaths per 1000 live births . the aim of this paper was to show how a deteriorating neonatal delivery system in nigeria may have , in part , been improved by the application of a novel recycled incubator technique ( rit ) . retrospective assessment of clinical , technical , and human factors in 15 nigerian neonatal centres was carried out to investigate how the application of rit impacted these factors . pre - rit and post - rit neonatal mortalities were compared by studying case files . effect on neonatal nursing was studied through questionnaires that were completed by 79 nurses from 9 centres across the country . technical performance was assessed based on 10-indices scores from clinicians and nurses . the results showed an increase in neonatal survival , nursing enthusiasm , and practice confidence . appropriately recycled incubators are good substitutes to the less affordable modern incubators in boosting neonatal practice outcome in low - income countries .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions Conflict of Interests Authors' Contributions

PMC3420829

there is a shortage of dentists working in australian rural locations.1,2 this problem will increase in severity as the cohort of dentists who qualified between 19651975 begin to retire , especially as many of them work in rural areas . it is essential to encourage new dental graduates to consider working in country areas to alleviate this problem . research literature in other health disciplines shows that a rural placement can increase rural recruitment.3,4,5 in 2005 the james cook university school of medicine in northern australia provided eight week rural internships for all 6 year medical students . the internships were evaluated via questionnaires , site visits , interviews and follow up teleconferences , including a compulsory debrief session . the students reported that the internship was one of the most important clinical experiences of the final year course and they were able to meet their academic curriculum learning objectives.3 the study did not examine whether the students took up rural employment post - graduation and the researchers noted that the long term destination of the students would be an important aspect of further investigation.3 a longitudinal study of medical students in western australia identified rural background , self - reported value of the experience , placement duration and voluntary attendance at the rural placement as some of the factors which could persuade students to work in a rural practice.6 a study in 2007 also concluded rural background is a key predictor for working in a rural medical practice.7 other studies add to the substantial evidence that having a rural background is the single most significant predictor of a subsequent rural career for medical practitioners.8,9,10 the timing of a rural placement is important , with a study showing that offering it to 3 year undergraduates instead of 4 year students led to lower graduate recruitment into rural sectors.11 a rural placement program in tasmania reported that it provided a positive influence on the students intention to work rurally after their graduation.12 despite these positive reports a review published in 200713 claimed that there is still no definitive evidence of the effectiveness of rural intern - ships and recommends isolating the factors which positively influence graduates working in rural regions by producing methodological , structured , rigorous , longitudinal studies which follow up graduates and control for the independent predictors of rural practice . the evidence of the influence of rural placements on dental student s future plans is more limited . nunn and freeman14 highlighted that undergraduate teaching in many parts of the world is focused on techniques which are suited for developed populations and education is providing less encouragement for undergraduates to practice in remote areas . they felt a significant clinical outreach experience away from the large centralized teaching hospitals might encourage students to work in a rural location . one example at the university of western australia incorporated an optional rural , remote and indigenous placement ( rrip).15 the program lasted from 20022005 with 143 students graduating in this period , 55% had a rural placement . there was a gradual increase in the proportion of students who had participated in the placement who then went on to work in a rural area within 12 months of graduation ; 26% in 2002 , 38% in 2003 , 60% in 2004 and 48% in 2005 . the majority ( 95% ) were very positive in their feedback and found the placements to be a valuable learning experience.15 melbourne dental school16 provided a 4 week rural outplacement in 20062007 for 70 students in groups of 6 to 10 to an outreach clinic in the small town of shepparton and questionnaires provided feedback of their experience . the study found 19.5% of students were considering rural practice post placement with only one student uninterested and 79.1% expressed definite affirmation . all the students gained confidence in performing most procedures during their placement and it helped prepare them for dentistry post - graduation . mascarenhas et al17 stated that community based experiences are focussed on patient care and time management skills rather than learning specific clinical skills . a placement program in south australia showed that dental students can make a significant contribution to the provision of public dental services in rural communities , in terms of reducing patient waiting lists , providing patient care and giving students the opportunity to acquire an appreciation of the factors that influence rural dental healthcare practice.18 a community based dental education program set in the united states19 reported that students in community clinics see approximately 6 or more patients per day , whereas traditional dental school clinics which are focused on education see approximately 2 to 3 patients per day . the benefits for students from community programs were noted as the acquirement of knowledge , skills and values that are not readily available in dental school based clinical training . they concluded that students care for more patients , gain teamwork skills and have the opportunity to treat a more diverse range of patients.19 an outreach scheme developed in the uk involved fourth year dental students seconded to primary care centers in socially deprived areas with inadequate general dental services and poor oral health.20 the students completed questionnaires prior to and after the program . qualitative and quantitative research methodology was used in the program assessment using likert scales and semi structured open questions . post outreach the students reported an increase in confidence , improved time management skills and an enhanced ability to identify certain clinical problems . although not a rural experience it did show that working in a primary care location was popular and improved clinical skills.20,21 the faculty of dentistry , university of sydney decided to implement a rural placement program drawing on the positive experiences of the universities of western australia , melbourne and manchester as part of a strategy to promote rural training funded by the australian department of health and ageing . this paper discusses the effectiveness of rural placements in dental undergraduate teaching in terms of the suitability of the clinical training provided , its effect on the students clinical skills and confidence and whether the program influences students to consider rural work postgraduation . with the approval of the ethics review committee ( rpah zone ) data reported here were collected from fourth final year bachelor of dentistry ( bdent ) undergraduates . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . a placement lasted one month at one of three rural clinics , working under supervision four clinical days per week with one day allowed for study . there were four one month placements at bowral ( 6 students per placement ) and bathurst and orange ( taking 2 students each . ) each placement site had a registered dentist acting as a clinical supervisor for the students who received a training course to learn the faculty s teaching protocols . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . a faculty staff member was on hand to respond to student queries and issues throughout the placements . pre placement meetings were set up to brief the students on the type of clinical care they would practice and to answer any concerns . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . the clinical supervisors were interviewed via phone and the faculty members were interviewed in person . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . non respondents were resent questionnaires via email and a follow up telephone call was made one week later to students who had still not responded . with the approval of the ethics review committee ( rpah zone ) data reported here were collected from fourth final year bachelor of dentistry ( bdent ) undergraduates . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . a placement lasted one month at one of three rural clinics , working under supervision four clinical days per week with one day allowed for study . there were four one month placements at bowral ( 6 students per placement ) and bathurst and orange ( taking 2 students each . ) each placement site had a registered dentist acting as a clinical supervisor for the students who received a training course to learn the faculty s teaching protocols . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . a faculty staff member was on hand to respond to student queries and issues throughout the placements . pre placement meetings were set up to brief the students on the type of clinical care they would practice and to answer any concerns . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . the clinical supervisors were interviewed via phone and the faculty members were interviewed in person . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . non respondents were resent questionnaires via email and a follow up telephone call was made one week later to students who had still not responded . the first part of the analysis provides a profile of the students socio - demographic profile including their rural background . data were entered into sas 9.1 for frequency , mean , percentages and paired t - tests were used to compare the various likert scale group data . open ended responses were analysed using framework analysis to identify trends and themes in the group responses . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . rural background has been broadly defined as any rural experience or rural exposure.12 students were asked what type of environment they had grown - up in and to comment on any rural experience they had encountered . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . there were more non - volunteers raised in small town areas ( 8.1% ) than the volunteers ( 3.2% ) . however there is not enough difference between the two groups to confound the results and no significance difference between the groups when a paired t - test was performed with both groups predominantly from a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . the area the volunteer group perceived their greatest improvement was in treatment skills with a 0.5 increase . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteer group reported a lower level of overall clinical ability compared with the pre - placement non - volunteers . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . the clinical treatment episodes undertaken by the students were entered into the nsw dental public service data collection information system for oral health ( isoh ) and the main categories of care are shown in table 5 . the most common clinical procedure in bathurst was dental extractions , whilst at bowral and orange restorative care was the most common procedure , followed by acute care at bowral and dental extractions at orange . the system recorded across all the placements a total of 712 episodes of restorative care , 500 episodes of acute care , 420 dental extractions , 160 treatment plans and prevention , 143 of periodontal care and 45 episodes of denture care and fixed prosthodontics . the mean number of episodes of care for each student across the three placements per placement was 59 . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . other procedures mentioned by several students were detecting caries , patient management , root canal therapy , acute care management , dealing with special care patients and cutting crown preparations . they reported that they were expected to complete patient treatment at a much faster pace than at the teaching metropolitan hospitals . according to the students they saw between 6 and 8 patients per day whereas at the university teaching hospitals non volunteers reported seeing approximately 3 to 4 patients per day . the non - volunteers reported completing more specialist dentistry such as prosthodontics and less basic clinical dentistry . non volunteers gave reasons for regretting not participating in the placement such as missing a valuable clinical experience , the opportunity to make new friends and making professional contacts . the minority of non - volunteers who had no regrets explained they felt being at the university teaching hospitals provided greater opportunity for more specialized clinical training such as fixed prosthodontics . the clinical supervisors reported the students increased in confidence , as well as improved clinical ability and clinical efficiency . supervisors also commented that students improved in their treatment planning , time managements and communication skills . they also confirmed the students saw between 68 patients per day as their clinical efficiency improved . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . the faculty members noted some of the returning students had stronger communication skills and confidence based on debriefing conversations . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteers were not asked this question in the follow up questionnaire as they had not had a rural experience to alter their perspective . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . the first part of the analysis provides a profile of the students socio - demographic profile including their rural background . data were entered into sas 9.1 for frequency , mean , percentages and paired t - tests were used to compare the various likert scale group data . open ended responses were analysed using framework analysis to identify trends and themes in the group responses . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . rural background has been broadly defined as any rural experience or rural exposure.12 students were asked what type of environment they had grown - up in and to comment on any rural experience they had encountered . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . there were more non - volunteers raised in small town areas ( 8.1% ) than the volunteers ( 3.2% ) . however there is not enough difference between the two groups to confound the results and no significance difference between the groups when a paired t - test was performed with both groups predominantly from a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . the area the volunteer group perceived their greatest improvement was in treatment skills with a 0.5 increase . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteer group reported a lower level of overall clinical ability compared with the pre - placement non - volunteers . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . the clinical treatment episodes undertaken by the students were entered into the nsw dental public service data collection information system for oral health ( isoh ) and the main categories of care are shown in table 5 . the most common clinical procedure in bathurst was dental extractions , whilst at bowral and orange restorative care was the most common procedure , followed by acute care at bowral and dental extractions at orange . the system recorded across all the placements a total of 712 episodes of restorative care , 500 episodes of acute care , 420 dental extractions , 160 treatment plans and prevention , 143 of periodontal care and 45 episodes of denture care and fixed prosthodontics . the mean number of episodes of care for each student across the three placements per placement was 59 . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . other procedures mentioned by several students were detecting caries , patient management , root canal therapy , acute care management , dealing with special care patients and cutting crown preparations . they reported that they were expected to complete patient treatment at a much faster pace than at the teaching metropolitan hospitals . according to the students they saw between 6 and 8 patients per day whereas at the university teaching hospitals non volunteers reported seeing approximately 3 to 4 patients per day . the non - volunteers reported completing more specialist dentistry such as prosthodontics and less basic clinical dentistry . non volunteers gave reasons for regretting not participating in the placement such as missing a valuable clinical experience , the opportunity to make new friends and making professional contacts . the minority of non - volunteers who had no regrets explained they felt being at the university teaching hospitals provided greater opportunity for more specialized clinical training such as fixed prosthodontics . the clinical supervisors reported the students increased in confidence , as well as improved clinical ability and clinical efficiency . supervisors also commented that students improved in their treatment planning , time managements and communication skills . they also confirmed the students saw between 68 patients per day as their clinical efficiency improved . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . the faculty members noted some of the returning students had stronger communication skills and confidence based on debriefing conversations . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteers were not asked this question in the follow up questionnaire as they had not had a rural experience to alter their perspective . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . the overall response rate from the students was excellent , with all the volunteers and a high percentage of the non - volunteers ( pre questionnaire - 67% and post - 65% ) completing the pre and post placement questionnaires . non respondents were followed up using email and mobile phones , with a mixture of phoning and sms texting to encourage them to complete the questionnaire . the use of a control group increased the validity of the findings and by recording information pre and post completion of the rural placements . the program targeted 4 year students11 and each placement lasted one month because the research concluded that this timeframe encouraged students to consider rural work post - graduation.6 rural background was measured as the literature identified it as a potential confounder.6,7,8,9,10 the two student groups were found to be not significantly different in terms of their rural background allowing for comparisons to be made . likert scale methods and some open ended semi structured questions were used to provide both quantitative and qualitative data as the literature review showed these methods are a valuable research tool.3,4,15,20 also clinical supervisors via phone interviews were asked semi structured questions regarding the clinical work completed to verify the student responses and isoh data . the data collection system allowed a large amount of data to be acquired in a short time . the likert scales and students self - perceived levels of treatment planning , time management , communication skills , clinical ability and treatment skills were all positive for the rural sample . our results are similar to the manchester20 and leeds studies21 which found that students increased in their confidence , time management and diagnosis skills . we also found that their self - perceived clinical skills increased , the students saw more patients per day in the rural clinic than at the dental school clinics and this was confirmed by the rural clinical supervisors . this supports the evidence by baiit19 which also found students saw approximately 6 patients per day in a community clinic compared to 2 and 3 at the dental school teaching clinics . the non - volunteers had a significantly higher mean pre - placement score ( 18.5 ) than the volunteers ( 17.3 ) . an explanation as to why the pre volunteers had a lower likert score across the five skills could be due to pre placement anxiety . the finding that greater improvement occurs in the volunteers from pre to post placement than the non - volunteers is important as it demonstrates the significant increase in clinical confidence of the volunteer students post placement . the volunteer and non - volunteer group were directly compared and there was a significantly ( p<.05 ) larger increase in skill level from pre to post placement for the volunteers than the non - volunteers despite starting with a lower pre placement self - perceived score . this increase may be due to the volunteers considerable clinical work undertaken whilst on the placement in seeing more patients per day and completing more basic clinical dentistry than at the metropolitan teaching hospitals . the supervisors commented that the students helped reduce the patient waiting lists at the orange and bathurst clinics supporting richards et al18 who felt that dental students can make a significant contribution to the public dental services waiting lists . the clinical data entered into the nsw information system for oral health ( isoh ) supports the reported responses from the students about their level of clinical activity , and is an independent verification that considerable practical primary care dentistry was undertaken . in our study 97% of the volunteers post placement felt they were more likely to work in a rural setting after graduation , demonstrating a dramatic change in attitude which supports the findings of western australia15 and melbourne16 who found students were encouraged to work rurally post placement . this shows the positive influence the rural placements had on the students attitude towards considering working in a rural location upon graduation . longitudinal follow up is important for monitoring the long term value of rural placements ; therefore a follow up of these students will be undertaken in 18 months time to record their working location . after the students returned from the rural placement , the non - volunteers were asked if they had any regrets based on what they had heard from the volunteers , more than half of the non - volunteers felt they had missed out on a valuable teaching experience and the chance to make and build contacts and friendships . this demonstrates the positive feedback from the volunteers post placement regarding the placement when reintegrated with the non - volunteers . self - report was used so there is potential for recall bias , judgement error , memory loss and providing socially desirable answers . however with this type of study self - report is probably the most practical way to collect data given the intensity of the dental undergraduates curriculum . the weakness with semi - structured questions and likert scales is that the students were somewhat restricted as they were asked a set of specific questions . an interview for each participant would have provided more detailed in depth responses , however this would have been very time consuming and labour intensive for both the student and interviewer . likert scale scores are open to some interpretation and there was a higher response rate from the volunteer group and this may have had some impact . the timing of the completion of questionnaires varied and this is a potential weakness , pre volunteers completed their questionnaire one week pre placement and within 14 days post placement . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the questions were based on the questionnaire design and placement evaluation set up by the manchester20 study , however the questionnaire was not a validated instrument and had nt been used previously . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . self - report was used so there is potential for recall bias , judgement error , memory loss and providing socially desirable answers . however with this type of study self - report is probably the most practical way to collect data given the intensity of the dental undergraduates curriculum . the weakness with semi - structured questions and likert scales is that the students were somewhat restricted as they were asked a set of specific questions . an interview for each participant would have provided more detailed in depth responses , however this would have been very time consuming and labour intensive for both the student and interviewer . likert scale scores are open to some interpretation and there was a higher response rate from the volunteer group and this may have had some impact . the timing of the completion of questionnaires varied and this is a potential weakness , pre volunteers completed their questionnaire one week pre placement and within 14 days post placement . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the questions were based on the questionnaire design and placement evaluation set up by the manchester20 study , however the questionnaire was not a validated instrument and had nt been used previously . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . the rural placement program provided an excellent clinical experience as students reported that their clinical confidence and skills had improved . they also reported an increased likelihood to consider working in a rural location after graduation .

objective : to evaluate the effectiveness of a rural clinical placement on students self - perceived clinical skills and work location choice post-graduation.methods:a one month rural placement program was introduced in 20082009 for 4th year dental undergraduates . student s views on the rural exercise were collected by pre and post self - completion questionnaires , which were distributed to rural placement students and to the students who did not participate in the placement . information was collected on self - reported skill levels in various clinical techniques using likert scales and future rural work intentions . clinical supervisors and university faculty members opinions on the students clinical work were also collected via interviews.results:the mean age of the respondents was 27 years and the majority were female ( 57% ) . all the placement students ( volunteers ) completed pre and post placement questionnaires and ( 67% ) of the non - placement students completed the pre questionnaire and ( 65% ) a post questionnaire . when the two groups were compared in terms of their likert scores , there was a significantly ( p<.02 ) larger increase in skill level in the rural group . clinical supervisors confirmed volunteers increased in confidence and ability in clinical skills post placement . the majority ( 96% ) of students who completed a placement reported that they were more likely to work in a rural environment after graduation.conclusions:the rural placement improved the self - perceived clinical skills of the volunteers and enhanced positive attitudes towards working in a rural location .

INTRODUCTION MATERIALS AND METHODS Sample and procedures RESULTS Data Analysis Socio-demographic profile Self-Analysis of the Students Clinical Skills using Likert Scales Clinical ISOH Scores Qualitative data on Clinical care Student Open Question Responses on Rural Clinical Experience Clinical Supervisor Responses Faculty Members Responses Students Rural Intentions DISCUSSION Limitations CONCLUSIONS

PMC3582042

health behaviour in school - aged children is a general health survey of preadolescent and adolescent children conducted in affiliation with who.6 in canada , cycle 6 of this survey ( 2009/10 ) involved administration of both student- ( n=26 078 ) and school - level administrator ( n=436 ) questionnaires . we combined these data with environmental measures describing school neighbourhoods that were obtained using geographic information systems . the survey involved a systematic multi - stage cluster sample design that involved students and schools from 11 of 13 canadian territories and provinces.6 the sample available for the current analysis was 19 576 students with complete data ( weighted sample 20 076 ) from 419 schools . implicit or explicit consent to participate was obtained from school boards , individual schools , parents / guardians and students , as per local jurisdictional requirements . the study protocol was approved by the general research ethics board of queen 's university ( study approval number : geduc-430 - 09 ) . based upon a standard self - report module , reports of active transportation injuries were identified for all participants for a 12-month period prior to survey administration.6 injuries that required medical attention and which occurred : ( 1 ) in the street / road / parking lot or while ( 2 ) biking / cycling or walking / running ( not for a sports team or exercise) were operationally defined as active transportation injuries . three levels of active transportation to school were identified : ( 1 ) youth who did not regularly engage in active transportation ; ( 2 ) youth who regularly engaged and lived near their school ; and ( 3 ) youth who regularly engaged but lived far from school . participants who reported that their usual mode of transportation to school was bus , train , streetcar , subway , or boat / ferry or car , motorcycle , or moped were placed in category 1 . students who reported regular active transportation ( by walking or bicycling ) with travel times either < 5 min for cycling or < 15 min for walking were placed in category 2 . those reporting greater lengths of time for walking or bicycling were placed in category 3 . potential individual - level confounders were gender , age , ethnicity , perceived family socioeconomic status , perceived residential neighbourhood safety and participation in organised sports . potential area - level confounders describing the school neighbourhood were : urban / rural geographic status , average precipitation levels as reported by environment canada , summary measures of total road lengths , street or road connectivity,7 speed limits in the 1 km buffer surrounding each school , and canada census of population estimates of median household incomes for 2006 ( pcensus for mappoint ; tetrad computer applications inc . an initial descriptive analysis was used to characterise the study sample using proportions and measures of central tendency . examination of the clustered and nested structure of the data revealed an intraclass correlation of 2.6% ( 0.026 ) at the school - level.8 we therefore modelled effects at individual- and school - levels while accounting for this clustering . multi - level logistic regression modelling was used with random intercepts permitted for each school . standardised weights ( mean=1.00 ) were calculated and applied to account for the fact that children from different provinces and territories , school board types , languages of instruction , and urban / rural geographic status had different probabilities of entering the sample . following bivariate analyses , multi - level models were built as follows : ( 1 ) the active transportation variable and all individual - level factors were entered as risk factors ( multivariate model 1 ) ; ( 2 ) backwards selection methods were performed using a change in estimate of > 10% for retention of individual - level variables9 ; then area - level variables were added to the retained individual - level factors ( multivariate model 2 ) ; and ( 3 ) backwards selection was reperformed for the final model , using the same change - in - estimate approach ( multivariate model 3 ) . conservatively , any covariate whose removal from the model caused a change in the estimate greater than 10% or was statistically significant ( p<0.05 ) was retained in model 3 . finally , the modelling process was repeated for two specific active transportation injury outcomes : ( 1 ) walking or running injuries and ( 2 ) cycling injuries . a priori , the study was powered to identify an or of 1.2 with > 80% power ( =0.05 ) for the main analysis . health behaviour in school - aged children is a general health survey of preadolescent and adolescent children conducted in affiliation with who.6 in canada , cycle 6 of this survey ( 2009/10 ) involved administration of both student- ( n=26 078 ) and school - level administrator ( n=436 ) questionnaires . we combined these data with environmental measures describing school neighbourhoods that were obtained using geographic information systems . the survey involved a systematic multi - stage cluster sample design that involved students and schools from 11 of 13 canadian territories and provinces.6 the sample available for the current analysis was 19 576 students with complete data ( weighted sample 20 076 ) from 419 schools . implicit or explicit consent to participate was obtained from school boards , individual schools , parents / guardians and students , as per local jurisdictional requirements . the study protocol was approved by the general research ethics board of queen 's university ( study approval number : geduc-430 - 09 ) . based upon a standard self - report module , reports of active transportation injuries were identified for all participants for a 12-month period prior to survey administration.6 injuries that required medical attention and which occurred : ( 1 ) in the street / road / parking lot or while ( 2 ) biking / cycling or walking / running ( not for a sports team or exercise) were operationally defined as active transportation injuries . three levels of active transportation to school were identified : ( 1 ) youth who did not regularly engage in active transportation ; ( 2 ) youth who regularly engaged and lived near their school ; and ( 3 ) youth who regularly engaged but lived far from school . participants who reported that their usual mode of transportation to school was bus , train , streetcar , subway , or boat / ferry or car , motorcycle , or moped were placed in category 1 . students who reported regular active transportation ( by walking or bicycling ) with travel times either < 5 min for cycling or < 15 min for walking were placed in category 2 . those reporting greater lengths of time for walking or bicycling were placed in category 3 . potential individual - level confounders were gender , age , ethnicity , perceived family socioeconomic status , perceived residential neighbourhood safety and participation in organised sports . potential area - level confounders describing the school neighbourhood were : urban / rural geographic status , average precipitation levels as reported by environment canada , summary measures of total road lengths , street or road connectivity,7 speed limits in the 1 km buffer surrounding each school , and canada census of population estimates of median household incomes for 2006 ( pcensus for mappoint ; tetrad computer applications inc . , an initial descriptive analysis was used to characterise the study sample using proportions and measures of central tendency . examination of the clustered and nested structure of the data revealed an intraclass correlation of 2.6% ( 0.026 ) at the school - level.8 we therefore modelled effects at individual- and school - levels while accounting for this clustering . multi - level logistic regression modelling was used with random intercepts permitted for each school . standardised weights ( mean=1.00 ) were calculated and applied to account for the fact that children from different provinces and territories , school board types , languages of instruction , and urban / rural geographic status had different probabilities of entering the sample . following bivariate analyses , multi - level models were built as follows : ( 1 ) the active transportation variable and all individual - level factors were entered as risk factors ( multivariate model 1 ) ; ( 2 ) backwards selection methods were performed using a change in estimate of > 10% for retention of individual - level variables9 ; then area - level variables were added to the retained individual - level factors ( multivariate model 2 ) ; and ( 3 ) backwards selection was reperformed for the final model , using the same change - in - estimate approach ( multivariate model 3 ) . conservatively , any covariate whose removal from the model caused a change in the estimate greater than 10% or was statistically significant ( p<0.05 ) was retained in model 3 . finally , the modelling process was repeated for two specific active transportation injury outcomes : ( 1 ) walking or running injuries and ( 2 ) cycling injuries . a priori , the study was powered to identify an or of 1.2 with > 80% power ( =0.05 ) for the main analysis . a weighted sample of 20 076 students from 419 schools with complete data was available for analysis . about a third ( 33.6% ) of the sample engaged in active transportation to school and 357 ( 1.8% of the sample ) incurred an active transportation injury ( table 1 ) . of these injuries , 68.9% occurred while cycling , 31.1% occurred while walking or running , 45.1% required medical treatment ( eg , placement of a cast or stitches ) and 40.6% caused the participant to miss at least 1 day of school or usual extracurricular activities . approximately one injury was reported for every 2900 h of exposure to active transportation . description of sample demographics , engagement in active transportation and the occurrence of active transportation injuries ( n=20 076 ) table 2 summarises bivariate , then adjusted models that describe the association between engagement in active transportation and the occurrence of related injury . ors can be interpreted as relative risks.10 model 3 provides final estimates for this relationship while controlling for potential confounders . a statistically significant positive association that followed a graded trend ( ptrend=0.02 ) was observed , with an adjusted 1.52-fold increase ( 95% ci 1.08 to 2.15 ) in the relative odds of active transportation injury for youth who regularly engage in active transportation over longer distances . two covariates were retained in the final model : age group ( or : 0.75 ; 95% ci 0.57 to 0.97 for ages 1415 vs 1113 ) and urban / rural status ( or : 1.64 ; 95% ci 1.14 to 2.36 for urban vs rural communities ; ptrend=0.008 ) . results of multi - level logistic regression analysis examining potential risks for active transportation injury associated with engagement in active transportation to school ( n=20 076 ) adjusted for individual - level variables ( gender , age , ethnicity , family socioeconomic status , perceived neighbourhood safety and participation in organised sports ) . adjusted for retained individual - level variables ( age ) and area - level variables ( urban / rural geographic status , street connectivity , speed limit surrounding school , % roads with speed limit 60 km / h , total length of roads , school neighbourhood median family income , total rain and total snow ) . adjusted for retained individual ( age ) and area - level ( urban / rural geographic status ) variables . the most important finding from this study was that as young people engaged in active transportation for longer distances , their risks for active transportation injury increased irrespective of their mode of active transportation . many health promotion interventions aim to increase participation in active transportation to school due to its inherent benefits to health.11 however , these same interventions do not necessarily consider the negative outcomes of active transportation to school such as injury . our findings therefore contribute to a more comprehensive understanding of this public health issue and associated health promotion messages . it is difficult to establish temporality in our observed effects due to the cross - sectional design . our use of self - reported measures of injury and active transportation may have contributed to non - differential misclassification leading to bias of the ors towards no effect . for example , it is quite possible that some of the events that were classified as active transportation injuries were actually recreational . our lack of detailed information about some relevant contextual factors ( eg , bicycle helmet laws , cycling infrastructure , pedestrian guards and crossings ) are a further limitation , and the analysis also does not account for the timing of injuries and whether they were experienced outside of school commuting times . finally , there is the possibility of selection bias , such that youth not attending school on the day of survey administration may have been more likely to have active transportation injuries which in turn are differentially related to more active transportation to school . if this bias existed , it would again bias our or estimates towards no effect . the two main strengths of this research were our use of a large nationally representative sample of students as well as our focus on the negative side effects of an important public health topic with obvious benefits to health . . negative health outcomes of active transportation remain understudied in the adolescent health promotion literature , and there is a need for future studies to evaluate the relative benefits ( eg , reduced obesity ) and potential harms ( eg , increased injury ) within the same analyses . public health interventions targeted at increasing active transportation to school in canadian youth should consider possible unintentional injury outcomes of active transportation . interventions aimed at increasing physical activity should not lose sight of possible injury - related outcomes . examples of interventions include the walking school bus where children travel together in large groups12 and environmental solutions that foster improvements to walking and cycling infrastructure.13 if well designed , these population health interventions could have a very positive impact on the physical health of canadian youth overall , while limiting the potential for associated injuries . the relationship between active transportation to school and active transportation injury was examined in a nationally representative sample of canadian youth using multi - level analytical methods . response relationship between active transportation to school and active transportation injury across increasing travel distances . we suggest that new and existing interventions promoting active transportation to school should further incorporate injury control strategies in order to continue encouraging physical activity in the safest possible manner . engagement in active transportation to school represents one possible strategy of promoting physical activity in groups of young people . while the positive effects of active transportation are obvious , possible negative effects of such practices in terms of unintentional injury this national study of young people from across canada aimed to understand the effects of engagement in active transportation to school on the risks for related injury . modest increases in risk for active transportation injury were evident , rising in accordance to distance travelled to school . injury does represent one possible negative consequence of what in general is a positive behaviour for the health of young people .

active transportation to school provides a means for youth to incorporate physical activity into their daily routines , and this has obvious benefits for child health . studies of active transportation have rarely focused on the negative health effects in terms of injury . this cross - sectional study is based on the 2009/10 canadian health behaviour in school - aged children survey . a sample of children aged 1115 years ( n=20 076 ) was studied . multi - level logistic regression was used to examine associations between walking or bicycling to school and related injury . regular active transportation to school at larger distances ( approximately > 1.6 km ; 1.0 miles ) was associated with higher relative odds of active transportation injury ( or : 1.52 ; 95% ci 1.08 to 2.15 ) , with a suggestion of a dose response relationship between longer travel distances and injury ( p=0.02 ) . physical activity interventions for youth should encourage participation in active transportation to school , while also recognising the potential for unintentional injury .

Methods Overview of study design and measures Participants Key measures Analysis Results Discussion Conclusion

PMC3110771

to successfully interact with the stimuli of our environment , we need to process selectively the information most relevant for our tasks . when stimuli are attended to their processing become more rapid , more accurate , and more detailed ( posner et al . , 1980 ; desimone and duncan , 1995 ; carrasco and mcelree , 2001 ; carrasco et al . , 2004 ; liu et al . , 2005 , 2009 ) . attention improves performance on several visual tasks , such as contrast sensitivity , speed and orientation discrimination as well as spatial resolution ( lee et al . , 1999 ; are limited , when the stimuli demanding attention for a perceptual task exceed system capacity , performance decreases . for example , in a visual search task in which an object ( target ) has to be detected amongst irrelevant items ( distractors ) , reaction times increase directly with distractor number ( unless the difference between the stimuli is so striking to make the target pop out from the cluttered scene ) . this correlation reflects the limited capacity of selective attention that prevents the observer from monitoring all items at the same time . similarly , when more than one perceptual task is performed at the same time , overall performance decreases because of the underlying processing limitations . this occurs even for simple tasks , such as naming a word or identifying the pitch of a tone ( pashler , 1992 ; pashler and o'brien , 1993 ; huang et al . , interference between concurrent perceptual tasks of the same sensory modality has been consistently reported in many psychological and psychophysical studies ( navon et al . , 1984 ; pashler , 1994 ; bonnel and prinzmetal , 1998 ; alais et al . , 2006b ) . however , the evidence for audiovisual cross - modal interference is conflicting ( duncan et al . bonnel and hafter ( 1998 ) found that in a identification task in which the sign of a change ( luminance in vision and intensity in audition ) had to be detected , performance in dual - task conditions were lower than in the single - task conditions regardless the interference was in the same or different modalities . spence et al . ( 2000 ) found that selecting an auditory stream of words presented concurrently with a second ( distractor ) stream , it is more difficult if a video of moving lips mimicking the distracting sounds it is also displayed . these psychophysical findings are not only congruent with some of the cognitive literature of the 1970s and 1980s ( taylor et al . , 1967 ; tulving and lindsay , 1967 ; alais et al . , 2006b ) , but also with recent neurophysiological and imaging results . 2004 ) examined the electrophysiological correlates for auditory interference with vision by an identification task of non - ambiguous complex stimuli such as faces and voices . their results suggest that cross - modal interactions occur at various different stages , involving brain areas such as fusiform gyrus , associative auditory areas ( ba 22 ) , and the superior frontal gyri . hein et al . ( 2007 ) showed with a functional magnetic resonance ( fmri ) study , that even without competing motor responses , a simple auditory decision interferes with visual processing at neural levels including prefrontal cortex , middle temporal cortex , and other visual regions . taken together these results imply that limitations on resources for vision and audition operate at a central level of processing , rather than in the auditory and visual peripheral senses . however , much evidence also supports the notion of independence of attentional resources for vision and audition ( allport et al . , 1972 ; triesman and davies , 1973 ; shiffrin and grantham , 1974 ; alais et al . , 2006b ; santangelo et al . , 2010 ) . for example , larsen et al . ( 2003 ) compared subjects accuracy for identification of two concurrent stimuli ( such as a visual and spoken letter ) relative to performance in a single - task . they found that the proportion of correct response was almost the same for all experimental conditions and , furthermore , in the divided - attention condition the probability to correctly report a stimulus in one modality was independent of whether the stimulus was correctly reported in the other modality . similarly , bonnel and hafter ( 1998 ) used an audiovisual dual - task paradigm to show that when identification of the direction of a stimulus change is capacity - limited ( see above ) , simple detection of visual and auditory patterns is governed by capacity - free processes , as in the detection task there was no performance drop compared with single - task controls . visual thresholds were unaffected by concurrent pitch discrimination of chords and vice versa . however , when two tasks were performed within the same modality , thresholds increased by a factor of around two for visual discrimination and four for auditory discrimination . in line with these psychophysical results , a variety of imaging studies suggests that attention can act unimodally at early levels including the primary cortices such as a1 and v1 ( jancke et al . , 1999a , b ; posner and gilbert , 1999 ; somers et al . , 1999 ) . most of the studies mentioned deal with dual - task conditions where both tasks are brief ( hundreds of milliseconds ) stimuli to be detected or discriminated . very few consider conditions in which one of the tasks must be performed by continuously monitoring a specific pattern over a temporal scale of seconds , even though this is a typical requirement for many everyday activities , such as reading or driving . we measure performance on the multiple object - tracking ( mot ) task of pylyshyn and storm ( 1988 ) , while asking subjects to perform simultaneously either a visual contrast discrimination task or an auditory pitch discrimination task . the results show strong within - modality interference , but very little cross - modality interference , strongly supporting the idea that in sustained tasks each modality has access to a separate pool of attentional resources . four naive subjects ( two males and two females , mean age 26 years ) , all with normal hearing and normal or corrected - to - normal visual acuity , served as subjects . all gave informed consent to participate to the study that was conducted in accordance with the guidelines of the university of florence . all visual stimuli were presented on a sony trinitron crt monitor ( screen resolution of 1024 768 pixels , 32 bit color depth , refresh rate of 60 hz , and mean luminance 68.5 cd / m ) subtending ( 40 30 ) at the subjects view distance of 57 cm . to create visual stimuli we used psychophysics toolbox ( version 2 ) for matlab ( brainard , 1997 ; pelli , 1997 ) on a mac g4 running mac osx 9 . auditory stimuli were digitized at a rate of 65 khz , and presented through two high quality loudspeakers ( creative mms 30 ) flanking the computer screen and lying in the same plane 60 cm from the subject . speaker separation was around 80 cm and stimuli intensity was 75 db at the sound source . the primary task was visual tracking of multiple moving objects ( mot ; pylyshyn and storm , 1988 ) . the mot task consisted of 12 disks ( diameter 0.9 ) moving across a gray background at 5/s . they moved in straight lines , and when colliding with other dots or the sides bounced appropriately ( obeying the laws of physics ) . at the start of each trial 35 disks were displayed in green ( xyy coordinates = 0.25,0.69 , 39.5 ) for 2 s to indicate that those were the targets whilst the remaining were displayed an isoluminant red ( xyy coordinates = 0.61 , 0.33 , 39.5 ) . the trial continued for 4 s ( tracking period ) , then disks stopped and four became orange ( xyy coordinates = 0.52,0.44 , 39.4 ; see movie s1 in supplementary material ) . the subjects task was to choose which of these was the target ( only one valid target turned orange on each trial ) . subjects were familiarized with the task during a training session of 50 trials before starting the experimental protocol . each experimental session had five trials per condition ( varying in the number of dots to track ) for a total of 15 trials per session . no feedback was provided , but subjects could check their overall performance at the end of each session . stimuli for the secondary visual task were luminance - modulated gratings of 0.5 s duration with a spatial frequency of 3 c / deg covering the entire screen . on each trial ( 4 s duration , during the dot tracking ) subjects were presented with a sequence of three gratings , ramped in and out within a raised cosine envelope ( over 20 ms ) , with an inter stimulus interval randomly chosen between 0.5 and 1.3 s. two out of three gratings had the same contrast ( 50% ) while the target grating ( that subjects had to detect ) , randomly first , second or third in the sequence , had more or less contrast . the size of the contrast difference ( ) was chosen from trial to trial by means of an adaptive staircase quest ( watson and pelli , 1983 ) that homed in on threshold ( 67% of correct responses ) . the auditory secondary stimulus was of a sequence of three tones with the same presentation duration and temporal spacing as the visual version , two reference stimuli of 880 hz with the target frequency differing from trial to trial by hz . in the dual - task condition subjects performed both the contrast or frequency discrimination task , and the mot task . to avoid possible biases for response order we counterbalanced subjects responding first to the mot task with those that responded for to the secondary task . four naive subjects ( two males and two females , mean age 26 years ) , all with normal hearing and normal or corrected - to - normal visual acuity , served as subjects . all gave informed consent to participate to the study that was conducted in accordance with the guidelines of the university of florence . all visual stimuli were presented on a sony trinitron crt monitor ( screen resolution of 1024 768 pixels , 32 bit color depth , refresh rate of 60 hz , and mean luminance 68.5 cd / m ) subtending ( 40 30 ) at the subjects view distance of 57 cm . to create visual stimuli we used psychophysics toolbox ( version 2 ) for matlab ( brainard , 1997 ; pelli , 1997 ) on a mac g4 running mac osx 9 . auditory stimuli were digitized at a rate of 65 khz , and presented through two high quality loudspeakers ( creative mms 30 ) flanking the computer screen and lying in the same plane 60 cm from the subject . speaker separation was around 80 cm and stimuli intensity was 75 db at the sound source . the primary task was visual tracking of multiple moving objects ( mot ; pylyshyn and storm , 1988 ) . the mot task consisted of 12 disks ( diameter 0.9 ) moving across a gray background at 5/s . they moved in straight lines , and when colliding with other dots or the sides bounced appropriately ( obeying the laws of physics ) . at the start of each trial 35 disks were displayed in green ( xyy coordinates = 0.25,0.69 , 39.5 ) for 2 s to indicate that those were the targets whilst the remaining were displayed an isoluminant red ( xyy coordinates = 0.61 , 0.33 , 39.5 ) . the trial continued for 4 s ( tracking period ) , then disks stopped and four became orange ( xyy coordinates = 0.52,0.44 , 39.4 ; see movie s1 in supplementary material ) . the subjects task was to choose which of these was the target ( only one valid target turned orange on each trial ) . subjects were familiarized with the task during a training session of 50 trials before starting the experimental protocol . each experimental session had five trials per condition ( varying in the number of dots to track ) for a total of 15 trials per session . no feedback was provided , but subjects could check their overall performance at the end of each session . stimuli for the secondary visual task were luminance - modulated gratings of 0.5 s duration with a spatial frequency of 3 c / deg covering the entire screen . on each trial ( 4 s duration , during the dot tracking ) subjects were presented with a sequence of three gratings , ramped in and out within a raised cosine envelope ( over 20 ms ) , with an inter stimulus interval randomly chosen between 0.5 and 1.3 s. two out of three gratings had the same contrast ( 50% ) while the target grating ( that subjects had to detect ) , randomly first , second or third in the sequence , had more or less contrast . the size of the contrast difference ( ) was chosen from trial to trial by means of an adaptive staircase quest ( watson and pelli , 1983 ) that homed in on threshold ( 67% of correct responses ) . the auditory secondary stimulus was of a sequence of three tones with the same presentation duration and temporal spacing as the visual version , two reference stimuli of 880 hz with the target frequency differing from trial to trial by hz . in the dual - task condition subjects performed both the contrast or frequency discrimination task , and the mot task . to avoid possible biases for response order we counterbalanced subjects responding first to the mot task with those that responded for to the secondary task . to evaluate the costs of dividing attention between sensory modalities , we measured subject performance for visual tracking alone , or with either an auditory or a visual secondary task . figure 1 shows the individual results for the three experimental conditions , plotting performance ( d ) in the dual - task conditions against single - task performance . each small symbol indicates individual subject performance in a given condition defined by the number of dots to track whilst large circles indicate the data averaged across subjects and conditions . it is quite clear that the concurrent visual task greatly reduced performance , shown by the average decrease in d from 2.48 to 1.50 , and also by the fact that all individual data lie below the equality line . the difference was highly significant ( one - tailed paired t - test : t11 = 6.98 , p < 0.001 ) . however , when the competing task was auditory rather than visual , there was no effect on tracking performance . average d was virtually unchanged ( 2.48 vs 2.28 ) , certainly not significant ( one - tailed paired t - test ; t11 = 1.07 , p = 0.30 ) . sensitivities for the mot task performed alone ( on the abscissa ) plotted against sensitivities for dual - task conditions ( on the ordinate ) . the 12 data points represent 4 subjects in 3 experimental conditions , defined by the number of dots to track ( from 3 to 5 ) . green symbols refer to the intra - modal condition ( secondary task contrast discrimination ) , red to the cross - modal condition ( auditory secondary task ) . small symbols refer to individual data ( different symbols shape indicates different number of dots to track : three dots triangles , four dots diamond , and five dots pentagon ) whilst large symbols to averages . there is a clear effect for intra - modal interference , but not for cross - modal interference . we also measured sensitivity for both the visual and auditory secondary tasks when performed alone and matched these results with those achieved in the dual - task condition . psychometric functions for auditory frequency discrimination ( left panel ) and visual contrast discrimination ( right panel ) for subjects f. g. performance in the auditory task was almost identical when frequency discrimination was performed alone ( black data points and lines ) or together with a visual mot task ( blue data points and lines ) as shown by the almost overlapping curves . however , when the two concurrent tasks were of the same sensory modality ( vision ) , subject performance was dramatically reduced by around a factor of 3 . auditory frequency discrimination is shown on the left , visual contrast discrimination on the right . it is obvious that the auditory discrimination was little affected by the concurrent visual tracking task . the two psychometric functions ( best fitting cumulative gaussian functions ) are virtually identical , yielding thresholds ( frequency yielding 66% correct target identification ) close to 67 hz in both conditions . however , visual contrast discrimination thresholds were much higher in the dual than in the single - task condition , 5.1 compared with 14.5 ( a factor of nearly three ) . figure 3 plots for all subjects the interference factor ( ratio of dual- to single - task thresholds ) for the within and between modality conditions . it is clear that the auditory task is relatively immune to interference ( average factor 0.05 ) , while performance for luminance contrast discrimination thresholds increased by a factor of more than 2.5 . subject performance on the secondary task , either auditory ( red bars ) or visual ( green bars ) . the interference factor is defined as the ratio between dual - task and single - task thresholds ( a value of one meaning no interference between modalities . in this paper we asked whether vision and audition share cognitive attentional resources in performing sustained tasks , particularly relevant for everyday functioning . as most previous research has been restricted to tasks spanning only a few milliseconds ( larsen et al . , 2003 ; alais et al . , 2006b ) , or conditions with fast streams of simple auditory or visual patterns ( duncan et al . , 1997 ) , our study provides new knowledge about attentional mechanisms in ecological situations , where prolonged monitoring of information is necessary . the results clearly indicate that under these conditions , vision and audition have access to separate cognitive resources . performance on a sustained task , typical of everyday requirement , was completely unaffected by a concurrent auditory discrimination task . the lack of interference did not reflect a bias in deploying attention on the visual primary task more than on the auditory task , as both tasks were performed as well as when they were presented alone . on the other hand , sharing attention between two tasks of the same sensory modality produces a robust decrease of performance for both primary and secondary tasks . that vision and audition have access to separate cognitive resources is consistent with imaging studies showing that attention can modulate responses in primary and secondary visual and auditory cortexes ( gandhi et al . if in both modalities attentional effects modulate neural responses at these early of sensory information processing , when the visual and auditory signals are relatively independent , it is reasonable that few interactions are seen between these two senses . our results are important not only for the psychophysical data on the role of sustained attention between modalities , but also because they establish guidelines in designing audio visual instrumentation . information should be divided as much as possible between modalities , to maximize on the attentional resources available . this becomes increasingly more important as more virtual - reality applications are developed and are used routinely in everyday life . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

our perceptual capacities are limited by attentional resources . one important question is whether these resources are allocated separately to each sense or shared between them . we addressed this issue by asking subjects to perform a double task , either in the same modality or in different modalities ( vision and audition ) . the primary task was a multiple object - tracking task ( pylyshyn and storm , 1988 ) , in which observers were required to track between 2 and 5 dots for 4 s. concurrently , they were required to identify either which out of three gratings spaced over the interval differed in contrast or , in the auditory version of the same task , which tone differed in frequency relative to the two reference tones . the results show that while the concurrent visual contrast discrimination reduced tracking ability by about 0.7 d , the concurrent auditory task had virtually no effect . this confirms previous reports that vision and audition use separate attentional resources , consistent with fmri findings of attentional effects as early as v1 and a1 . the results have clear implications for effective design of instrumentation and forms of audio visual communication devices .

Introduction Materials and Methods Subjects Stimuli and procedure Results Discussion Conflict of Interest Statement Supplementary Material

PMC3981440

long qt syndrome ( lqts ) is a rare inherited arrhythmogenic disease characterized by susceptibility to life - threatening arrhythmias and sudden cardiac death . anomalous origin of coronary arteries is also a relatively rare congenital malformation and has been reported as the cause of angina pectoris , arrhythmia , syncope and fatal myocardial infarction . the only relevant feature in her history was palpitation episodes especially during swimming for a few years and she was unaware of her grade 1 hypertension . there was no sudden death or cardiac arrest or syncope both in her and her family 's medical history . on her electrocardiogram ( ecg ) we determined long qt pattern with qtc prolongation of 482 msn ( figure 1a ) . she was given 50 mg metoprolol daily and underwent 24 h holter ecg monitorization which revealed no arrhythmia record . according to her medical history and qt prolongation pattern we classified this case as romano - ward long qt syndrome type 1 . we planned a six month follow up visit but 2 months after the first visit she was admitted to our emergency room with palpitation and chest pain during sleep . on her admission ecg there was no ischemic changes and no arrhythmia but we hospitalized the patient because of typical angina . because of dynamic t wave changes and typical angina we performed a coronary computed tomography angiography ( cta ) ( figure 1b ) to rule out coronary artery disease . we did not prefer diagnostic coronary angiography because there were no risk factors and troponin levels were negative during follow up . coronary cta showed an anomalous origin of right coronary artery with mechanical compression of the anomalous right coronary artery between the aorta and pulmonary root , which causes a moderate stenosis . because of this moderate stenosis and angina we performed a myocardial perfusion scintigraphy and we continued -blocker therapy for the patient and did not plan icd because there was no previous cardiac arrest , syncope and / or documented ventricular tachycardia , also her qtc was under 500 ms . ecg recording from the mother ( 67-year - old ) and the son ( 5-year - old ) of the patient also revealed lqts ( figures 1c and 1d ) . but these relatives had no history of arrhythmic episodes . figure 1a)electrocardiogram ( ecg ) recording of the patient ; b ) right coronary artery with anomalous origin from the aorta partially running between the aorta and pulmonary artery where moderate stenosis is seen ( arrow ) . ao , aorta ; pa , pulmonary artery ; c ) ecg recording from the mother of the patient ; d ) ecg recording from the son of the patient . a)electrocardiogram ( ecg ) recording of the patient ; b ) right coronary artery with anomalous origin from the aorta partially running between the aorta and pulmonary artery where moderate stenosis is seen ( arrow ) . ao , aorta ; pa , pulmonary artery ; c ) ecg recording from the mother of the patient ; d ) ecg recording from the son of the patient . lqts is an inherited arrhythmogenic disease characterized by susceptibility to life - threatening arrhythmias , sudden cardiac death . two major forms of lqts have been identified , one transmitted as an autosomal dominant trait ( romano ward syndrome ) , the second is an autosomal recessive disease ( jervell and lange - nielsen syndrome ) . therapy for lqts is directed toward reduction of the incidence of syncope and sudden death . the data to guide management comes from large registries and referral centers with a bias toward patients with severe disease . the pacemaker function is also used in those with pause - dependent or bradycardia - induced ventricular tachycardia . an important clinical dilemma has been deciding with certainty who should or should not have a defibrillator . the american college of cardiology / american heart association / european society of cardiology have issued guidelines , which include the use of icds , for the management of lqts . prophylactic b - blockade has a class i indication for all individuals with abnormal prolongation of the qt interval , regardless of symptoms . icds have a class i indication in secondary prevention for those surviving cardiac arrest , class iia for those with symptoms or syncope while taking b - blockers , and class iib for primary prevention in those with possible high - risk characteristics . if the patient has syncope despite full dose -blockade icd implantation should be considered with the final decision being based on the individual patient characteristics ( age , sex , previous history , genetic subgroup including sometimes mutation - specific features , presence of ecg signs including 24 h holter recordings indicating high electrical instability ) . for this case we did not plan icd because there was no previous cardiac arrest , syncope and/or documented ventricular tachycardia in her medical history , also measured qtc was under 500 ms . if genetic analysis is not available it is best to avoid very strenuous exercise and keep stress at bay . breathing exercises , meditation , and yoga are all ways to help manage stress . eating foods high in potassium , such as bananas , or take potassium supplements symptomatic patients must also stay away from jobs that might threaten the lives of other people ( pilot , bus driver etc . ) anomalous origin of coronary arteries has been reported as the cause of angina pectoris , arrhythmia , syncope and fatal myocardial infarction . the incidence of anomalous origin of the right coronary artery ( rca ) out of the right sinus of valsalva ranges from under 0.010.09% . the exact mechanism , associated to cardiac events , is unclear in cases of coronary artery with anomalous origin and no obvious obstructive lesion . it might be related to mechanical compression of the anomalous coronary artery between the aorta and pulmonary root or great vessels , especially during exercise . surgical repair is recommended , especially with anomalous origin of the left coronary artery ( lca ) . however , there is controversy concerning the treatment of anomalous right coronary artery ( rca ) with interarterial course due to its relatively high incidence and the fact that it leads to few , if any , clinical problems . all two entities are associated with an increased risk for developing malignant tachyarrhythmia and sudden cardiac death . this may be conflicting enough to overlook coronary artery disease or lqts itself mimicking coronary artery disease . further - more this case emphasizes that these two conditions may coexist in the same patient . if angina or ecg changes occur , one of coronary imaging techniques must be chosen to investigate coronary artery disease according to the patient 's risk factors and coronary ct angiography seems like the best option in low risk group . surgical repair or icd implantation were not necessary for our patient but detection of coronary artery disease or coronary artery anomalies causing ischemia may change medical treatment as life saving .

anomalous origin of coronary arteries is also a relatively rare congenital malformation and has been reported as the cause of angina pectoris and arrhythmia . long qt syndrome ( lqts ) is a rare inherited arrythmogenic disease characterized by susceptibility to life - threatening arrhytmias andsudden cardiac death . we present a 36-year - old patient in whom two rare anomalies coexist and treated succesfully with -blocker therapy .

Introduction Case Report Discussion

PMC2896090

bioinformatics is a vast and complex multidisciplinary research area where numerous tools have been developed over the years to analyse constantly growing amounts of data . since 1998 , the european bioinformatics institute ( embl ebi ) has provided public access to various mainstream sequence analysis applications ( 1,2 ) . these include sequence similarity search services ( http://www.ebi.ac.uk/tools/similarity.html ) , such as fasta ( 3 ) , blast ( 4,5 ) and interproscan ( 6 ) and multiple sequence alignment tools ( http://www.ebi.ac.uk/tools/sequence.html ) , such as clustalw ( 7 ) , t - coffee ( 8) , muscle ( 9 ) , kalign ( 10 ) and mafft ( 11 ) . these services are provided via a perl - cgi job dispatcher framework for managing job submission and result representation . the popularity of these services has made it necessary to redesign the system in order to minimize maintenance and enhance the integration of features requested by users . a new and modular framework , called jdispatcher , has been developed to improve the accessibility and quality of the services relevant to the biological community . jdispatcher is aimed at both novice and expert users and exposes novel methods of obtaining annotations and visualizing sequence analysis results through one uniform and consistent interface . these services are available interactively over the web and via soap and rest interfaces for systematic or programmatic use . the new framework provides input validation to assure successful job submissions , offers new visualization features to assist in the interpretation of results and uses the ebi search engine , eb - eye ( 12 ) , to integrate relevant annotations . a user can submit sequences using web forms that contain all supported parameters and their possible values . the different tools have been grouped into categories based on their purpose ( table 1 ) . table 1.tools available in the jdispatcher frameworkcategorytoolsequence similarity searches ( sss)psisearch , psiblast , ncbiblast , wublast , fasta , ssearch , ggsearch and glsearchmultiple sequence alignments ( msa)clustalw2 , tcoffee , kalign , muscle , mafft , and prank tools available in the jdispatcher framework within a category , the tools share the same interface design , which uses well established usability patterns , such as wizard - like steps to guide the user through the submission process . it makes use of decision - trees to validate all the parameters required to warrant successful job submissions . if the validation fails , the user is notified about which specific parameters or data are invalid , and the job is not submitted . alternatively , jdispatcher assigns a unique job identifier and sends a request to a workload management system for the job to be executed . the identifier is then used to keep track of the tasks and to retrieve the results when they become available . the results of an analysis are made available using various representations ( e.g. html tables , xml files , images , etc . ) . in order to produce these representations , each result is converted into a generic category - specific model that is used by a renderer that generates the requested output . the renderers are specific to the model and not to the tool , and thus are available across all the tools in a category . the availability of multiple views of the same data helps the user to interpret and compare results from different tools within a category . sequence search algorithms produce limited hits annotation . with the new framework it is possible to navigate hits and access related information . figure 1 shows the summary table of an ssearch of mouse glomulin ( uniprotkb / swiss - prot glmn_mouse ) , which is essential for the development of the vascular system , against the uniprotkb / swiss - prot database ( 13 ) . each column heading has clickable arrows that allow the user to sort the results according to the values in the columns [ e.g. sequence length , score , percentage identity , positives and e()-value ] . each match is enriched with links to cross - references and related information in various data resources ( e.g. gene expression , genomic sequences , structures , function , ontologies and literature citations ) . optionally , the alignment from the search , and/or the full - annotation for the selected matches can be displayed . figure 1.summary table view of the results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch . summary table view of the results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch . figure 2 shows the visual output obtained from searches using ssearch and ncbi blast of the glomulin sequence against uniprotkb / swiss - prot using default parameters . comparison of the two images reveals notable differences in the sequence matches reported by the two search methods . for example , differences in the aligned regions between glomulin and aberrant root formation protein 4 for arabidopsis ( alf4_arath ) are clearly visible in both ; ssearch identifies two mon2 homologues at e()-values < 1 ( mon2_xenla and mon2_human ) , which may indicate there is a structural relationship between glmn at the c - terminus of the mon2 homologues , although these may not share related functions . figure 2.comparisons between the visual output results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch and ncbi blast , respectively . comparisons between the visual output results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch and ncbi blast , respectively . determining which functional domains and families a protein belongs to is critical to the understanding of the biological processes it may be involved in . this is important for the characterization of existing drug targets as well as in the identification of novel ones . family and domain functional predictions have been built into the framework , using pre - calculated matches from the interpro consortium ( 14 ) data . this enables users , not only to search for sequence similarities when using the uniprot databases , but also to characterize the sequence query in terms of domain architectures that may elicit its function . figure 3 shows functional predictions for a hypothetical bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . the hypothetical sequence has several good homologues , all belonging to the gpcr rhodopsin - like superfamily , which are clearly seen . this indicates the query protein could represent a potential target for receptor - binding studies . figure 3.functional prediction view of the results obtained when comparing the sequence of putative bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . functional prediction view of the results obtained when comparing the sequence of putative bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . in both , the visual output and functional predictions result representations , the matches are coloured , from red to blue , according to e()-value , using a relative scale , from the most to the least significant hits within the result . an absolute scale , which ranges from e ( ) = 0 to e()=1.0 , is also available . these aim to aid the user in deciding whether weak similarities may be biologically significant . these images are available in scalable vector graphics ( svg ) , portable network graphic ( png ) and jpeg output , providing wide compatibility . the raw result and processed forms , such as the summary table content and xml formats are downloadable for further processing by the user . the examples above illustrate how , from a single sequence similarity search , it is possible to access related sources of annotation , determine visually which results are relevant and infer gene and protein functional associations , using the jdispatcher framework . web services technologies have opened up important opportunities for the analysis of life sciences data . it is now well established that sharing resources , across geographically distributed networks , is advantageous to scientists and bioinformaticians through the re - use of generic services , such as those presented in this article . the new jdispatcher framework provides multiple front - ends : in addition to the web interface , soap and rest apis ( http://www.ebi.ac.uk/tools/webservices/ ) have been implemented to offer programmatic access using accepted web services standards . the soap and rest apis cater for users requiring systematic access to a wide range of sequence similarity search and multiple sequence alignment services , which can be built into local analytical workflows and pipelines ( e.g. taverna ( 15 ) , triana ( http://www.trianacode.org/ ) , knime ( www.knime.org ) ( 16 ) and pipeline pilot ( http://accelrys.com/products/scitegic/index.html))typical usage scenarios include the characterization of novel genomes and proteomes and the analysis of data derived from meta - genome experiments . using the apis , complex applications can be developed in various programming languages , which include : c / c++ , c # , java , perl , php , python and ruby , or scripting environments such a bash , csh , batch and powershell . this allows integration of services into existing and/or new applications that require access to fast sequence database searching or multiple sequence alignment methods . to facilitate this type of usage , the services provide extensive meta - information describing the available parameters , including their possible values and descriptions of their purpose . typical applications of the jdispatcher framework services include : providing an alternative interface for specialist usage targeted at a specific community ; integrating a service into an existing data portal to provide analysis services ; and enhancing analysis results by directly connecting the result with the data . these are of importance to service providers and users of pipelines who may not have the resources to run and maintain the infrastructure required to support equivalent functionality . jdispatcher is aimed at both novice and expert users and exposes novel methods of obtaining annotations and visualizing sequence analysis results through one uniform and consistent interface . these services are available interactively over the web and via soap and rest interfaces for systematic or programmatic use . the new framework provides input validation to assure successful job submissions , offers new visualization features to assist in the interpretation of results and uses the ebi search engine , eb - eye ( 12 ) , to integrate relevant annotations . a user can submit sequences using web forms that contain all supported parameters and their possible values . the different tools have been grouped into categories based on their purpose ( table 1 ) . table 1.tools available in the jdispatcher frameworkcategorytoolsequence similarity searches ( sss)psisearch , psiblast , ncbiblast , wublast , fasta , ssearch , ggsearch and glsearchmultiple sequence alignments ( msa)clustalw2 , tcoffee , kalign , muscle , mafft , and prank tools available in the jdispatcher framework within a category , the tools share the same interface design , which uses well established usability patterns , such as wizard - like steps to guide the user through the submission process . it makes use of decision - trees to validate all the parameters required to warrant successful job submissions . if the validation fails , the user is notified about which specific parameters or data are invalid , and the job is not submitted . alternatively , jdispatcher assigns a unique job identifier and sends a request to a workload management system for the job to be executed . the identifier is then used to keep track of the tasks and to retrieve the results when they become available . the results of an analysis are made available using various representations ( e.g. html tables , xml files , images , etc . ) . in order to produce these representations , each result is converted into a generic category - specific model that is used by a renderer that generates the requested output . the renderers are specific to the model and not to the tool , and thus are available across all the tools in a category . the availability of multiple views of the same data helps the user to interpret and compare results from different tools within a category . sequence search algorithms produce limited hits annotation . with the new framework it is possible to navigate hits and access related information . figure 1 shows the summary table of an ssearch of mouse glomulin ( uniprotkb / swiss - prot glmn_mouse ) , which is essential for the development of the vascular system , against the uniprotkb / swiss - prot database ( 13 ) . each column heading has clickable arrows that allow the user to sort the results according to the values in the columns [ e.g. sequence length , score , percentage identity , positives and e()-value ] . each match is enriched with links to cross - references and related information in various data resources ( e.g. gene expression , genomic sequences , structures , function , ontologies and literature citations ) . optionally , the alignment from the search , and/or the full - annotation for the selected matches can be displayed . figure 1.summary table view of the results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch . summary table view of the results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch . figure 2 shows the visual output obtained from searches using ssearch and ncbi blast of the glomulin sequence against uniprotkb / swiss - prot using default parameters . comparison of the two images reveals notable differences in the sequence matches reported by the two search methods . for example , differences in the aligned regions between glomulin and aberrant root formation protein 4 for arabidopsis ( alf4_arath ) are clearly visible in both ; ssearch identifies two mon2 homologues at e()-values < 1 ( mon2_xenla and mon2_human ) , which may indicate there is a structural relationship between glmn at the c - terminus of the mon2 homologues , although these may not share related functions . figure 2.comparisons between the visual output results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch and ncbi blast , respectively . comparisons between the visual output results obtained when searching the sequence of mouse glomulin against the uniprotkb / swiss - prot database using ssearch and ncbi blast , respectively . determining which functional domains and families a protein belongs to is critical to the understanding of the biological processes it may be involved in . this is important for the characterization of existing drug targets as well as in the identification of novel ones . family and domain functional predictions have been built into the framework , using pre - calculated matches from the interpro consortium ( 14 ) data . this enables users , not only to search for sequence similarities when using the uniprot databases , but also to characterize the sequence query in terms of domain architectures that may elicit its function . figure 3 shows functional predictions for a hypothetical bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . the hypothetical sequence has several good homologues , all belonging to the gpcr rhodopsin - like superfamily , which are clearly seen . . figure 3.functional prediction view of the results obtained when comparing the sequence of putative bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . functional prediction view of the results obtained when comparing the sequence of putative bioactive lysophospholipid that was compared against uniprotkb / swiss - prot using ncbi blast . in both , the visual output and functional predictions result representations , the matches are coloured , from red to blue , according to e()-value , using a relative scale , from the most to the least significant hits within the result . an absolute scale , which ranges from e ( ) = 0 to e()=1.0 , is also available . these aim to aid the user in deciding whether weak similarities may be biologically significant . these images are available in scalable vector graphics ( svg ) , portable network graphic ( png ) and jpeg output , providing wide compatibility . the raw result and processed forms , such as the summary table content and xml formats are downloadable for further processing by the user . the examples above illustrate how , from a single sequence similarity search , it is possible to access related sources of annotation , determine visually which results are relevant and infer gene and protein functional associations , using the jdispatcher framework . web services technologies have opened up important opportunities for the analysis of life sciences data . it is now well established that sharing resources , across geographically distributed networks , is advantageous to scientists and bioinformaticians through the re - use of generic services , such as those presented in this article . the new jdispatcher framework provides multiple front - ends : in addition to the web interface , soap and rest apis ( http://www.ebi.ac.uk/tools/webservices/ ) have been implemented to offer programmatic access using accepted web services standards . the soap and rest apis cater for users requiring systematic access to a wide range of sequence similarity search and multiple sequence alignment services , which can be built into local analytical workflows and pipelines ( e.g. taverna ( 15 ) , triana ( http://www.trianacode.org/ ) , knime ( www.knime.org ) ( 16 ) and pipeline pilot ( http://accelrys.com/products/scitegic/index.html))typical usage scenarios include the characterization of novel genomes and proteomes and the analysis of data derived from meta - genome experiments . using the apis , complex applications can be developed in various programming languages , which include : c / c++ , c # , java , perl , php , python and ruby , or scripting environments such a bash , csh , batch and powershell . this allows integration of services into existing and/or new applications that require access to fast sequence database searching or multiple sequence alignment methods . to facilitate this type of usage , the services provide extensive meta - information describing the available parameters , including their possible values and descriptions of their purpose . typical applications of the jdispatcher framework services include : providing an alternative interface for specialist usage targeted at a specific community ; integrating a service into an existing data portal to provide analysis services ; and enhancing analysis results by directly connecting the result with the data . these are of importance to service providers and users of pipelines who may not have the resources to run and maintain the infrastructure required to support equivalent functionality . the modularity of this new framework reduces maintenance overheads and simplifies the addition of tools and features . keeping the result data model and the renderers separate provides the flexibility to add additional representations to all functionally related tools . the presented visualization examples highlight important insights in the understanding of existing and new nucleotide and protein sequences from both genomes and metagenome experiments and suggest novel ways in which these data can be interpreted . academic and commercial laboratories can integrate the jdispatcher framework services with their local analytical pipelines or workflows . these represent an important contribution to the growing number of available services in bioinformatics and have been submitted to the biocatalogue ( 17 ) ( www.biocatalogue.org ) , a registry of freely available web services in the life sciences . the european commission under felics [ contract number 021902 ( rii3 ) , within the research infrastructure action of the fp6 structuring the european research area programme ] ; core funding from the european molecular biology laboratory ; european patent office . funding for open access charge : embl .

the embl - ebi provides access to various mainstream sequence analysis applications . these include sequence similarity search services such as blast , fasta , interproscan and multiple sequence alignment tools such as clustalw , t - coffee and muscle . through the sequence similarity search services , the users can search mainstream sequence databases such as embl - bank and uniprot , and more than 2000 completed genomes and proteomes . we present here a new framework aimed at both novice as well as expert users that exposes novel methods of obtaining annotations and visualizing sequence analysis results through one uniform and consistent interface . these services are available over the web and via web services interfaces for users who require systematic access or want to interface with customized pipe - lines and workflows using common programming languages . the framework features novel result visualizations and integration of domain and functional predictions for protein database searches . it is available at http://www.ebi.ac.uk/tools/sss for sequence similarity searches and at http://www.ebi.ac.uk/tools/msa for multiple sequence alignments .

INTRODUCTION JDispatcher framework Results representation Web Services CONCLUSIONS FUNDING

PMC5024354

the drag exerted by the earth surface on the atmospheric flow has long been recognized to play an important role in the general circulation of the atmosphere [ holton , 2004 ] . this follows from considerations related to the angular momentum budget , which leads to a picture of angular momentum fluxes in the free troposphere balancing surface drag and explains the presence of surface easterlies in the tropics and surface westerlies in the midlatitudes . although it is clear that the magnitude of the surface stress controls the strength of the surface easterlies and westerlies , it has also , more surprisingly , been shown to affect the location of the surface westerly jet in the midlatitudes [ robinson , 1997 ; chen et al . , 2007 ] . moreover , the drag exerted by topography is particularly important for the northern hemisphere ( nh ) winter extratropical circulation . the stationary rossby ( or planetary ) waves forced by the flow over the himalayas and rockies were shown to explain the winter mean longitudinal distribution of 500 hpa heights in the nh midlatitudes [ charney and eliassen , 1949 ; held et al . , the drag exerted by orographically generated gravity waves has also been shown to affect the nh winter zonal mean flow both in the troposphere and the lower stratosphere since the early days of weather and climate prediction [ wallace et al . , 1983 ; palmer et al . , 1986 ; these early studies emphasized the importance of parameterizing orographic gravity wave effects in general circulation models ( gcms ) in order to reproduce the observed mean patterns of the nh winter circulation . later studies demonstrated that the orographic gravity wave drag affects the zonal mean flow by changing the resolved planetary waves [ mclandress and mcfarlane , 1993 ; shaw et al . the parameterization of such orographic lowlevel blocking effects influences the representation of the nh winter circulation in both weather forecast and climate simulations [ lott and miller , 1997 ; zadra et al . , 2003 ; sandu et al . albeit recognized as being important for both weather forecasts and climate simulations , the representation of drag processes associated with surface stress is a major source of uncertainty in gcms . parameters controlling their strength are poorly constrained and often tuned to obtain the desired model behavior , i.e. , forecast skill [ sandu et al . the uncertainty not only stems from the lack of observational constraints , but also from the fact that surface stress has received relatively little attention compared to other processes such as clouds , convection , or radiation . in the last couple of years , the interest in the surface momentum budget has increased , particularly in the framework of the world meteorological organization 's working group for numerical experimentation ( wgne ) and of a workshop on the momentum budget and its role in weather and climate organized in march 2015 at eynsham hall , united kingdom . in 2014 , wgne set up a model intercomparison exercise of subgrid ( or unresolved ) surface stress , the wgne drag project . this exercise aims to compare the total subgrid surface stress ( tot ) and its partitioning among the parameterizations that most models use to represent it : the boundary layer scheme and the subgrid orography scheme ( section 2 will provide more details on the representation of subgrid surface stress in models ) . monthly mean zonal and meridional subgrid surface stresses from the first 24 h of shortrange forecasts performed for january and july 2012 were gathered from 12 modeling groups [ zadra , 2015 ] . since the models forecasts were starting from similar initial states , the angular momentum fluxes in the free atmosphere can be presumed to be similar and one would then expect similar total surface stresses . first results of this exercise suggest that while the intermodel spread is relatively small over the oceans , tot is highly modeldependent over land [ zadra , 2015 ] . as the subgrid , or parameterized stress characterizes the unresolved part of the flow , the intermodel spread found over land is due in part to differences in horizontal resolution . nevertheless , the zonally averaged magnitude of tot can still differ by as much as 20% over land even for models of comparable resolution , such as the integrated forecast system ( ifs ) of the european centre for mediumrange weather forecasts ( ecmwf ) and the unified model ( um ) of the metoffice ( figure 1 ) . these differences in the zonal average stress are mostly related to differences in stress over orography ( not shown ) . moreover , the partition of tot between the boundary layer and subgrid orography schemes is also modeldependent . the um for example has almost twice as much subgrid orography stress and up to 50% less boundary layer stress than ifs in the nh ( figure 1 ) . finally , the wgne drag project also revealed that the diurnal cycle of tot is quite different among the models ( not shown ) . zonal averages of the magnitude of the total subgrid surface stress tot ( n / m ) and of its components for ifs ( black , scale on left ) and um ( blue , scale on left ) , from the results submitted for the wgne drag project for january 2012 , and relative difference between um and ifs ( red , scale on right ) . for ifs , the boundary layer stress ( left ) represents the sum of the stresses from the turbulence and turbulent orographic form drag schemes ; for um it represents the stress from the turbulence scheme which represents the orographic form drag implicitly through an orographic enhancement of the roughness ( see section 2 for details ) . for comparison with the results from the wgne drag project , the magnitude of the stress used to construct the zonal averages is computed in this plot only from monthly mean diurnal averages of the u and v components of the surface stress ( derived from the first 24 h of the daily 00utc forecasts ) . the zonal averages include only land grid cells and were smoothed using a 5 running mean . the ongoing wgne drag project emphasizes the importance of better constraining the representation of subgrid surface stress in models , especially in regions with orography . it also highlights the need to better understand how the schemes contributing to surface stress , and their uncertainties , affect weather and climate predictions . the present study looks into a particular aspect of this broader question , namely how some of these schemes impact the representation of the nh winter circulation on different time scales . more precisely , our study aims to address two of the questions raised by the wgne drag project , namely ( i ) whether differences in tot of the order of those revealed by the wgne drag project matter for the representation of the nh winter circulation , and ( ii ) whether the partitioning of tot between the different schemes in a particular model is also important for predicting certain circulation aspects . as most of the uncertainty in surface stress comes from regions with orography , we focus on the impacts of two of the schemes which represent orographic drag processes . to answer these questions , we use the ifs to perform both 10 day weather forecasts and seasonal integrations , in which we mimic an increase in tot that is comparable to the difference between the ifs and the um shown in the wgne drag project . this increase is simulated in two ways , namely by changing a poorly constrained parameter either in the boundary layer or in the subgrid orography scheme . both changes concern the representation of subgrid orographic drag exerted on the flow at low levels . the representation of the subgrid surface stress in the ifs is described in section 2 . section 4 discusses the impacts of the changes to subgrid orographic drag on the representation of the nh winter circulation in short and mediumrange forecasts . particular emphasis is put on whether these impacts are different depending on which of the parameterizations is modified . the reason is that different schemes parameterize different dragrelated processes . in the ifs the boundary layer scheme represents stress associated with unresolved elements of the earth surface from horizontal scales less than 5 km , while the subgrid orography scheme ( sgo ) represents stress associated with horizontal scales between 5 km and the model resolution . the 10 day weather forecasts and the seasonal integrations discussed in this study are performed at horizontal resolutions of approximately 32 , respectively , 75 km at the equator ( section 3 ) . at their turn , each of these two schemes represents several processes . thus , the boundary layer scheme uses a turbulence parameterization ( turb hereafter ) to represent turbulent drag associated with surface elements such as land use or vegetation . the turb scheme is based on monin obukhov similarity and uses prescribed roughness length tables to describe the characteristic height of these surface elements [ ifs documentation , 2015 , part iv ] . the boundary layer scheme also includes a turbulent orographic form drag parameterization ( tofd hereafter ) to represent drag associated with subgrid orography elements with horizontal scales less than 5 km such as hills or individual mountains [ beljaars et al . , 2004 ] . the tofd stress is formulated in terms of the variance of the terrain slope , which is derived from an empirical representation of the height spectrum in the scale range of 5 km to a few meters ( this spectrum is derived from a 1 km resolution elevation data set ) . the vertical distribution of the stress is prescribed on model levels according to the formulation proposed by wood et al . . other models ( e.g. , the um of the metoffice ) do not have a special parameterization for turbulent orographic form drag , but represent it by artificially enhancing the surface roughness length over orography , so that the orographic form drag is implicitly represented by the turbulence parameterization . the impact of either the tofd parameterization or the orographic enhancement of roughness is poorly documented in the literature [ wallace et al . , 1983 ] , although it has been shown to affect the nh wintertime circulation to some extent [ sandu et al . the sgo scheme [ lott and miller , 1997 ] also represents different types of drag . if the airflow has enough energy to pass over the socalled effective subgrid mountain height , orographic gravity waves are generated . the subgrid stress associated with the propagation and breaking of these waves is represented by the gravity wave drag component of the sgo scheme ( gwd hereafter ) . at low levels , where the flow does not have enough energy the stress induced by this process is described by the lowlevel blocking component of the sgo scheme ( block hereafter ) . the depth over which the flow is blocked , and goes around the mountain , controls to a large extent the effect of the block scheme . this blocking depth is equal to the subgrid mountain height minus the depth of the layer where the flow goes over the mountain ( and generates gravity waves ) . the depth of this latter layer is defined by a critical froude number which makes both the gravity wave amplitude and the blocking depth stability dependent . for strong flow and/or weak stability , the gravity wave amplitude is large and the blocking depth is small , whereas for weak flow and very stable stratification the gravity wave amplitude is small and the blocking height large . other models use similar schemes to represent gravity wave drag and lowlevel blocking effects [ e.g. , mcfarlane , 1987 ; webster et al . , 2003 ; brown and webster , 2004 ] , and some include an orographic lift component , which describes a force perpendicular to the flow that depends on the wind direction relative to the orientation of the subgrid orography [ lott , 1999 ] . the contributions of the gwd and block schemes to the subgrid surface stress can not be disentangled in ifs , so they are presented combined hereafter as the sgo contribution to tot . special diagnostics were added for the purpose of this study within the boundary layer scheme in order to separate the contributions of the turb and tofd schemes to tot . figures 2 and 3 show how much each of these schemes contributes to tot in shortrange forecasts performed with the ifs for february 2014 ( the experimental setup is described in more detail in section 3 ) . in zonal average , the tofd and sgo schemes contribute fairly equally over land to the magnitude of tot ( figure 2 ) . their contribution is comparable to that of the turb scheme at latitudes with a fair amount of orography ( e.g. , mid to high latitudes in the nh ) . however , the maps shown in figure 3 indicate that the schemes act in different regions . the tofd and sgo act as expected in regions with orography , while the turb scheme acts predominantly in regions with increased synoptic activity ( i.e. , storm tracks ) and its contribution to tot over orography is small . these figures only illustrate 24 h averages of the monthly mean magnitude of the surface stress , ignoring thus differences in the diurnal cycle . however , as it will be shown in the next section , the stress produced by the various schemes has also different diurnal cycles . zonal averages of the magnitude of tot and of the contributions of the turb , tofd , and sgo schemes to tot ( n / m ) for the ctl experiment . these zonal averages are computed from monthly means of daily surface stress magnitude ( derived from the first 24 h of the daily 00utc forecasts performed for february 2014 ) , include only land grid cells and were smoothed using a 5 running mean . magnitude of the surface stress due to the turb , tofd , and sgo schemes ( n / m ) for the ctl experiment . these fields represent monthly means of the daily surface stress magnitude , derived from the first 24 h of the daily 00utc forecasts performed for february 2014 . the wgne drag project demonstrated that tot and its partition among the different schemes is largely modeldependent , particularly in regions with orography [ zadra , 2015 ] . moreover , individual model results heavily depend on the assumptions and choices of parameter values made within the schemes contributing to tot . given that it is currently not possible to constrain either tot or its orographic component with observations on global , and even on regional scale , the assumptions and parameter choices often rely on a rather pragmatic optimization approach . in previous work [ sandu et al . , 2013a , 2013b ] , we showed that the mediumrange forecast skill for the geopotential height during the nh winter can be improved by combining changes to the turb scheme concerning the degree of turbulent diffusion in stable layers with changes to certain parameters of the tofd and block schemes . in the present study , we look in more detail at the individual impacts of the tofd and block schemes on the nh winter circulation . these two schemes are likely the most poorly constrained and less documented in terms of impacts on the largescale circulation . given that they represent similar processes contributing to the surface stress ( orographic drag at low levels ) , these schemes also constitute good candidates for exploring the role of the partitioning of tot between different schemes . the forecasts start from the ecmwf operational analysis and are performed with a vertical resolution of 137 levels at a linear spectral truncation of t639 corresponding to approximately 32 km at the equator . second , seasonal integrations are performed with the same number of vertical levels , but at a lower spectral resolution , i.e. , t255 corresponding to approximately 75 km at the equator . these runs are initialized with the ecmwf interim reanalysis erai [ dee et al . , 2011 ] . they start on 1 august for every year between 1984 and 2014 and are integrated over a period of 7 months . this setup allows to examine the climate of the model during the winter season , decemberjanuaryfebruary ( djf ) , and its response to changes in subgrid orographic drag . the most recent operational ifs cycle ( cy41r1 operational since may 2015 ) is used for all experiments . for each of these two types of runs , we perform a control experiment with the reference model , an experiment with a larger magnitude of the stress resulting from the tofd scheme and an experiment with a larger magnitude of the stress resulting from the block scheme . the 10 day forecast experiments will be denoted as ctl , high tofd ( htofd ) , and high block ( hblock ) hereafter , while the seasonal integrations will be denoted as ctlcl , htofdcl , and hblockcl . the magnitude of the stress resulting from each scheme is enhanced by increasing the value of one of its poorly constrained constant factors , tofd for the tofd scheme and cd in the block scheme . these factors scale the stress ( ) in the two schemes as follows : ( 1)tofdtofdu|u|ftofd blockcdu|u|fblock , where is the air density , u the horizontal wind vector , ftofd is a function of the standard deviation of the filtered subgrid orography , and fblock is function of stability , the characteristics of the subgrid orography ( i.e. , standard deviation , slope , and anisotropy ) and the direction of the flow with respect to the mountain ridges ( a detailed description of the two schemes is included in ifs documentation [ 2015 , chap . 3.4 and 4 , part iv ] ) . the changes are such that they lead to a similar increase in the zonally averaged magnitude of tot ( defined as in the caption of figure 2 ) in the short range of the htofd and hblock forecasts . furthermore , this increase is similar to the difference between the ifs and the um ( figure 1 ) . thus , the increase in zonal mean tot in the htofd and hblock experiments is of approximately 1015% in the nh midlatitudes ( figure 4 ) , while the difference between the ifs and um in the wgne drag project was of about 1520% ( figure 1 ) . also note that an increase in the stress from any of the schemes leads to a reduction in near surface wind , which further results in a small reduction of the stress produced by the other schemes ( figure 4 ) . in the htofd experiment for example , the tofd stress increases by approximately 60% , while the turb and sgo stress each decrease by 10% . zonal averages of the magnitude of tot ( right ) and of the contributions of the turb , tofd , and sgo schemes to tot ( from left to right ) ( n / m ) for the ctl experiment ( black , scale on left ) . the green and red curves , scale on right , show the relative change of tot and of its components in the experiments htofd and hblock with respect to the ctl . the zonal averages are computed as in figure 2 . by design the zonally averaged magnitude of tot increases by a similar amount in the nh midlatitudes in the htofd and hblock experiments . however , a zoom on the tot increase in the himalaya region reveals that the tofd scheme enhances the stress over a larger area than the block scheme , which seems to act more near the edges of the mountain chain ( figure 5 ) . furthermore , figure 6 reveals that the diurnal cycle of the increase in tot is of opposite phase in the htofd and hblock experiments . thus , the increase in tot is maximum for htofd and minimum for hblock between 6 and 12 utc ( about 1218 local time ) . the boundary layer is mostly unstable ( or less stable ) and the nearsurface wind has a daily maximum due to strong turbulent mixing ( not shown ) . since the tofd scheme responds to wind speed only , it will reflect the diurnal cycle of the surface wind . however , the block scheme responds not only to wind speed , but is also strongly modulated by stability mainly through the depth of the blocked layer . when the wind speed is high and stability is weak , the flow the layer where the flow goes over the mountain is deep and the remaining layer where the flow is blocked is shallow . monthly mean difference in the daily magnitude of tot ( n / m ) between the htofd ( left ) , respectively , hblock ( right ) and the ctl experiment . time evolution of the monthly mean differences in the magnitude of tot ( n / m ) between the htofd ( green ) , respectively , hblock ( red ) and the ctl experiment during the first 5 days of the forecasts performed for february 2014 , integrated over the himalaya area showed in figure 5 . full lines indicated 6 h averages , and dashed lines indicate diurnal averages of the difference in the magnitude of tot . we examine the impacts of these two seemingly similar , yet different , ways of increasing tot in a particular model . we seek to understand the consequences of these changes on the nh winter circulation , first on daily time scales and then at longer time ranges . changes in surface pressure have traditionally been used to illustrate orographyrelated effects on the nh winter circulation since the first studies describing the parameterization of orographic gravity wave drag in gcms [ wallace et al . , 1983 ; palmer et al . , 1986 ; the impact of enhancing the orographic drag at low levels in the htofd and hblock 10 day forecast experiments is therefore first assessed in terms of surface pressure changes at short and medium ranges ( 15 days ) . the impacts on the representation of the nh circulation are then discussed in terms of zonal wind , temperature , and geopotential height . the enhancement of tot , simulated by increasing either the tofd or the block stress , leads to changes in the mean surface pressure in a matter of hours ( figure 7 ) . thus , pressure gradient changes across the regions with the largest mountain chains ( himalayas and the rocky mountains ) , as well as an increase in surface pressure over europe , can already be observed within the first 6 h of the forecasts . during the first 24 h these changes amplify and extend to larger spatial scales in both experiments , although they are always stronger for the htofd forecasts . mean change in surface pressure ( hpa ) in the experiments htofd ( left ) and hblock ( right ) with respect to the ctl , at lead time 6 ( top ) , 24 ( middle ) , and 120 ( bottom ) hours of the daily 00utc forecasts performed for february 2014 . the shortrange response in surface pressure over europe is particularly intriguing because it does not clearly resemble the pattern of the pressure changes over north america and east asia . this raises the question whether this is a local effect due to the alps or greenland or it is due to nonlocal effects caused by the changes in orographic drag over the rockies or the himalayas . more generally , the question is whether these changes in surface pressure in the short range are purely local orographic effects or are nonlocal effects due to impacts of the orographic drag on the zonal flow . to answer this question we performed additional daily 10 day forecasts for february 2014 . the changes made in the original htofd and hblock experiments were this time applied only in selected regions of the globe . for each of the two schemes , three new experiments were thus run in which the stress was enhanced over regions covering north america , greenland and europe , and east asia , illustrated by the green boxes in figure 8 . mean change in surface pressure ( hpa ) in the experiments in which the tofd , respectively , the block stress , are enhanced only in the regions demarked by the dark green lines , with respect to the ctl , at lead time 24 of the daily 00utc forecasts performed for february 2014 . the dark green hatches ( from 1 to 0 m / s , by 0.25 m / s ) indicate the deceleration of surface westerlies on the west side of the mountain ranges . the changes in surface pressure observed after 24 h over north america and east asia in the new experiments ( figure 8 , top , bottom ) are virtually identical with those obtained in the original htofd and hblock experiments ( figure 7 ) . the changes observed in the new experiments are also mainly due to a locally induced meridional pressure gradient ( figure 8 , middle ) . the decrease in surface pressure seen east / south of the alps in the experiment where the tofd stress is enhanced over greenland and europe only ( figure 8 , middle ) is outweighed however in the original htofd experiment ( figure 7 ) by the increase in pressure induced by the changes in stress over east asia ( figure 8 , bottom ) . as suggested by zadra et al . , the local response in surface pressure over the largest mountain chains can be understood in terms of geostrophic balance . the induced meridional pressure gradient across the rockies , alps , and , respectively , himalayas , corresponds to a westward geostrophic zonal wind anomaly , that is a deceleration of the midlatitude surface westerlies caused by the enhancement of the stress over orography . a decrease in the zonal surface wind of 1 to 0.2 m s can be indeed seen on the west side of the mountain chains for all experiments ( green hatches in figure 8) . that changing the drag exerted by orography on the flow at low levels leads to a rapid response in surface pressure is hardly a surprise , given our current understanding ( zadra et al . , 2003 ) . what is interesting instead is that although the changes made to the two schemes lead to a similar zonally averaged change in tot , the magnitude of the response in surface pressure is rather different ( figure 7 ) . one possibility is that differences in the spatial distribution and the diurnal cycle of tot ( figures 5 and 6 ) lead to a different response in surface pressure . the other possibility is that this response is damped to some extent in the hblock experiment due to the interactions between the gwd and block parameterizations . indeed , the lowlevel blocking and gravity wave drag are both represented in the sgo scheme . therefore , it is impossible to tell a priori whether a change of the stress at low levels , such as that simulated in the hblock experiment , does not also affect the orographic gravity waves . if this was the case , the changes in surface pressure obtained in the hblock experiment could be due at least in part to changes in the propagation and breaking of the gravity waves . to investigate this possibility , we repeated the ctl and hblock experiments while turning off the gwd part of the sgo scheme . the difference in mean surface pressure between these two new experiments matches closely that between the original hblock and ctl experiments shown in figure 7 . this suggests that the response obtained in the short range of the hblock forecasts is actually due to lowlevel blocking effects . after 5 days , the changes to the tofd scheme lead to an overall increase of the surface pressure at high latitudes and a decrease at low latitudes . the impacts on the zonal mean wind and temperature are also qualitatively similar , yet much stronger in the htofd experiment ( not shown ) . these results corroborate the findings of zadra et al . , who discussed in detail the impacts of introducing the lowlevel blocking parameterization of lott and miller in the gem model used at the canadian meteorological centre . this study demonstrated that the introduction of the blocking scheme leads to a weakening of the midlatitude westerlies in the nh during wintertime , the flow being affected up to approximately 150 hpa by day 5 of the forecasts ( see their figure 6 ) . it also induces a change in zonal mean temperature at latitudes higher than 40n characterized by a lowlevel warming and upper level cooling that also reaches 100 hpa by day 5 ( see their figure 9 ) . relative difference of root mean square error ( rmse ) of 500 and 1000 hpa geopotential height , between the htofd ( black ) , respectively , hblock ( red ) , and the ctl forecasts , as a function of lead time . when error bars do not cross the zero line , the performance of the respective experiment is significantly worse / better ( 95% interval ) than the ctl . for both the experiments and the ctl forecasts the rmse was computed with respect to the analysis from which the forecasts started . a positive difference in rmse indicates a deterioration of the model performance in the experiment with respect to the ctl . the effects obtained when increasing the tofd stress are stronger in all circulation aspects examined so far . the impact on the forecast performance is hence bigger in the htofd experiment ( figure 9 ) . what is surprising however is that the change made to the tofd stress considerably degrades the ability of the model to reproduce the nh circulation in the entire troposphere , while that made to the block stress leads to a slight improvement of the model performance . as illustrated by the increase , respectively , the reduction of the root mean square error of the geopotential height at 1000 and 500 hpa in figure 9 , these impacts are significant up to day 5 for the htofd , and day 10 for the hblock experiment . the enhancement of tot , simulated by increasing either the tofd or the block stress , leads to changes in the mean surface pressure in a matter of hours ( figure 7 ) . thus , pressure gradient changes across the regions with the largest mountain chains ( himalayas and the rocky mountains ) , as well as an increase in surface pressure over europe , can already be observed within the first 6 h of the forecasts . during the first 24 h these changes amplify and extend to larger spatial scales in both experiments , although they are always stronger for the htofd forecasts . mean change in surface pressure ( hpa ) in the experiments htofd ( left ) and hblock ( right ) with respect to the ctl , at lead time 6 ( top ) , 24 ( middle ) , and 120 ( bottom ) hours of the daily 00utc forecasts performed for february 2014 . the shortrange response in surface pressure over europe is particularly intriguing because it does not clearly resemble the pattern of the pressure changes over north america and east asia . this raises the question whether this is a local effect due to the alps or greenland or it is due to nonlocal effects caused by the changes in orographic drag over the rockies or the himalayas . more generally , the question is whether these changes in surface pressure in the short range are purely local orographic effects or are nonlocal effects due to impacts of the orographic drag on the zonal flow . to answer this question we performed additional daily 10 day forecasts for february 2014 . the changes made in the original htofd and hblock experiments were this time applied only in selected regions of the globe . for each of the two schemes , three new experiments were thus run in which the stress was enhanced over regions covering north america , greenland and europe , and east asia , illustrated by the green boxes in figure 8 . mean change in surface pressure ( hpa ) in the experiments in which the tofd , respectively , the block stress , are enhanced only in the regions demarked by the dark green lines , with respect to the ctl , at lead time 24 of the daily 00utc forecasts performed for february 2014 . the dark green hatches ( from 1 to 0 m / s , by 0.25 m / s ) indicate the deceleration of surface westerlies on the west side of the mountain ranges . the changes in surface pressure observed after 24 h over north america and east asia in the new experiments ( figure 8 , top , bottom ) are virtually identical with those obtained in the original htofd and hblock experiments ( figure 7 ) . the changes observed in the new experiments are also mainly due to a locally induced meridional pressure gradient ( figure 8 , middle ) . the decrease in surface pressure seen east / south of the alps in the experiment where the tofd stress is enhanced over greenland and europe only ( figure 8 , middle ) is outweighed however in the original htofd experiment ( figure 7 ) by the increase in pressure induced by the changes in stress over east asia ( figure 8 , bottom ) . as suggested by zadra et al . , the local response in surface pressure over the largest mountain chains can be understood in terms of geostrophic balance . the induced meridional pressure gradient across the rockies , alps , and , respectively , himalayas , corresponds to a westward geostrophic zonal wind anomaly , that is a deceleration of the midlatitude surface westerlies caused by the enhancement of the stress over orography . a decrease in the zonal surface wind of 1 to 0.2 m s can be indeed seen on the west side of the mountain chains for all experiments ( green hatches in figure 8) . that changing the drag exerted by orography on the flow at low levels leads to a rapid response in surface pressure is hardly a surprise , given our current understanding ( zadra et al . , what is interesting instead is that although the changes made to the two schemes lead to a similar zonally averaged change in tot , the magnitude of the response in surface pressure is rather different ( figure 7 ) . one possibility is that differences in the spatial distribution and the diurnal cycle of tot ( figures 5 and 6 ) lead to a different response in surface pressure . the other possibility is that this response is damped to some extent in the hblock experiment due to the interactions between the gwd and block parameterizations . indeed , the lowlevel blocking and gravity wave drag are both represented in the sgo scheme . therefore , it is impossible to tell a priori whether a change of the stress at low levels , such as that simulated in the hblock experiment , does not also affect the orographic gravity waves . if this was the case , the changes in surface pressure obtained in the hblock experiment could be due at least in part to changes in the propagation and breaking of the gravity waves . to investigate this possibility , we repeated the ctl and hblock experiments while turning off the gwd part of the sgo scheme . the difference in mean surface pressure between these two new experiments matches closely that between the original hblock and ctl experiments shown in figure 7 . this suggests that the response obtained in the short range of the hblock forecasts is actually due to lowlevel blocking effects . after 5 days , the changes to the tofd scheme lead to an overall increase of the surface pressure at high latitudes and a decrease at low latitudes . the impacts on the zonal mean wind and temperature are also qualitatively similar , yet much stronger in the htofd experiment ( not shown ) . these results corroborate the findings of zadra et al . , who discussed in detail the impacts of introducing the lowlevel blocking parameterization of lott and miller in the gem model used at the canadian meteorological centre . this study demonstrated that the introduction of the blocking scheme leads to a weakening of the midlatitude westerlies in the nh during wintertime , the flow being affected up to approximately 150 hpa by day 5 of the forecasts ( see their figure 6 ) . it also induces a change in zonal mean temperature at latitudes higher than 40n characterized by a lowlevel warming and upper level cooling that also reaches 100 hpa by day 5 ( see their figure 9 ) . relative difference of root mean square error ( rmse ) of 500 and 1000 hpa geopotential height , between the htofd ( black ) , respectively , hblock ( red ) , and the ctl forecasts , as a function of lead time . when error bars do not cross the zero line , the performance of the respective experiment is significantly worse / better ( 95% interval ) than the ctl . for both the experiments and the ctl forecasts the rmse was computed with respect to the analysis from which the forecasts started . a positive difference in rmse indicates a deterioration of the model performance in the experiment with respect to the ctl . the effects obtained when increasing the tofd stress are stronger in all circulation aspects examined so far . the impact on the forecast performance is hence bigger in the htofd experiment ( figure 9 ) . what is surprising however is that the change made to the tofd stress considerably degrades the ability of the model to reproduce the nh circulation in the entire troposphere , while that made to the block stress leads to a slight improvement of the model performance . as illustrated by the increase , respectively , the reduction of the root mean square error of the geopotential height at 1000 and 500 hpa in figure 9 , these impacts are significant up to day 5 for the htofd , and day 10 for the hblock experiment . in the previous section we showed that the partitioning of tot between the tofd and block schemes matters for the representation of the nh winter circulation in 10 day weather forecasts . in this section we investigate if this partition is also important at longer time ranges . we do that by analyzing the mean changes in circulation over the 30 winter ( djf ) seasons simulated in the seasonal integrations described in section 3 . given that the runs start on 1 august , the examined lead time is between 4 and 7 months , which is similar to lead times of relevance for seasonal forecast integrations . in both the htofdcl and hblockcl experiments , the mean changes in surface pressure over these 30 djf seasons ( figure 10 , top ) resemble those after 5 days of integration in the htofd experiment ( figure 7 ) in that they show an increase in surface pressure at high latitudes and a decrease in the midlatitudes . however , this change in the meridional pressure gradient is particularly strong over the north atlantic , where it resembles a negative phase of the north atlantic oscillation . although these changes seem at first sight stronger in the htofdcl experiment , the regions in which they are statistically significant ( according to a 95% significance level computed with a regular paired t test ) are less wide spread than in the hblockcl experiment . mean change in surface pressure ( hpa ) ( top row ) and zonal wind at 200 hpa ( m / s ) ( bottom row ) in htofdcl and hblockcl with respect to the ctlcl experiment over the 30 djf seasons ( 19842014 ) . the horizontal white pattern ( gridded areas ) indicate 90 ( 95)% statistical significance according to a paired t test . the effects of orographic lowlevel drag on the upper tropospheric zonal winds are illustrated in figure 10 ( bottom ) . the flow changes suggest that the increase in tot affects the subtropical jets in both experiments , especially over the north atlantic and north pacific , where the southward displacement of the storm tracks is most pronounced . interestingly , there are important differences between the responses over europe , where climate models can show large biases in storm track location [ zappa et al . , changes in the zonal mean zonal wind are also seen in the stratosphere in both experiments ( figure 11 ) . albeit weaker , the impacts on stratospheric winds are similar to those obtained by sigmond and scinocca when enhancing the orographic gravity wave momentum flux ( see their figure 3 ) . their study showed that increased orographic gravity wave momentum flux produces a basic state with weaker midlatitude winds in the wintertime lower stratosphere . furthermore , it demonstrated that the deceleration of the lower stratospheric winds is related to changes in the resolved waves . thus , an increased gravity wave drag strengthens the barrier to the equatorward propagation of planetary waves occurring in the midlatitude lower stratosphere . consequently , the equatorward eliassenpalm ( ep ) flux is reduced and the planetary wave driving of the mid to highlatitude stratosphere is increased , leading to a deceleration of the polar stratospheric winds . by downward control arguments changes in the stratospheric circulation eventually affect the tropospheric circulation [ haynes et al . , 1991 ] . mean change in zonal mean zonal wind ( m / s , contours ) in htofdcl ( left ) and hblockcl ( right ) with respect to the ctlcl experiment over the 30 djf seasons ( 19842014 ) . the horizontal white pattern ( gridded areas ) indicate 90 ( 95)% statistical significance according to a paired t test . our experiments show a deceleration of the zonal winds on the poleward flank of the stratospheric polar vortex and a slight acceleration on its equatorward flank ( figure 11 ) . these changes however appear to be statistically significant only in the hblockcl experiment . in the htofdcl experiment , the only significant changes are found in the lower troposphere and midlatitude stratosphere . in order to understand if these changes in stratospheric winds can also be explained by changes in the resolved wave driving , we performed an ep flux analysis similar to that of sigmond and scinocca . a transformed eulerian mean ( tem ) momentum balance was used to analyze the forcing of the zonally averaged flow by transient plus stationary eddies , following andrews et al . ; and the components of the ep flux , which is a direct measure of the total forcing of the zonalmean state by the eddies [ edmon et al . , 1980 ] , the resulting ep flux arrows for the ctlcl experiment are displayed in figure 12 ( left ) . ( left ) epflux ( arrows ) and zonal mean zonal wind ( contours , m / s ) in the ctlcl experiment for the 30 djf seasons ( 19842014 ) . ( center / right ) change in epflux ( arrows ) in htofdcl , respectively , hblockcl with respect to the ctlcl experiment over the 30 djf seasons . for clearer visualization the ep flux arrows have been scaled by the relative ranges of the two axes of the plot ( 3.14 radians by 10 pa ) . the visibility of vectors in the stratosphere has been further enhanced by using a scaling factor proportional to the squareroot of pressure and by applying a magnifying factor ( of 3 ) above 100 hpa . a budget for resolved wave driving is presented for the stratospheric box between 100 and 10 hpa and between 45n and 90n . red numbers across the box represent differences of ep fluxes integrated over the box boundaries between the htofdcl / hblockcl and the ctlcl experiment , while the red numbers in the box represent the differences in time and areamean momentum deposition , or ep flux divergence , associated with the resolved waves ( all wave numbers ) within the box ( 104 kg ms4 ) . we then integrated the ep flux following kushner and polvani [ 2004 , equation ( 7 ) ] over a box bounded by 100 and 10 hpa and 4590n ( green lines in figure 12 , middle , right ) . the red numbers on the sides of the box represent the difference in the integrated ep flux through the respective boundary , between each experiment and the ctlcl . the values in figure 12 suggest that for both experiments the amount of upward ep flux from the troposphere into the stratosphere across the 100 hpa boundary increases , and so does the equatorward ep flux across the 45n boundary . in the hblockcl experiment the upward ep flux through the 10 hpa surface decreases slightly , while in the htofdcl it increases by an amount comparable to the increase across the 45n boundary . overall , the hblockcl experiment shows thus an increased momentum deposition , or ep flux convergence , in the polar stratosphere , while in the htofdcl experiment this resolved wave driving changes little compared to the ctlcl experiment . this larger momentum deposition explains why the deceleration of winds at latitudes north of 60 n is stronger ( and statistically more robust ) in the hblockcl experiment than in the htofdcl one ( figure 11 ) . the differences in ep flux also explain the deceleration of the zonal winds above 30 hpa observed around 30n in both experiments . the stronger deceleration observed in the htofdcl experiment appears to be associated with a stronger ep flux convergence at 2030 hpa between 30n and 40n . the analysis of the differences in various aspects of the nh winter circulation over the 30 djf seasons suggests that the partitioning of the subgrid orographic lowlevel drag between the tofd and block schemes is also important at longer time scales . while both schemes affect the circulation in the entire atmosphere , the details of these effects depend on which scheme represents the drag . the block scheme leads to effects somewhat similar , albeit weaker , than those of the gwd scheme discussed in sigmond et al . and sigmond and scinocca . in contrast to those studies , the increased highlatitude stratospheric wave drag in hblockcl is caused by more upward wave activity entering the region ( figure 12 ) , rather than by less equatorward wave activity leaving the region [ see sigmond and scinocca , 2010 , figure 6 ] ; in other words , the changes are induced from below rather than from within the stratosphere . the turbulent orographic form drag on the other hand affects more strongly the lower troposphere and only partly the lower stratosphere . finally , note that the main contribution to the ep flux in the stratosphere is due to wave numbers 15 , while the main contribution to the tropospheric ep flux is by wave numbers greater than 5 ( not shown ) . therefore , the orographic form drag mainly affects the synoptic eddies while the lowlevel blocking affects the largescale waves . the representation of surface stress is important for accurately simulating the largescale circulation in both weather forecasts and climate predictions . the first results of the ongoing wgne drag project revealed that stateoftheart weather forecast models differ significantly in terms of the magnitude of the total subgrid surface stress , especially over land and more particularly over orography . furthermore , models also differ in the partitioning of this stress among the different parameterization schemes . in the present study we tried to answer two questions raised by this intercomparison . we investigated the impact of changing tot in a particular model by an amount comparable to the intermodel spread found in the wgne drag project . the discussion focuses on changes in the representation of the nh wintertime circulation in 10 day weather forecasts and seasonal integrations . furthermore , we examined the impact of varying the partitioning of this increase in tot between two schemes representing subgrid orographic drag , the turbulent orographic form drag and the lowlevel blocking schemes . these schemes have been chosen as the representation of orographic drag processes is still uncertain , and their impacts have been only poorly documented in the literature . the experiments designed to address these questions consisted in increasing the zonal mean tot by 1015% in the nh midlatitudes , via an increase in either the tofd or the block contributions . such an increase in tot was shown to significantly affect various aspects of the nh winter circulation both in 10 day forecasts and in longer integrations , irrespectively of which scheme is modified . thus , changes in surface pressure are seen in all experiments and at all examined time scales , ranging from a few hours to a few months . a meridional surface pressure gradient across the large mountain chains is visible after a few hours of integration . these changes are induced through geostrophic balance as a response to the deceleration of the midlatitude surface westerlies caused by the enhancement of orographic stress . at longer time ranges the enhanced orographic stress also results in changes in the zonal wind , temperature , and geopotential at all time scales . the amplitude and robustness of the changes in surface pressure , winds and temperature depend however on the particular scheme which is modified . interestingly , in 10 day forecasts the increase in tofd stress has a much stronger impact on the circulation than that in block stress , while the impact is of comparable magnitude for the seasonal integrations . more surprisingly , although both simulate an increase in stress over orography the changes to the tofd and block schemes have opposite impacts in terms of mediumrange forecast skill . the tofd changes lead to a considerable degradation of the geopotential height skill in the troposphere ( up to 200 hpa ) up to day 5 , while the block changes slightly improve the performance throughout the entire forecast range . the seasonal integrations suggest that the two schemes have also different effects in terms of the model climate for the djf season . the increase in block stress results in a significant deceleration of the zonal wind on the poleward flank of the stratospheric polar vortex by changing the resolved wave driving of the mid to highlatitude stratosphere . this was confirmed by an ep flux analysis similar to that performed by sigmond and scinocca . the changes to the tofd scheme on the other hand mainly affect the lower troposphere and the midlatitude stratosphere . in summary , these results suggest that the representation of the nh winter circulation is sensitive not only to changes in the total subgrid ( orographic ) stress , but also to the partitioning of this stress among different schemes . the reasons for the different circulation response to the partitioning of the total surface stress remain to be understood . the different impacts of the two schemes discussed here could be related to inherent differences in their formulations , i.e. , stability versus non stability dependence , the diurnal cycle and the horizontal scales on which they act .

abstracta recent intercomparison exercise proposed by the working group for numerical experimentation ( wgne ) revealed that the parameterized , or unresolved , surface stress in weather forecast models is highly modeldependent , especially over orography . models of comparable resolution differ over land by as much as 20% in zonal mean total subgrid surface stress ( tot ) . the way tot is partitioned between the different parameterizations is also modeldependent . in this study , we simulated in a particular model an increase in tot comparable with the spread found in the wgne intercomparison . this increase was simulated in two ways , namely by increasing independently the contributions to tot of the turbulent orographic form drag scheme ( tofd ) and of the orographic lowlevel blocking scheme ( block ) . increasing the parameterized orographic drag leads to significant changes in surface pressure , zonal wind and temperature in the northern hemisphere during winter both in 10 day weather forecasts and in seasonal integrations . however , the magnitude of these changes in circulation strongly depends on which scheme is modified . in 10 day forecasts , stronger changes are found when the tofd stress is increased , while on seasonal time scales the effects are of comparable magnitude , although different in detail . at these time scales , the block scheme affects the lower stratosphere winds through changes in the resolved planetary waves which are associated with surface impacts , while the tofd effects are mostly limited to the lower troposphere . the partitioning of tot between the two schemes appears to play an important role at all time scales .

Introduction Representation of Subgrid Surface Stress in the IFS Experimentation Impacts in Short and MediumRange Weather Forecasts ShortRange Response in Surface Pressure Impacts on the NH Circulation in MediumRange Forecasts Impacts at Longer Time Range Conclusions

PMC4691994

the minor salivary glands are important components of the oral cavity , present in most parts of the mouth , and their secretions directly bathe the tissues . individual glands are usually in the submucosa or between muscle fibers and consist of groups of secretory end pieces made up of mucous acinar cells and serous or seromucous demilune cells . we report a case of ulceration in the palatal salivary gland presented with intermittent heavy bleeding from the lesion . a 50-year - old male patient reported with the complaint of bleeding from the swelling in the palate for 4 months . the patient had consulted a doctor who had diagnosed it to be bleeding from a swelling in the palate . on intraoral examination , a small , well - defined , solitary papule measuring about 4 mm in diameter was present in the midpalatal region , on the left side of midpalatal raphe , along a line running from the right second premolar to the left second premolar . well defined , solitary papule with central ulceration in the palate on palpation , it was nontender and soft . the lesion showed some compressibility . upon compressing , serous discharge exudate from the central ulcerated area . on asking the patient to spit following gargling with water , heavy bleeding the case was provisionally diagnosed as ulcerative sialadenitis of minor salivary gland with considering minor salivary gland adenoma , adenomatoid hyperplasia , and nonhealing tuberculous ulcer as a differential diagnosis . histopathological examination showed hyperkeratotic stratified squamous epithelium and mucous acini collection in the underlying connective tissue . a minimal amount of chronic inflammatory cells and endothelial lined blood vessels with red blood cells were also seen . histopathological features were suggestive of a chronic nonspecific ulcer . based on the clinical features , histopathology report and other investigations , the final diagnosis was given as ulcerative sialadenitis of the palatal salivary gland . hyperkeratotic stratified squamous epithelium and mucous acini collection in the underlying connective tissue . minimal amount of chronic inflammatory cells and endothelial lined blood vessels with red blood cells were also seen ( h and e , 10 ) despite their name , minor salivary glands have an important role in the physiology and pathology of the oral cavity . they secrete more or less continuously , providing a steady flow of fluid , and organic substances to protect the oral tissues . because individual glands open directly onto the mucosal surface , their secretions create and regulate the local environment . the effect of smoking on the oral cavity has been well studied for quite a number of years . the glandular area of the hard palate mucosa shows papular elevations ( up to 23 mm in height ) with central umbilications with or without pigmentation of the surrounding mucosa . the central umbilication could be like a red spot in the center of a greyish or pale elevated papule . in mild stomatitis nicotina lesions red dots over blanched elevated areas can be seen , and in severe cases , papular lesions up to 05 cm . in diameter or more with umbilications up to 23 mm . in diameter . ulcerative sialadenitis of the minor salivary gland has not been mentioned in the literature so far . inflammation of the minor salivary gland openings due to smoking and some kind of traumatic irritation would have resulted in ulceration . the ulcerated friable mucosa would have developed the tendency to bleed on applying negative pressure . a further enlightenment on this condition could not be done as this is the first case to be reported on ulcerative sialadenitis of the minor salivary gland

minor salivary glands have an important role in the physiology and pathology of the oral cavity though they have been neglected at times . smoking has a direct effect on the palatal minor salivary glands , and their most common presentation is stomatitis nicotina . we report a case of ulceration in the palatal salivary gland presented with intermittent heavy bleeding from the lesion .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

PMC3353617

in 1990 , ramirez introduced a new procedure to close abdominal wall hernia ( awh ) , called components separation technique ( cst ) . thanks to endoscopy , surgical repair possibilities have risen , reducing the operative trauma and preserving vascular and neuronal anatomical structures . this report aims to describe a single port endoscopic approach for cst to repair the abdominal wall of a patient undergoing surgery for abdominal aneurysm and already subject to placement of a mesh for awh . we performed endoscopic - assisted cst , using a single - port access with a gasless technique . cst is a useful procedure to close large abdominal wall incisional hernia avoiding the use of mesh , notably under contamination , when prosthetic material use is contraindicated . the endoscopic - assisted cst produces same results than the conventional open separation technique and also minimised tissue trauma that ensures blood supply and prevents postoperative wounds complications . the described single port method was found to be safe and effective to close large midline abdominal hernias when a primary open or laparoscopic closure is not feasible or when patients have been previously treated with abdominal meshes . large midline abdominal wall defects reconstruction keeps being a great challenge to surgeon due to technical difficulties and relatively high recurrence rate and morbidity . morbidity and recurrence rate are high when several conditions coexist : contamination or infection after relaparotomy or after previous abdominal prosthetic repair . the open or laparoscopic repair by using prosthetic materials is still the most frequent procedure performed , even if contraindicated in contaminated field . furthermore , patients with this condition sometime need major surgical intervention for neoplasm or aortic diseases . in 1990 , ramirez et al . introduced a new procedure to close abdominal wall hernia , called the components separation technique ( cst ) . this technique is based on autologous tissue reconstruction of the abdominal wall and it is performed through bilateral separation and advancement of muscular layers , in order to bridge the fascial gap , avoiding prosthetic material use . anterolateral abdominal wall is composed of multiple overlapping muscle layers : the external oblique , the internal oblique , the transversus muscles and the rectus muscles , which are enveloped by the anterior and posterior sheath , fused in the midline at the linea alba . a superficial and deep vascular system form the blood supply of the abdominal wall structures coming from the epigastric , superior and inferior and the intercostals arteries . described limitations of this technique were complications against the skin and the subcutaneous tissue , caused by surgical interruption of perforating vessels during exposure of oblique muscle . thanks to endoscopy , the possibility of surgical repair has increased and thereby reducing the operative trauma and preserving the anatomical structures . lowe et al . demonstrated that the endoscopic - assisted cst minimised tissue trauma and preserved blood supply of the skin , with less morbidity . this contribution aims to illustrate a different endoscopic approach for cst and to evaluate this technique feasibility in a case of abdominal reconstruction after relaparotomy with mesh section for an abdominal aneurysm repair . we performed the endoscopic - assisted cst , as reported in the literature , but with an original modification consisting of a single - port access gasless technique . the patient undergoing this procedure needed to repair of an abdominal aortic aneurysm . at the same time , he presented a large mesh positioned above the muscolar fascia for hernia resulting from a previous surgery for left hemicolectomy . surgery was performed under general anesthesia , with patient supine and the surgical approach was through a median laparotomy resulting in the total section of the mesh . the vascular surgeon provided to repair an abdominal aortic aneurysm ( 8 cm long ) , but after the surgery it was impossible closer the muscolar walls and to remove the mesh because of tissue adhesion . then , in order to close the abdominal incision , a 1.5 cm skin incision was made 5 cm caudally to the arch of the ribs and 5 cm caudo - laterally to the midline [ figure 1 ] . the operating 30 telescope ( storz seps endoscope ; storz tuttlingen , germany ) , which has a lifting handle and a single 5-mm operating port , was inserted into the subcutaneous plane . aponeurosis of the external oblique muscle was divided , for a length of 18 cm , using an ace harmonic scalpel ( ethicon endo - surgery , inc . , cincinnati , oh ) , 2 cm laterally to the border of rectus abdominis muscle . the surgeon used the left hand within the abdomen cavity in order to control the fascial border and , during the procedure , to assess abdominal wall release . under video - endoscopic control , the external oblique muscle was separated from the internal oblique muscle ( into the avascular plane present between these muscles ) up to anterior axillary line , through blunt dissection . this step is performed by twisting the scope ( laterally and medially ) , that is manually driven by means of trans - abdominal illumination [ figure 2 ] . the separation is essential since the fibrous interconnections between both muscles prevent optimal medial shift of the rectus abdominis muscle . at the end of the procedure , the fascia is closed with a running polydioxanone suture ( psd - loop , johnson and johnson , ltd . ) , without tension and also the edges of the mesh move closer with prolene suture . a skin incision of 1.5 cm is made 5 cm caudal to the arch of the ribs and 5 cm caudolateral to the midline and the endoscope is inserted the external oblique muscle was separated from the internal oblique muscle in the avascular plane between both muscles to the midaxillary line by blunt dissection with the scope manually driven by means of trans - abdominal illumination the abdominal wall cst allows ventral defects closure by transposition of the abdominal wall muscles . cst abdominal wall mobilisation produces a release that is , for each ipsilateral mucles group , about 4 cm long in the upper abdomen , 3 cm in the lower abdomen and 8 cm in the waist . if additional advancement is needed ( rarely ) , the rectus muscle posterior fascia can be freed , allowing 2 cm of additional advancement . cst is a useful procedure to close a large abdominal wall incisional hernia , avoiding the use of mesh . it is also particularly notable under contaminate condition , when use of prosthetic material is contraindicated or in complex conditions such as that described in this report . the patient was prior surgically treated and presented a mesh perfectly adherent whose replacement would have been messy and complex . in a recent randomised controlled trial , comparing the results after cst and prosthetic repair of abdominal wall hernia , cst achieved favorable outcomes . in 2000 , lowe et al . modified the original cst in a minimally invasive technique . thanks to introduction of endoscopic approach and through the use of a balloon distension in order to create a subcutaneous space and to transect the external oblique aponeurosis , preserving the cutaneous perforating vessels . later on , some authors demonstrated that the endoscopic - assisted cst produces same results than open conventional separation technique ; moreover , it minimises tissue trauma that ensures the blood supply and it prevents postoperative wound complications . these procedures result in prolonged operative time , and the use of a single port access with a gasless technique may decrease the operative time . it seems to us that the described single port method , even if performed in a single case , is safe and effective to close large midline abdominal hernias when a primary open or laparoscopic closure is not feasible , or when patients have been previously treated with abdominal meshes .

background : in 1990 , ramirez introduced a new procedure to close abdominal wall hernia ( awh ) , called components separation technique ( cst ) . thanks to endoscopy , surgical repair possibilities have risen , reducing the operative trauma and preserving vascular and neuronal anatomical structures . this report aims to describe a single port endoscopic approach for cst to repair the abdominal wall of a patient undergoing surgery for abdominal aneurysm and already subject to placement of a mesh for awh.methods:we performed endoscopic - assisted cst , using a single - port access with a gasless technique.conclusion:cst is a useful procedure to close large abdominal wall incisional hernia avoiding the use of mesh , notably under contamination , when prosthetic material use is contraindicated . the endoscopic - assisted cst produces same results than the conventional open separation technique and also minimised tissue trauma that ensures blood supply and prevents postoperative wounds complications . the described single port method was found to be safe and effective to close large midline abdominal hernias when a primary open or laparoscopic closure is not feasible or when patients have been previously treated with abdominal meshes .

BACKGROUND: METHODS: CONCLUSION: INTRODUCTION STANDARD TECHNIQUE IMPROVEMENTS BENEFITS

PMC4738997

during endurance running events , there are many factors that can influence the pace of the runner . for example , changes in terrain , elevation , environmental temperature , and fatigue can all influence the pace that can be maintained . likewise , a runner may strategize to maintain a constant ( e.g. , target pace ) or variable pace ( e.g. , run - walk strategy ) . understanding variability of pacing may lead to a better understanding of factors that influence marathon performance and help runners either complete a marathon successfully or improve race performance . specifically , the intriguing aspect of investigating variability of pace is that the runner , theoretically , selects a pace to maintain homeostasis . however , if the wrong pace is selected ( e.g. , too fast ) fatigue will result and the runner will slow down or possibly not finish the distance event . variability in pacing has been studied in respect to short- and middle - distance running ( e.g. , 3,000 m to 10 km ) ( 1 , 2 , 4 , 5 , 6 , 7 , 9 , 10 ) . these studies have mostly focused on the influence of pacing on metabolic measures related to performance but some studies do provide insight into variability of pacing . for example , billat ( 2 ) reported that the coefficient of variation in velocity was 1%5% during a 3000 m run . ( 4 ) demonstrated that fatigue did not increase variability of velocity during a distance run ( 1280 m ) . despite research like this , there is only limited research on the variability of pace during long distance events such as a marathon . ( 5 ) reported that elite runners completing a marathon had very little change in 5 km pace during a marathon suggesting low variability of pace . it makes sense that elite runners attempt to maintain a constant pace throughout the marathon . likewise , lambert et al . ( 8) reported that the top - ten runners completing a 100 km ultra - marathon race had less changes in pace than runners finishing between 11th and 77th place . although it makes sense to study pacing strategies of elite athletes , the majority of marathon participants are non - elite runners . information on pacing strategies for this population would be helpful in designing appropriate training programs , for example . therefore , the purpose of this study was to describe the variability of pacing during a marathon for non - elite runners . a second purpose was to determine if there is a relationship between variability of pacing and marathon performance for this same population . it was hypothesized that there is a non - linear relationship between variability of pace and marathon performance in a such that slower runners will experience less variation in pace compared to mid - range finshers . faster runners attempting to maintain a pace throughout the race will also have less variability of pace compared to mid - range finishers . velocity data during a marathon were obtained by downloading publicly available global position system ( gps ) data from the garmin connect web page . each data set included at least : marathon location , marathon date , speed , and position data . a total of 311 gps profiles from 2 races ( las vegas and san diego marathons ) were downloaded for analysis . these races were selected due to the number of gps profiles available via garmin connect . subject - specific descriptive information ( e.g. , age , gender , height , weight , or ethnicity ) were not available . the study was determined to be exempt from requiring consent from human subjects since de - identified secondary data were used . all gps profiles were inspected prior to analysis and it was determined that 26 profiles were not suitable for analysis resulting in 285 profiles ( 116 for race 1 , 169 for race 2 ) used for analysis . profiles were removed either due to large gaps in the gps profile or the presence of negative velocity values . of the 285 profiles , 13 had additional data beyond the finish line evident by a dramatic drop in velocity as well as extension of the position data beyond the finish line . data beyond the finish line location ( as determined by position coordinates ) were removed from the profiles . two ( 2 ) profiles from race 1 and 11 files from race 2 were edited for this reason . all gps profiles used for analysis were resampled to yield the same sample rate ( 0.15 hz ) using a custom program ( matlab , mathworks , version 6.1 ) . variability of pacing was determined by calculating the coefficient of variation of velocity ( velcov ) . the velocity data in the gps profile were used to calculate velcov using the formula : velstdev = the standard deviation of velocity over the duration of the marathon . each marathon finish time was placed into one of three finish time bins : bin 1 : < 3.9 hrs , bin 2 : 3.9 4.6 hrs , bin 3 : > 4.6 hrs . these bins were defined in order to have a similar number of data sets per bin . frequency distributions of marathon finish time and velcov per race were generated with velcov data tested for normality ( kolmogorov - smirnov test ; spss 17.0 ) . a 2 ( race ) 3 ( bin ) analysis of variance ( anova ) was used to compare the dependent variable ( velcov ) between races and the marathon bin finish times . post - hoc tests ( least square difference ) were computed if the omnibus f - ratio was found to be significant to compare velcov between bins . non - parametric tests were used in the case that the data were not normally distributed . finally , linear and quadratic regression lines were generated predicting marathon finish time from velcov using all 285 data sets . velocity data during a marathon were obtained by downloading publicly available global position system ( gps ) data from the garmin connect web page . each data set included at least : marathon location , marathon date , speed , and position data . a total of 311 gps profiles from 2 races ( las vegas and san diego marathons ) were downloaded for analysis . these races were selected due to the number of gps profiles available via garmin connect . subject - specific descriptive information ( e.g. , age , gender , height , weight , or ethnicity ) were not available . the study was determined to be exempt from requiring consent from human subjects since de - identified secondary data were used . all gps profiles were inspected prior to analysis and it was determined that 26 profiles were not suitable for analysis resulting in 285 profiles ( 116 for race 1 , 169 for race 2 ) used for analysis . profiles were removed either due to large gaps in the gps profile or the presence of negative velocity values . of the 285 profiles , 13 had additional data beyond the finish line evident by a dramatic drop in velocity as well as extension of the position data beyond the finish line . data beyond the finish line location ( as determined by position coordinates ) were removed from the profiles . two ( 2 ) profiles from race 1 and 11 files from race 2 were edited for this reason . all gps profiles used for analysis were resampled to yield the same sample rate ( 0.15 hz ) using a custom program ( matlab , mathworks , version 6.1 ) . variability of pacing was determined by calculating the coefficient of variation of velocity ( velcov ) . the velocity data in the gps profile were used to calculate velcov using the formula : velstdev = the standard deviation of velocity over the duration of the marathon . each marathon finish time was placed into one of three finish time bins : bin 1 : < 3.9 hrs , bin 2 : 3.9 4.6 hrs , bin 3 : > 4.6 hrs . these bins were defined in order to have a similar number of data sets per bin . descriptive statistics were calculated for velcov and marathon finish time for each race . frequency distributions of marathon finish time and velcov per race were generated with velcov data tested for normality ( kolmogorov - smirnov test ; spss 17.0 ) . a 2 ( race ) 3 ( bin ) analysis of variance ( anova ) was used to compare the dependent variable ( velcov ) between races and the marathon bin finish times . post - hoc tests ( least square difference ) were computed if the omnibus f - ratio was found to be significant to compare velcov between bins . non - parametric tests were used in the case that the data were not normally distributed . finally , linear and quadratic regression lines were generated predicting marathon finish time from velcov using all 285 data sets . the frequency distributions of velcov and for marathon finish time for each race are presented in figure 1 . velcov was 16.9 6.4% and 16.8 6.6% for race 1 and race 2 ( table 1 ) . marathon finish time was 4.27 0.80 hours for race 1 and 4.40 0.86 for race 2 . it was determined that velcov was not normally distributed for either race 1 or race 2 ( p<0.01 ) . non - parametric tests and parametric tests were conducted with both analyses yielding identical results . velcov was not influenced by the interaction of race and bin ( p>0.05 ) and was not different between races ( race 1 16.6 6.3% ; race 2 16.7 6.5% ; table 1 ; p>0.05 ) . velcov was influenced by bin finish time ( p<0.05 ) . using post - hoc tests , it was determined that velcov was lower in bin 1 vs. bin 2 ( p<0.05 ) , lower in bin 1 vs. bin 3 ( p < 0.05 ) , and lower in bin 2 vs. bin 3 ( p<0.05 ) for both races ( table 1 ) . marathon time was not influenced by an interaction of race and bin ( p>0.05 ) and was not different between races ( table 1 ; p>0.05 ) . marathon time was different between bins ( p<0.05 ) with bin 1 containing the fastest times and bin 3 the slowest . both linear and quadratic regression lines predicting marathon finish time from velcov were significant ( figure 2 , p<0.05 ) . this study set out to describe the variability of marathon pace for non - elite runners . using 285 gps data sets from two different marathons , it was determined that the velcov was 16.7 6.5% for marathon finish times of 4.35 0.84 hours . a second goal of this study was to determine if there was a relationship between marathon finish time and velcov . first , we placed each marathon finish time in a specific bin ( i.e. , < 3.9 hours , 3.9 4.6 hours , > 4.6 hours ) and compared velcov between bins for each race . using this approach , we determined that velcov was different between marathon finish times such that velcov was greater for slower finish times for both races . the second approach we used was to build linear and quadratic regression lines to predict marathon finish time from velcov . using this approach , we determined that both linear and quadratic lines fit the data ( r2 = 0.46 for both lines ) such that velcov increased with slower marathon finish times . originally , we hypothesized that the slower marathon times would have less variability of pace than the average marathon finish time . based upon the two approaches we took , it seems that this was not the case and instead variability of pace continued to increase with slower marathon finish times . the few studies that have reported variability of pacing have either been of shorter distances ( e.g. , 2 ) or of elite runners ( e.g. , 5 , 8) . for example , billat ( 2 ) reported that velcov of middle- and long - distance running to be in the range of 1%5% for 3,000 m to 10 km for competitive runners . ( 5 ) indicates that the marathon race winners had low variability of pacing ( approximately less than 20 s difference between 5 k splits ) as did the 100th place finishers ( range of about 3 minute difference between fastest and slowest 5 k splits ) . lambert et al . ( 8) observed velcov as low as 5.4% for the top ten finishers of a 100 km ultra - marathon race and as high as 19.6% for the 61st67th place finishers . in the present study , variability of pace was greater ( 16.7 6.5% ) compared to billat ( 2 ) and ely et al . ( 5 ) but comparable to some of the data presented by lambert et al . the reason for the difference between studies is likely related to the level of competitive athlete studied . in our case , the subjects were not elite runners . considering all 285 data sets , the mean marathon finish time was 4.35 0.84 hours . given the result that velcov was influenced by marathon time , it does makes sense that the velcov will be higher among noncompetitive marathon distance runners compared to elite marathoners with finishing times under 3 hours . for example , using the linear and quadratic regression equations , velcov would be between 3.04% ( quadratic ) and 3.00% ( linear ) for a 3 hour marathon . however , the gps device does contain some noise in the signal and we had to discard 26 profiles from analysis due to different errors in the signal . we considered that velcov may have been influenced by the noise in the signal and therefore we smoothed the data set using a low - pass filter ( 4th order , zero phase - lag , cutoff frequency = 0.15/4 hz ) to remove any high frequency noise ( e.g. , intermittent high - velocity spikes ) . the smoothed data sets were then compared using the same statistical procedures as the original data and it was determined that the outcome of the analysis was the same regardless of which data sets were used . therefore , we concluded that the noise did not influence the outcome of the study . changes in elevation may have an effect on velcov since runners tend to change their velocity while running up or downhill . the elevation profiles for each race are illustrated in figure 3 and are normalized to the starting elevation in order to emphasize the change in elevation ( vs. the actual elevation ) . from this illustration , it seems that the changes in elevation were not dramatic between races or even within a race which coincides with our observation that variability of pace was not different between races . nevertheless , it is hypothesized that races with either more frequent or greater changes in elevation would result in a greater velcov than what was observed in this study . there does seem to be a relationship between velcov and marathon time since velcov was different across the marathon finish time bins for each race and because the data were fit with both linear and quadratic regression lines . ( 8) also observed an increase in variability of pace with longer finishing times during a 100 km race . it makes sense that velcov is low for fast marathon ( 5 ) or ultra - marathon ( 8) times since runners are trying to maintain as fast a velocity possible over the entire distance in an attempt to achieve a faster finishing time . large velcov over the course of the race would mean the runner is undergoing large changes in velocity which would seem detrimental to elite marathon performance . it also makes sense that velcov is greater for slower marathon times since the runner does not have the same physical capacity as the elite marathon runner to maintain a consistent pace . for example , a runner who ran for a period of time but then needed to walk to recover from the exertion would have a greater velcov then a runner who could maintain a consistent pace over the same period of time . however , it is not clear why velcov continued to increase for longer marathon times ( i.e. , > 4.6 hrs ) . originally , it was thought that these runners would have low variability of pace since the capacity to achieve a fast pace would be lower and therefore any change velocity in velocity would also be lower . for example , if a runner was trying to maintain a 13 min / mi pace ( about a 5 hour 40 minute marathon ) had to walk their pace may only change slightly to 15 min / mi while walking . however , if a faster runner trying to maintain an 8 min / mi pace ( about a 3 hour 30 min pace ) had to walk , the change in pace would be much larger . this might mean that runners with slower marathon times had more frequent vs. larger changes in pace compared to faster runners . that being said , the importance of a quadratic regression equation fitting the marathon finish time - velcov relationship is that there would be a maximum of this function . however , in the case of the quadratic regression equation that best fit the data , the maximum of the parabola would occur at velcov 92% with a marathon finish time of 7.74 hours . although we had a few observations above or near a 7 hour marathon time , the greatest velcov was under 50% . therefore , it seems unlikely that a plateau or decrease in velcov would be observed even if longer marathon times were studied . the linear or quadratic regression equations explained 46% of the variance in marathon finish time by variability of pace . this obviously means that there is a wide range of possible values of velcov for a given marathon finish time . nevertheless , it seems that a greater variability of pace is associated with slower marathon performance . that being said , there might be some advantages to using a more variable pace ( e.g. , run - walk strategy ) that are overlooked in this analysis . for example , it might be that using a planned variable pace which includes specific intervals of running followed by specific intervals of walking may allow a runner to minimize the impact of fatigue in a way that the marathon can be successfully completed . we did not attempt to separate planned run - walk strategies in this analysis even though we suspect that some of the profiles do reflect this approach . we suspect that variation of pace is related to fitness and/or fatigue . for example , cade et al . ( 3 ) reported that 11 of 21 marathoners studied adopted a walk / run / walk pacing strategy in the last six miles due to high body temperature and/or lower levels of blood glucose . although we did not examine variability of pace at different sections of the marathon , there might be some value in determining if pace varies within a race . to gain some insight into this , we fit each velocity vs. time profile with a linear line of best fit . both races had velocity vs. time slopes that were negative ( race 1 : 0.100.1 m / s / s ; race 2 : 0.130.1 m / s / s ) ; meaning that , on average , runners slowed their pace over the course of the marathon . we compared the slopes between races and bins and determined that there was no difference in slope between races or bins . this is an indication that the amount of slowing of pace was independent of the actual pace maintained . future research is needed to determine if the amount of variation in pace is different for different stages of the race . we observed that non - elite marathon runners change their pace in such a way that those with slower marathon finish times had more variability of pace than those with faster finish times . we also observed that , on average , runners slow their pace over the course of the marathon . for runners interested in achieving a faster marathon performance , he / she would likely benefit by training at low variability of pace . however , many marathoners may simply want to complete the event vs. achieve a fast marathon time and it is important to match training specificity with the marathon goal . in this case , the training regimen of slower runners should include increased variability of pace .

the purpose of this study was twofold : 1 ) to describe variability of pacing during a marathon and 2 ) to determine if there is a relationship between variability of pacing and marathon performance . publically available personal global positioning system profiles from two marathons ( race 1 n = 116 , race 2 n = 169 ) were downloaded ( http://connect.garmin.com ) for analysis . the coefficient of variation of velocity ( velcov ) was calculated for each profile . each profile was categorized as finishing in under 3.9 hours , between 3.9 and 4.6 hours , or longer than 4.6 hours . linear and quadratic lines of best fit were computed to describe the relationship between marathon finish time and velcov . a 2 ( race ) 3 ( bin ) analysis of variance ( anova ) was used to compare the dependent variable ( velcov ) between races and the marathon bin finish times . velcov was not influenced by the interaction of finish time bin and race ( p>0.05 ) and was not different between races ( race 1 : 16.6 6.4% , race 2 : 16.8 6.6% , p>0.05 ) . velcov was different between finish time categories ( p<0.05 ) for each race such that velcov was lower for faster finish times . using combined data from both races , linear ( marathon finish time = marathon finish time = 0.09velcov + 2.9 , r^2 = 0.46 ) and quadratic ( marathon finish time = 0.0006 velcov 2 + 0.11 velcov + 2.7 , r^2 = 0.46 ) lines of best fit were significant ( p<0.05 ) . slower marathon finishers had greater variability of pace compared to faster marathoner finishers .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC5005044

hip pain and injuries commonly occur during sporting activities that involve physical contact , strain , and pressure placed on the hip joint . hip arthroscopy is a minimally invasive surgery frequently performed in athletes to restore function of the joint and help return athletes to sport . many studies have demonstrated that arthroscopic management of femoroacetabular impingement ( fai ) leads to high patient satisfaction and high rate of rts in elite athletes [ 17 ] . determined that arthroscopic treatment of hip injuries in elite athletes might result in improved hip outcomes at minimum 1-year follow - up . compared athletes versus non - athletes diagnosed with fai , showing significantly higher outcomes for athletes at 6-week follow - up . however , there are few descriptive studies that outline the rts of a cohort of athletes at minimum 2-year follow - up . the purpose was to compare athletes who returned to sport to those who did not in terms of patient reported outcome ( pro ) scores and sports related movements , using the hip outcome score - sports specific subscale ( hos - sss ) as the primary outcome measurement . we hypothesize patients who returned to their sport will demonstrate higher hos - sss scores and increased abilities to perform sport - specific movements such as jumping or making cutting / lateral movements . pro scores were collected pre- and post - operatively at 3 months , 1 year and minimum 2-year follow - up . we collected pro scores at clinic visits , through encrypted emails , or by telephone calls . pro scores used included the modified harris hip score ( mhhs ) , the non - arthritic hip score ( nahs ) , the hip outcome score activities of daily living ( hos - adl ) and the hos - sss . the hos - sss represents a patient s self - reported ability to perform specific - related sports movements such as cutting / lateral movements . all four questionnaires were used , as it has been reported that there is no conclusive evidence for the use of a single pro questionnaire for patients undergoing hip arthroscopy [ 9 , 10 ] . additional pro scores were used in addition to the hos - sss to assess baseline and post - operative abilities to perform abilities of daily living and to see if differences between patients who did and did not retun to sport were based on more sports specific movements of if differences were present in more general scores . the questionaire also included : sport played before hip pain , primary sport and sport ability level after surgery . visual analog scale ( vas ) pain scores were collected . as an a priori power analysis , we calculated that we would need at least 51 patients in each group to rule out type ii error when comparing pro scores , assuming a 10-point difference between groups and a standard deviation of 20 points in both groups . between september 2008 and april 2012 there were 475 procedures for which the patient did not report whether he or she played a sport prior to surgery and 304 procedures for which the patient reported no involvement in sports prior to surgery . of the remaining 321 procedures , for which patients did report playing a sport prior to surgery , the hos - sss and whether the patient returned to sport after surgery were recorded for 52% ( 168/321 ) hips . we obtained 2-year follow - up for 49% ( 158/321 ) hips in 148 patients which constituted our study cohort . a two - tailed student s t - test was performed to compare pre- and post - operative pro data for the cohort . changes from pre- to post - operative pro scores and vas were caluculated ( delta ) , and a t - test was used to compare changes between groups . a test was used to compare categorical data such as abilities to perform sports - specific movements . in the overall cohort of 148 amateur and professional athletes ( 158 hips ) who underwent hip arthroscopies 60 patients ( 65 hips ) comprised the non - return - to - sports ( nrts ) group . twenty five patients ( 17% ) did not rtss due to hip - related issues . thirty two patients ( 22% ) did not rtss due to non - hip - related issues such as lifestyle transitions , advancing from high school to college , a desire to avoid further injury , and co - morbid conditions . the remaining 88 patients ( 93 hips ) who reported returning to sports constituted the rts group . the average ages were 30.7 ( range , 13.261.4 ) years for the rts group and 30.4 ( range , 14.859 ) years for the nrts group . the rts group contained 29 male patients ( 30 hips , 32% ) and 59 female patients ( 63 hips , 68% ) , while the nrts group contained 32 female patients ( 35 hips , 54% ) and 28 male patients ( 30 hips , 46% ) . there were no statistically significant differences between groups in terms of gender , age or body mass index ( bmi ) ( table i ) . in the overall cohort , the remainder of the cohort was split evenly between patients who played at a high school or college level and patients who played recreationally . table i.demographic information for each group , by number of hipsrtsnrtsp - valuetotal count9365male30300.08female63350.08age30.731.10.86height ( inches)67.567.90.61weight ( pounds)157.5161.00.60bmi30.724.00.90time to 2-year follow - up ( months)2.12.10.27 demographic information for each group , by number of hips we recorded the patients level of play in four approximate levels : recreation , high school , college and professional . of patients in the nrts group , 38.5% were recreational athletes , 33.8% were high school - level athletes , 15.4% were college - level athletes and 12.3% were professional athletes prior to surgery . of patients in the rts group , 36.3% were recreational athletes , 27.5% were high school - level athletes , 23.8% were college - level athletes , and 12.5% were professional athletes prior to surgery . a test did not show a significant difference between groups in terms of the preoperative level of play . at the time of latest follow - up , 33.8% of patients in the rts group reported a decreased level of play , while 66.2% patients reported an equivalent or increased level of play , compared with their level prior to surgery . the 10 most commonly played sports were established as distinct categories , while one additional category represented all other activities reported . table ii.sports played by patients in each group , by number of patientsrtsnrtsp - valuerunning32180.37soccer10110.26volleyball1130.12softball750.79tennis950.67basketball760.70football380.06baseball560.54hockey530.88swimming620.56other33230.30 sports played by patients in each group , by number of patients there were no statistically significant differences between groups related to type of arthroscopic procedures performed ( table iii ) . each group reported one conversion to total hip replacement and no revisions were noted . table iii.procedures for each group , by number of hipsrtsnrtsp - valuefemoral osteoplasty58410.928acetabuloplasty63450.843ligamentum teres debridement38270.932synovectomy14100.955femoral chondroplasty00acetabular chondroplasty00removal of loose body13150.141iliopsoas release41340.308trochanteric bursectomy400.238labralrepair57390.870base refixation23100.155combined230.682simple stitch32260.473debridement31180.451excision100.857resection100.857reconstruction240.383none140.183capsularrepair / plication54280.0635release39360.096partial capsulotomy010.857none00 procedures for each group , by number of hips there were no significant preoperative differences between groups for vas , or any of the four pro measures , including hos - sss . both groups demonstrated statistically significant improvements from preoperative to 3-month , 1- and minimum 2-year follow - up scores based on vas and the four pro measures , including hos - sss ( p < 0.001 ) ( fig . 1 and table iv ) however , all four pro scores were significantly higher in the rts group at minimum 2-year follow - up compared with the nrts group ( p < 0.01 ) . the vas score was also significantly lower in the rts group than the nrts group at minimum 2-year follow - up ( p < 0.01 ) . furthermore , 1-year scores were significantly better in the rts group for all four pro measures ( p < 0.05 ) but not vas average scores for the rts and nrts groups are shown in dark grey and light grey , respectively . braces indicate statistically significant differences in delta value , calculated from preoperative to 2-year time points , ( between the rts and nrts groups ) . table iv.preoperative and minimum 2-year outcome scores for each group , by number of hipsrtsnrtsp - value rts versus nrtsp - value pre to 2-years ( rts)p - value pre to 2 years ( nrts)mhhspre66.864.90.412 years86.178.40.00263.0 105.3 10nahspre65.459.90.0532 years86.976.80.000237.5 104.4 10hos - adlpre69.766.20.212 years88.578.80.000381.5 100.00015hos - ssspre44.941.90.442 years76.062.00.00196.8 103.9 10vaspre5.255.320.852 years1.593.352.2 102.0 104.3 10satisfaction2 years8.457.282.2 10p - values are provided for the differences between groups , as well as for the differences between preoperative and minimum 2-year scores , for each group . average scores for the rts and nrts groups are shown in dark grey and light grey , respectively . braces indicate statistically significant differences in delta value , calculated from preoperative to 2-year time points , ( between the rts and nrts groups ) . preoperative and minimum 2-year outcome scores for each group , by number of hips p - values are provided for the differences between groups , as well as for the differences between preoperative and minimum 2-year scores , for each group . delta values calculated between the preoperative and minimum 2-year scores were significantly different between the rts and nrts groups for mhhs , hos - adl and hos - sss , but not for nahs ( p = 0.14 ) . pre- and post - operative baseline and 2-year follow - up averages are reported in table iv . the vas delta values between the two groups were also significantly different ( p < 0.001 ) . in the rts group , the mean vas improved from 5.4 preoperatively to 1.8 at 2-year follow up , while in the nrts group , the mean vas improved from 5.4 preoperatively to 3.4 at 2-year follow up . ability to perform sports - related movements was assessed by analyzing data from questions within the hos - sss . these sport - specific movements were graded at 2-year follow - up from 0 to 4 . a grade of 0 showcased the ability to perform the action with no difficulty , whereas a grade of 4 demonstrated the inability to perform said action with varying degrees of difficulty in between . for the rts group , 91 ( 98% ) completed surveys compared with 65 ( 100% ) from the nrts group . we found that , at minimum 2-year follow - up , patients in the rts group were significantly better able to perform the following sport - specific movements ( p < 0.05 ) : running one mile , jumping , landing from a jump , stopping quickly and cutting / lateral movements ( fig . 2.ability of patients in rts and nrts groups to perform sports - related activities , as reported via hos - sss . subplots indicate frequency of hos - sss responses within each group , ranging from 0 ( no difficulty to perform sports - related activity ) to 4 ( unable to perform activity ) . ability of patients in rts and nrts groups to perform sports - related activities , as reported via hos - sss . subplots indicate frequency of hos - sss responses within each group , ranging from 0 ( no difficulty to perform sports - related activity ) to 4 ( unable to perform activity ) . we illustrate our findings of hip arthroscopy evaluating rts capabilities in a general hip arthroscopy cohort . we found that arthroscopic hip surgery was associated with significant improvements in all pro scores . the rts group demonstrated significantly higher pro scores than the nrts group at 1- and minimum 2-year follow - up . similarly , vas pain scores were significantly more improved in rts patients than in nrts patients , at minimum 2-year follow - up . additionally , rts patients had higher self - reported abilities to perform several sport - specific actions such as cutting / lateral movements and running 1 mile . fai is a common cause for pain and discomfort in patients , whether athletes or not [ 1 , 2 , 6 , 8 , 11 , 12 ] . abnormal axial , torsional and rotational forces placed on the hip joint result from these pathologies [ 13 ] . recently implicated as a possible factor in early onset osteoarthritis , fai causes abnormal and distressing motions of the lower extremities . many studies have hypothesized that early treatment of fai using arthroscopic modes of surgery may prevent further hip damage as well as aid in an early return to activities [ 24 , 8 ] . although this study includes a diverse group of atheltes undergoing hip arthroscopy primarily for labral tears , the majority of this patients have a component of fai . the data contained here can be used to counsel fai patients on the potential to rtss following hip arthroscopy . it is clear that not all patients are able to rts , or chose not to , following hip arthroscopy , and of those who do approximately one - third return at a decreased level of play . previous studies on rts have included different designs and measures [ 13 , 14 ] . compared hip arthroscopy outcomes between 80 athletes and 42 non - athletes diagnosed with fai at minimum 1-year follow - up . no differences between athletes and non athletes were noted at 1-year follow up , and mhhs increased from a mean of 6180 , nahs increased from a mean of 6785 . this study differs in that we only included athletes in the study , and compared patients who continued in their sport to patients who did not . we observed very similar changes in mhhs and nahs scores at 2-year follow up ( table iv ) . interestingly , there did not seem to be a significant change in mean pro scores from 3 months to 2 years . investigated rtss in a general cohort of 153 patients that underwent open surgical hip dislocation for fai . within their cohort 126 patients acknowledged sports participation prior to surgery , and 107 ( 85% ) acknowledged sports participation after surgery . however , eight patients started participating in sports after surgery that did not participate prior to surgery , and 19 ( 12.4% ) stopped participating in sports . at a mean 5-year follow - up they found an hos - sss score of 75 , and a hos - adl score of 89 . in this study , we found 25 of 148 patients ( 17% ) did not rtss for hip - related issues . the studies utilized different methods of collecting sports participation data and clearly the surgical procedures differ ; however , the percentage of patients that discontinued sports secondary to persistent hip disability was similar between studies . one might expect those patients who participate in high level sports , are female , are older , have a higher bmi or have a more complex surgical procedure to be at risk for not returning to sports . however , this study found no difference in preoperative level of competition , gender , age , bmi , types of sports and surgical procedures and whether the patient returned to sports or not . we did note football , a high impact and high - level sport , showed a near statistically significant difference in patients not returning to sport . however , we do nt believe the study is powered to detect such differences , and this should be the topic of further investigation . capsular closure at the time of hip arthroscopy has recently gained attention as a potential variable that may impact long term outcomes . frank et al . recently demonstrated improved hos - sss scores in patients undergoing capsular closure at 6 months , 1 and 2.5 years compared with a group of patients not undergoing complete closure ( frank and nho ajsm 2014 ) . interestingly in this study , the rts group had 54 ( 58% ) patients treated with capsular repair and/or plication compared with 28 ( 44% ) from the nrts group . with the number of patient available the difference trended towards statistical significance ( p = 0.06 ) . those that receive capsular repair / plication may have a better outcome than those who do not . there are numerous studies in the literature , such as those discussed above , assessing rtss in professional athletes . professional athletes may be more inclined to rts due to their limited window of opportunity to maintain their livelihood and may be influenced by monetary considerations as opposed to recreational athletes who may decide not to rts for the sake of prolonging hip longevity [ 3 , 16 , 17 ] . yet , even among professional athletes , confounding variables such financial incentives , age and talent , which differ among them , often play a role in rtss as well . our study addresses a wide continuum of athletic activity , from recreational to professional , that represents the majority of patients who are candidates for arthroscopic intervention of the hip . we belive the results of this study are applicable to the general athletic population that a hip arthroscopist may encounter in practice . in our study , we defined two cohorts , one consisting of patients that reported involvement in athletic activities post - surgery and the other consisting of patients that did not . although there is no accepted gold standard in quantifying rtss , we believe that using pre- and post - operative comparisons of both pros and sports - specific activity provides valuable information in comprehensively assessing successful rtss . our study addressed a mixed cohort of patients who provided sports - related information and other outcomes at minimum 2-year follow - up . the cohort is limited to patients who chose to indicate participation in sports , which may represent selection bias . the study was a retrospective review of prospectively collected data , and we had complete sport related information on 158 of 321 ( 49% ) hips . additionally , our cohort was not broken down into categories indicating level of play , such as recreational versus professional , as this information was not consistently available . responses indicating sports - related abilities were also self - reported , and therefore have the potential for reporting bias . a common limitation not unique to our study is the incomplete ability to assess the many confounding factors that influence and affect an individual s decision to rtss . in a general cohort of hip arthroscopy patients , patients who indicated rtss demonstrated significantly higher pro scores and abilities to perform several sport - related movements , compared with patients who did not . this work was supported by the american hip institute , though no funding was received directly for this work . dr . domb reports : consulting fees from arthrex inc . , stryker mako surgical corp . , amplitude , and pacira ; royalties from arthrex inc . , orthomerica , and djo global ; and boardmembership at the aana learning center committee . dr . domb is a boardmember at the american hip institute , which funds research and is the institute at which this study was conducted . dr . domb reports research support given to the american hip institute from arthrex inc . breg , ati , and pacira ; however , no direct support was given for this study in particular .

previous studies assessed elite athletes return to sport ( rts ) after hip arthroscopy , but few investigated a cohort including athletes from all levels of sport . this study compared athletes who returned to sport to those who did not , based on four patient - reported outcome ( pro ) scores , including the hip outcome score sports specific subscale ( hos - sss ) . between september 2008 and april 2012 , hip arthroscopies were performed on 157 patients ( 168 hips ) who reported playing a sport preoperatively and indicated their level of sports activity post - operatively . two - year follow - up was available for 148 ( 94% ) amateur and professional athletes with a total of 158 hips . of these 60 cases ( 65 hips ) did not return to sports ( nrts ) and were in the nrts group . the remaining 88 cases ( 93 hips ) constituted the rts group . the modified harris hip score , non - arthric hip score , hip outcome - activities of daily living ( hos - adl ) , and hos - sss were used to assess outcomes . the hos - sss was used to assess specific sport - related movement . both groups demonstrated significant improvement at 2 years post - operatively in visual analog score and four pro scores ( p < 0.001 ) . there was no significant preoperative differences in hos - sss scores between groups ; however , the rts group had significantly higher hos - sss scores at 1 and 2 years post - surgery . post - operatively , the rts group had significantly better ability to jump , land from a jump , stop quickly and perform cutting / lateral movements ( p < 0.05 ) . in summary , patients who indicated rtss demonstrated significantly higher pro scores and abilities to perform several sport - related movements , compared with patients who did not .

INTRODUCTION METHODS RESULTS DISCUSSION LIMITATIONS CONCLUSION FUNDING CONFLICT OF INTEREST STATEMENT

PMC3339708

the state - wide study by the university of ulm on family friendliness in medical school found a mixed picture at the freiburg faculty from the perspective of those interviewed . on the one hand , some specific aspects of the freiburg faculty are singled out for praise by the interviewees , for example the flexibility introduced through the block structure at the second stage of studies since the introduction of the new licensure act in 2004 . at the same time , however , students are still calling for improvements in some areas as balancing medical school and children still constitutes a challenge for many of the respondents , not least of all because of the peculiarities of the medical curriculum . expressing in school grades , the family friendliness of the faculty this result puts freiburg , along with heidelberg and tbingen , into midfield across the state . around three percent of students in freiburg it is striking that many student parents ( 60% ) have already some form of professional training prior to starting medical school . the average age of students with children is about 30 and over 90% of respondents are married or cohabiting ( see table 1 ( tab . 1 ) ) . financial problems play a lesser role than timing issues in balancing medical school and caring for children . a large proportion of respondents ( 71% ) appear to have problems balancing medical school and family life in freiburg , both in the preclinical stage of studies ( 82% ) and , although not as many , the clinical stage of studies ( 68% ) . the perception is that the main detrimental factors regarding compatibility in freiburg are the number of compulsory courses , the high number of afternoon events , the relatively rigid timetable and the exam cluster in a short period of time at the end of the semester ( exam week ) . due to the structure of medical studies , these are problems which are of course also criticised on other campuses but they appear to be particularly strongly felt in freiburg according to the survey . points seen as positive by the majority are the block structure and flexible group assignments at the second stage of studies . similarly , a number of special solutions for students with children are highlighted as being positive , such as consideration for the preferred location when assigning places for the practical year . to improve the situation of student parents , the primary demand is fully customised and flexible timetabling . a majority of respondents also are in favour of primarily offering compulsory courses in the mornings , with lectures mainly offered in the afternoon . furthermore , there are wishes for announcing course lists and timetables for each subject as early as possible to enable them to plan ahead . and finally , special regulations regarding options for making up for missed classes caused through childcare issues , in particular if due to the illness of a child , are advocated . on this point , 90% of respondents call for greater flexibility regarding compulsory attendance , 71% for options to re - sit missed dates or exams and 93% of respondents think that the introduction of a parent passport makes sense . the introduction of a parent passport was already planned at medical faculties in ulm and frankfurt am main last year . in addition , in conflict situations in education a parent passport can help reaffirm the rights of student parents . the evaluation of the results also showed that with regard to certain points the respondents have a great need for advice ( e.g. regarding study organisation , childcare facilities or a part - time studying ) . however , it is noticeable at the same time that only a small proportion of respondents ( 31% ) actually take advantage of advisory services run by the office of the dean regarding study organisation . in contrast , 71% state they would make use of individual advisory services on study organisation with a child , 70% of respondents did not know that such an offer exists . this clearly shows that in future existing advisory services must be more clearly advertised by the academic dean s office . the most frequent requests from students were for an information booklet , a dedicated office for advisory services and coordinating the various advisory services within the university . and finally , the inadequate provision of childcare places and the very rigid and inadequate opening times of the care facilities within the university are criticised . the minimum number of compulsory courses is fixed through the medical licensure act ( appo ) and in contrast to other courses , is very high . apart from a large amount of theoretical knowledge , a medical career requires a multitude of different practical skills and a high level of social skills . these extensive requirements are reflected in a very time - intensive curriculum ( according to appo ) . thus reconciling family life and medical studies nevertheless , the number of compulsory courses in the curriculum in freiburg is higher than prescribed by the appo . the office of the dean of studies is currently looking into reducing the number of compulsory hours in general as part of the planned curriculum reform . the lectures offered are usually optional . the design of the freiburg curriculum in the first and second stage of studies is , with only a few exceptions , structured in such a way that the main lectures are offered in the morning and courses and practical training in the afternoon . this , according to the present study , is a major problem for students with children , especially as childcare is normally only on offer until early afternoon , with the courses and the practical training in some cases running into evening hours . the dean s office in freiburg takes these issues affecting students with children very seriously . it has not been possible , however , to develop solutions together with the institutes and clinics that meet the requirements of all parties involved . timetables and clinical routines for years have been structured so that patient care and routine processes occur mainly in the mornings , making simultaneous teaching in small groups impossible . the needs of staff with children are certainly also taken into account , who themselves prefer core working hours to be in the mornings . since changing the times of the main lectures also seems difficult , the dean s office is prioritising the way of offering more lectures online as video podcasts , thus enabling flexible access to these teaching events . whether it will be possible to offer some courses and practical training for students with children in the mornings in the long term , will certainly be tested in the context of implementing the new curriculum . with the introduction of the new licensure act , the medical faculty in freiburg has opted for the introduction of block teaching in the second phase of studies . also , any number of courses can be chosen and thus balanced with childcare , work on md theses or other obligations . a disadvantage of the block structure , however , is that all teaching units of a subject must be offered in a short period of time . many subjects strive to provide students with children options for changing but this is not always possible , especially not when such requests are made short - term . both the winter and the summer semester in freiburg run over 14 weeks in the clinical section of studies . the extreme stress on the students during the exam week ( usually 6 to 8 exams in one week ) has already been modified in the past so that dates during the study week are also available for exams . in addition , many of the small subjects hold their exams immediately after their block of classes during the semester . the trend towards holding examinations in small subjects immediately after the block has been taught is strongly supported by the dean s office . compulsory attendance or regular lecture attendance and any possible ways of making good missed dates are covered in the study regulations in freiburg . regular attendance of a course is defined as having been present for at least 85% of the total teaching time . if the absence exceeds 15% due to circumstances beyond the control of a student , the course director , in agreement with the dean of studies , will decide of measures to compensate for missing dates . this wording was deliberately chosen so that the personal circumstances of students can be taken into consideration . it thus lies within the discretion of each subject area when or how missed classes can be retaken or compensated for in other ways . when compensating for missed practical coursework , it is key to note that in medical school - unlike in the humanities - especially the high proportion of practical work can not be compensated through other tasks such as papers or homework . the successful participation in obligatory practical coursework by the students is essential for the benefit of future patients . the core message of the survey conducted by the university hospital ulm is confirmed by a study carried out by the centre of competence for the evaluation of teaching in medicine baden - wrttemberg in the winter semester 2008/09 on the study and live situations of medical students in freiburg . 32% of students with children stated here childcare is causing them study - related problems . again , time related issues regarding the compatibility of studies and childcare were identified as a particular challenge for students with children . the respondents hopes for a flexible and individual design of medical studies are juxtaposed with the relatively fixed structure of medical school . the understandable desire of students with children for individual and flexible timetabling can not always be met due to the high numbers of students enrolled in the medical programme , although the office of the dean of studies preferentially allocates places to student mothers and fathers . it would undoubtedly constitute a structural improvement if appropriate timetable software could be installed which would allow for more personal requests being met than is possible with the traditional manual model . for this to work , however , it would be necessary for the subject areas to agree to central timetabling . the results of the ulm study showed a surprising lack of knowledge about the advice services offered by dean s office regarding study planning for students with children . these services are explicitly referred to during the introductory events at the beginning of the first pre - clinical and the first clinical term . since this does not appear to be sufficient , in future the existing advisory services will be made visually clearer in the introductory powerpoint presentation . in addition , prompted by the results of the survey on family - friendliness , simultaneously a website will be set up containing information services on the homepage of the dean s office especially for students with children . it will contain all of the services offered by the dean s office and the appropriate links to the medical faculty and the university . any additional services , for example a coordinating office or social services for students with children can , however , not be offered by the dean s office with current staffing levels . this would incur personnel and operating costs that exceed the current budget of the dean s office . overall , the medical faculty is keen to improve the study environment for students with children . but currently only an insufficient number of child and infant care places are available at the university and the university hospital . it would certainly be desirable to resolve this situation through appropriate offers by the university and university hospitals , especially in view of the increasing number of female medical students ( 60 to 70% of the total cohort ) . a first success in this regard was achieved by the equal opportunities officer at the faculty of medicine as recently a nursing room in the women s clinic for children under 6 months can now also be used by female medical students . as the centre of competence for the evaluation of teaching in medicine baden - wrttemberg had already carried out its survey regarding the study and live situations of medical students in freiburg during the winter semester 2008/2009 , the medical faculty was not surprised by the results of the ulm study on family - friendliness . the existing predominant block structure , the flexible assignment to groups in the clinical section and the advantages in the placements for the practical year , all of which were already introduced in the context of the new medical licensure act in 2004 , received a correspondingly positive assessment from the students in the ulm survey on family friendliness . the results of this survey have also encouraged the faculty of medicine to retain the tried and tested block structure in the last two years of clinical studies as part of current curriculum developments in medical degree courses following fundamental basic training in theory and practice in the first year of clinical studies in order to ensure the continuity of the flexibility achieved to date . in addition , the existing information strategy of the dean s office has been revisited in terms of students with children . this is to be further improved in order to facilitate the students access to information . a planned website with specific information and advisory services for students with children on the homepage of the dean s office will serve as a basis . existing advisory services offered by the academic dean s office must be more clearly referred to in future , as the survey points to an information deficit in this area . but the problem of a lack of human resources in the dean s office remains . and given the extremely large number of medical students , some 2,200 , and a proportion of 60 - 70% of female students which is tending towards increasing , these problems will further intensify in the coming years .

the survey on family - friendly study organisation in medical schools conducted by the university hospital in ulm has identified a need for improvement in various respects in freiburg . due to the specific structure of medical school and the high amount of mandatory lectures , students with children face serious problems in balancing family life and their studies at the same time . on the other hand , the freer , modular structure of the clinical curriculum in freiburg has been mainly rated as positive by the interviewees . in order to improve the situation of students with children , the interviewees favour a more flexible curriculum in general as well as an increase in information and advice services offered by the faculty.in the first place , the results of the study encourage us to maintain the modular structure in the final two clinical years in freiburg in view of current curriculum developments . additionally , we aim to offer targeted support to students with children . furthermore , a wider range of e - learning supported lectures is to help students manage their studies with childcare and family obligations .

Summary of Study Results for the Faculty of Freiburg Structure of Medical Studies in Freiburg Discussion of Results and Outlook Conclusion Note Competing interests

PMC3173638

epicrania fugax ( ef ) is a novel syndrome first described by pareja et al . in 2008 in ten patients that complained of a very brief unilateral pain paroxysms , starting in posterior cranial regions and rapidly spreading to ipsilateral eye , forehead or nose , along a linear or zigzag trajectory . in some cases , pain was accompanied by autonomic signs such as conjunctival injection , lacrimation or rhinorrhea . to this first series , 15 new patients with the same clinical features have been added , so reinforcing the proposal of ef as a new headache variant or a new headache syndrome [ 24 ] . recently , two patients who fulfilled all the characteristics for ef except the direction of radiation have been reported . since the first description of bref in february 2010 , we made , in an outpatient headache clinic located in a tertiary hospital , a prospective search of the patients complaining of brief pain paroxysms running front to back , from anterior to posterior cephalic regions . as we searched for a novel headache syndrome a detailed history was obtained in all the cases , including precipitant event and the coexistence of other types of headache . the characteristics of pain paroxysms were carefully evaluated , including temporal features ( duration and frequency ) and spatial features ( site of origin , trajectory , and site of ending ) . other characteristics such as pain quality , pain intensity ( assessed with a visual analogical scale ) , or pain accompaniments , especially autonomic signs have been included . subsequently , a complete physical and neurological examination was performed , including inspection , palpation and sensory examination of the stemming area , as well as palpation of the supraorbital , infraorbital , supratrochlear , minor occipital and greater occipital nerves . computed tomography or magnetic resonance imaging of the head , and routine blood work - up with erythrocyte sedimentation rate and immunological screening we also made a prospective search of patients with a clinical picture from march 2008 , which would be comprised under the headings or epicrania fugax with forward radiation , as was described by pareja at al . we compared the characteristics of both groups with a chi - square test and also fisher s exact test , if necessary . through a one - year period , we assessed nine patients ( four males , five females ) , with pain paroxysms comparable to bref . mean age at onset was 43.4 13.1 years ( range 2662 ) . demographic and clinical features of these nine patients are shown in table 1 . through a three - year period , 18 patients ( 6 males , 12 females ) with fref were attended in our headache clinic . comparison of demographic and clinical characteristics in bref and fref patients is summarized in table 2.table 1demographic and clinical features of nine bref patientspatient no.123456789sex ( m / f)fmfmmffmfage ( years)654639525828533329age at onset623939525826533329other headachesnomigrainemigrainetensin - typemigrainenononomigraineinterictal painnononononocircumscribedcircumscribednonopain paroxysmshead sideleftrightleftleftrightleftrightrightleftsite of origineyeforeheadeyeeyeeyefrontalfrontalfrontaleyesite of endoccipitaloccipitaloccipitaloccipitalparietalparietaloccipitaloccipitaloccipitaltrajectorylinearlinearlinearlinearlinearlinearlinearlinearlinearcharacterstabbingstabbingburningstabbingpressingstabbingelectricstabbingstabbingduration ( sec)551031533155intensity ( vas)6710459975accompanimentsnonononotearing itching eyerhinorrhoea itching eyenonoitching eyetriggersnonononophysical activitytouchnononofrequency8/day12/week1/week1/day1/day20/day20/day12/day1/daytemporal patternchronicremittingremittingchronicremittingchronicchronicchronicchronictherapyamitriptylinenonoamitriptylinegabapentinlamotriginecarbamazepinegabapentinegabapentineresponsecompletecompletenocompletecompletepartialpartialm male , f female , vas visual analogical scale ( 0 : no pain , 10 : the worst imaginable pain)table 2main features of bref and fref patientsbref ( n = 9)fref ( n = 18)sex ( f / m)5/412/6age at onset ( mean sd)43.4 13.142.5 17.7duration sec ( mean sd)7.1 4.95.7 4.3frequency / day ( mean sd)7 8.49.9 15.4intensity vas ( mean sd)6.9 2.16.8 2.1autonomic signs3/910/18interictal pain2/910/18triggers2/94/18preventative requirement7/912/18bref backward radiation epicrania fugax , fref forward radiation epicrania fugax m male , f female , sd standard deviation , sec seconds vas visual analogical scale ( 0 : no pain , 10 : the worst imaginable pain ) demographic and clinical features of nine bref patients m male , f female , vas visual analogical scale ( 0 : no pain , 10 : the worst imaginable pain ) main features of bref and fref patients bref backward radiation epicrania fugax , fref forward radiation epicrania fugax m male , f female , sd standard deviation , sec seconds vas visual analogical scale ( 0 : no pain , 10 : the worst imaginable pain ) we found no differences between bref and fref , respectively , in age at onset ( 43.4 13.1 vs. 42.5 17.7 years ) , and female / male ratio ( 5/4 vs. 12/6 ) . a complete examination of stemming points did not reveal trophic changes in any of the patients and sensory disturbances were appreciated in 8 out of 12 patients , who suffered interictal pain . palpation of pericranial nerves did not detect abnormal findings , and laboratory tests and imaging studies were also normal in all the cases . pain paroxysms were strictly unilateral in all bref patients ( in five cases on the left and in four on the right ) . four cases described a history of migraine and one of tension - type headache , but ef was considered as quite different from the other headaches . pain started in eye ( n = 5 ) , forehead ( n = 1 ) or frontal region ( n = 3 ) , and inmediately spread backwards along a linear trajectory to occipital ( n = 7 ) or parietal ( n = 2 ) scalp . in two of the patients , there was an interictal pain in stemming point located in a circumscribed area , resembling nummular headache paroxysms were described as stabbing in most of the patients ( n = 6 ) , or as electric , burning or pressing ( 1 patient each ) . there was no significant difference between bref and fref , respectively , in paroxysms duration ( 7.1 4.9 vs. 5.7 4.3 s ) , pain intensity in a 010 visual analogical scale ( 6.9 2.1 vs. 6.8 2.1 ) , frequency of episodes per day ( 7 8.4 vs. 9.9 15.4 ) , presence of triggers ( 22.2 vs. 22.2% ) and prophylactic treatment requirement ( 77.7 vs. 66.6% ) . patients in bref group presented less frequently interictal pain in stemming point ( 22.2 vs. 55.5% ) and accompanying autonomic signs ( 33.3 vs. 55.5% ) , but in both the cases without statistical significance . in 2008 , pareja et al . described a headache whose features did not fit any of the acknowledged headaches and they named it as epicrania fugax . this headache was framed within epicranias , a term proposed to group all the headaches that apparently stem from the superficial or extracranial structures , including the scalp and the layers of the skull . common to this group of pain syndromes are a focal location or a sequence of multidirectional paroxysms , shortage of autonomic signs , and a dysesthesic area . regarding ef , there are several entities to which differential diagnosis have to be considered . for instances , psh is a primary headache syndrome , with a female preponderance , characterized by short ( 3 s or less ) stabbing pain paroxysms in a localized area that start and end in the same place [ 10 , 11 ] . frequency of paroxysms is highly variable and they usually occur with an irregular or sporadic temporal pattern . though the consecutive stabs may sporadically shift from one region to another giving an illusion of movement , clinical radiation observed in ef is definitely not typical for psh . nummular headache ( nh ) resembles ef in the presence of a focal painful area . pain in nh is typically continuous and , when paroxysms are superimposed , they begin and end in situ [ 9 , 13 ] . eight of our patients ( two in bref and six in fref groups ) had a nummular type head pain in the stemming area between ef paroxysms , as it has been previously described [ 3 , 14 ] . lacrimation , rhinorrhoea and itching eye are autonomic signs that appears in some of our patients . these signs are typical of other headache syndromes such as sunct ( shortlasting , unilateral , neuralgiform headache attacks with conjunctival injection and tearing ) or suna ( shortlasting , unilateral , neuralgiform headache attacks with cranial autonomic features ) . these headaches are also present with painful neuralgiform attacks in the orbital and periorbital regions , triggered by tactile stimuli on trigeminal and extrageminal territories , and lasting from 2 s to 10 min . however , the dynamic component of pain , either posterior anterior or anterior posterior , in fref and bref are inherent qualities of epicrania fugax , not described in sunct or suna . unlike sunct , suna can be diagnosed with the presence of just one cranial autonomic feature and the attacks have a wider range of duration . thus , some of ef patients could be diagnosed of suna but the presence of a dynamic component of pain in ef is a quality not described for suna [ 1618 ] . this is the essential attribute of ef : a fast and ample movement of the pain through one side of the head , no matter where the stemming point is . origin of this pain is probably peripheral according to the existence of a stemming or trigger zone in a focal area , and the stabbing or electric character of the pain . paroxysms of pain can originate in the terminal branches of supraorbital nerve ( son ) or greater occipital nerve ( gon ) , and eventually extend to the peripheral fibers or branches . it has been proposed that the spreading of the pain is due to the electric transmission or paracrine diffusion of chemical mediators . central mechanisms in which trigeminal afferents are implicated can not be excluded , mainly in those patients presenting with autonomic signs . we report nine patients with the same features described for ef , except the direction of radiation . when we compare them with a group of patients fulfilling the characteristics of ef , we do not find differences between both the groups . our report reinforces ef as an independent headache syndrome and proposes bref as a not infrequent variant of ef . another option is to in considered bref and fref as the same entity with the different presentations .

epicrania fugax ( ef ) is a novel syndrome , described as a paroxysmal and brief head pain , starting in posterior cranial regions and rapidly spreading forward ipsilateral eye , nose or forehead . two patients with comparable clinical features stemming from frontal scalp to ipsilateral posterior regions have been recently described and proposed as backward radiation epicrania fugax ( bref ) . we report a new series of nine bref and compare their clinical characteristics with 18 forward radiation ef ( fref ) . since first description of bref in february 2010 we have assessed nine patients ( four males , five females ) with this clinical picture at an outpatient headache office in a tertiary hospital . comparison is established with 18 fref patients ( 6 males , 12 females ) , attended since the publication of first series of ef in march 2008 . we found no differences between bref and fref , respectively , in age at onset ( 43.4 13.1 vs. 42.5 17.7 years ) , female / male ratio ( 5/4 vs. 12/6 ) , pain intensity ( 6.9 2.1 vs. 6.8 2.1 in a 010 visual analogical scale ) , duration ( 7.1 4.9 vs. 5.7 4.3 s ) and frequency of episodes per day ( 7 8.4 vs. 9.9 15.4 ) . patients in bref group presented less frequently interictal pain in stemming point ( 22.2 vs. 55.5% ) and accompanying autonomic signs ( 33.3 vs. 55.5% ) , but without statistical significance in both the cases . this series reinforces the proposal of ef as a new headache variant or a new headache syndrome . clinical picture of brief pain paroxysms starting in the anterior scalp and radiating backwards does not fit known headaches or neuralgias and might correspond to a reverse variant of ef , clinical characteristics of which are comparable to fref .

Introduction Methods Results Discussion Conclusion

PMC3981410

the presence of a draining sinus along the anterior border of the sternocleidomastoid muscle in the neck usually indicates a second or third branchial cleft sinus or fistula . a heterotopic salivary gland is a rare lesion , which may present at the same site as a cutaneous discharging sinus . the rarity of this congenital lesion , together with the fact that there are no available reports of this lesion from india , stimulated us to report this case to raise awareness of this condition . a 10-year old female presented to the otolaryngology department of our hospital with a draining sinus in her right lower neck . since birth her parents had noticed an intermittent clear mucoid discharge from an opening located in her lower neck . this was preceded by mild swelling or fullness in the lower neck , which was not associated with her having eaten . there was no history suggestive of any infection at any time in the form of pain , redness or pus discharge . on examination , there was a pin - point opening at the anterior border of the sternocleidomastoid muscle approximately 1 cm above the right sternoclavicular joint , but there was no swelling or thickening around the opening . a sinugram using iohexol , a non - ionic contrast dye , was obtained to delineate the sinus / fistula tract ( figure 1 ) . after injecting methylene blue solution to delineate the tract , an elliptical incision was made . the dissection revealed a descending branch and a larger posterosuperio tract . in the middle of the tract , a dilated structure of approximately 1 cm containing mucoid collection and embedded within the muscle the tract ascended further for another 1 cm and ended blindly in a superficial plane . the specimen was made up of a tubular structure 5 cm in length and 0.5 cm in its average diameter , dilated in its middle portion surrounded by muscle . microscopic examination revealed mucinous salivary gland tissue embedded in striated muscle and inflammatory cells were seen ( figure 2 ) . heterotopic salivary gland tissue ( hsgt ) consists of salivary tissue outside of the major and minor salivary glands . heterotopic salivary gland tissue is found most commonly in the periparotid and intra - parotid lymph nodes . the neck , at the anterior border of the sternocleidomastoid muscle near the sternoclavicular joint is also an area of salivary gland heterotopia . klimko and horanyi reported the first histologically documented case of hsgt in the lower neck in 1958 . the other rare locations in the head and neck region include the mandible , maxilla , middle neck , larynx , hypopharynx , middle ear and thyroglossal duct . they are usually asymptomatic incidental findings but can present as a mass or a fistula . benign and malignant neoplasms can arise in such heterotopic salivary tissue , which may sometimes be the initial presenting feature . recognition of salivary gland as a heterotopia is based on the anatomic location of the tissue , and microscopically by the association and relationship to surrounding tissues . for instance , a salivary gland surrounded by skeletal muscle or in a ( extraparotid ) lymph node is heterotopic . as salivary glands do not migrate during development , like parathyroids or the thymus , it is presumed that most heterotopias of salivary glands in neck arise de novo from the endoderm of the pharynx . in the lower neck , it is believed that the tissue originates from a pre - cervical sinus of his or a cervical vesicle , a distinct embryologic structure that forms in the lower part of the neck between the second branchial arch and the upper thoracic wall , which normally disappears before birth . this is thought to occur by heteroplasia or anomalous differentiation of ectodermal cells within the remnant of the pre - cervical sinus to salivary tissue . there are rare reports of occurrence of heterotopic salivary tissue in association with branchial cleft fistula which support the belief that the heterotopic glands arise through errors of development in the branchial apparatus . so hsgt should be looked for in every cervical anomaly related to any branchial apparatus derived lesion . clinically , they are located along the anterior border of sternocleidomastoid muscle with a cutaneous opening draining mucoid or serous discharge , which resembles branchial cleft fistulas or sinuses . the opening may be anywhere , in mid or lower neck . in our case , it was near the sternoclavicular joint . this made us doubt it was a branchial cleft fistula where the opening is usually at the junction of upper 2/3 and lower 1/3 . other differentiating features are the absence of a history suggestive of infection and an increase in the discharge at the time of meals , mastication or menstruation in case of heterotopic salivary gland sinuses . cases generally present at birth or by early childhood , but they are rarely diagnosed in adulthood . congenital abnormalities of the branchial apparatus can manifest as different abnormal conditions in the neck , including a cyst ( a small , partial malformation with no external opening ) , a sinus ( a malformation with one superficial opening ) , or a fistula ( a complete malformation with both internal and external communications ) . fistulas of the second branchial cleft are the most common , accounting for as many as 90% of all branchial cleft fistulas . the tract crosses superiorlylateral to the common carotid artery , the glossopharyngeal nerve , and the hypoglossalnerve , and lies between the internal and external carotid arteries . fistulas of the third branchial cleft , which are rare , also appear at the anterior border of the sternocleidomastoid muscle . here surgery for heterotopic salivary gland fistula is limited in extent compared to surgical removal of a branchial fistula . in our patient , another horizontal incision was given superiorly to facilitate the dissection but the tract soon ended and appeared as if some fiber of strap muscle had been removed . a sinugram / fistulogram is helpful in accurate pre - operative diagnosis and differentiating hsgt from branchial cleft fistulas . with the fistulogram , the surgeon can delineate the course of the tract , which may be superficial in a heterotopic salivary gland with an arborizing pattern resembling a sialogram or deep between carotid arteries in a banchial fistula . it also shows the distal end structure , which may be a cyst , an internal communication with the pharynx in case of a brachial cleft fistula , or a heterotopic salivary gland . the fistulogram or a sinugram can give information regarding the width of the tract and any ectasia or dilatation , a feature of hsgt , as was present in our case . therefore , the above mentioned clinical features and a pre - operative fistulogram may help to differentiate between various draining fistulas of neck present at the anterior border of sternocleidomastoid .

we report a case of congenital heterotopic salivary gland with draining sinus in the lower neck on the right side of a 10-year - old female , which we initially thought to be a branchial fistula . heterotopic salivary glands are rare lesions in the neck and when present appear very similar to branchial cleft sinus or fistula . this congenital lesion is rare . this is probably the first report from india . it is important to report this case to raise the awareness of this condition .

Introduction Case Report Discussion

PMC2246070

recent work shows that diffusion magnetic resonance imaging ( dmri ) can help recovering complex white matter fiber bundles . however this is still an open problem due to the structural complexity of the fiber bundles , which can have crossing configurations . diffusion tensor imaging ( dti ) is restricted in these conditions due to the hypothesis that the diffusion within a voxel follows a gaussian distribution , a model that can not model intravoxel crossings . q - ball imaging ( qbi ) , a recent high - angular resolution diffusion imaging ( hardi ) technique , overcomes this limitation by reconstructing a diffusion orientation distribution function ( odf ) , a spherical function that has its maxima agreeing with the underlying fiber populations . the odf reconstruction from qbi is attractive because it is model - free and has been recently shown possible with a regularized and analytical solution , which produces a robust and very fast odf reconstruction . in fact , the odf estimation is , in practice , as fast as a standard least - square diffusion tensor ( dt ) estimation . efficient segmentation of fiber tracts in dmri images is an important problem in neuroimaging problem because it has many potential applications . for example , it could potentially provide important information on diseases that affect fiber tracts . alteration of the fiber tracts may provide new biomarkers in white matter pathologies and segmentation of these tracts can also improve our understanding of the functional role these tracts have and the cognitive consequences of their disruption . the goal of this work is to provide a segmentation method that can separate the main white matter fiber bundles in the brain . we propose a new method that can segment fiber bundles and deal with fiber crossings while also requiring a minimum number of hypothesis from the data and a small number of algorithmic parameters . spectral embedding and clustering methods have recently proved to be effective in image segmentation [ 4 , 5 ] . however , classical approaches require restrictive hypotheses that are difficult to meet in real applications . for instance , n - cuts and laplacian eigenmaps require data within each cluster to be uniformly sampled , which produces artifacts when this hypothesis is not met . moreover , classical approaches for image segmentation also assume that the scale within each cluster is the same using a single - scale parameter for the whole dataset . in order to overcome these limitations this method looses the dependence on the sampling of the elements to cluster . moreover , we propose to use an adaptive scale - space parameter in order to deal with space - scale differences across different clusters . finally , we propose two approaches to automatically determine the number of clusters by analyzing the spectra of the image embedding . another contribution of this paper is to show that the q - ball odf clustering using diffusion maps can reproduce the dt clustering using n - cuts on simple synthetic images without crossings . on more complex data with crossings , we show that our method succeeds to separate fiber bundles and crossing regions on synthetic data , where the dt - based methods generate artifacts and exhibit wrong number of clusters . finally , we successfully segment the fiber bundles in a real - human brain dataset in different regions with fibers crossing . the main goal of this work is to produce a segmentation algorithm able to segment white matter fiber bundles from dmri data . in order to represent intravoxel crossings with the odf , we need at least 15 real coefficients when a spherical harmonic basis is used [ 3 , 8 , 9 ] . this leads to 3d images with a high dimensional element at each voxel . this high dimensionality makes previous diffusion imaging segmentation approaches based on level set methods such as [ 1012 ] computationally expensive . moreover , these methods require an initialization step . in order to perform the segmentation in an initialization - free manner and with a lower - dimensionality image , we use spectral clustering methods [ 4 , 5 ] which perform dimensionality reduction before performing the segmentation and do not need initialization . the segmentation is then performed on the statistics within each cluster and the fiber crossings can be identified . in this section , we present the three main parts of our algorithm . first , the estimation of the q - ball diffusion odf and its compact representation using spherical harmonics . second , the metric used to measure distances between q - ball odfs . last , the diffusion aps spectral clustering technique used to segment the odf image into the background and the different fiber bundles . qbi reconstructs the diffusion odf directly from the hardi measurements on a single sphere by the funk - radon transform ( frt ) . in practice , the frt value at a given spherical point is the great circle integral of the signal on the sphere defined by the plane through the origin with normal vector . the odf is intuitive because it has its maximum(a ) aligned with the underlying population of fiber(s ) . however , computing statistics on a large number of discrete odf values on the sphere is computationally heavy and infeasible to integrate into a segmentation algorithm of the whole brain . a more compact representation of the odf is thus needed . in [ 3 , 8 , 9 , 13 ] a simple analytic spherical harmonic ( sh ) reconstruction of the odf is proposed . for completeness of the article , we now review and develop the main parts of our regularized analytical odf reconstruction solution . the idea is to first estimate hardi signal on the sphere with a regularized spherical harmonics approximation and then do a simple linear transformation of the harmonics to obtain the desired regularized odf . the sh , normally indicated by ym ( denotes the order and m the phase factor ) , are a basis for complex functions on the unit sphere . explicitly , they are given as follows : ( 1)ym(,)=2+14(m)!(+m)!pm(cos)eim , where ( , ) obey physics convention ( [0,],[0,2 ] ) and pm is an associated legendre polynomial . for k=0,2,4, , and m=k, ,0, ,k , we define the new index j:=j(k , m)=(k2+k+2)/2+m and define our modified basis y with elements yj such that ( 2)yj={2re(ykm)if km<0,yk0if m=0,2img(ykm)if 0<mk , where re(ym ) and img(ym ) represent the real and imaginary parts of ym , respectively . symmetry is ensured by choosing only even order sh and the ratios in front of each term also ensure that the modified basis is real and orthonormal with respect to the inner product f , g=f*gd , where denotes integration over the unit sphere and f * is the complex conjugate of f for f and g complex functions on the sphere . we thus approximate the signal at each of the n gradient directions i as ( 3)s(i,i)=j=1rcjyj(i,i ) , where r=(+1)(+2)/2 is the number of terms in the modified sh basis y of order . letting s be the n 1 vector representing the input signal for every encoding gradient direction , c the r 1 vector of sh coefficients cj , and b is the n r matrix constructed with the discrete modified sh basis ( 4)b=(y1(1,1)y2(1,1)yr(1,1)y1(n,n)y2(n,n)yr(n,n ) ) . we can write the set of equations as an overdetermined linear system s = bc . we want to solve for the sh series coefficients cj , where cj=s(,)yj(,)d. at this point , instead of simply evaluating the integrals directly as done in or performing a straightforward least - squared minimization as in [ 15 , 16 ] , we add local regularization directly into our fitting procedure . this is to be able to use a high - order estimation without overmodeling the small perturbations due to noise in the input diffusion mri signal . we thus define a measure , e , of the deviation from smoothness of a function f defined on the unit sphere as e(f)=(bf)2d , where b is the laplace - beltrami operator . using the orthonormality of the modified sh basis , where we have yi(,)yj(,)d=ij , the above functional e can be rewritten straightforwardly [ 3 , 13 ] as ( 5)e(f)=b(pcpyp)b(qcqyq)d=j=1rcj2(j)2((j)+1)2=ctlc , where l is simply the rr matrix with entries (j)2((j)+1)2 along the diagonal ( (j ) is the order associated with the jth coefficient , that is , for j=1,2,3,4,5,6,7, (j)=0,2,2,2,2,2,4 , ) . therefore , the quantity we wish to minimize can be expressed in matrix form as ( 6)m(c)=(sbc)t(sbc)+ctlc , where is the weight on the regularization term . the coefficient vector minimizing this expression can then be determined just as in the standard least - squares fit ( =0 ) from which we obtain the generalized expression for the desired spherical harmonic series coefficient vector ( 7)c=(btb+l)1bts . from this sh coefficient vector we can recover the signal on the q - ball for any ( , ) as s(,)=j=1rcjyj(, ) . intuitively , this approach penalizes an approximation function for having higher - order terms in its modified sh series . this eliminates most of the higher - order terms due to noise while leaving those that are necessary to describe the underlying function . however , obtaining this balance depends on choosing a good value for the parameter . we use the l - curve numerical method and experimental simulations to determine a good smoothing parameter [ 3 , 13 , 18 ] . here , =0.006 is used as in [ 3 , 13 , 18 ] . the true diffusion orientation distribution function ( odf ) in a unit direction u , (u ) , is given by the radial projection of the probability distribution function ( pdf ) of the diffusing water molecule . tuch showed that this diffusion odf could be estimated directly from the raw hardi signal s on a single sphere of q - space by the funk - radon transform ( frt ) ( figure 1 ) . in [ 3 , 13 ] , we showed how this frt can be evaluated analytically with an elegant corollary to the funk - hecke theorem . the final odf reconstruction on the sphere then becomes a simple linear transformation of the sh coefficients cj describing the input hardi signal s , ( 8)(,)=j=1r2p(j)(0)cjfjyj(, ) , where fj are the sh coefficients describing the odf and p(j)(0)=(1)/2(135((j)1)/246(j ) ) because (j ) is always even in our modified sh basis . we see that the shs are eigenfunctions of the funk - radon transform with eigenvalues depending only on the order of the sh series . hence , by using an sh estimation of the hardi signal , we have showed that the qbi can be solved analytically . this was also showed in [ 8 , 9 ] . an important contribution in favor of our approach is that this solution can be obtained while imposing a well - defined regularization criterion . the accuracy of the modified sh series approximation with the laplace - beltrami smoothing was established in and our regularized odf solution was also shown to have better fiber detection properties and shown to be more robust to noise than similar solutions [ 8 , 9 ] . once the odf are computed , we want to capture similarities and dissimilarities between two odfs , that is , two spherical functions ,s2 that can be represented by real - sh vectors of length r , f={f1, ,fr } and f={f1, ,fr}r , as shown in ( 8) in the previous section . since the odfs come from real physical diffusion measurements they are bounded and form an open subset of the space of real - valued 2 spherical functions with an inner product , defined as ( 9),=(,)(,)d=(i=1rfiyi(,)j=1rfjyj(,))d. again , because of the orthonormality of the spherical harmonic basis , the cross - terms cancel and the expression is simply ( 10),=j=1rfjfj. therefore , the induced 2 norm =, giving us the distance metric between two odfs is ( 11)=j=1r(fjfj)2 . the euclidean distance was also used successfully for odf segmentation in and for dti segmentation in even though more appropriate metrics exist such as the j - divergence [ 11 , 20 ] and riemannian geodesic distances . for instance , since the odf can be viewed as a probability distribution function ( pdf ) of fiber orientations , one can use the kullback - leibler distance between two pdfs , as done in . however , in that case the problem quickly blows up computationally because one needs to use all n discrete hardi data on the sphere instead of the r sh coefficients ( rn ) . we now want to segment white matter fiber bundles in a q - ball image . one of the open questions in q - ball image analysis and clustering is that which metric should be used to compare q - ball odfs . here , we describe a clustering algorithm that infers an embedding and a metric to compare odf images . we derive an affinity measure incorporating the euclidean distance and the spatial location distance between odfs . as mentioned in , the euclidean distance within this embedding actually represents an intrinsic metric of the data , which can be used to perform statistics in the embedded space and can thus be used to segment q - ball odf images into white matter fiber bundles . in recent years , spectral manifold learning and clustering techniques [ 4 , 6 , 2123 ] have become one of the most popular modern clustering family of methods . they are simple to implement , they can be solved efficiently by standard linear algebra software , and they very often outperform traditional manifold learning and clustering algorithms such as the classical principal component analysis ( pca ) and k - means algorithms . moreover , due to the dimensionality reduction properties , they are especially well suited to work with high - dimensional data . these techniques have been recently used to cluster various types of images [ 4 , 5 ] and white matter fiber tracts . in our case , we perform the spectral clustering for two different types of elements : the dt and the odf . in the dt case , the element is represented by a 6-dimensional vector corresponding to the upper ( or lower ) triangular part of the dt 33 symmetric matrix . in the odf case , the element is represented by the 15-dimensional vector corresponding to the 4th - order spherical harmonic odf estimation . each element to be clustered is represented as a node in a graph and the edges joining the vertex are a measure of affinity between the elements . this affinity measure lies between 0 and 1 , 0 being the less affine case . a spectral decomposition of this graph is taken by calculating the eigenvalue decomposition ( evd ) of the graph laplacian . then a low - dimensional euclidean manifold embedding is inferred from this decomposition . preservation of the distance relationship : after a distance is defined between elements , the learned manifold should preserve the distance relation.uniform sampling of the elements : the density of the extracted elements changes if and only if these elements belong to anatomically different bundles.convexity of the elements : if two elements are in the dataset , almost all of the intermediate tracts obtained by the interpolation that can be inferred from the metric used to build the affinity matrix are in the dataset . preservation of the distance relationship : after a distance is defined between elements , the learned manifold should preserve the distance relation . uniform sampling of the elements : the density of the extracted elements changes if and only if these elements belong to anatomically different bundles . convexity of the elements : if two elements are in the dataset , almost all of the intermediate tracts obtained by the interpolation that can be inferred from the metric used to build the affinity matrix are in the dataset . it is not easy to guarantee that the data to be embedded and clustered will adhere to these hypotheses . donoho and grimes , in , analyze when a spectral embedding algorithm is able to recover the true parameterization of a set of images . as medical images represent the discretization of a continuous space , hypotheses 1 and 3 are plausible . however , there is no indication that within a fiber bundle the distribution of the elements ( dt or odf ) are uniformly sampled . moreover , in it is shown that different sampling frequencies within one cluster leads the n - cuts and laplacian eigenmaps methods to subdivide the cluster in several parts . in order to overcome this limitation and to be resilient to sampling frequency differences within a cluster , we use the diffusion maps spectral embedding technique . we now describe the three steps involved in the diffusion maps algorithm in turn . step 1 ( computing the affinity matrix).letting x represent the set of all odf elements to cluster , the main idea is to look for a representation between the elements of x that is more representative than r ( recall that odfs are r ) and reduces the dimensionality of the problem . with keeping this in mind , a fairly good way of representing any set of elements with an affinity function a : xx>0 , is a weighted graph , g(x , e , w( ) ) , where the weight of the edge between two vertices represents the affinity of the elements connected by this edge . more formally , for an edge , e=(fi , fj)e , the weight of the edge is w(e)=a(fi , fj ) . hence , each element of the adjacency matrix of g or conversely the affinity matrix of ( x , a( ) ) is ( 12)aij = a(fi , fj ) . taking this in account , the weighted graph g(x , e , w( ) ) can be also noted as g(x , a ) . letting x represent the set of all odf elements to cluster , the main idea is to look for a representation between the elements of x that is more representative than r ( recall that odfs are r ) and reduces the dimensionality of the problem . with keeping this in mind , a fairly good way of representing any set of elements with an affinity function a : xx>0 , is a weighted graph , g(x , e , w( ) ) , where the weight of the edge between two vertices represents the affinity of the elements connected by this edge . more formally , for an edge , e=(fi , fj)e , the weight of the edge is w(e)=a(fi , fj ) . hence , each element of the adjacency matrix of g or conversely the affinity matrix of ( x , a( ) ) is ( 12)aij = a(fi , fj ) . taking this in account , the weighted graph g(x , e , w( ) ) can be also noted as g(x , a ) . usually , a distance function d( ) instead of an affinity function is given . the distances can be easily converted into affinities by applying a kernel to the distance function ( 13)a(fi , fj)=e(d(fi , fj)2/ij2 ) , where is an adaptive scale - space parameter that may depend on the elements fi and fj . in this work , the adaptive scale - space parameter is taken following . a neighbor - number k is given as parameter to the algorithm and then ij2=d(fi , fik)d(fj , fjk ) , where fik is the kth closest neighbor according to the distance function d(, ) of element fi . this choice of a scaling parameter for each point allows self - tuning of the point - to - point distances according to the local statistics of the neighborhoods surrounding points i and j. as in image segmentation , the spatial position of each element is important , the spatial dependency should be incorporated into the affinity matrix . following [ 5 , 31 ] , we use markovian relaxation to incorporate this information . in order to represent the affinity of all the elements that can be reached within one spatial step , the affinity matrix is modified in the following way : ( 14)a1={aijif coords(fi)coords(fj)21,0 in any other case , where coords ( f ) are spatial coordinates of element f in the image ( 15)p1=1maxld(a1)ll{maxld(a1)lld(a1)iiif i = j , a1 in any other case , where d(a1 ) is a diagonal matrix with d(a1)ii=ja1ij , usually called the row - sum or degree matrix of a1 . then , obtaining the affinities of elements that can be reached within s spatial steps is enough to elevate p1 to the power of s , ps=(p1)s as stated in . moreover , s can be chosen to be the smallest positive integer which results in nonzero elements in the whole matrix in order to represent the weakest connected induced graph . the diagonal adjustment forces the inherent random walk to a uniform steady state , hence every part of the markov field will be explored at the same speed . for the sake of clarity , ps will be referred to as affinity matrix a in the rest of the paper . step 2 ( performing the embedding).the algorithm must embed the elements of x into an n - dimensional euclidean space y(x ) . this is done by applying eigenvalue decomposition to the laplacian of the affinity matrix . as in [ 6 , 7 , 27 ] , this is done by performing the spectral decomposition of the graph laplacian of the graph induced by a , ( 16)=d(a)a|x||x| , where |x| is number of elements to be clustered.in order to overcome the necessity of hypothesis 2 , we prenormalize the affinity matrix as done in . this is done by normalizing the weight of each edge of the graph , aij , by the probability density of both elements relating through the edge , ( 17)(ap)ij = aijp(i)p(j ) , where p( ) , the probability density function of the elements in x , is not known but can be approximated up to a multiplication factor by(18)p(i)=kaik=kaki.due to the necessity of having a uniform behavior of the clustering algorithm without minding the scale of the affinity measure taken , a doubly stochastic matrix normalization is performed : ( 19)ads = d(ap)1/2apd(ap)1/2 |x||x| . as ads is a double stochastic symmetric matrix , the eigenvalue decomposition of ( 16 ) can be calculated by taking the singular value decomposition ( svd ) ( 20)vsvt = ads|x||x| . finally , the euclidean coordinates yi of an element fix in the n - dimensional embedding manifold are ( 21)y(fi)=yi=1vi0(1vi1, ,nvin)t , fix , where ( 22)v=(v0v|x|1)|x||x| is the eigenvector column matrix and the corresponding eigenvalues are , 1=01|x|10 . the first eigenvector v0 is not taken into account as a component in the embedding because it is constant and hence meaningless as shown in [ 6 , 7 , 27 ] . the algorithm must embed the elements of x into an n - dimensional euclidean space y(x ) . this is done by applying eigenvalue decomposition to the laplacian of the affinity matrix . as in [ 6 , 7 , 27 ] , this is done by performing the spectral decomposition of the graph laplacian of the graph induced by a , ( 16)=d(a)a|x||x| , where |x| is number of elements to be clustered . in order to overcome the necessity of hypothesis 2 , we prenormalize the affinity matrix as done in . this is done by normalizing the weight of each edge of the graph , aij , by the probability density of both elements relating through the edge , ( 17)(ap)ij = aijp(i)p(j ) , where p( ) , the probability density function of the elements in x , is not known but can be approximated up to a multiplication factor by(18)p(i)=kaik=kaki . due to the necessity of having a uniform behavior of the clustering algorithm without minding the scale of the affinity measure taken , a doubly stochastic matrix normalization is performed : ( 19)ads = d(ap)1/2apd(ap)1/2 |x||x| . as ads is a double stochastic symmetric matrix , the eigenvalue decomposition of ( 16 ) can be calculated by taking the singular value decomposition ( svd ) ( 20)vsvt = ads|x||x| . finally , the euclidean coordinates yi of an element fix in the n - dimensional embedding manifold are ( 21)y(fi)=yi=1vi0(1vi1, ,nvin)t , fix , where ( 22)v=(v0v|x|1)|x||x| is the eigenvector column matrix and the corresponding eigenvalues are , 1=01|x|10 . the first eigenvector v0 is not taken into account as a component in the embedding because it is constant and hence meaningless as shown in [ 6 , 7 , 27 ] . step 3 ( clustering).once the embedding has been performed , several techniques have been proposed for the clustering step [ 4 , 6 , 32 ] . once the embedding has been performed , several techniques have been proposed for the clustering step [ 4 , 6 , 32 ] . the first step in this process is to determine the number of clusters , this can be done in two ways . the first , as in , is choosing the number of clusters according to the elbow . for instance , if the slope of the eigenvalue plot changes noticeably at eigenvector i , the number of clusters should be i+1 . the second way is reordering the affinity matrix rows and columns following the second eigenvector as proved in , which shows the block structure of the matrix as squared blocks along the matrix diagonal . their commended number of dimensions for the embedding is the same as the number of clusters . finally , the clustering is performed by running a k - means clustering algorithm on the space spanned by y(x ) . a formal justification for this approach can be found in [ 6 , 32 ] . synthetic datawe generate synthetic hardi data using the multitensor model which is simple and leads to an analytical expression of the odf [ 2 , 18 ] . for a given b - factor and noise level , we generate the diffusion - weighted signal ( 23)s(ui)=k=1n1nexp(b uitdk()ui)+noise , where ui is the ith gradient direction on the sphere , n is the number of fibers , and 1/n is the volume fraction of each fiber . in practice , we use n=81 from a 3rd - order tessellation of the icosahedron , b=3000 s / mm , and n=1 or 2 . dk( ) is the diffusion tensor with standard eigenvalues [ 3,3,1.7 ] 10 mm / s oriented in direction , which agree with reported physiological values . finally , we add complex gaussian noise with standard deviation of 1/35 , producing a signal with signal - to - noise ratio of 35 . we generate synthetic hardi data using the multitensor model which is simple and leads to an analytical expression of the odf [ 2 , 18 ] . for a given b - factor and noise level , we generate the diffusion - weighted signal ( 23)s(ui)=k=1n1nexp(b uitdk()ui)+noise , where ui is the ith gradient direction on the sphere , n is the number of fibers , and 1/n is the volume fraction of each fiber . in practice , we use n=81 from a 3rd - order tessellation of the icosahedron , b=3000 s / mm , and n=1 or 2 . dk( ) is the diffusion tensor with standard eigenvalues [ 3,3,1.7 ] 10 mm / s oriented in direction , which agree with reported physiological values . finally , we add complex gaussian noise with standard deviation of 1/35 , producing a signal with signal - to - noise ratio of 35 . we generate three synthetic data example , two simple examples : one with a ring of sinusoidal - shaped fibers , one with fibers with different sizes and scales , and the other with complex crossing areas simulating the u-fibers ( corticocortical fibers ) that can occur in the brain . these synthetic datasets help understand the behavior of the different spectral clustering methods when confronted with simple and complex fiber geometries . human brain datadiffusion - weighted data and high - resolution t1-weighted images were acquired on a whole - body 3 tesla magnetom trio scanner ( siemens , erlangen ) equipped with an 8-channel head array coil . the spin - echo echo - planar - imaging sequence , te=100 ms , tr=12 s , 128 128 image matrix , fov = 220 220 mm , consists of 60 diffusion encoding gradients with a b - value of 1000 s / mm . seven images without any diffusion weightings are placed at the beginning of the sequence and after each block of 10 diffusion - weighted images as anatomical reference for offline motion correction . the measurement of 72 slices with 1.7 mm thickness ( no gap ) covered the whole brain . random variations in the data were reduced by averaging 3 acquisitions , resulting in an acquisition time of about 45 minutes . additionally , fat saturation was employed and we used 6/8 partial fourier imaging , a hanning window filtering , and parallel acquisition ( generalized autocalibrating partially parallel acquisitions , reduction factor = 2 ) in the axial plane . diffusion - weighted data and high - resolution t1-weighted images were acquired on a whole - body 3 tesla magnetom trio scanner ( siemens , erlangen ) equipped with an 8-channel head array coil . the spin - echo echo - planar - imaging sequence , te=100 ms , tr=12 s , 128 128 image matrix , fov = 220 220 mm , consists of 60 diffusion encoding gradients with a b - value of 1000 s / mm . seven images without any diffusion weightings are placed at the beginning of the sequence and after each block of 10 diffusion - weighted images as anatomical reference for offline motion correction . the measurement of 72 slices with 1.7 mm thickness ( no gap ) covered the whole brain . random variations in the data were reduced by averaging 3 acquisitions , resulting in an acquisition time of about 45 minutes . additionally , fat saturation was employed and we used 6/8 partial fourier imaging , a hanning window filtering , and parallel acquisition ( generalized autocalibrating partially parallel acquisitions , reduction factor = 2 ) in the axial plane . the brain is peeled from the t1-anatomy , which was aligned with the talairach stereotactical coordinate system . the 21 images without diffusion weightings distributed within the whole sequence were used to estimate motion correction parameters using rigid - body transformations , implemented in . the motion correction for the 180 diffusion - weighted images was combined with a global registration to the t1 anatomy computed with the same method . the registered images were interpolated to the new reference frame with an isotropic voxel resolution of 1.72 mm and the 3 corresponding acquisitions and gradient directions were averaged . distance functions between elements to clusterin order to implement the diffusion maps spectral clustering method a distance function for each data type is chosen . this distance function is used to calculate the affinity matrix as expressed by ( 13 ) . in the dt case , following , we use the riemannian tensor distance . in the odf case we use the distance shown in ( 11 ) . in order to implement the diffusion maps spectral clustering method a distance function for each data type this distance function is used to calculate the affinity matrix as expressed by ( 13 ) . in the dt case , following , we use the riemannian tensor distance . in the odf case diffusion maps versus n - cutsthe first experiment shows the difference in performance between the diffusion maps and n - cuts approach . the n - cut algorithm does not perform the sampling - based normalization described by ( 17 ) and is thus sensitive to sampling frequency differences within the clusters . in order to show this sampling hypothesis problem , we used a ring fiber bundle with different sampling frequencies . within the ring , the fibers have a sinusoidal shape and the frequency of the modulating sine function is 4 times bigger in the lower half of the ring . more formally , the fibers follow the angular function o()=+(1/8)sin(),0<2 , where =8 for the upper half of the ring and =32 for the lower half . the results of both clustering techniques are shown in figure 2 , where the background has been masked out . figure 2(a ) shows the plot of the first 10 eigenvalues for the n - cuts method , shown in figures 2(b ) and 2(c ) , and the slope between the line joining 0 and 1 and the line joining 1 and 2 changes drastically . figure 2(d ) shows the plot of the first 10 eigenvalues for the diffusion maps method whose clustering results are shown in figure 2(e ) . the n - cuts exhibits frequency - dependent clustering artifacts while the diffusion maps method clearly shows two clusters . in the diffusion maps , the clustering has correctly segmented the background and the ring . the first experiment shows the difference in performance between the diffusion maps and n - cuts approach . the n - cut algorithm does not perform the sampling - based normalization described by ( 17 ) and is thus sensitive to sampling frequency differences within the clusters . in order to show this sampling hypothesis problem , we used a ring fiber bundle with different sampling frequencies . within the ring , the fibers have a sinusoidal shape and the frequency of the modulating sine function is 4 times bigger in the lower half of the ring . more formally , the fibers follow the angular function o()=+(1/8)sin(),0<2 , where =8 for the upper half of the ring and =32 for the lower half . the results of both clustering techniques are shown in figure 2 , where the background has been masked out . figure 2(a ) shows the plot of the first 10 eigenvalues for the n - cuts method , shown in figures 2(b ) and 2(c ) , and the slope between the line joining 0 and 1 and the line joining 1 and 2 changes drastically . figure 2(d ) shows the plot of the first 10 eigenvalues for the diffusion maps method whose clustering results are shown in figure 2(e ) . the n - cuts exhibits frequency - dependent clustering artifacts while the diffusion maps method clearly shows two clusters . in the diffusion maps , the clustering has correctly segmented the background and the ring . odf versus dt imagesin figure 3 , a single fiber scenario with no fiber crossing is shown . the dt - based and odf - based image clustering produce the same results . hence , odf clustering reproduces dt - based results on a simple fiber population example.finally , figure 4 shows a fiber crossing scenario with two overlapping fiber bundles that have different geometries . segmentation was performed over the dt and the odf image shown in figure 5 . moreover , the odf n - cuts segmentation exhibits artifacts not present in the odf diffusion maps segmentation . the odf diffusion maps effectively identify the two different fiber bundles as well as the fiber crossing areas . in figure 3 , a single fiber scenario with no fiber crossing is shown . the dt - based and odf - based image clustering produce the same results . hence , odf clustering reproduces dt - based results on a simple fiber population example . finally , figure 4 shows a fiber crossing scenario with two overlapping fiber bundles that have different geometries . segmentation was performed over the dt and the odf image shown in figure 5 . moreover , the odf n - cuts segmentation exhibits artifacts not present in the odf diffusion maps segmentation . the odf diffusion maps effectively identify the two different fiber bundles as well as the fiber crossing areas . the real - data experiment presented in this section shows the segmentation and labeling of a cropped axial and coronal slice . the cropped slices were chosen by an expert in regions of known fiber crossings where the dt model is normally limited . hence , our segmentation algorithm is confronted with elements that have different orientation and different diffusion characteristics . in order to show that odf data segments the white matter fiber bundles better than the dt data in real cases , we analyze the evolution of the affinity matrix as the scale - space parameter changes in the axial cropped slice shown in figure 6 . affinity matrices were computed with varying scale - space parameter between 1/5 , 1/10 , 1/20 , and 1/40 of the quantity of elements ( |x| ) to cluster , respectively . in order to show the block structure of the affinity matrices , it can be seen in figure 7 that as the scale diminishes , the dt data shows a high correlation between all the elements of the slice . this makes clustering very difficult because the blocks are small and highly correlated . on the other hand , this block structure shows a high correlation of the elements within each block and a low interblock correlation , giving a much better input to the clustering algorithm than the dt data . in figure 6 , the location of the cropped axial slice is shown in the axial slice , figure 6(a ) , and coronal slice , figure 6(b ) . as it can be seen in the segmented and labeled axial slice , figure 8 , the segmentation also allows to identify and label some of the main white matter structures , corpus callosum ( cc ) , anterior corona radiata ( acr ) , forceps major ( fmajor ) and forceps minor ( fminor ) . in figure 9 , the location of the cropped coronal slice is shown in the axial slice , figure 9(a ) , and coronal slice , figure 9(b ) . as it can be seen in the segmented and labeled coronal slice , figure 9(c ) , the segmentation allows to identify and label main white matter structures : corpus callosum ( cc ) , cingulum ( cg ) , corona radiata ( cr ) , superior longitudinal fasciculus ( slf ) . note that the segmentation is resilient to crossing areas such as seen at the interface between cr and cc . we have presented an algorithm to perform q - ball imaging segmentation of white matter fiber bundles . the proposed method combines state - of - the - art hardi reconstruction and state - of - the - art spectral clustering techniques . our algorithm is initialization - free and has only two parameters . a scale - space parameter and the number of regions ( clusters ) are to be found . regarding this number of clusters parameter , we have proposed to estimate it automatically . we have introduced a spectral embedding technique that does not require uniform sampling of the elements . to do so , the affinity measure used incorporates an euclidean distance measure between the spherical harmonic coefficients describing the q - ball odfs and also incorporates the spatial location distance between odfs . the affinity measure and the metric induced in the embedded space is then used to cluster q - ball odf images into multilabel segmentation representing the fiber bundles . spectral embedding has already been applied to dmri ( e.g. , ) . however , to our knowledge , this is the first work using the diffusion maps that avoids the high dependence on element sampling . we have illustrated that the odfs are the desirable elements to use for clustering in the white matter because the classical dt model is limited in regions of fiber crossings . the odf is even more attractive because of the recent analytical spherical harmonic solution to the odf reconstruction [ 3 , 8 , 9 , 13 ] . the analytical solution is in fact as fast as a standard dt least - square estimation . in this work , we believe that we have used the state - of - the - art odf reconstruction method , which is regularized , robust and very simple to implement . the spectral embedding performed by the diffusion maps technique is at the heart of our segmentation algorithm . whereas other spectral embedding techniques have a tendency to produce artifacts in the presence of different sampling characteristics within a cluster , the technique used in this work greatly reduces this tendency by performing the simple linear algebra calculation shown in ( 17 ) . spectral embedding techniques produce a representation of the embedded data based on element - to - element affinities . this leads to the fundamental issue : how to choose the affinity measure ? it is a challenge to find a measure that incorporates similarities between elements as well as the spatial location difference between elements . for similarities between elements , we chose the euclidean distance between spherical harmonic coefficients describing the odfs . this approach is simple and very efficient because it allows to process the odfs directly on the sh coefficients . the euclidean distance has also been used successfully in a level set segmentation framework and it would be interesting to compare our spectral clustering approach against it . for spatial location difference , we chose markovian relaxation in order to be consistent with the graph theoretical representation of the diffusion maps technique . although this way of representing the distance involves an artificial elimination of all the nonneighboring relations of the odf elements in the affinity matrix and an adjustment of the diagonal elements , we believe that the resulting affinity relations represent the affinity better . the affinity of two neighboring elements at the beginning of the markovian relaxation algorithm is represented by a function of the euclidean distance between them . this affinity can be interpreted as the probability that a random walker has of going from the first element to the second . the affinity of two elements at the end of the relaxation is the probability of a random walker starting from one element and reaching the second in a certain number of steps . we believe that there is room for improvement in this last part of the algorithm . in the first place , the k - means algorithm needs an explicit number of clusters to find . this can be heuristically determined by analyzing the eigenvalue plot or the reordered affinity matrix structure , as shown in this work . however , an automatic method that could find the number of clusters would considerably improve the algorithm . in the second place , the k - means algorithm and its variants , for instance , k - medians , k - medioids , search for isotropic clusters in the embedding space . this could also improve the last clustering phase of our algorithm finally , in order to analyze the importance of the difference between our diffusion maps algorithm and the widely used n - cuts , we used synthetic simulations . in these simulations , we generated a synthetic image with a single cluster within which the sampling of the elements changed . we showed that when this sampling changes , the n - cuts algorithm produces artifacts while our diffusion maps method does not . as uniform sampling within a cluster is a difficult property to guarantee in the white matter fiber bundles , our diffusion maps method is better suited for this task . we thus believe that diffusion maps are the right clustering method to be used on dmri processing problems . , we have shown that in order to perform spectral clustering on complex dmri with crossing fiber bundles , an hardi technique such as q - ball imaging is better than the classical dti technique . this is because the odf reconstructed from qbi is able to recover multiple crossing fiber populations . second , a diffusion maps - based technique for image segmentation was introduced to reduce artifacts arising from the widely used n - cuts image segmentation . we have illustrated the advantages of the odf diffusion maps segmentation algorithm , and showed on a real dataset that our algorithm is able to identify important and complex white matter fiber bundles . finally , the diffusion maps technique has been shown to be more robust to sampling frequency variations within each object to be segmented . in order to cluster the elements , we have used an adaptive scale - space parameter and we have used markovian relaxation in order to incorporate spatial dependencies . overall , the approach is theoretically sound with the graph - based representation which lies at the heart of spectral clustering methods . therefore , we have an algorithm to perform fiber bundle clustering for a single brain . it is now important to study the behavior over several subjects in order to assess the reproducibility of the algorithm . in time , this will enable to perform multisubject statistics within bundles in the embedded space . this will help characterize the white matter fiber bundles of several subjects and study if the alteration of these segmented tracts can provide new biomarkers for white matter diseases .

white matter fiber clustering aims to get insight about anatomical structures in order to generate atlases , perform clear visualizations , and compute statistics across subjects , all important and current neuroimaging problems . in this work , we present a diffusion maps clustering method applied to diffusion mri in order to segment complex white matter fiber bundles . it is well known that diffusion tensor imaging ( dti ) is restricted in complex fiber regions with crossings and this is why recent high - angular resolution diffusion imaging ( hardi ) such as q - ball imaging ( qbi ) has been introduced to overcome these limitations . qbi reconstructs the diffusion orientation distribution function ( odf ) , a spherical function that has its maxima agreeing with the underlying fiber populations . in this paper , we use a spherical harmonic odf representation as input to the diffusion maps clustering method . we first show the advantage of using diffusion maps clustering over classical methods such as n - cuts and laplacian eigenmaps . in particular , our odf diffusion maps requires a smaller number of hypothesis from the input data , reduces the number of artifacts in the segmentation , and automatically exhibits the number of clusters segmenting the q - ball image by using an adaptive scale - space parameter . we also show that our odf diffusion maps clustering can reproduce published results using the diffusion tensor ( dt ) clustering with n - cuts on simple synthetic images without crossings . on more complex data with crossings , we show that our odf - based method succeeds to separate fiber bundles and crossing regions whereas the dt - based methods generate artifacts and exhibit wrong number of clusters . finally , we show results on a real - brain dataset where we segment well - known fiber bundles .

1. INTRODUCTION 2. METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSIONS

PMC3425797

environmental tobacco smoke ( ets ) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness ( ahr ) in susceptible individuals and animals [ 15 ] . epidemiological studies show that the probability of developing or exacerbating asthma increases in ets exposure patients [ 310 ] . the nervous system , including the nerves supplying the airways , is highly susceptible to environmental influences . the airways are innervated through autonomic and sensory nerve fibers [ 12 , 13 ] . sensory innervation in the airways is important in the pathogenesis of inflammation associated with asthma . substance p ( sp ) , a member of the tachykinin family , releases from sensory nerve fibers and has potent effects on airway smooth muscle tone , vascular permeability to protein and mucus secretion [ 14 , 15 ] . recent studies have shown that sp plays an important role in antigen- and irritant - induced ahr [ 2 , 16 , 17 ] . furthermore , the studies including ours found that increased sp in the airway is involved in cigarette smoke exposure - induced ahr and airway inflammation [ 2 , 1825 ] . however , the role of sp in cigarette smoke exposure - enhanced airway responsiveness is not clear . recent studies found that sp also acts as a neuromodulator increasing the tracheal smooth muscle contractions to electrical field stimulation ( efs ) [ 2630 ] . both of watson and hall 's studies found that exogenous and endogenous sp facilitated efs - induced tracheal contractions , but had no effect on contractions induced exogenous ach [ 26 , 27 ] . nadel 's group further showed that sp potentiated airway smooth contraction to efs in a concentration - dependent fashion [ 28 , 29 ] . tournoy et al . also found that efs - induced airway smooth muscle contraction was decreased in nk1 receptor knockout mice compared with wild - type control . all of these studies suggest that sp enhances efs - induced airway smooth contraction due to increasing the release of ach ( figure 1 ) . thus , we hypothesized that ets exposure increases sp from sensory nerves or changes nk receptor , which facilitates ach release from cholinergic nerve and enhances airway smooth muscle responses ( figure 1 ) . the enhanced airway smooth muscle response may contribute to increased susceptibility to ahr . the present experiments characterize the role of sp on ets - enhanced airway smooth muscle responses . icr mice ( harlan , indianapolis , in , usa ) were housed with access to food and water ad libitum in an fda - approved facility . all procedures were performed in accordance with the recommendations of the guide for the care and use of laboratory animals , published by the national institutes of health , and the protocols were approved by the wvu animal care and use committee no . the animals were treated humanely and with regard for alleviation of suffering . by classical definition , environmental tobacco smoke ( ets ) is a diluted mixture of the smoke given off by the burning end of a tobacco product ( side stream smoke , ~85% ) and the smoke exhaled by smokers ( mainstream smoke , ~15% ) . based on previous ets exposure studies [ 1 , 31 , 32 ] , we used sidestream tobacco smoke ( ss ) as a surrogate to ets . the two - month - older mice were exposed to either ss or filtered air ( fa ) for 6 h / day for 1 day or 5 consecutive days . the trachea and lung tissues were collected 16 h after ss or fa exposure . a major goal of the current study was to understand possible neuronal mechanisms by ss that affect lung function . the ss exposure protocol and methods used in this study have been described in our recent publication . briefly , mice were randomly placed in an exposure chamber ( bioclean , duoflo model h 5500 , lab products inc ) that measured 1.92 1.92 0.97 m ( 3.58 m ) . sidestream smoke from marlboro filter cigarettes ( phillip morris , richmond , va , usa ) was introduced into the exposure chamber at a rate of four cigarettes every 15 minutes for 6 hours using a smoking machine ( rm 1/g , heinr borgwaldt gmbh , hamburg , germany ) . at the end of the 6 h exposure period , the exhaust fan unit was turned on to rapidly lower the level of smoke in the exposure chamber . the concentrations of carbon monoxide in the exposure chamber were monitored and kept to an average of about 50 parts per million ( ppm ) , relative humidity was about 50% , and temperature was about 23c . total suspended particulate concentration was about 1.1 mg / m , similar to exposure levels used by others to approximate the cloud of particulates surrounding a person during active smoking . fresh tracheas from mice 16 h after fa or ss exposure were cut into 3 mm wide rings , mounted in holders , and maintained in gassed ( 95% o2 - 5% co2 ) modified krebs - henseleit solution at 37c with a composition ( in mm ) as follows : 113 nacl , 4.8 kcl , 2.5 cacl2 , 1.2 mgso4 , 24 nahco3 , 1.2 kh2po4 , and 5.7 glucose , ph 7.4 . each tracheal ring was loaded into a pair of stainless - steel hooks , suspended in tissue holders . each holder was anchored in a 10-ml water - jacketed organ bath , and the top steel hook was attached to a force - displacement transducer connected to a recorder ( gould instruments , valley view , oh , usa ) . the rings were equilibrated for 60 min at a resting tension of 0.25 g , determined by preliminary studies , during which time the modified krebs - henseleit solution in the baths was changed every 15 min . after equilibration , methacholine ( mch ) responses were constructed by adding 10 m mch to the bath and sp cumulative concentration - response curves were constructed by adding a series of concentrations of sp to the bath in half - log - increment concentrations ranging from 10 to 10 m. after concentration - response curves were completed , electrical - field - stimulation- ( efs- ) induced responses were obtained with a stimulator ( model s48 , grass instruments , richmond , va , usa ) . frequency - response curves were constructed by increasing the frequency from 0.3 to 30 hz using a submaximum voltage of 120 v , 0.2-ms pulse duration , and 10 s train duration . between each stimulation period , efs - induced contractions were normalized by the percent response of each tissue to 10 m mch . in some experiments , atropine ( 10 m ) was added to krebs solution to verify that the responses elicited by efs were mediated by the release of ach . to determine the possible involvement of endogenously released sp , some experiments were given cp-99994 ( final concentration 10 m ) , sp receptor antagonist , which was incubated at least 30 min before addition of sp or efs . the procedures for immunohistochemical quantification of airway nerves have been described previously [ 1 , 33 , 34 ] . briefly , in a separate group of mice - exposed using the same ss exposure protocol , tracheas were removed 16 h after ss or fa exposure , fixed in picric acid - formaldehyde fixative for 3 h , and rinsed three times with a 0.1 m phosphate - buffered saline containing 0.15% triton x-100 . the tracheas were dissected and frozen in isopentane , cooled with liquid nitrogen , and stored in airtight bags at 80c . the tracheas were oriented with the dorsal surface uppermost so the tracheal muscle would be sectioned in a coronal plane . cryostat sections ( 12 m thick ) were collected on gelatin - coated coverslips and dried at room temperature . the cryostat sections on coated coverslips were covered with sp antibody diluted 1 : 100 , incubated at 4c for overnight , and rinsed with a 1% bovine serum albumin + phosphate - buffered saline containing 0.15% triton x-100 three times , with 5 min being allowed for each rinse . the sections were then covered with fluorescein isothiocyanate - labeled goat antirabbit antibody diluted 1 : 100 , incubated at 37c for 45 min , and rinsed . after all immunohistochemical procedures were conducted , the coverslips were mounted with fluoromount and observed with a fluorescence microscope equipped with fluorescein ( excitation wavelengths 455500 nm , emission wavelengths > 510 nm ) . controls consisted of testing the specificity of primary antiserum by absorption with 1 g / ml of the specific antigen . nonspecific background labeling was determined by omission of primary antiserum . for measuring nerve fiber density in tracheal smooth muscle , we collected images of control , sp nerve fibers in series under the zeiss lsm 510 confocal microscope . a series of images representing all of the tracheal smooth muscles in a section was collected in digital files and saved to an internal database and measured using optimus software . we selected regions of smooth muscle using the rhodamine channel to avoid possible bias created by the presence or absence of nerve fibers . the smooth muscle regions were outlined to measure total cross - sectional area of smooth muscle . the microscope was then switched to reveal nerve fibers in the fluorescein channel , and the image was digitally captured . then nerve fiber area was standardized to the total cross - sectional area of smooth muscle . the final value of nerve fiber density is expressed as % of dividing the sp nerve fiber area by the total area of smooth muscle . at least 10 measurements were made for each section , and 15 sections were measured in each animal . the concentration of sp ( 392500 pg / ml ) in each sample was assayed using the sp immunoassay system ( r&d systems , minneapolis , mn , usa ) according to manufacturer 's instructions . all samples were running in duplicate . unless otherwise stated , values are means se . contractions elicited by efs and sp are expressed as a percentage of the contraction elicited by mch 10 m. the half - maximal effective concentration ( ec50 ) values for sp were calculated using a four - parameter logistic curve fit ( sigmoidal , sigmaplot 2000 ) and were presented with 95% confidence intervals . force development was expressed by normalizing force ( g ) divided by the wet weight of the tissue . nerve fiber density was expressed as % area of sp - immunoreactive nerve fibers in the total area of the smooth muscle . statistical analyses of immunohistochemistry and ec50 were performed using student 's t - test . statistical analysis of efs was performed using two - way repeated - measures analysis of variance . one factor between the groups was ss exposure ; the other factor within the group was efs or sp effect . when the main effect was considered significant at p < 0.05 , pairwise comparisons were made with a post hoc analysis ( fisher 's least significant difference ) . p < 0.05 was considered significant , and n represented the number of animals studied . mch chloride and atropine sulfate were obtained from sigma chemical ( st . louis , mo , usa ) . sp and sp antibody were obtained from peninsula ( belmont , ca , usa ) . fluorescein isothiocyanate - labeled goat antirabbit antibody was obtained from icn immunobiologicals ( costa mesa , ca , usa ) . the initial experiments were intended to find if ss exposure increases airway smooth muscle contraction to mch . there was no significant difference in the tracheal smooth muscle contraction to mch ( 10 m ) between 1 day - ss - exposed ( n = 16 ) mice and fa exposed mice ( n = 16 ) ( figure 2 ) . although 5 days of ss exposure slightly increased smooth muscle contraction to mch , there was no significant difference in airway responses to mch between 5 days of ss - exposed ( n = 30 ) mice and fa exposed mice ( n = 30 ) ( figure 2 ) . the next experiments were intended to figure out the effect of ss on airway responsiveness to sp and efs . the cumulative dose - response curve for sp was not significantly shifted to the left after 1 day of ss exposure ( figure 3(a ) ) . there is no significant difference between ec50 values in ss exposure ( 6.17 0.11 ) and fa exposure ( 5.96 0.14 ) . also the frequency - response curve to efs was not significantly altered 1 day of ss exposure ( figure 3(b ) ) . however , the cumulative dose - response curve for sp was markedly shifted to the left after 5 days exposure to ss ( figure 3(c ) ) , there is significant difference between ec50 values in ss exposure ( 6.65 0.09 ) and fa exposure ( 6.01 0.11 ) . also , a leftward shift in the frequency - response curve to efs was observed after 5 days of ss exposure ( figure 3(d ) ) , and contractions produced by efs at 10 hz and 30 hz were significantly increased after 5 days of ss exposure . the pretreatment with atropine ( final concentration 10 m ) totally abolished efs - induced airway smooth muscle contraction in both fa and ss - exposed mice ( figures 3(b ) and 3(d ) ) , indicating that efs - induced airway smooth muscle contraction mainly involves endogenously acetylcholine ( ach ) release . to ensure sufficient reduction of sp effects , the effective dosage of nk1 receptors antagonist , cp-99994 , was tested first . in separate experiments , the different concentrations of cp-99994 were tested by sp cumulative doses ( 10 m to 10 m ) . the sp cumulative dose - response curves were significantly shifted to the right in a concentration - dependent manner by cp-99994 ( figure 4 ) . ec50 values for sp are 6.04 0.14 ( control ) , 5.83 0.12 ( cp-99994 10 m ) , 5.64 0.11 ( cp-99994 10 m ) , and 5.56 0.11 ( cp-99994 10 m ) . there is significant difference in ec5 between control and cp-99994 10 m. the combination of cp-99994 ( 10 m ) and nk2 receptors antagonist sr48968 ( 10 m ) virtually abolished sp - induced airway smooth muscle contraction , indicating that cp-99994 10 m is effective dose to block sp - induced airway smooth muscle contraction . thus , next experiment , cp-99994 10 m was chosen to test the role of sp on 5 of days ss - enhanced airway smooth muscle responses . cp-99994 ( final concentration 10 m ) was incubated at least 30 min before using sp or efs . cumulative concentration response curve for sp and the efs - stimulated contractions in saline pretreated groups after 5 of days ss exposure ( figures 5(a ) and 5(c ) ) demonstrated similar changes as those described above , 5 days of ss exposure . the contractions produced by efs at 10 hz and 30 hz were significantly increased in saline pretreated groups after 5 days of ss exposure . the tracheal smooth muscle contractions to efs at 10 and 30 hz were not significantly altered in the cp-99994 treatment group ( 10 hz : 21.44 2.54% ; 30 hz : 25.8 g 2.31% ) compared with the saline group ( 10 hz : 23.36 2.28% ; 30 hz : 27.89 2.29% ) in fa exposed mice . however , cp-99994 significantly inhibited the ss - enhanced smooth muscle responses to efs at 10 and 30 hz ( figure 5(d ) ) . there are no significant differences in efs - induced tracheal smooth muscle contractions between 5 of days ss - exposed mice and fa exposed mice after cp-99994 treatment . also , the enhanced airway responses to sp were total abolished by cp-99994 treatment ( figure 5(b ) ) . sp nerve fibers in the tracheal smooth muscle of mice were analyzed based on the immunohistochemical localization by fluorescein . the sp nerve fibers were mainly localized on tracheal smooth muscle and epithelium ( figure 6(a ) ) . there is no significant difference in density of sp nerve fibers in the airway smooth muscle between 1 day of ss exposure and fa exposure ( figures 6(a ) and 6(b ) ) . however , the density of sp nerve fibers in the airway smooth muscle was significantly increased after 5 days of ss exposure ( figures 6(a ) and 6(b ) ) . these findings suggest that 5 days of ss exposure increases sp content in tracheal smooth muscle . elisa data showed that sp levels in lung were significantly elevated in 5 days of ss - exposed mice compared with fa exposure ( figure 6(c ) ) . the results obtained from the current study show that exposure to ss significantly enhances tracheal smooth muscle responsiveness in the mice , as evidenced by elevated contractility to sp and efs . the elevation of airway smooth muscle responses by ss exposure was attenuated by treatment with a nk1 receptor antagonist , indicating that sp played a key role in the enhancement of smooth muscle contractile responses . our previous study showed that exposure to irritant changes sp airway innervation and enhances tracheal smooth muscle responsiveness [ 25 , 35 ] , suggesting that sp may contribute to irritant - enhanced smooth muscle responsiveness . the current results from immunohistochemistry found that ss exposure changes sp innervation of tracheal smooth muscle , directly indicating that increased sp level in airway involved ss - enhanced smooth muscle responsiveness . sp localized in the peripheral endings of sensory nerves innervating the lung and airways originates in nerve cell bodies located in sensory ganglia [ 37 , 38 ] . stimulation of sensory nerves by inhalation of irritants is known to trigger the release of sp from afferent endings [ 2 , 25 , 39 , 40 ] . the data in the present study found that ss - enhanced - airway smooth muscle contractions to efs were attenuated by sp receptor antagonist , directly indicating that sp is involved . however , the role of sp in smoke exposure - enhanced airway responsiveness is not clear . recent studies found that sp acts as a neuromodulator altering airway responsiveness to efs [ 2630 ] . watson and hall 's studies found that exogenous and endogenous sp facilitated electrical field stimulation ( efs)-induced tracheal contractions , but had no effect on contractions induced exogenous ach [ 26 , 27 ] . nadel 's group further showed that sp potentiated airway smooth contraction to efs in a concentration - dependent fashion [ 28 , 29 ] . thus , one possible explanation for our finding is that enhanced sp in airway after ss exposure acted as mediator , which altered airway responsiveness to efs . our result found that administration of atropine completely blocks airway smooth muscle responsiveness to efs in both fa and ss - exposed animals , demonstrating that smooth muscle contraction to efs in mice trachea is entirely atropine sensitive and totally depends on endogenous ach release from parasympathetic nerve terminals . it also indicates that there is no sp release in efs - induced airway responses . furthermore , our mch experiments found that ss exposure did not increase airway contractions to mch and suggested enhanced sensitivity of airway smooth muscle was not involved in ss exposure . thus the logical explanation is that enhanced sp acts as a neuromodulator increasing the release of acetylcholine ( ach ) from parasympathetic nerve . although the exact mechanism of enhanced ach release from parasympathetic nerve was not determined in the present study , the finding that the sp receptor antagonist cp-99994 significantly attenuated the effect of ss on efs responses in trachea suggests the idea that ss exposure induces sp upregulation . the enhancing sp may associate with nk1 receptor on the surface of cholinergic neurons , which alters the sensitivity of cholinergic neurons or activates cholinergic neurons and facilitates ach release from cholinergic nerve . our data also found that contractions of airway smooth muscle to efs at 10 and 30 hz were slightly decreased in the cp-99994 treatment group ( 10 hz : 21.44 2.54% ; 30 hz : 25.89 2.31% ) compared with the saline treatment group ( 10 hz : 23.36 2.28% ; 30 hz : 27.89 2.29% ) after fa exposure . study that efs - induced airway smooth muscle contraction was decreased in nk1 receptor knockout mice compared with of nk1 receptor wild - type controls supports that normal level of sp in airway smooth muscle involved airway responses to efs , although there is no direct effect of sp on efs - induced airway responses . the present experiment also showed that sp dose responses are enhanced after 5 days ss exposure . one possibility is that sensitivity of airway smooth muscle to sp is enhanced by ss exposure . however , it is unlikely that the sensitivity of airway smooth muscle was enhanced due to sensitivity of airway smooth muscle to mch that was not significantly changed . alternatively , an altered density and/or expression of the nk receptors or properties of nk receptors ( e.g. , affinity ) on the airway smooth muscle may be involved by ss exposure . our experiments used three concentrations of cp 99994 and detected that even high - dose cp 99994 ( 1 m ) only partially but incomplete inhibited exogenous sp - induced airway contraction , suggesting that mice airway may contain a heterogeneous population of receptors . furthermore , our experiment uses combination with nk1 and nk2 receptor antagonist ( sr 48968 ) treatment and found that combination with nk1 and nk2 receptor antagonist treatment virtually eliminated the response to sp in airway , indicating both nk1 and nk2 receptors mediate the contractile response to sp . thus , the possible explanation is that ss exposure alters density and/or expression of the nk receptors or properties of nk receptors ( e.g. , affinity ) on the airway smooth muscle , which affect airway repose to sp . the previous studies including ours have found that childhood exposure to ets is a significant risk factor for exacerbation of asthma , but studies of smoking in adults are less conclusive . there are evidence that mainstream cigarette smoking exposure may decrease the incidence of some chronic inflammatory and have a beneficial effect of smoking on airway responsiveness [ 4144 ] . these studies found that exposure to mainstream cigarette smoke attenuates airway responses to ach , methacholine [ 4143 ] and neurokinin a , also decreased airway inflammation comparing with nonsmoking animals . however , it has also been reported that mainstream cigarette smoking or ets exposure can enhance allergic airway inflammation [ 45 , 46 ] and increase airway responses to ach [ 45 , 47 ] , endothelin [ 48 , 49 ] . the low levels of ets or mainstream cigarette smoke promote allergic sensitization and increase airway responsiveness [ 46 , 50 ] , whereas high levels of mainstream cigarette smoke exposure associated with direct smoking suppress allergic airway inflammation and airway hyperresponsiveness [ 4143 , 50 ] . in our present experiment , we used low levels of sidestream tobacco smoke ( ss ) as a surrogate to ets instead of mainstream cigarette smoke , and found ss exposure significantly enhances tracheal smooth muscle responsiveness sp and efs , supporting that low levels of ets enhance the development of allergic airway inflammation . one of the significant findings in the current results from immunohistochemistry found that ss enhances sp expression in airway nerves . these data provide direct evidence that sp nerve fibers in airway smooth muscles were increased by 5 days of ss exposure . clinic data demonstrated that sp nerve fiber density was increased in airway smooth muscle of severe asthmatics . a more recent study showed that the sp nerve fiber density was increased in airway epithelium from human subjects with persistent nonproductive cough . these studies show that increased levels of sp in human airway sensory nerves may contribute to alteration of airway responses . in conclusion , our results show that 5 days of ss exposure increases sp nerve fibers innervating tracheal smooth muscle . at the same time , smooth muscle responses to efs and sp are increased in tracheal segments . administration of cp-99994 , an antagonist of the nk1 receptor , attenuates the ss exposure - enhanced tracheal smooth muscle responses to efs , indicating that the enhanced responses are dependent on sp increase from sensory nerves or change of nk receptors density in the airway smooth muscle . combining immunohistochemistry finding that sp nerve fibers were increased in tracheal smooth muscle after 5 days of ss exposure , the current study suggests that the increased sp production by ss exposure may contribute ss - enhanced smooth muscle responsiveness in mice trachea .

environmental tobacco smoke ( ets ) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness ( ahr ) in susceptible individuals and animals , but the underlying mechanism is not fully understood . substance p ( sp ) release from sensory nerve fibers has been linked to ahr . the present experiments characterize the role of sp in tracheal smooth muscle on ets - increased airway responses . the mice were exposed to either sidestream tobacco smoke ( ss ) , a surrogate to ets , or filtered air ( fa ) for 1 day or 5 consecutive days . contractions of tracheal smooth muscle to sp and electrical field stimulation ( efs ) were not significantly altered in 1 of day ss - exposed mice . however , 5 of days ss exposure significantly increased airway smooth muscle contractions to sp and efs . administration of cp-99994 , an antagonist of the neurokinin ( nk)1 receptor , attenuates the ss exposure - enhanced tracheal smooth muscle responses to efs . furthermore , the immunohistochemistry showed that sp nerve fibers were increased in tracheal smooth muscle after 5 of days ss exposure . these results suggest that the increased sp production may contribute to ss - enhanced smooth muscle responsiveness in mice trachea .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

PMC3699708

the online version of this article ( doi:10.1007/s10858 - 013 - 9739 - 5 ) contains supplementary material , which is available to authorized users . modern multidimensional nmr spectroscopy allows successful determination of three - dimensional structure for double- or triple - labeled proteins with molecular weights of up to 50 kda ( kay 2005 ) . typically protein structure elucidation can be split into two main parts ( wthrich 1986 ) : sequence - specific assignment of chemical shifts , and extraction of distances between proton pairs from noesy spectra . the precision of determined structures depends primarily on a number of observed noe contacts and their proper assignment . the main difficulty arises due to the ambiguity of noe assignment since chemical shifts are derived from nmr measurements with a certain level of precision . it was shown that in larger systems less than 10 % of noe cross peaks can be assigned unambiguously ( mumenthaler et al . several dedicated protocols for automatic noe assignment were developed ( gronwald et al . 2002 ; herrmann et al . 2002b ; nilges and odonoghue 1998 ) . here we report an nmr measurement that allows to dramatically decrease the ambiguity of assignment of noe between aromatic and aliphatic protons . it was shown previously ( jee and guntert 2003 ) that chemical shifts of aromatic side chains are crucial for noe assignment . these residues are buried in the hydrophobic core of a protein and give rise to many long - range noe contacts . therefore , the ability to obtain unambiguous contacts among aromatic and aliphatic protons can be a valuable tool for determination of protein structure with improved precision . the intrinsic uncertainty of noe assignment due to flips of aromatic rings and frequently degenerated chemical shifts is alleviated by introduction of pseudo - atoms or other solutions ( wthrich et al . although three - dimensional noesy methods are most commonly applied ( zhang et al . 1994 ; muhandiram et al . 1993 ) , the full potential of n , c isotope labeling can be utilized with double - heteronuclear - edited 4d spectra ( luan et al . it should be emphasized that the reasonable resolution of these spectra can be preserved by employing non - uniform sampling ( nus ) of indirect time domains . due to the strict requirements regarding high dynamic range and linearity of peak intensities , nus noesy spectra are particularly demanding for processing methods ; this is reflected by a limited number of applications published to date ( luan et al . diagonal - free noesy experiments have attracted a considerable attention since the spectral reconstruction from sparse data is less severe for spectra with a limited dynamic range of signal amplitudes . recently , a non - uniformly sampled ( nus ) 4d n , n - edited trosy - noesy - trosy experiment based on the orthogonal spin state selection was reported ( werner - allen et al . the utility of an alternative approach to the diagonal peak suppression based on the straightforward diagonal subtraction ( wu et al . 2004 ) was also demonstrated for methyl methyl noesy spectroscopy ( wen et al . 2012 ) . in these methods , however , the sensitivity per unit of time is reduced by the non - negligible factor of 2 . 2012 ) is an example of the most straightforward approach to the acquisition of diagonal - free noesy spectra . of the particular relevance we propose a novel four - dimensional aliphatic aromatic noesy experiment in which diagonal peaks are efficiently suppressed by the selective manipulation of c spins of different kinds . the major aim of this experiment is to retain high sensitivity of the method while achieving a reasonable level of selectivity and associated diagonal peak suppression . the proposed method complements the set of recently reported diagonal - free 4d aliphatic amide ( stanek et al . 2012 ) , amide amide ( werner - allen et al . 2010 ) and aliphatic aliphatic ( wen et al . 2012 ) nus noesy experiments . two possible solutions are discussed here : aliphatic - to - aromatic ( fig . 1b ) hmqc - noesy - hsqc . in both cases the optimized hmqc scheme ( stanek et al . 2012 ) was incorporated for t1 ( h ) and t2 ( c ) evolution as it utilizes the multiple - quantum line narrowing effect for c h coherences ( grzesiek et al . 1995 ) . in the previous work on 3d h(aro)-noesy - ch3,hn and 3d c(aro)-noesy - ch3,hn ( xia et al . 2001 ) the refocused inept was utilized for the selection of protons coupled to aromatic c spins . in this case selective transfer can be achieved employing shaped inversion pulses and pulsed field gradients ( pfgs ) that suppress undesired coherences in the subsequent z - filtration . we have found that the same task is far more demanding using hmqc . in this case pfgs can not be used to distinguish aromatic and aliphatic mq h c coherences as both have equal coherence order after initial 1/2jch delay and hard excitation pulse . if the selective excitation pulse is employed pfgs can be useful , however , sensitivity loss of a factor of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sqrt 2 $ $ \end{document } is incurred and additional time is required for gradient encoding when c magnetization is transverse . we thus conclude that the optimal solution includes phase cycling of selective excitation pulses . despite these difficulties hmqc is preferred over refocused inept owing to ( 1 ) the apparently slower relaxation of mq coherences and ( 2 ) the possibility to incorporate both magnetization transfer delays ( 1/jch in total ) for the shared - time proton evolution ( t1 ) that leads to increased sensitivity ( stanek et al . 1pulse sequence scheme for ( a ) c(aliph)hmqc - noesy - c(aro)hsqc and ( b ) c(aro)hmqc - noesy - c(aliph)hsqc . 90 and 180 hard pulses are represented by filled and open bars , respectively . shaped pulses are represented as follows : ( a ) 40 ppm selective pulse of the iburp-2 profile ( geen and freeman 1991 ) ( 659 s duration , 7.6 khz peak r.f . field ) is shown as wide open bell - shaped pulse ( denoted e ) . wide grey rectangular pulses ( a and c ) are of duration of 61 s and peak r.f . 4.1 khz ( calibrated to give null excitation 90 ppm off -resonance ) . ( b ) wide grey bell - shaped pulse denotes 80 ppm - selective hyperbolic secant adiabatic pulse ( silver et al . narrow grey bell - shaped pulses ( f and h ) utilize gaussian profile ( bauer et al . 1984 ) ( truncated at 1 % , duration of 178 s , peak r.f . note that for some hardware the shaped 90 pulses ( a , c , f and h ) may require small angle phase adjustment to compensate 0th order phase shift with respect to refocusing pulses ( b and g ) which are applied at full power . n refocusing pulse enclosed in dashed - line box ( sequence b ) is optional and may be used if delay complementing to half - dwell time , = [ ( sw3 ) pw180(n)]/2 , is positive . refocusing of caliph - c couplings in t2 ( a ) or t3 ( b ) may be considered if max . the composite 34.2x123x197.6x288.8x pulse ( d and i ) was used in hmqc for broadband inversion of c spins ( shaka 1985 ) . watergate ( piotto et al . 1992 ) with 3 - 9 - 19 pulses separated by ( 3.2 khz ) was employed for experiment ( a ) . c composite pulse decoupling was performed employing wurst scheme ( kupe and freeman 1995 ) . /2 pulses were 7.1 , 14.3 and 31 s for h , c and n , respectively . 1 , 2 and 3 are incremented for quadrature detection in t1 , t2 and t3 using states ( 1 ) or states - tppi ( 2 , 3 ) method . four - step phase cycle is as follows : 1 = 45 ; 2 = x , x ; 3 = 2(x ) , 2(x ) ; 4 = 2(x ) , 2(x ) ; 5 = 135 ; rec = x , delays are set as follows : a = 1.79 ms ( 4 jchaliph ) , b = 1.56 ms ( 4 jcharom ) . noesy mixing time m = 150 ms was used . for the semi - constant time evolution in t1 ( h ) the delays 1 , 2 and 3 are t1/2 , t1(1 2/t1,max)/2 and (1 t1/t1,max ) , where = 2a , or = 2b for sequences ( a ) and gradient levels and durations are : g1 ( 2 ms , 6.5 gs / cm ) , g2 ( 2 ms , 14.2 gs / cm ) , g3 ( 0.5 ms , 1.77 gs / cm ) , g4 ( 2 ms , 11.3 gs / cm ) , g5 ( 2 ms , 12.9 gs / cm ) , g6 ( 0.5 ms , 5.4 gs / cm ) . proton carrier frequency was set on resonance with water ( 4.68 ppm ) , carbon carrier was set to 35 ppm and switched to 125 ppm as indicated by the vertical arrow ; n carrier was set to 117 ppm and shifted to 162 ppm at the beginning of noesy mixing period . for the aliphatic - to - aromatic noesy ( a ) 4,400 sampling points ( t1,t2,t3 ) were randomly chosen from 120 84 30 cartesian grid according to gaussian probability distribution , p(t ) = exp[(t / tmax)/2 ] , = 0.5 , with poisson disk restrictions ( kazimierczuk et al . 2008 ) . maximum evolution times of 15 ( t1 ) , 6 ( t2 ) and 5 ms ( t3 ) were achieved in the indirectly detected dimensions . spectral widths of 8 , 14 , 6 and 12 khz were set in 1 , 2 , 3 and 4 dimensions , respectively . in the full 4d spectrum any residual diagonal peaks can be folded in c ( 2 and 3 ) dimensions without the risk of overlap and misinterpretation with genuine peaks . the only requirement is to ensure proper h ( 1 ) spectral width to avoid aliasing in this indirect dimension . the restriction of c spectral widths is very practical as it saves vast amounts of disk space . the total experimental time was 57 h pulse sequence scheme for ( a ) c(aliph)hmqc - noesy - c(aro)hsqc and ( b ) c(aro)hmqc - noesy - c(aliph)hsqc . 90 and 180 shaped pulses are represented as follows : ( a ) 40 ppm selective pulse of the iburp-2 profile ( geen and freeman 1991 ) ( 659 s duration , 7.6 khz peak r.f . field ) is shown as wide open bell - shaped pulse ( denoted e ) . wide grey rectangular pulses ( a and c ) are of duration of 61 s and peak r.f . 4.1 khz ( calibrated to give null excitation 90 ppm off -resonance ) . ( b ) wide grey bell - shaped pulse denotes 80 ppm - selective hyperbolic secant adiabatic pulse ( silver et al . narrow grey bell - shaped pulses ( f and h ) utilize gaussian profile ( bauer et al . 1984 ) ( truncated at 1 % , duration of 178 s , peak r.f . note that for some hardware the shaped 90 pulses ( a , c , f and h ) may require small angle phase adjustment to compensate 0th order phase shift with respect to refocusing pulses ( b and g ) which are applied at full power . n refocusing pulse enclosed in dashed - line box ( sequence b ) is optional and may be used if delay complementing to half - dwell time , = [ ( sw3 ) pw180(n)]/2 , is positive . refocusing of caliph - c couplings in t2 ( a ) or t3 ( b ) may be considered if max . evolution time of aliphatic c spins exceeds 9 ms . the composite 34.2x123x197.6x288.8x pulse ( d and i ) was used in hmqc for broadband inversion of c spins ( shaka 1985 ) . watergate ( piotto et al . 1992 ) with 3 - 9 - 19 pulses separated by ( 3.2 khz ) was employed for experiment ( a ) . c composite pulse decoupling was performed employing wurst scheme ( kupe and freeman 1995 ) . /2 pulses were 7.1 , 14.3 and 31 s for h , c and n , respectively . 1 , 2 and 3 are incremented for quadrature detection in t1 , t2 and t3 using states ( 1 ) or states - tppi ( 2 , 3 ) method . four - step phase cycle is as follows : 1 = 45 ; 2 = x , x ; 3 = 2(x ) , 2(x ) ; 4 = 2(x ) , 2(x ) ; 5 = 135 ; rec = x , x , x , x . delays are set as follows : a = 1.79 ms ( 4 jchaliph ) , b = 1.56 ms ( 4 jcharom ) . for the semi - constant time evolution in t1 ( h ) the delays 1 , 2 and 3 are t1/2 , t1(1 2/t1,max)/2 and (1 t1/t1,max ) , where = 2a , or = 2b for sequences ( a ) and ( b ) , respectively ( stanek et al . 2012 ) . gradient levels and durations are : g1 ( 2 ms , 6.5 gs / cm ) , g2 ( 2 ms , 14.2 gs / cm ) , g3 ( 0.5 ms , 1.77 gs / cm ) , g4 ( 2 ms , 11.3 gs / cm ) , g5 ( 2 ms , 12.9 gs / cm ) , g6 ( 0.5 ms , 5.4 gs / cm ) . proton carrier frequency was set on resonance with water ( 4.68 ppm ) , carbon carrier was set to 35 ppm and switched to 125 ppm as indicated by the vertical arrow ; n carrier was set to 117 ppm and shifted to 162 ppm at the beginning of noesy mixing period . for the aliphatic - to - aromatic noesy ( a ) 4,400 sampling points ( t1,t2,t3 ) were randomly chosen from 120 84 30 cartesian grid according to gaussian probability distribution , p(t ) = exp[(t / tmax)/2 ] , = 0.5 , with poisson disk restrictions ( kazimierczuk et al . maximum evolution times of 15 ( t1 ) , 6 ( t2 ) and 5 ms ( t3 ) were achieved in the indirectly detected dimensions . spectral widths of 8 , 14 , 6 and 12 khz were set in 1 , 2 , 3 and 4 dimensions , respectively . in the full 4d spectrum any residual diagonal peaks can be folded in c ( 2 and 3 ) dimensions without the risk of overlap and misinterpretation with genuine peaks . the only requirement is to ensure proper h ( 1 ) spectral width to avoid aliasing in this indirect dimension . the restriction of c spectral widths is very practical as it saves vast amounts of disk space . the total experimental time was 57 h here , the selection of either aliphatic ( fig . 1b ) proton - carbon mq coherences is accomplished solely by band - selective excitation of c spins in conjunction with the four - step phase cycle of two /2 pulses ( a , f , h , respectively ) . therefore , the choice of selective /2 pulses is of the particular relevance . apparently , it is quite straightforward not to excite aromatic c spins ( the sequence shown in fig . more advanced pulses such as gaussian cascade q5 ( emsley and bodenhausen 1992 ) or e - burp ( geen and freeman 1991 ) could be utilized as well , however , these pulses are ( 1 ) of significantly larger duration , ( 2 ) more sensitive to miscalibration and ( 3 ) require high peak r.f . field . it was experimentally verified that these shortcomings offset theoretical benefits of high quality pulses and significant loss of sensitivity is incurred . for the aromatic - hmqc ( fig . due to the use of shaped /2 c pulses in hmqc the refocusing pulse is obligatory for correct phase properties of signals in 2 dimension . however , no additional selectivity improvement can be achieved by replacement of this pulse by a band - selective one . similarly , the subsequent carbon inversion pulse in hmqc ( denoted d and i , respectively ) should be sufficiently broadband . a shaped pulse here would only result in the t1-dependent jch evolution of unaffected coherences during total time of 1 + 2 the resulting signals would be impossible to suppress using neither phase cycling nor pulsed field gradients . all nmr experiments were performed for the e32q mutant of human s100a1 protein , for which resonance assignments are available in bmrb under accession code 17857 . the high resolution 3d structure based on the standard 3d c- and n - edited noesy - hsqc spectra ( zhang et al . 2011 ) , this protein mutant also exists in solution as a homodimer built up of noncovalently attached subunits ( molecular weight of the monomer is 10 kda ) . nmr sample contained 1.0 mm n , c labeled protein ( the monomer concentration ) in 90 % /10 % h2o / d2o , 50 mm tris - d11 , 1.0 mm edta , 0.1 mm nan3 and 50 mm nacl with ph adjusted to 7.2 . the experiments were performed at 37 c on a 700 mhz agilent / varian spectrometer equipped with the performa xyz pfg unit and the 5 mm h , c , n triple - resonance probehead . 3d noesy - hsqc and 4d hmqc - noesy - hsqc spectra were analyzed with sparky ( goddard and kneller ) . in both cases , peak picking and inspection of resulting peak lists conventionally sampled 3d noesy - c - hsqc spectrum was acquired in 36 h and processed using nmrpipe program ( delaglio et al . 1995 ) . to derive noesy interproton distance constrains from the 4d c(ali),c(aro)-edited noesy , automatic noesy assignment procedure ( herrmann et al . the final validation of obtained constraints was performed with xplor - nih program ( schwieters et al . the processing of four - dimensional nus data was accomplished by the home - written ssa ( signal separation algorithm ) software package , which can be downloaded free of charge for non - commercial purposes from the website http://nmr.cent3.uw.edu.pl ( 64-bit versions for linux ) . artefact suppression in ssa software ( cleaner4d program ) was intentionally disabled by setting maximum number of iterations to zero . this is equivalent to the use of raw zero - augmented fourier transformation and limits cleaner4d to data conversion . the actual computation of spectral data was performed using reconstructor4d program ( also from ssa package ) that implements ( a ) automatic choice between fft and dft whenever beneficial , ( b ) multi - threaded computations and ( c ) memory - mapped and asynchronous input output disk operations to improve overall performance . the total processing time on a pc equipped with intel i5 3.3 ghz cpu running under ubuntu 11 was 2 min and 6 min 30 s using 4 and 1 threads , respectively . final spectrum stored in sparky format ( single - precision ) was of size of 10.1 gb . ssa software supports native agilent / varian as well as nmrpipe format as input data . two possible solutions are discussed here : aliphatic - to - aromatic ( fig . 1b ) hmqc - noesy - hsqc . in both cases the optimized hmqc scheme ( stanek et al . 2012 ) was incorporated for t1 ( h ) and t2 ( c ) evolution as it utilizes the multiple - quantum line narrowing effect for c h coherences ( grzesiek et al . 1995 ) . in the previous work on 3d h(aro)-noesy - ch3,hn and 3d c(aro)-noesy - ch3,hn ( xia et al . 2001 ) the refocused inept was utilized for the selection of protons coupled to aromatic c spins . in this case selective transfer can be achieved employing shaped inversion pulses and pulsed field gradients ( pfgs ) that suppress undesired coherences in the subsequent z - filtration . we have found that the same task is far more demanding using hmqc . in this case pfgs can not be used to distinguish aromatic and aliphatic mq h c coherences as both have equal coherence order after initial 1/2jch delay and hard excitation pulse . if the selective excitation pulse is employed pfgs can be useful , however , sensitivity loss of a factor of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sqrt 2 $ $ \end{document } is incurred and additional time is required for gradient encoding when c magnetization is transverse . we thus conclude that the optimal solution includes phase cycling of selective excitation pulses . despite these difficulties hmqc is preferred over refocused inept owing to ( 1 ) the apparently slower relaxation of mq coherences and ( 2 ) the possibility to incorporate both magnetization transfer delays ( 1/jch in total ) for the shared - time proton evolution ( t1 ) that leads to increased sensitivity ( stanek et al . 1pulse sequence scheme for ( a ) c(aliph)hmqc - noesy - c(aro)hsqc and ( b ) c(aro)hmqc - noesy - c(aliph)hsqc . 90 and 180 hard pulses are represented by filled and open bars , respectively . shaped pulses are represented as follows : ( a ) 40 ppm selective pulse of the iburp-2 profile ( geen and freeman 1991 ) ( 659 s duration , 7.6 khz peak r.f . field ) is shown as wide open bell - shaped pulse ( denoted e ) . wide grey rectangular pulses ( a and c ) are of duration of 61 s and peak r.f . 4.1 khz ( calibrated to give null excitation 90 ppm off -resonance ) . ( b ) wide grey bell - shaped pulse denotes 80 ppm - selective hyperbolic secant adiabatic pulse ( silver et al . narrow grey bell - shaped pulses ( f and h ) utilize gaussian profile ( bauer et al . 1984 ) ( truncated at 1 % , duration of 178 s , peak r.f . note that for some hardware the shaped 90 pulses ( a , c , f and h ) may require small angle phase adjustment to compensate 0th order phase shift with respect to refocusing pulses ( b and g ) which are applied at full power . n refocusing pulse enclosed in dashed - line box ( sequence b ) is optional and may be used if delay complementing to half - dwell time , = [ ( sw3 ) pw180(n)]/2 , is positive . refocusing of caliph - c couplings in t2 ( a ) or t3 ( b ) may be considered if max . the composite 34.2x123x197.6x288.8x pulse ( d and i ) was used in hmqc for broadband inversion of c spins ( shaka 1985 ) . watergate ( piotto et al . 1992 ) with 3 - 9 - 19 pulses separated by ( 3.2 khz ) was employed for experiment ( a ) . c composite pulse decoupling was performed employing wurst scheme ( kupe and freeman 1995 ) . /2 pulses were 7.1 , 14.3 and 31 s for h , c and n , respectively . 1 , 2 and 3 are incremented for quadrature detection in t1 , t2 and t3 using states ( 1 ) or states - tppi ( 2 , 3 ) method . four - step phase cycle is as follows : 1 = 45 ; 2 = x , x ; 3 = 2(x ) , 2(x ) ; 4 = 2(x ) , 2(x ) ; 5 = 135 ; rec = x , delays are set as follows : a = 1.79 ms ( 4 jchaliph ) , b = 1.56 ms ( 4 jcharom ) . noesy mixing time m = 150 ms was used . for the semi - constant time evolution in t1 ( h ) the delays 1 , 2 and 3 are t1/2 , t1(1 2/t1,max)/2 and (1 t1/t1,max ) , where = 2a , or = 2b for sequences ( a ) and gradient levels and durations are : g1 ( 2 ms , 6.5 gs / cm ) , g2 ( 2 ms , 14.2 gs / cm ) , g3 ( 0.5 ms , 1.77 gs / cm ) , g4 ( 2 ms , 11.3 gs / cm ) , g5 ( 2 ms , 12.9 gs / cm ) , g6 ( 0.5 ms , 5.4 gs / cm ) . proton carrier frequency was set on resonance with water ( 4.68 ppm ) , carbon carrier was set to 35 ppm and switched to 125 ppm as indicated by the vertical arrow ; n carrier was set to 117 ppm and shifted to 162 ppm at the beginning of noesy mixing period . for the aliphatic - to - aromatic noesy ( a ) 4,400 sampling points ( t1,t2,t3 ) were randomly chosen from 120 84 30 cartesian grid according to gaussian probability distribution , p(t ) = exp[(t / tmax)/2 ] , = 0.5 , with poisson disk restrictions ( kazimierczuk et al . 2008 ) . maximum evolution times of 15 ( t1 ) , 6 ( t2 ) and 5 ms ( t3 ) were achieved in the indirectly detected dimensions . spectral widths of 8 , 14 , 6 and 12 khz were set in 1 , 2 , 3 and 4 dimensions , respectively . in the full 4d spectrum any residual diagonal peaks can be folded in c ( 2 and 3 ) dimensions without the risk of overlap and misinterpretation with genuine peaks . the only requirement is to ensure proper h ( 1 ) spectral width to avoid aliasing in this indirect dimension . the restriction of c spectral widths is very practical as it saves vast amounts of disk space . the total experimental time was 57 h pulse sequence scheme for ( a ) c(aliph)hmqc - noesy - c(aro)hsqc and ( b ) c(aro)hmqc - noesy - c(aliph)hsqc . 90 and 180 shaped pulses are represented as follows : ( a ) 40 ppm selective pulse of the iburp-2 profile ( geen and freeman 1991 ) ( 659 s duration , 7.6 khz peak r.f . field ) is shown as wide open bell - shaped pulse ( denoted e ) . wide grey rectangular pulses ( a and c ) are of duration of 61 s and peak r.f . 4.1 khz ( calibrated to give null excitation 90 ppm off -resonance ) . ( b ) wide grey bell - shaped pulse denotes 80 ppm - selective hyperbolic secant adiabatic pulse ( silver et al . narrow grey bell - shaped pulses ( f and h ) utilize gaussian profile ( bauer et al . 1984 ) ( truncated at 1 % , duration of 178 s , peak r.f . note that for some hardware the shaped 90 pulses ( a , c , f and h ) may require small angle phase adjustment to compensate 0th order phase shift with respect to refocusing pulses ( b and g ) which are applied at full power . n refocusing pulse enclosed in dashed - line box ( sequence b ) is optional and may be used if delay complementing to half - dwell time , = [ ( sw3 ) pw180(n)]/2 , is positive . refocusing of caliph - c couplings in t2 ( a ) or t3 ( b ) may be considered if max . evolution time of aliphatic c spins exceeds 9 ms . the composite 34.2x123x197.6x288.8x pulse ( d and i ) was used in hmqc for broadband inversion of c spins ( shaka 1985 ) . watergate ( piotto et al . 1992 ) with 3 - 9 - 19 pulses separated by ( 3.2 khz ) was employed for experiment ( a ) . c composite pulse decoupling was performed employing wurst scheme ( kupe and freeman 1995 ) . /2 pulses were 7.1 , 14.3 and 31 s for h , c and n , respectively . 1 , 2 and 3 are incremented for quadrature detection in t1 , t2 and t3 using states ( 1 ) or states - tppi ( 2 , 3 ) method . four - step phase cycle is as follows : 1 = 45 ; 2 = x , x ; 3 = 2(x ) , 2(x ) ; 4 = 2(x ) , 2(x ) ; 5 = 135 ; rec = x , x , x , x . delays are set as follows : a = 1.79 ms ( 4 jchaliph ) , b = 1.56 ms ( 4 jcharom ) . for the semi - constant time evolution in t1 ( h ) the delays 1 , 2 and 3 are t1/2 , t1(1 2/t1,max)/2 and (1 t1/t1,max ) , where = 2a , or = 2b for sequences ( a ) and ( b ) , respectively ( stanek et al . 2012 ) . gradient levels and durations are : g1 ( 2 ms , 6.5 gs / cm ) , g2 ( 2 ms , 14.2 gs / cm ) , g3 ( 0.5 ms , 1.77 gs / cm ) , g4 ( 2 ms , 11.3 gs / cm ) , g5 ( 2 ms , 12.9 gs / cm ) , g6 ( 0.5 ms , 5.4 gs / cm ) . proton carrier frequency was set on resonance with water ( 4.68 ppm ) , carbon carrier was set to 35 ppm and switched to 125 ppm as indicated by the vertical arrow ; n carrier was set to 117 ppm and shifted to 162 ppm at the beginning of noesy mixing period . for the aliphatic - to - aromatic noesy ( a ) 4,400 sampling points ( t1,t2,t3 ) were randomly chosen from 120 84 30 cartesian grid according to gaussian probability distribution , p(t ) = exp[(t / tmax)/2 ] , = 0.5 , with poisson disk restrictions ( kazimierczuk et al . maximum evolution times of 15 ( t1 ) , 6 ( t2 ) and 5 ms ( t3 ) were achieved in the indirectly detected dimensions . spectral widths of 8 , 14 , 6 and 12 khz were set in 1 , 2 , 3 and 4 dimensions , respectively . in the full 4d spectrum any residual diagonal peaks can be folded in c ( 2 and 3 ) dimensions without the risk of overlap and misinterpretation with genuine peaks . the only requirement is to ensure proper h ( 1 ) spectral width to avoid aliasing in this indirect dimension . the restriction of c spectral widths is very practical as it saves vast amounts of disk space . the total experimental time was 57 h here , the selection of either aliphatic ( fig . 1b ) proton - carbon mq coherences is accomplished solely by band - selective excitation of c spins in conjunction with the four - step phase cycle of two /2 pulses ( a , f , h , respectively ) . therefore , the choice of selective /2 pulses is of the particular relevance . apparently , it is quite straightforward not to excite aromatic c spins ( the sequence shown in fig . more advanced pulses such as gaussian cascade q5 ( emsley and bodenhausen 1992 ) or e - burp ( geen and freeman 1991 ) could be utilized as well , however , these pulses are ( 1 ) of significantly larger duration , ( 2 ) more sensitive to miscalibration and ( 3 ) require high peak r.f . field . it was experimentally verified that these shortcomings offset theoretical benefits of high quality pulses and significant loss of sensitivity is incurred . for the aromatic - hmqc ( fig . due to the use of shaped /2 c pulses in hmqc the refocusing pulse is obligatory for correct phase properties of signals in 2 dimension . however , no additional selectivity improvement can be achieved by replacement of this pulse by a band - selective one . similarly , the subsequent carbon inversion pulse in hmqc ( denoted d and i , respectively ) should be sufficiently broadband . a shaped pulse here would only result in the t1-dependent jch evolution of unaffected coherences during total time of 1 + 2 the resulting signals would be impossible to suppress using neither phase cycling nor pulsed field gradients . all nmr experiments were performed for the e32q mutant of human s100a1 protein , for which resonance assignments are available in bmrb under accession code 17857 . the high resolution 3d structure based on the standard 3d c- and n - edited noesy - hsqc spectra ( zhang et al . 2011 ) , this protein mutant also exists in solution as a homodimer built up of noncovalently attached subunits ( molecular weight of the monomer is 10 kda ) . nmr sample contained 1.0 mm n , c labeled protein ( the monomer concentration ) in 90 % /10 % h2o / d2o , 50 mm tris - d11 , 1.0 mm edta , 0.1 mm nan3 and 50 mm nacl with ph adjusted to 7.2 . the experiments were performed at 37 c on a 700 mhz agilent / varian spectrometer equipped with the performa xyz pfg unit and the 5 mm h , c , n triple - resonance probehead . 3d noesy - hsqc and 4d hmqc - noesy - hsqc spectra were analyzed with sparky ( goddard and kneller ) . in both cases , peak picking and inspection of resulting peak lists was performed in approximately 30 min . conventionally sampled 3d noesy - c - hsqc spectrum was acquired in 36 h and processed using nmrpipe program ( delaglio et al . 1995 ) . to derive noesy interproton distance constrains from the 4d c(ali),c(aro)-edited noesy , automatic noesy assignment procedure ( herrmann et al . 2002a ) implemented in program cyana 3.0 ( guntert et al . the final validation of obtained constraints was performed with xplor - nih program ( schwieters et al . the processing of four - dimensional nus data was accomplished by the home - written ssa ( signal separation algorithm ) software package , which can be downloaded free of charge for non - commercial purposes from the website http://nmr.cent3.uw.edu.pl ( 64-bit versions for linux ) . artefact suppression in ssa software ( cleaner4d program ) was intentionally disabled by setting maximum number of iterations to zero . this is equivalent to the use of raw zero - augmented fourier transformation and limits cleaner4d to data conversion . the actual computation of spectral data was performed using reconstructor4d program ( also from ssa package ) that implements ( a ) automatic choice between fft and dft whenever beneficial , ( b ) multi - threaded computations and ( c ) memory - mapped and asynchronous input output disk operations to improve overall performance . the total processing time on a pc equipped with intel i5 3.3 ghz cpu running under ubuntu 11 was 2 min and 6 min 30 s using 4 and 1 threads , respectively . final spectrum stored in sparky format ( single - precision ) was of size of 10.1 gb . ssa software supports native agilent / varian as well as nmrpipe format as input data . the performance of proposed selective noesy experiments was first verified on conventionally sampled homonuclear 2d spectra ( fig . 2 ) . for both possibilities considered here , namely aliphatic - to - aromatic ( fig . 2d ) excellent suppression of undesired autocorrelation ( i. e. aliphatic aliphatic and aromatic aromatic ) signals was obtained . noteworthy is that in the non - selective versions of these experiments ( i.e. without use of shaped pulses ) substantial amount of spectral artefacts appear that may distort relevant region of aromatic aliphatic cross - peaks ( see , e.g. , pseudo - diagonals in fig . pulses and decoupling schemes at high static magnetic fields the simultaneous acquisition of aromatic aliphatic and aliphatic aromatic noesy cross - peaks is hardly achievable ( fig . 2b , e ) . thus , selection of one possibility is not lossy in terms of structural information gained.fig . 2the comparison of homonuclear ( 1 ( h)2 ( h ) ) versions of ( a ) selective aliphatic - to - aromatic , ( b ) non - selective aliphatic - to - aromatic ( see fig . 1b ) , ( d ) selective aromatic - to - aliphatic and ( e ) non - selective aromatic - to - aliphatic noesy experiments . spectra ( a ) and ( d ) were acquired using pulse sequences shown in fig . non - selective versions ( b ) and ( e ) utilize the same pulse sequences as ( a ) and ( d ) , respectively , with all the shaped pulses replaced by hard ones . spectra ( b ) and ( e ) show that non - selective experiments suffer from many spectral artefacts due to poor broadband performance of apart from sign inversions across the spectrum , intense axial peaks as well as phase - distorted pseudo - diagonals are present ( e ) . selective experiments ( a , d ) achieve to filter - out virtually all diagonal signals with only slight decrease of intensity of cross - peaks . ( c ) superimposed 1d cross - sections across 1 ( h ) from spectra ( a , blue curves ) and ( b , green curves ) for 2 coordinates indicated by the vertical arrows ( 13 ) . ( f ) 2 ( h , direct dimension ) cross - sections of spectra ( d , blue ) and ( e , green curves ) at 1 coordinates indicated by the horizontal arrows ( 13 ) . the cross - sections are plotted using the same intensity scale , thus they enable direct comparison of experimental sensitivity between selective and non - selective versions of experiments ( a vs. b , and d vs. e ) . for each experiment 120 spectral width of 8 khz in the indirect 1 ( h ) dimension was set the comparison of homonuclear ( 1 ( h)2 ( h ) ) versions of ( a ) selective aliphatic - to - aromatic , ( b ) non - selective aliphatic - to - aromatic ( see fig . 1b ) , ( d ) selective aromatic - to - aliphatic and ( e ) non - selective aromatic - to - aliphatic noesy experiments . spectra ( a ) and ( d ) were acquired using pulse sequences shown in fig . 1a , b , respectively . non - selective versions ( b ) and ( e ) utilize the same pulse sequences as ( a ) and ( d ) , respectively , with all the shaped pulses replaced by hard ones . spectra ( b ) and ( e ) show that non - selective experiments suffer from many spectral artefacts due to poor broadband performance of apart from sign inversions across the spectrum , intense axial peaks as well as phase - distorted pseudo - diagonals are present ( e ) . selective experiments ( a , d ) achieve to filter - out virtually all diagonal signals with only slight decrease of intensity of cross - peaks . ( c ) superimposed 1d cross - sections across 1 ( h ) from spectra ( a , blue curves ) and ( b , green curves ) for 2 coordinates indicated by the vertical arrows ( 13 ) . ( f ) 2 ( h , direct dimension ) cross - sections of spectra ( d , blue ) and ( e , green curves ) at 1 coordinates indicated by the horizontal arrows ( 13 ) . the cross - sections are plotted using the same intensity scale , thus they enable direct comparison of experimental sensitivity between selective and non - selective versions of experiments ( a vs. b , and d vs. e ) . for each experiment 120 spectral width of 8 khz in the indirect 1 ( h ) dimension was set the sensitivity of noesy experiments is a critical issue . . 2 do not indicate any substantial signal degradation resulting from use of selective pulses . indeed , the closer inspection of 1d traces showing noesy cross - peaks ( fig . 2c , f ) reveals only minor decrease of sensitivity accompanied by virtually complete suppression of diagonal peaks . aromatic cross - peaks . although quite imprecise and susceptible to presence of unrelated spectral artefacts ( as in fig . 2a , d ) are approximately up to 10 % less intense than their non - selective counterparts ( fig . 2b , e ) . in the context of non - uniform sampling employed for four - dimensional experiment a rather moderate price for elimination of strong diagonal peaks and associated spectral artifacts 1a , b ) seem to provide equivalent structural information , there is a number of practical considerations involved . since aliphatic protons are typically far more populated than aromatic ones high resolution in aliphatic h dimension is especially desired . this suggests to prefer the aromatic - to - aliphatic variant which provides the optimal resolution of directly detected h dimension . in the alternative aliphatic - to - aromatic spectrum the indirectly detected h dimension features lower resolution , however , the assignment is supported by additional c chemical shift . noteworthy , aliphatic h and c nuclei exhibit a broader distribution of chemical shifts than aromatic ones , thus simplifying the assignment task . to sum up frequently , samples in h2o / d2o solution are used also for acquisition of noesy spectra in order to avoid data inconsistency due to different sample preparation and isotopic effects . in this case the evident advantage of c , c - separated noesy is that watergate ( piotto et al . 1992 ) can be employed without suppression of relevant resonances . on the other hand , for the c , c - separated noesy presaturation of hdo signal it is also worth mentioning that in the case of the c , c - noesy the detection of aromatic protons decreases requirements on c decoupling during acquisition . as broadband decoupling at high b0 field can be quite detrimental for the probehead circuits , the c , c - edited noesy might be easier to implement on some hardware ( e.g. , cryoprobes ) . the most decisive argument in favor of the c , c - separated noesy is its higher sensitivity . although it is not manifested in 2d h h versions of compared experiments ( fig . 2a , d show similar intensity of cross - peaks ) , the direct comparison of four - dimensional c , c - edited spectra indicated that c , c - edited experiment is considerably more sensitive . as the effect of shaped pulses is similar for both sequences , the most likely reason for different sensitivity of these experiments is the different attenuation of transverse relaxation rate observed for multiple quantum aromatic and aliphatic c h coherences stored during c t2 evolution period . the distinguishing feature of selective aliphatic aromatic noesy is the absence of strong diagonal signals which provide no additional structural information . this has a crucial implication that resulting spectra acquired using non - uniform sampling are not distorted by sampling artefacts , or their impact can be neglected in comparison to thermal noise ( see the flat baselines of noise in fig . 3c , d ) . therefore , more advanced processing schemes such as ssa or clean do not have a particular advantage over zero - augmented fourier transformation ( kazimierczuk et al . 2012 ) . the practical consequence is that a full 4d spectral matrix can be rapidly reconstructed , usually in a few minutes , depending mostly on hard disk performance ( see experimental section ) . additionally , the s / n of most intense cross - peaks found in the indirect 3d cubes 13 shown in fig . 3a , b confirms the above mentioned difference in overall sensitivity of two variants of selective noesy.fig . 3a , b maximum signal - to - noise ratio measured for peaks in the individual 3d cubes ( indirect domains 1 2 3 ) in each point of directly detected dimension ( 4 ) . the plots show the ratio of the most intense spectral point ( absolute value ) to the noise level in the corresponding 3d cube ( c , d ) . assuming that cross - peaks do not overlap in the 4d spectrum these plots reflect the overall sensitivity of experiment . considerable sensitivity advantage of the aliphatic - to - aromatic ( a , c ) over aromatic - to - aliphatic ( b , d ) noesy is apparent . noise levels ( c , d ) in the 3d cubes for each point of directly detected dimension were measured as the median of absolute values of all spectral intensities . from the uniformity of noise level across directly detected dimension one can conclude that advanced procedures for nus artifact suppression are not beneficial in this case , and that the both experiments are sensitivity - limited . the scale was cut for intense water resonance ( at 4.68 ppm ) a , b maximum signal - to - noise ratio measured for peaks in the individual 3d cubes ( indirect domains 1 2 3 ) in each point of directly detected dimension ( 4 ) . the plots show the ratio of the most intense spectral point ( absolute value ) to the noise level in the corresponding 3d cube ( c , d ) . assuming that cross - peaks do not overlap in the 4d spectrum these plots reflect the overall sensitivity of experiment . considerable sensitivity advantage of the aliphatic - to - aromatic ( a , c ) over aromatic - to - aliphatic ( b , d ) noesy is apparent . noise levels ( c , d ) in the 3d cubes for each point of directly detected dimension were measured as the median of absolute values of all spectral intensities . from the uniformity of noise level across directly detected dimension one can conclude that advanced procedures for nus artifact suppression are not beneficial in this case , and that the both experiments are sensitivity - limited . the scale was cut for intense water resonance ( at 4.68 ppm ) e32q mutant of human s100a1 protein was used to demonstrate the advantage of 4d c(ali),c(aro)-edited noesy in identification of noes between aromatic and aliphatic protons . after synchronization of the 3 and 4 axes with the aromatic region of c hsqc , the peak picking can be performed manually . majority of these correlations can be unambiguously assigned by visual comparison of particular aliphatic planes of 4d noesy and the aliphatic region of c - hsqc spectrum.fig . 4schematic illustration of reliable cross - peak assignment strategy using 4d c(ali),c(aro)-edited noesy spectrum . in the top left panel a plane of 4d c(ali),c(aro)-edited noesy at tyr 74 h/c chemical shifts is presented with expanded methyl region of the spectrum on the right side . in the bottom left panel 2d h corresponding cross peaks in the cross - section of 4d spectrum and the methyl region of hsqc are connected by arrows . relevant methyl - aromatic cross - peaks can be readily assigned ( even manually ) as the ambiguity is removed by additional aliphatic - c chemical shift ( 2 ) schematic illustration of reliable cross - peak assignment strategy using 4d c(ali),c(aro)-edited noesy spectrum . in the top left panel a plane of 4d c(ali),c(aro)-edited noesy at tyr 74 h/c chemical shifts is presented with expanded methyl region of the spectrum on the right side . in the bottom left panel 2d h corresponding cross peaks in the cross - section of 4d spectrum and the methyl region of hsqc are connected by arrows . relevant methyl - aromatic cross - peaks can be readily assigned ( even manually ) as the ambiguity is removed by additional aliphatic - c chemical shift ( 2 ) it should be emphasized that most of proton chemical shift degeneracies observed in 3d c(aro)-edited noesy has been removed in 4d c(ali),c(aro)-edited noesy providing 21 new reliable structural constraints . the cross peak present in the 3d noesy spectrum splits into two noe correlations in the 4d spectrum . 5an example showing removal of proton chemical shift degeneracy by addition of aliphatic carbon-13 dimension . in the standard 3d c - edited noesy - hsqc spectrum ( top left panel ) only two cross peaks are observed for h proton of phe 15 . in the bottom aromatic noesy is presented , and one of the cross peaks splits into two . on the right panel the corresponding fragment of previously determined 3d structure of s100a1 e32q ( pdb code 2lhl ) is shown . only the e70 hf15 h and k27 hf15 h interactions were marked an example showing removal of proton chemical shift degeneracy by addition of aliphatic carbon-13 dimension . in the standard 3d c - edited noesy - hsqc spectrum ( top left panel ) only two cross peaks are observed for h proton of phe 15 . in the bottom left panel a cross section from 4d aliphatic aromatic noesy is presented , and one of the cross peaks splits into two . on the right panel the corresponding fragment of previously determined 3d structure of s100a1 e32q ( pdb code 2lhl ) is shown . only the e70 hf15 h and k27 hf15 h interactions were marked all constraints obtained from the 4d noesy spectrum were carefully validated against the previously determined structure of the e32q s100a1 protein ( pdb accession code 2lhl ) . as demonstrated for h/c of phe 15 , all nuclei contributing to observed noes are in spatial proximity ( cf . the right panel of fig . 6 ) . additionally , it was verified that most cross - peaks identified in the 4d spectrum were assigned consistently with peaks present in its 3d counterpart . the only few differences concern stereospecific assignment of noe contacts and peaks that have not be assigned in 3d spectrum due to ambiguity.fig . 6 in the top left panel the slice of 3d c - edited noesy - hsqc for h/c of phe 15 is presented . in the bottom left panel corresponding plane of 4d c(ali),c(aro)-edited noesy is presented . in the right panel a fragment of 3d structure derived previously is presented . residues are color - coded as are the lines connecting corresponding peaks in the spectra , while phe 15 is presented in black in the top left panel the slice of 3d c - edited noesy - hsqc for h/c of phe 15 is presented . in the bottom left panel corresponding plane of 4d c(ali),c(aro)-edited noesy is presented . in the right panel a fragment of 3d structure derived previously is presented . residues are color - coded as are the lines connecting corresponding peaks in the spectra , while phe 15 is presented in black at present manual noesy cross peak assignment is rarely performed . therefore , the standard automatic protocol for assignment of noe correlations , implemented in cyana software , was applied for the analysis of the 3d c(aro)-edited noesy - hsqc and 4d c(ali),c(aro)-edited noesy spectra . it seems surprising that 3d spectrum resulted in a slightly larger number of constraints than the 4d one . it should be borne in mind , however , that most of 3d - derived noe correlations in larger proteins are heavily degenerated . current assignment algorithms deduce such a distance constraint on the basis of chemical shifts and the supposed proton spatial proximity in the tentative structure model . consequently , a number of correlations are , to some extent , speculative and estimation of their intensities ( necessary for distance calibration ) is putative . on the other hand , most of 4d - derived noe correlations it might be of crucial importance for de novo structure determination of an unknown system . as shown in table 1 some weak correlations visible in 3d noesy were missing in the 4d spectrum due to its intrinsically lower sensitivity , however , these include mostly weak intraresidual h most importantly , the number of essential long range and intersubunit constraints was not affected.table 1the summary of noe constraints derived from standard 3d c - edited noesy hsqc ( tuned to aromatic carbons ) and 4d c(ali),c(aro)-edited noesy3d c(aro)-edited noesy hsqc4d c(ali),c(aro)-edited noesynumber of common constraints from 3d and 4d spectraconstraints between aromatic and aliphatic protons156131111intraresidual ( i j 1)635044medium range ( 1 < i j < 5)221410long range ( i j 5)454437intersubunit2623204d spectrum provided 20 additional constraints that could not be assigned in the 3d spectrum . a number of weak noe contacts present in 3d spectrum were not detected in 4d noesy , including mostly intraresidual hh , hh and hh noe contacts . nevertheless , the number of most relevant long range and intersubunit constraints remained virtually equal the summary of noe constraints derived from standard 3d c - edited noesy hsqc ( tuned to aromatic carbons ) and 4d c(ali),c(aro)-edited noesy 4d spectrum provided 20 additional constraints that could not be assigned in the 3d spectrum . a number of weak noe contacts present in 3d spectrum were not detected in 4d noesy , including mostly intraresidual hh , hh and hh noe contacts . nevertheless , the number of most relevant long range and intersubunit constraints remained virtually equal the final structural calculations were performed with xplor - nih program to confirm if the noe distance constraints from automatic procedure were calibrated correctly . the substitution of constraints derived from 3d noesy with constraints from 4d c(ali),c(aro)-edited noesy did not lead to any constraint violations during calculations , or noticeable differences between obtained structures . aromatic c , c - edited 4d noesy experiments . as these experiments inherently lack the diagonal signals , significant spectral artifacts originating from either non - uniform sampling , hardware instability or r.f . thus , the applications of proposed experiment is not limited to small - sized proteins . owing to high spectral resolution and selection of a subset of noe contacts it may become a valuable tool for three - dimensional structure elucidation of large proteins . full benefits of the novel method presented here can be achieved when applied in conjunction with previously reported 4d amide amide , amide

a band - selective aromatic aliphatic c , c - edited four - dimensional noesy experiment is proposed here . its key advantage is the absence of auto - correlation signals which makes it very attractive for joint use with non - uniform sampling . it is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses ( for either aromatic-13c or aliphatic-13c spins ) . the method was applied to the sample of e32q mutant of human s100a1 protein , a homodimer of total molecular mass ~20 kda . high - resolution 4d spectra were obtained from ~1.5 % of sampling points required conventionally . it is shown that superior resolution facilitates unambiguous assignment of observed aliphatic aromatic cross - peaks . additionally , the addition of aliphatic-13c dimension enables to resolve peaks with degenerated aliphatic 1h chemical shifts . all observed cross - peaks were validated against previously determined 3d structure of e32q mutant of s100a1 protein ( pdb 2lhl ) . the increased reliability of structural constraints obtained from the proposed high - resolution 4d 13c(ali),13c(aro)-edited noesy can be exploited in the automated protocols of structure determination of proteins.electronic supplementary materialthe online version of this article ( doi:10.1007/s10858 - 013 - 9739 - 5 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Selective 4D HMQC-NOESY-HSQC experiments Experimental Results and discussion Conclusions Electronic supplementary material

PMC3270573

the assessment of olfactory function is an essential step for the diagnosis and treatment of olfactory disabilities . in particular , the quantification of the olfaction functional level is necessary to assess the recovery from or the progression of the smell dysfunction . the link between some types of smell impairments and the early detection of neurodegenerative ( albers et al . , 2006 ; barrios et al . , 2007 ; doty , 2008 ) or psychiatric ( atanasova et al . , 2008 ) diseases also emphasizes the need of precise olfactory quantification procedures . different protocols like the connecticut chemosensory clinical research center ( cccrc ) test ( cain , 1989 ) , the university of pennsylvania smell identification test ( upsit ) ( doty et al . , 1984 ) , sniffin ' sticks ( burghart , wedel , germany ) ( hummel et al . , 1997 ) , t&t olfactometry , or the odor stick identification test for japanese ( osit - j ) ( zusho , 1983 ) are employed throughout the world to assess the sense of smell in patients with different cultural backgrounds and disabilities . these tests consist of different odor containers that are presented to a patient to quantify specific odor thresholds , and to evaluate odor identification and discrimination . a given test sample is typically employed in the clinic and then replaced by a new one after a certain period of use . in many cases the sample is opened , used , and closed again over an extended period of time which can range from days to several months . thus , there is a need to assess control quality of these test samples and to establish a protocol to quantify their degradation over time . artificial noses are devices that use one or multiple sensors to mimic the sense of smell . the sensor reacts to an odorant and generates a signal that can be used for characterization , discrimination , or recognition purposes . the first artificial nose with a tin - oxide sensor was reported in the early 1980s . a wide range of sensor technologies have been developed since them to build many different types of artificial noses : electrical , gravimetric , optical , etc . , [ for a review of sensor technologies for artificial noses see ( stitzel et al . , 2011 ) ] . the applications of artificial noses cover areas such as safety , security , food , and beverage quality control , environmental monitoring , medical diagnostics , etc . , ( dymerski et al . , 2011 ; stitzel et al . , 2011 ) . in the following sections we propose the use of low - cost artificial noses to quantitatively characterize olfactory tests for clinical studies , and we also discuss the need to establish a map between human and machine olfaction for research and clinical studies . this test consists of two parts : the butanol threshold test and the identification test . for the purposes of this study we will mainly concentrate on the butanol threshold test . the test was manufactured at hospital fundacin de alcorcn , madrid with the collaboration of the pharmacy unit following the guidelines of the original article . the threshold test employs aqueous dilutions of 1-butanol where successive dilutions differ by a factor of three . the test solutions were stored in 125 ml polyethylene bottles containing 60 ml of solution which are presented to patients during the test . the bottle closure has a pop - up spout that fits to both nostrils ( see figure 1 ) . to sample a bottle , the person places the spout into both nostrils and then sniffs . there are two common ways to present the stimulus : the ascending method of limits ( amls ) procedure and the single staircase ( ss ) procedure . in the aml , the odorant and the water are presented sequentially from low to high concentration and the point of transition between no detection and detection is estimated . in the ss method , the concentration of the stimulus is increased following trials in which correct detection occurs . in both methods , the concentration at which this occurs marks the threshold . the connecticut chemosensorial clinical research center test used in this study . the brown bottles contain the butanol threshold test , while the white plastic jars contain the identification test . recently , toledano and colleagues have described a short olfactory test based on cccrc that predicts how severe is the smell loss ( toledano et al . , 2009 ) . this short test consists on determining the lowest concentration of butanol that the patient can detect four times in a row . patients detecting beyond the butanol dilution number 3 have a high probability of normal olfaction . the identification kit is composed of ten 180 ml opaque plastic jars containing 5 g of the substance in sachet - like packets of stimuli . based on the performance of anosmic patients , we can ascertain that seven stimuli appeal almost exclusively to the sense of smell ( baby powder , chocolate , cinnamon , coffee , mothballs , peanut butter , and bar soap ) and one appeals to the trigeminal sense as well ( vicks ) . in addition to the names on the list , responses of no sensation and do not know are permitted . the examiner provides corrective feedback whenever the participant makes an error . if the participant exhibits some evidence of function , but nevertheless makes mistakes , the examiner presents missed items for a second time . this allows the patient to rectify mistakes and thereby decreases the possibility of cognitive errors . in such cases , the score for the test comprises the number of olfactory items out of seven correctly identified and a notation regarding the ability to perceive trigeminal stimulation . the outcome of the threshold and the identification tests is combined into a composite score , an average of the two tests . as previously defined ( toledano et al . , 2003 ) , a score of 5 points or more indicates normosmia , a score between 2 and 4.5 points indicates reduced olfactory function in terms of hyposmia , and a score of less than 2 points indicates functional anosmia . to categorize progression of olfactory function , a subjective improvement and a normal score in butanol , threshold and composite , respectively , its correlation with previously established tests of olfactory function ( e.g. , the 12-item cross - cultural smell identification test [ cc - sit , a subtest of the upsit ] and the sniffin ' sticks test ) has been demonstrated in various studies ( kobal et al . , 2000 ; toledano et al . , 2007 ) . during the last decades artificial nose technology has provided many successful examples of industrial applications [ for recent reviews see ( dymerski et al . , 2011 ; stitzel et al . , the possibilities of potential use in medical and clinical studies have also increased . in this section we discuss a preliminary study on the use of a low - cost , portable artificial nose to quantitatively characterize the cccrc test . for the study reported in this paper we used an odor analysis platform capable of managing up to 16 samples with on / off control electrovalves and three auxiliary devices such as pumps , heaters , and/or mixers ( see figure 2 ) . although the platform can be equipped with a wide variety of sensor arrays , we aimed to study the performance of inexpensive sensors that could be used in future widely distributed control quality devices . this small sensor also provides a good combination of high sensitivity to low concentrations of odorants , long life , low power consumption , and robustness . the sensor is comprised of a metal oxide semiconductor layer formed on an alumina substrate of a sensing chip together with an integrated heater . in the presence of an odorant , the sensor 's conductivity increases as a function of the concentration . a simple electrical circuit converts the change in conductivity to an output signal that corresponds to the odorant . the signal generated by the sensor is not amplified , it is only conditioned with an lmc6484 amplifier in voltage follower mode and sent to the analog - to - digital ( a / d ) converter module to be acquired and stored in a computer . the voltage range of this signal is 05 v. analysis platform used in this study with the tgs2600-b00 ( figaro engineering inc . ) chemoresistive sensor . from the control point of view , the main component used in this platform is an 8-bit pic18f2455 microcontroller ( microchip technology inc . ) . this microcontroller is ideal for low - cost , low power ( nano - watt ) , and connectivity applications . the firmware was programmed with the mplab ide development environment provided by the same company . the computer application software was developed with ide dev - c++ ( gnu license ) using windows apis . the equipment is connected to a pc via serial bus , by a proprietary protocol based on eia-485 . for this application we used the rotary vane pump 01-k g - lc ( rietschle thomas company ) in continuous vacuum mode ( 20 mbar max . ) all the instrumentation used in this study is characterized by its compactness and low price , which are important factors for a wide dissemination of quality control in olfactory tests . since the goal of our experiments is to assess the degradation of well - known odorants , we used the peak of the sensor signal to characterize the dilutions / odorants in a comparative manner ( the peak corresponding to an aged olfactory test vs. the peak corresponding to a new test ) . this is a simple and straightforward measurement that does not require further processing of the signal . the performance of the tgs2600-b00 sensor has a moderate ambient temperature / humidity dependence as described in the manufacturer 's worksheet ( http://www.figarosensor.com/products/2600pdf.pdf ) . all experiments reported here were performed at a room temperature between 23c and 24c and a humidity of 3035% . the acquisition of the signal was performed with the following protocol : the sensor was exposed to room air with no heating ( ov ) for 5 s , followed by 100 s at maximum heating ( 4.8 v ) to clean the sensor ( heater resistance is 83 ) . after this cleaning phase , we took 5 s of absorption of the odorant ( 4.8 v for heating , this duration was chosen to avoid saturation ) , and finally 100 s of desorption ( room air again at maximum heating ) . five consecutive measurements of all odorants were taken following this protocol to avoid history dependent effects . this test consists of two parts : the butanol threshold test and the identification test . for the purposes of this study we will mainly concentrate on the butanol threshold test . the test was manufactured at hospital fundacin de alcorcn , madrid with the collaboration of the pharmacy unit following the guidelines of the original article . the threshold test employs aqueous dilutions of 1-butanol where successive dilutions differ by a factor of three . the test solutions were stored in 125 ml polyethylene bottles containing 60 ml of solution which are presented to patients during the test . the bottle closure has a pop - up spout that fits to both nostrils ( see figure 1 ) . to sample a bottle , the person places the spout into both nostrils and then sniffs . there are two common ways to present the stimulus : the ascending method of limits ( amls ) procedure and the single staircase ( ss ) procedure . in the aml , the odorant and the water are presented sequentially from low to high concentration and the point of transition between no detection and detection is estimated . in the ss method , the concentration of the stimulus is increased following trials in which correct detection occurs . in both methods , the concentration at which this occurs marks the threshold . the connecticut chemosensorial clinical research center test used in this study . the brown bottles contain the butanol threshold test , while the white plastic jars contain the identification test . recently , toledano and colleagues have described a short olfactory test based on cccrc that predicts how severe is the smell loss ( toledano et al . , 2009 ) . this short test consists on determining the lowest concentration of butanol that the patient can detect four times in a row . patients detecting beyond the butanol dilution number 3 have a high probability of normal olfaction . the identification kit is composed of ten 180 ml opaque plastic jars containing 5 g of the substance in sachet - like packets of stimuli . based on the performance of anosmic patients , we can ascertain that seven stimuli appeal almost exclusively to the sense of smell ( baby powder , chocolate , cinnamon , coffee , mothballs , peanut butter , and bar soap ) and one appeals to the trigeminal sense as well ( vicks ) . in addition to the names on the list , responses of no sensation and do not know are permitted . the examiner provides corrective feedback whenever the participant makes an error . if the participant exhibits some evidence of function , but nevertheless makes mistakes , the examiner presents missed items for a second time . this allows the patient to rectify mistakes and thereby decreases the possibility of cognitive errors . in such cases , the score for the test comprises the number of olfactory items out of seven correctly identified and a notation regarding the ability to perceive trigeminal stimulation . the outcome of the threshold and the identification tests is combined into a composite score , an average of the two tests . as previously defined ( toledano et al . , 2003 ) , a score of 5 points or more indicates normosmia , a score between 2 and 4.5 points indicates reduced olfactory function in terms of hyposmia , and a score of less than 2 points indicates functional anosmia . to categorize progression of olfactory function , a subjective improvement and a normal score in butanol , threshold and composite , respectively , its correlation with previously established tests of olfactory function ( e.g. , the 12-item cross - cultural smell identification test [ cc - sit , a subtest of the upsit ] and the sniffin ' sticks test ) has been demonstrated in various studies ( kobal et al . during the last decades artificial nose technology has provided many successful examples of industrial applications [ for recent reviews see ( dymerski et al . , 2011 ; stitzel et al . , the possibilities of potential use in medical and clinical studies have also increased . in this section we discuss a preliminary study on the use of a low - cost , portable artificial nose to quantitatively characterize the cccrc test . for the study reported in this paper we used an odor analysis platform capable of managing up to 16 samples with on / off control electrovalves and three auxiliary devices such as pumps , heaters , and/or mixers ( see figure 2 ) . although the platform can be equipped with a wide variety of sensor arrays , we aimed to study the performance of inexpensive sensors that could be used in future widely distributed control quality devices . this small sensor also provides a good combination of high sensitivity to low concentrations of odorants , long life , low power consumption , and robustness . the sensor is comprised of a metal oxide semiconductor layer formed on an alumina substrate of a sensing chip together with an integrated heater . in the presence of an odorant , the sensor 's conductivity increases as a function of the concentration . a simple electrical circuit converts the change in conductivity to an output signal that corresponds to the odorant . the signal generated by the sensor is not amplified , it is only conditioned with an lmc6484 amplifier in voltage follower mode and sent to the analog - to - digital ( a / d ) converter module to be acquired and stored in a computer . the voltage range of this signal is 05 v. analysis platform used in this study with the tgs2600-b00 ( figaro engineering inc . ) , the main component used in this platform is an 8-bit pic18f2455 microcontroller ( microchip technology inc . ) . this microcontroller is ideal for low - cost , low power ( nano - watt ) , and connectivity applications . the firmware was programmed with the mplab ide development environment provided by the same company . the computer application software was developed with ide dev - c++ ( gnu license ) using windows apis . the equipment is connected to a pc via serial bus , by a proprietary protocol based on eia-485 . for this application we used the rotary vane pump 01-k g - lc ( rietschle thomas company ) in continuous vacuum mode ( 20 mbar max . ) all the instrumentation used in this study is characterized by its compactness and low price , which are important factors for a wide dissemination of quality control in olfactory tests . since the goal of our experiments is to assess the degradation of well - known odorants , we used the peak of the sensor signal to characterize the dilutions / odorants in a comparative manner ( the peak corresponding to an aged olfactory test vs. the peak corresponding to a new test ) . this is a simple and straightforward measurement that does not require further processing of the signal . the performance of the tgs2600-b00 sensor has a moderate ambient temperature / humidity dependence as described in the manufacturer 's worksheet ( http://www.figarosensor.com/products/2600pdf.pdf ) . all experiments reported here were performed at a room temperature between 23c and 24c and a humidity of 3035% . the acquisition of the signal was performed with the following protocol : the sensor was exposed to room air with no heating ( ov ) for 5 s , followed by 100 s at maximum heating ( 4.8 v ) to clean the sensor ( heater resistance is 83 ) . after this cleaning phase , we took 5 s of absorption of the odorant ( 4.8 v for heating , this duration was chosen to avoid saturation ) , and finally 100 s of desorption ( room air again at maximum heating ) . five consecutive measurements of all odorants were taken following this protocol to avoid history dependent effects . the quality control assessment of a clinical olfactory test can use the quantitative comparison of the artificial nose characterization of a brand new test and that of a test that has been used for a certain period of time . here , we propose a simple quality analysis that consists on the quantitative assessment by the artificial nose of a dilution in the test as compared with the corresponding next dilution of a new test . a given olfactory kit fails the quality test when the response of the artificial nose for a dilution is close to the response obtained for the next dilution in the new test , which contains a lower concentration of the odorant . as mentioned above , a short version of the cccrc kit consists in the presentation of dilution number 3 . patients who can not smell this dilution have a very high probability of olfactory dysfunction ( toledano et al . , 2009 ) . to illustrate and test the proposed quality protocol with the artificial nose , we will use dilutions number 2 , 3 , and 4 of the cccrc threshold kit . dilution 2 has a higher concentration of butanol than dilution 3 , while dilution 4 has a lower concentration . figure 3 shows the response of the artificial nose to these dilutions corresponding to a one year old test ( labeled as o for old ) and to a new test ( labeled as n ) . this figure illustrates that , after one year , the sensor signal that corresponds to the old dilution number 2 ( 2o ) differs only slightly from the corresponding to the new dilution ( 2n ) and remains far from the signal of the new dilution number 3 ( 3n ) . however , the signal corresponding to the old dilution number 3 ( 3o ) is nearly as close to the signal corresponding to new dilution number 4 ( 4n ) as to the new dilution number 3 ( 3n ) . thus , as sensed by the artificial nose , dilution 3o could correspond to the next dilution ( lower concentration ) of a new test . in this situation , a quality control criteria based in the relative distance between the peaks of the signals would result in discarding the old test kit . response of the artificial nose to three dilutions of the cccrc kit for two different sets : a kit that has been used for over one year ( dilutions labeled as 2o , 3o , and 4o ) and a new one ( dilutions labeled as 2n , 3n , and 4n ) . for each dilution we show the mean of five measurements ( middle trace ) together with the standard deviation ( upper and lower traces ) . note that the artificial nose signal for dilution 3o is nearly as close to 3n as to 4n . a quality control criteria based in the relative distance between the peaks of the signals would result in discarding the old test kit . for each dilution , the maximum peak is reached a little after the stimulation is stopped . we remind that the protocol that we follow uses five consecutive measurements of the same odorant to avoid history dependent effects between different odorants . note that we use the same desorption period for all of them and odorants that reach a higher maximum also start from a slightly higher baseline in this protocol . we have also addressed the analysis with the artificial nose of the identification kit that the subject receives after the threshold kit in the cccrc test . the purpose of this second kit is the evaluation of the ability to identify an odor . thus , the analysis of the artificial nose in this case is oriented to determine the stability of the odorant over time . if the odorant changes significantly over the lifespan of the kit ( typically one year ) , this could result in the lack of identification or in a wrong identification of the odorant by the subject . figure 4 shows the result of this analysis for two odorants ( coffee and peanut butter ) . the aged tests ( used clinically for more than one year ) show a larger intensity as sensed by the artificial nose . the increase in signal amplitude observed in these aged jars is probably due to a degenerative chemical reaction or the presence of bacteria in the sample and in the pad because of the prolonged use of the kit . the rest of the odorants in the identification kit present a normal degradation as sensed by the electronic nose . response of the artificial nose to two samples ( peanut butter left panel and coffee right panel ) of the identification part of the cccrc kit . for each sample we show the mean of five measurements ( middle trace ) together with the standard deviation ( upper and lower traces ) . note that , as sensed by the artificial nose , the old peanut butter ( op ) and coffee jars ( oc ) have higher odor intensity than the ones that contain a new test ( np and nc , respectively ) . the precise quantification of olfactory perception is an essential step for the evaluation , diagnosis , and treatment of smell disorders and contributes to the success of a corresponding therapy . in the last decades several olfactory tests have been proposed to quantify odor thresholds and to evaluate human odor identification and discrimination . in this paper we have argued that olfactory tests could benefit from quality control performed by artificial noses to assess and guarantee their validity over their lifespan . we have proposed a protocol to test the quality of a threshold clinical kit by comparing the response of the artificial nose to a given dilution of the test with the immediate next dilution of a new test . we have validated this protocol with the cccrc threshold kit using an inexpensive chemoresistive sensor , which is a requirement for the successful dissemination of a quality control device for clinical purposes . the protocol does not need a complex processing of the sensor signal such as the use of classification or clustering algorithms required in many applications with artificial noses ( hierlemann and gutierrez - osuna , 2008 ; muezzinoglu et al . , 2010 ) . as illustrated in figures 3 and 4 , the response of the sensor to the odorants in the cccrc kit is highly reproducible . the comparative nature of the protocol ( the results of the artificial nose on an old test are always compared with those of a new test ) reduces the dependence of the results on sensor drift and environmental conditions such as temperature and humidity . human olfaction and machine olfaction have different mechanisms to sense smell , and thus the quantitative description of an odorant can also be very different for human and artificial noses ( burl et al . , 2001 ; schiffman et al . , 2002 ; lewis , 2004 ; haddad et al . , 2008 , 2010 ) . when artificial noses are used to characterize or compare human smell performance , the signals obtained with these devices need to be calibrated and matched to what is considered a normal perception by a human . in the context of clinical olfactory tests where the smell of odorants takes place under controlled conditions , the quantitative description of an odor by an artificial nose can be compared to an established reference to assess the quality of the test . the results shown in this paper indicate that this is possible , and that an inexpensive chemoresistive sensor can be used to characterize the threshold part of the connecticut chemosensorial clinical research center test . we have also reported that some odorants of the identification part of the cccrc seem to be potentiated as sensed by the artificial nose while others tend to degrade as the test grows older . the analysis of these results suggests that the conditions to keep the odorants in the identification test could be improved . further efforts to build a map between human and machine olfaction can largely contribute to the assessment of odor perception and to develop novel protocols for odor impairment diagnosis and treatment . artificial noses can be used to assess the intensity , steadiness , or temporal evolution of an odorant acting as a stimulus in a perception experiment . these experiments can be carried out with a standard feedback from the self - assessment of smell perception by the subject or through a more quantitative protocol that takes into account levels of brain activity in eeg ( lorig , 2000 ) and/or fmri setups ( lombion et al . , 2009 ) ( which can largely contribute to a quantitative map between human and machine olfaction ) . in all cases , the assessment of odor temporal structure and intensity by artificial noses will improve the quantification and the standardization criteria in these protocols . in addition , artificial noses can also be used in closed - loop experiments with olfactometers and other stimulation devices to implement neural activity - dependent stimulation protocols . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

clinical olfactory tests are used to address hyposmia / anosmia levels in patients with different types of olfactory impairments . typically , a given test is employed clinically and then replaced by a new one after a certain period of use which can range from days to several months . there is a need to assess control quality of these tests and also for a procedure to quantify their degradation over time . in this paper we propose a protocol to employ low - cost artificial noses for the quantitative characterization of olfactory tests used in clinical studies . in particular , we discuss a preliminary study on the connecticut chemosensorial clinical research center test kit which shows that some odorants , as sensed by an artificial nose , seem to degrade while others are potentiated as the test ages . we also discuss the need to establish a map of correspondence between human and machine olfaction when artificial noses are used to characterize or compare human smell performance in research and clinical studies .

Introduction Materials and methods Connecticut Chemosensorial Clinical Research Center Test Artificial noses for quality control of olfactory tests Results Discussion Conflict of interest statement

PMC5091051

lifestyle factors such as exercise can reduce the risk of age - related cognitive decline and neurodegeneration . this study focused on exercise as a lifestyle change for enhanced cognitive function depends on a neurobiological understanding . the neurogenesis in the brain of adult animals and humans overturned the long - held dogma that the adult brain has no capacity for generating new neurons ( altman and das , 1965 ; deng et al . , 2010 ) . in recent studies , exercise has emerged as the most effective and successful way for optimal aging . exercise in the healthy adults brings behavioral benefits , including significant increments in memory , attention , processing speed , and executive functions ( smith et al . , 2010 ) . regular engagement of exercise in midlife is associated with decreased risk of dementia later in life , presenting that exercise might have preventive effects on age - related cognitive decline ( hamer and chida , 2009 ) . abundant evidences from animal studies have reported that an enhancement in adult hippocampal neurogenesis may underlie the beneficial effects of exercise on cognition . indeed , exercise not only increased hippocampal neurogenesis but also improved morris water maze performance ( cho et al . , 2013 ; heo et al . , therefore , exercise can upregulate the neuronal cell proliferation process and increase the ability to maintain neuronal plasticity . three - month regular exercise increased the volume of blood in the dentate gyrus as evaluated by functional magnetic resonance imaging and improved the cognitive scores in humans ( pereira et al . , 2007 ) . exercise undoubtedly increased cerebral blood flow , angiogenesis , and the permeability of blood brain barrier ( black et al . 2014 ) . positive relationship between angiogenesis and neurogenesis was presented in the animal studies . for example , the improvement of cognition through 3-month exercise is conjectured as a result of enhanced hippocampal neurogenesis via increased hippocampal angiogenesis in the human brain ( pereira et al . , 2007 ) . these positive influences of exercise in the brain recommend that physical exercise and activity could be applied as a scheme to prevent the age - related cognitive dysfunction . age - related decline in hippocampal neurogenesis has been associated with decrease in neurotrophins such as brain - derived neurotrophic factor ( bdnf ) , insulin - like growth factor 1 ( igf-1 ) , and vascular endothelial growth factor ( vegf ) ( lai et al . , 2000 ; bndf , igf-1 , and vegf have a crucial role in exercise - induced adult hippocampal neurogenesis . thus , these neurotrophin factors might be worked for mediating exercise - induced adult hippocampal neurogenesis . here , this study was reviewed the functional and molecular role of physical exercise in cognitive function through neural plasticity , neurotrophic factors , and adult hippocampal neurogenesis . this study discussed the exercise - induced hippocampal neurogenesis and its relationship with cognitive enhancement , and concluded with a brief analysis to optimize the impact of exercise on brain function . exercise - induced improvements in learning and memory have been associated with exercise - dependent neural synaptic plasticity and hippocampal neurogenesis ( baek et al . , 2012 ; cho et al . , 2013 ; heo et al . , long - term potentiation ( ltp ) in a neurophysiological model of learning and memory was assessed in hippocampal slices from running mice . however , a significantly greater ltp was shown in the hippocampal dentate gyrus of running mice compared to the sedentary mice ( van praag et al . , 1999 ) . the enhancement in ltp following exercise is consistent with an increment of bdnf in the hippocampus , which can mediate neural plasticity ( cowansage et al . , 2010 ) . exercise may adjust the neural plasticity of the hippocampal dentate gyrus though the increased neurogenesis . newborn granule cells may have a unique role in synaptic plasticity of the hippocampus , and the role of these cells can be increased with exercise ( vivar et al . , the enhanced plasticity was appeared in a specific time of maturation process and was dependent on increased synaptic expression of nr2b containing n - methyl - d - asparate ( nmda ) receptors ( ge et al . , 2007 ) . ltd induced by low - frequency stimulation is relatively unaffected by exercise ( vasuta et al . , 2007 ) . however , ltd depends on the activation of nr2b containing nmda receptors in running mice ( vasuta et al . , 2007 ) . neural plasticity has been associated with morphological changes that occur in response to neural activity ( ngerl et al . some studies suggested that morphological changes in dentate gyrus of hippocampus have been observed with exercise ( eadie et al . exercise significantly increased the total length , complexity , and spine density of granule cell dendrites ( eadie et al . , 2005 ) . also exercise enhanced dendritic complexity in all zones of the granule cell layer such as subgranular , inner and outer granule cell zones ( redila and christie , 2006 ) . additionally 2-month exercise caused morphological changes not only in the dentate gyrus but also in the entorhinal cortex and ca1 pyramidal cells ( stranahan et al . there was no difference in spine density of new neurons between young and aged mice , suggesting that fewer cells proliferated in aging brain may be functionally equivalent to those generated at early period ( morgenstern et al . , 2008 ) . exercise modifies the morphology of dentate granule cells and other parameters related to memory function , and may also affect the rate of integration of newborn granule cells into the hippocampal circuitry ( vivar et al . , neurotrophic factors , including bdnf , igf-1 , and vegf are essential regulators for the effects of physical exercise on brain plasticity during development and adulthood ( mcallister et al . , 1999 ) . bdnf is a critical mediator for the beneficial effect of physical exercise on brain function because it supports neural survival , growth , and synaptic plasticity ( cowansage et al . , 2010 ) . indeed , bdnf is considered to be the most downstream factor mediating the upregulation of hippocampal neurogenesis by exercise ( yau et al . , 2014 ) . ( 1995 ) were the first reporting a positive correlation between physical exercise and bdnf mrna levels . both bdnf gene and protein expression are increased in the hippocampus after short- or long - period of exercise ( abel and rissman , 2013 ; marlatt et al . , 2012 ) , and remained at least 2 weeks after exercise has ended ( berchtold et al . , 2010 ) . bdnf is a neuroprotective growth factor that enhances synaptic plasticity and memory through its actions on the tyrosine receptor kinase b ( trkb ) ( mattson et al . , 2004 ) . 2008 ) and central bdnf in rodents ( neeper et al . , 1995 ) . blocking bdnf expression prevented the enhancement of cognitive function following physical exercise ( vaynman et al . the effects of exercise on synaptic plasticity are related to bdnf ( messaoudi et al . , it has been estimated that the brain is the major source of circulating bdnf ( 70%80% ) both at rest and during exercise ( rasmussen et al . , 2009 ) . in blood , more than 90% of the bdnf is stored in platelets and is released during clotting processes . therefore , serum is likely to reflect stored and circulating bdnf in the blood ( fujimura et al . , 2002 ) , while plasma seems to reflect only freely circulating bdnf ( lommatzsch et al . , 2005 ) . for that reason , peripheral bdnf and other factors that regulate platelet storage and release should be considered for making inferences between peripherally measured bdnf and central expression . particularly , these factors may influence the acute changes in peripheral bdnf levels following single bout of aerobic exercise ( griffin et al . , 2011 ) . other neurotrophins , such as igf-1 and vegf have been reported as essential factors in exercise - induced adult hippocampal neurogenesis ( trejo et al . , 2008 ) . igf-1 was shown to be involved in memory , plasticity , and neurogenic processes , especially in the aging brain ( maher et al . , 2006 ) . for instance , lack of igf-1 results in memory loss and ltp impairment ( trejo et al . , 2007 ) . increased igf-1 production following exercise may interact with bdnf to modulate synaptic plasticity ( mattson et al . , 2004 ) . igf-1 receptor activity also appears to be closely associated with the expression of bdnf following physical exercise . for example , igf-1 signaling via the igf-1 receptor is necessary for the exercise - induced bdnf expression . blockade of igf-1 prevented the exercise - induced upregulation of bdnf ( ding et al . on the other hand , blocking bdnf pathway prevented the exercise - induced upregulation of igf-1 in the hippocampus . based on these results , it seems that igf-1 and bdnf interact to mediate changes in hippocampal function following exercise ( gomez - pinilla et al . , interestingly , it has been reported that adult hippocampal neurogenesis occurs near the local microvasculature of hippocampus ( fabel et al . , 2003 ) . both igf-1 and vegf in the periphery are increased by exercise and mediate stimulation of neurogenesis and angiogenesis in the brain ( fabel et al . , 2003 ) . peripheral igf-1 is necessary for exercise - induced vessel remodeling in the brain ( lopez - lopez et al . , 2004 ) . also , vegf level in the brain is associated with exercise - induced angiogenesis ( ding et al . , 2006 ) . the enhancement of brain vegf has mitotic activity specific to vascular endothelial cells , affecting proliferation , survival , and migration ( ferrara and davis - smyth , 1997 ) . blockade of peripheral vegf restricted the increment in neurogenesis by exercise ( fabel et al . , 2003 ) . in addition , exercise for 50 days increased density of blood vessels in the dentate gyrus , and enhanced spatial working memory in the water maze ( clark et al . , 2009 ) . exercise increased cerebral blood volume with neurogenesis ( van der borght et al . , 2009 ) and improved cognitive function ( pereira et al . , 2007 ) . however , van praag et al . ( 2007 ) reported that an increment in angiogenesis is not necessarily correlated with increased neurogenesis . new neuron production can be regulated by many different extrinsic and intrinsic factors . in the healthy brain , at least two constitutive neurogenic regions exist ( ortega - perez et al . , 2007 ) : the subventricular zone ( svz ) of olfactory bulb and the subgranular zone ( sgz ) of hippocampal dentate gyrus . neurons born in the sgz differentiate and integrate into the local neural network as granule cells of the dentate gyrus . in the dentate gyrus of the hippocampus the origin of adult hippocampal neurogenesis is adult neural stem cells , which can grow into functionally matured neurons through a development process that is divided into three main stages cell proliferation , differentiation , and functional maturity over 4 to 6 weeks ( duan et al . , 2008 ) . about 9,000 new cells are generated each day in the rodent hippocampus of which about 80%90% differentiated into neurons ( cameron and mckay , 2001 ) . in humans , approximately 700 new neurons are added to the adult hippocampus each day from a study reporting the presence of positive staining for 5-bromo-2-deoxyuridine ( a thymidine analog ) in the svz and the dentate gyrus region of postmortem brain sections ( eriksson et al . , however , adult neurogenesis is dependent on the production of new neurons and declines with aging ( yau et al . , enhanced hippocampal neurogenesis is one of the most notable effects of exercise in the brain ( clark et al . , 2009 ) , and might be a key mechanism intermediating exercise - related improvement in the brain functions . adult hippocampal neurogenesis is doubled by exercise ( van praag et al . , 1999 ) . exercise influences on all aspects of new neuronal maturation , including cell proliferation , survival , and differentiation in the dentate gyrus ( speisman et al . , 2013 ) . furthermore , exercise - enhanced adult hippocampal neurogenesis plays an important role in learning , memory , and neural plasticity ( van praag et al . , 1999 ) . in rodents , voluntary running increased the proliferation of cells in the sgz of both young and aged animals ( van praag et al . , 1999 ) . moreover , voluntary running for three weeks enhanced the survival of adult - born neurons in the hippocampus ( muotri et al . , 2009 ) . in addition , voluntary running increased the amplitude of ltp in the dentate gyrus and improved the hippocampus - dependent task in the morris water maze ( van praag et al . , 2005 ) , indicating that increased neurogenesis correlates with improved cognition . mild exercise , but not of intense exercise , is effective for the enhancing adult hippocampal neurogenesis , especially the number of mature neurons that improve the spatial memory ( inoue et al . , 2015 ) . , 2013 ) and several neurological disorders causing cognitive decline ( zhao et al . , 2008 ) . neurogenesis declines as early as middle - age , and may refer the age - related decline of cognitive function ( erickson and barnes , 2003 ) . however , the effect of exercise on neurogenesis is retained over the lifespan . neuroimaging studies indicated that elderly humans with higher aerobic fitness have larger hippocampal volumes and better scores in cognition tasks ( erickson et al . , 2009 ) . young and aged rodents performed running wheel exercise showed enhanced plasticity and better performances on hippocampus - dependent tasks ( van praag et al . , 2005 ) . short- and long - term exercise improved memory function and prevented hippocampal impairments in alzheimer disease ( ad ) model ( parachikova et al . , 2008 ) . also , physical exercise is beneficial for ameliorating some of the neuropathological and behavioral deficits in parkinson disease animal models ( yau et al . , 2014 ) . exercise promoted the preservation of tyrosine hydroxylase ( th ; the rate - limiting enzyme during the synthesis of dopamine)-positive fibers in the striatum and th - positive neurons in the substantia nigra ( tajiri et al . , 2010 ) . several clinical studies reported that physical exercise can improve motor function and cognitive performance in parkinson disease patients ( petzinger et al . , 2013 ) . these findings suggest that exercise may represent a noninvasive therapeutic intervention to attenuate cognitive decline in neurodegenerative diseases . the present study presented evidences suggesting that exercise - induced improvements in cognitive functions are correlated with neurogenesis , synaptic plasticity , and neurotrophins in the hippocampus . exercise has a positive impact on the aged brain with neurodegenerative disorders that are associated with cognitive decline . within the hippocampus , the most noticeable changes with exercise are increased production of new neurons and neurotrophic factors . understanding of neurobiological mechanisms of exercise on brain functions and behaviors may lead to the development of novel therapeutic strategy for neurodegenerative disorders .

the functions of adult hippocampal neurogenesis have been extensively investigated during the past decade . numerous studies have shown that adult neurogenesis may play an important role in the hippocampal - dependent learning and memory . this study evaluated the influence of exercise on hippocampal neurogenesis , neural plasticity , neurotrophic factors , and cognition . areas of research focused on enhancing effect of exercise for adult hippocampal neurogenesis and protective role of exercise against brain diseases . the present study suggests that exercise improves brain functions and prevents decline of cognition across the lifespan . understanding of neurobiological mechanisms of exercise on brain functions may lead to the development of novel therapeutic strategy for neurodegenerative disorders .

INTRODUCTION EXERCISE AND NEURAL PLASTICITY EXERCISE AND NEUROTROPHIC FACTORS EXERCISE AND ADULT HIPPOCAMPAL NEUROGENESIS CONCLUSIONS

PMC5371702

fungal infections are one of the most severe problems in clinical practice , especially in hematology and oncology units . fungemia can be either a complication connected with the malignancy itself or an adverse effect of the oncological treatment ( chemo- , radio- and corticotherapy ) . furthermore , it can influence the final result of treatment . in the group of patients with oncological diseases , allogenic bone marrow transplantations , relapse of leukemia , pancytopenia lasting longer than ten days , undergoing corticotherapy and broad - spectrum antibiotic therapy , immunosuppressive treatment and graft versus host disease ( gvhd ) are the main factors predisposing to fungal infections . pathogens mainly responsible for invasive fungal infections ( ifi ) are candida spp . and aspergillus spp . the first kind often develops as a concurrent illness during acute mucosa inflammation , infections connected with central venous catheters and the use of broad - spectrum antibiotics , and pre - existing colonization with the pathogen in at least one body area . this is why pulmonary aspergillosis is one of the most frequent infections caused by aspergillus spp . ( 87% of patients ) , followed by sinusitis and rhinitis ( 16% ) , and less frequently encephalitis ( 8% ) . the risk factors of aspergillosis are neutropenia lasting for longer than two weeks , staying in an endemic region , tuberculosis and cytomegalic virus infection . risk factors promoting fungal infections the frequency of fungal infections is still growing , and the prophylactic and empiric antifungal treatment using extended spectrum drugs leads to development of resistance . unfortunately , such infections are not easy to diagnose , especially in their early stages . this is mainly connected with their nonspecific nature similar to virus and bacterial infections , affecting immunocompromised patients , few symptoms usually limited to pyrexia , decreased options of diagnostic testing in patients in a serious condition , and false negative results of performed diagnostic tests . it has been determined that the median mortality rate among patients , after chemotherapy , caused by invasive yeast infections is 39% and it varies among different types of fungi . as an example , the mortality rate in aspergillus spp . infections is 49.3% and it can rise to 86.7% in patients after hematopoietic cell transplantation . for patients with candidiasis the mortality rate is about 1436% , for invasive fusariosis it is much higher , and varies from 66 to 75% , and for scedosporiosis the estimated mortality rate is between 65 and 100% [ 911 ] . every patient with neutropenia and long lasting pyrexia who is not getting better after antibiotic therapy should be suspected of fungal infection , and there is an urgent need to perform laboratory tests . diagnostic procedures covering microscopic , microbiologic , serologic and genetic testing help to decide which pharmacological treatment would be the best [ 2 , 3 , 12 ] . for over 40 years , the treatment of fungal infections was based on amphotericin b. the numerous adverse effects , especially nephrotoxicity , forced scientists to search for other drugs . in the 1980s , a new generation azole antifungal drugs entered therapeutic use . depending on the amount of nitrogen atoms in the azole ring , the new pharmacotherapeutic group has been divided into imidazoles ( with 2 nitrogen atoms ) represented by ketoconazole , miconazole , clotrimazole , tioconazole , econazole , isoconazole , and triazoles ( 3 nitrogen atoms ) the first generation with fluconazole and itraconazole , the second generation with voriconazole and posaconazole [ 13 , 14 ] . the goal of this article is to present and compare the clinical pharmacology of the new generation triazole drugs voriconazole ( approved for therapy in 2002 ) and posaconazole ( approved for therapy in 2006 ) . the mode of action of voriconazole and posaconazole is the inhibition of fungal cytochrome p450 mediated 14-alpha lanosterol demethylation , which causes damage in the structure and the loss of cell membrane function . voriconazole is a broad spectrum antifungal agent indicated for the treatment of candidiasis also caused by fluconazole - resistant c. glabrata and c. krusei . moreover , it can be used in aspergillus ( a. flavus , a. fumigatus , a. terreus , a. niger , a. nidulans ) , cryptococcus neoformans , fusarium , scedosporium , penicillium , alternaria , blastomyces dermatidis , blastoschizomyces capitatus , cladosporium , coccidioides immitis , conidiobolus coronatus , madurella mycetomatis , exserohilum rostratum , exophiala spinifera , fonsecaea pedrosoi , paecilomyces lilacinus , phialophora richardsiae , scopulariopsis brevicaulis , and trichosporon infections . it has a greater activity against most molds compared with amphotericin b [ 1319 ] . the main indications for treatment with voriconazole are invasive aspergillosis ( first line treatment [ 15 , 20 ] ) , candidiasis in non - neutropenic patients , fluconazole - resistant severe invasive candida infections ( including c. krusei ) and also invasive scedosporium spp . and fusarium spp . infections ( first line treatment ) . the drug can be used in children older than two years , and in adults . it is classified as c in the fda pregnancy categorization , which means that the drug can be used in pregnant women only when the benefits for the mother outweigh the risk for the fetus . no greater effectiveness of voriconazole over fluconazole has been established . a decrease in the mortality rate compared with amphotericin b treatment in the group of patients with invasive aspergillosis has been observed . voriconazole is available on the market in the form of film - coated tablets , oral suspension , and as an infusion . a contraindication for the use of voriconazole is hypersensitivity to the drug itself or other azoles . during the treatment , according to numerous adverse drug reactions ( adr ) , the duration of antifungal therapy should be as short as possible . in cases of treatment posaconazole is also a broad spectrum antifungal agent indicated in first line prophylaxis of invasive aspergillus spp . and candida spp . infections . however , its range of action is very similar to voriconazole , and so posaconazole can also be used in the treatment of zygomycetes , coccidioides and chromoblastomyces infections . cases of effective treatment of the central nervous system , pulmonary system and the treatment in cases of ramichloridium mackenziei , fusarium proliferatum , and rhizopus infections have been published [ 2123 ] . the drug is available on the market in the form of film - coated tablets , oral suspension , and as an infusion . the intravenous formulation is available in the usa , the european union and canada , where it has been approved by the proper medicine agencies . in contrast to the previously described voriconazole , the molecule of posaconazole has a lipophilic character which leads to differences in administration of both drugs . the drug was developed for prophylaxis and treatment in patients with immunodeficiency , hematological ailments and persons after bone marrow transplantation treated with high doses of immunosuppressive drugs to prevent gvhd [ 15 , 20 , 24 ] . posaconazole is used in primary treatment of oral candidiasis in patients with immunodeficiency where a limited response to local treatment is expected . other indications are pharmacotherapy and prophylaxis of invasive aspergillosis refractory to amphotericin b and itraconazole and in the case of intolerance to the first line treatment , also with voriconazole . as the second line treatment , posaconazole is used in fusariosis ( in the case of intolerance to amphotericin b ) , chromoblastomycosis ( in the case of intolerance to itraconazole ) , coccidioidomycosis ( in the case of intolerance or refractory to amphotericin b , itraconazole and fluconazole ) . unfortunately , molds resistant to posaconazole and voriconazole have already been isolated , mainly in the candida spp . and aspergillus spp . strains [ 2426 ] . the main mechanism of resistance is the change of functional groups in the target protein cyp51 [ 15 , 17 , 24 ] . it has to be remembered that this is not the only mechanism of resistance to azoles , and that in fungi , always several mechanisms are involved at the same time . apart from the lack or weakness of drug penetration resulting from changes in the composition of sterols and phospholipids in the fungal cell , changes in the efflux process of the drug , defects in the drug metabolizing cytochrome p450 enzymes and changes of the drug itself play a great role in the development of resistance . in contrast to the bacterial one , the resistance to azoles in fungi may or may not affect cross - resistance to other drugs from the same group . the main treatment indications for voriconazole and posaconazole are shown in table 2 [ 16 , 24 ] . the best absorption is achieved when voriconazole is administered twice daily in the first 24 h via the loading dose and after the first day via the maintenance dose [ 16 , 20 , 28 , 29 ] . lower voriconazole concentrations have been reported after oral administration , which is why careful drug monitoring needs to be performed in cases of switch from an intravenous ( i.v . ) to an oral formulation . the distribution volume is 4.6 l / kg , which proves a good penetration of the drug into tissues . voriconazole is metabolized in the liver via the cyp2c9 , cyp3a4 and cyp2c19 isoenzymes , forming non - active metabolites . the genetic polymorphism of cyp2c19 isoenzyme has a great influence on inter - individual variability of pharmacokinetic parameters [ 16 , 31 ] . table 3 presents the main information about the mentioned isoenzyme , its alleles , the influence of genotype on the metabolism of voriconazole and measured serum levels . cyp2c19 isoenzyme and the influence of its mutations on the metabolism of voriconazole and the drug serum levels pm poor metabolism ; i m intermediate metabolism ; em extensive metabolism ; um ultra rapid metabolism the median half - life of voriconazole is approximately 6 h depending on the dose . multiple dosing or i.v . voriconazole is metabolized mainly in the liver ; only 2% of the administered dose is excreted in an unchanged form in the urine . dolton et al . suggest that there is no relation between body weight and dose requirements . however , some cases of side effects in obese patients treated with doses determined according to actual weight have been reported , whereas there have been no adverse drug reactions ( adr ) if voriconazole was administered in weight - independent dosage regimens [ 30 , 33 , 34 ] . the dose should be elevated in children because of the more frequent inter - individual variability in the pediatric population . way of administration is preferred in children with low body mass and absorption disturbances [ 16 , 20 ] . for the adult population , no effective method of pk / pd modeling has been established , which may be associated with a limited number of data on the pharmacokinetics of voriconazole in the group of patients suffering from invasive fungal infections . in adults , a loading dose of 400 mg every 12 h and a maintenance dose of 200 mg every 12 h is recommended . for comparison , two pharmacokinetic models have been established for the pediatric population . in the independent studies performed by karlsson et al . and neely et al . a loading dose of 7 mg / kg body mass for i.v . and 200 mg every 12 h for the oral formulation has been established [ 36 , 37 ] . kuo et al . stated that the best model describing the pharmacokinetics of voriconazole in the pediatric population is a linear model . posaconazole pharmacokinetics are linear , best described by a one - compartment pharmacokinetic model with first order elimination . after the oral administration , the absorption of the drug depends on the dose regimen and the amount of fat in the food . co - administration with ppi ( proton pump inhibitors ) and metoclopramide and also inflammatory states such as mucositis reduce the bioavailability of oral posaconazole formulations . after the administration of 800 mg of posaconazole in two doses , every 12 hours , the parameter is 98% ; after dividing the dose into four smaller doses every 6 h it rises to 220% . the saturation of absorption has been observed after using doses above 800 mg . after a single dose administration , the time needed to reach maximal concentration ( cmax ) is about 5 - 8 hours . the lipophilic posaconazole is characterized by a distribution rate of about 525 l / kg which suggests distribution in the extravascular space and penetration into the intracellular space [ 24 , 40 , 41 ] . intravenous posaconazole formulations are well tolerated when administered in a single dose of 300 mg during 30 minutes . however , careful infusion site monitoring is recommended . the metabolites formed by the p450 enzymes make up only a small amount of circulating metabolites [ 40 , 41 ] . the half - life is about 35 h , and the steady state is achieved after 57 days of administration [ 20 , 24 , 41 ] . studies carried out in the pediatric population from 8 to 17 years of age showed a similar safety profile to the one observed in adults . nonetheless , the number of patients was small , so there is a need for more similar studies to prove the hypothesis . in elderly patients , an increase of the cmax and auc ( area under the curve ) of about 29% was observed , what may suggest the need of dose regimen modification in this group of patients . this is considered to be an indication for administration of the drug on an empty stomach . studies have shown 2.5-fold higher auc values in patients with hepatic disturbances ( child - pugh classification a and b ) , which suggests the need of drug regimen modification in this group of patients . an influential problem is the absence of clinical data from patients with fatal hepatic impairment . among patients with renal failure , there is no need of drug regimen modification , but cyclodextrin , an intra - venous formulation excipient , can cumulate in the organism . the influence of this phenomenon has not been explained yet hence caution in the use of i.v . posaconazole is better absorbed when it is administered with high fat meals in four doses of 200 mg . its pharmacokinetics are influenced by coexistent ailments affecting the gastrointestinal motility ( diarrhea , mucosa inflammation ) and gvhd . studies on healthy volunteers did not show that gender and ethnicity affect the pharmacokinetics [ 24 , 40 , 41 ] . it has been demonstrated that in elderly patients , the serum concentration of posaconazole is higher due to a lower apparent volume of distribution . however , hepatic disturbances ( 2-fold higher -glutamyl transpeptidase [ ggt ] ) and drug administration through a nasogastric tube can be an indication for dose regimen modification [ 14 , 44 ] . the most frequently occurring adr are visual disturbances ( 2335% ) demonstrated as color vision change , blurred vision , scotoma and photophobia . these reactions appear at the time of administration and return to normal after about 30 minutes . moreover , skin reactions such as rash ( usually mild severity ) , gastric disturbances , hepatic disturbances ( elevation of ast , alt , alkaline phosphatase , ggt , lactate dehydrogenase , bilirubin ) can appear . in about 10% of patients , abnormal heart rhythm can be observed . there have also been cases of squamous cell carcinoma with unknown etiology reported [ 45 , 46 ] . adverse drug reactions that occur more often during treatment with voriconazole than posaconazole are sinusitis , hypoglycemia , hypokalemia , depression , hallucinations , anxiety , headache and dizziness , peripheral edema , thrombophlebitis , hypotension , acute respiratory distress syndrome , pulmonary edema , jaundice , backache , acute renal failure and hematuria [ 1416 ] . the treatment with posaconazole is often ( 1/100 to < 1/10 ) connected with the following adr : neutropenia , electrolyte dysfunction , anorexia , paresthesia , dizziness , drowsiness , headache , vomiting , nausea , stomachache , diarrhea , dyspepsia , dry mouth , flatulence , elevated alt , ast , alkaline phosphatase , ggt , lactate dehydrogenase , bilirubin , pyrexia , asthenia , tiredness . the most frequent adr of voriconazole and posaconazole are presented in table 4 [ 16 , 24 ] . voriconazole can increase serum levels of astemizole , terfenadine , cisapride , pimozide and quinidine . the drug is also connected with the increase of sirolimus and ergot alkaloids plasma concentrations . voriconazole increases the serum levels of cyclosporine a and tacrolimus , so reduction of the dose of these drugs when co - administered is recommended . the administration of voriconazole with statins may lead to rhabdomyolysis , with benzodiazepines can result in prolongation of sedative action , and with vincristine and vinblastine can result in neurotoxicity . the administration of voriconazole with an enzymatic inductor such as phenytoin and rifabutin is not recommended . the need for co - administration makes it necessary to increase the triazole maintenance dose . the drug is one of the cyp3a4 inhibitors , which creates a need for dose adjustment in co - administration with many other drugs [ 14 , 16 , 24 , 28 ] . in table 5 voriconazole and posaconazole drug interactions leading to changes in plasma concentrations of antifungal agents are presented [ 16 , 24 , 28 ] . voriconazole and posaconazole drug interactions the main drug interactions and recommendations for co - administration of voriconazole and posaconazole with other drugs are demonstrated in table 6 [ 16 , 24 ] . according to the pharmacokinetics of voriconazole and posaconazole , adverse drug reactions and other factors influencing the serum levels , therapeutic drug monitoring ( tdm ) is required for optimization of treatment of invasive fungal infections , but there is only a limited amount of research on the topic . state in their article the urgent need for tdm enrollment , and point out the areas with lack of information . the study carried out by soler - palacn et al . suggested the need for inclusion of therapeutic drug monitoring in the routine procedures among the pediatric population . in the case of posaconazole treatment , tdm is needed only in high - risk patients , to determine compliance [ 4951 ] . the british society for medical mycology laid down guidelines for tdm of antifungal agents . according to the publication , therapeutic drug monitoring should be performed in the majority of patients receiving voriconazole or posaconazole . in table 7 , the most important information connected to tdm of antifungal agents is presented [ 28 , 44 ] . therapeutic drug monitoring of voriconazole and posaconazole in conclusion , among numerous similarities , the clinical pharmacology of voriconazole and posaconazole exhibits some differences as well , as demonstrated in this article . table 8 is a summary of all similarities and differences between the two drugs discussed in this article [ 16 , 20 , 24 , 35 ] .

despite greater knowledge and possibilities in pharmacotherapy , fungal infections remain a challenge for clinicians . as the population of immunocompromised patients and those treated for their hematologic ailments increases , the number of fungal infections grows too . this is why there is still a quest for new antifungal drugs as well as for optimization of pharmacotherapy with already registered pharmaceutics.voriconazole and posaconazole are broad - spectrum , new generation , triazole antifungal agents . the drugs are used in the pharmacotherapy of invasive aspergillosis , candida and fusarium infections . voriconazole is also used in infections caused by scedosporium . posaconazole is used in the treatment of coccidioidomycosis and chromoblastomycosis . besides some similarities , the two mentioned drugs also show differences in therapeutic indications , pharmacokinetics ( mainly absorption and metabolism ) , frequency and severity of adverse drug reactions , drug drug interactions and dosage . as both of the drugs are used in the treatment of invasive fungal infections in adults and children , detailed knowledge of the clinical pharmacology of antifungal agents is the main factor in pharmacotherapy optimization in treatment of fungal infections.the goal of the article is to present and compare the clinical pharmacology of voriconazole and posaconazole as well as to point out the indications and contraindications of using the drugs , determine factors influencing their pharmacotherapy , and provide information that might be helpful in the treatment of fungal infections .

Introduction Mode of action and therapeutic indications Pharmacokinetic properties and dosage regimen Factors influencing the pharmacokinetics Adverse drug reactions Drug interactions Therapeutic drug monitoring of voriconazole and posaconazole

PMC4620296

it is , therefore , important that the nursing home is not only a place where care is delivered , but also a place where one lives and feels truly at home making use of a wide array of architectural and technological solutions [ 1 , 2 ] . this also applies to nursing home residents who are bed - ridden due to their declining health and physical fitness . bed - ridden residents are confined to bed because of illness or infirmity , especially for a long or indefinite period . the percentage of nursing home residents who are permanently or sometimes bed - ridden was estimated by the netherlands institute for social research to be about 25% , with the highest percentages found in psychogeriatric wards . to date , not much research has been done on people who are bed - ridden or staying in bed for prolonged periods of time , both in long - term care and hospitals , and how they perceive the space around them . annemans et al . cited the work by van der geest and mommersteeg , who concluded that the role of the bed in the general hospital experience is largely under researched : van der geest and mommersteeg [ 5 , pages 11 - 12 ] , [ t]he importance of the bed for health is obvious [ , and ] beds in hospitals and other medical settings have a special relation to health . [ hospital ] beds are not meant to provide privacy but rather to allow for optimal access to the patient by clinical staff . hospital beds are surrounded by medical care providers and medical equipment , facilitating permanent surveillance and the possibility of intervention . in addition , annemans et al . showed that persons who are bed - ridden , including patients in hospitals , have a different experience and perception of the space around them . for instance , while being transported through the hospital , the bed is an important mediator between the patient and his or her environment . annemans et al . concluded that most research on beds and transport seems often restricted to functional matters such as organisation and ergonomics . the situation in well - facilitated nursing homes , in terms of design and technology , is assumed to be similar to that of hospitals . , a nursing home is a place of residence , and many residents deal with the consequences of dementia syndrome affecting one 's perception and abilities , resulting in further limitations to one 's resilience and autonomy . given the transitions in dutch long - term care , it is expected that the number of bed - ridden nursing home residents , both with a somatic and a psychogeriatric background will increase . this is a direct consequence of the increased threshold for admission , which is related to one 's health status and the ability to carry out activities of daily living independently or with the help of home care services . the way long - term care settings are designed for bed - ridden nursing home residents is different from that for residents who have a degree of mobility . this is because the space bed - ridden residents are able to access and use independently is so limited . apart from guidance concerning fire safety that exists on a national level , there is practically no design guidance for this group of residents . the design and construction of nursing homes are a complex and dynamic process , as the very design of these buildings forces us to consider the technology - human interface directly in terms of living - space , ethics , and social priorities . moreover , the design process includes a large variety of stakeholders , who further add to the complexity . technology and architectural solutions can support the well - being , activities of daily living , and quality of life of older residents and support and optimize the work processes of healthcare professionals and professionals in the domain of maintenance [ 911 ] . in order to meet the needs of future nursing home residents , in particular those who are bed - ridden , new creative and inclusive approaches are needed to come up with new and innovative design solutions . in addition , annemans et al . mentioned that spatial aspects , for instance , that of the direct environment of a bed - ridden resident , can not be studied isolated from the activities going on in the spaces considered . depending on what is happening and who is involved , each experience is shaped differently . the aim of this study was to explore and generate design concepts for the direct environment of bed - ridden residents of nursing homes through interdisciplinary participatory design sessions . these design concepts should consist of innovative environmental enrichment scenarios , which , in this study , concern the enhancement of a person 's physical or social environment . delhoofen stated that , in the concept phase of a design process , also called the creative phase , creativity is allowed . creativity , on the one hand , is an important source of inspiration for any dynamic organization , and , on the other hand , it is a capricious and uncontrollable phenomenon which defies any formalization and set of rules . creativity is hard to manage , even though this is often necessary to find new solutions for complex problems . according to delhoofen , creativity is the ability to ignore restrictive frameworks of thinking and to find original solutions for both everyday and new problems . creativity is not the same as innovation , but creativity is necessary to come to new innovations . we define innovation as the creation of new products and the improvement and differentiation of products . a qualitative methodology called participatory action research was used for this study and it provides a tool to deal with the complexity of the design challenge and stakeholder - related needs [ 1318 ] . according to madden et al . , participative frameworks have been shown to be useful in design of environments , and participatory action research has a long history of use with disadvantaged groups in order to assist them to improve their living conditions , with researchers and the research subjects working collaboratively to develop solutions for community issues . moreover , participatory action research involves practitioners as both subjects and co - researchers [ 13 , page 613 ] . according to howard and somerville , participatory action research is a common methodology among designers but is not often framed as a codesign practice . in this study , participatory action research is integrated with activities of making , such as scenario writing and storyboarding , performance ethnography , and collaborative design and prototyping [ 2022 ] . according to sanders and stappers , making is an important activity for designers , as one can bring insights to the surface . prototypes can play a number of roles , such as evoking a focused discussion in a team , the testing of a hypothesis , and confronting others as the theory is not hidden in abstraction . other types of visualisation such as scenarios and storyboards can be made to allow designers to experience , test , transform , develop , and complete early ideas . sanders and stappers [ 19 , page 6 ] stated that in the very earliest phase of the design process , the focus is on using making activities for making sense of the future . here , making activities are used as vehicles for collectively ( e.g. designers and codesigners together ) exploring , expressing and testing hypotheses about future ways of living . this is why the current study focuses on scenarios and prototyping as a way to think about the future of nursing home care for bed - ridden residents . according to the map of design research by sanders and stappers [ 19 , 23 ] , the current research can be classified as user - centred design , more than participatory design research , as end users ( bed - ridden nursing home residents ) are not included in the study but seen as the subject . at the same time , care professionals , who are end - users of the solutions identified in this study , are also stakeholders who benefit from these solutions . work sessions were held in the evening of 18 june 2013 at the educational hospital wards of the fontys school of people and health studies , eindhoven , the netherlands ( figure 1 ) . the large nursing rooms ( containing 4 beds ) were about 50 m and the small rooms ( containing 1 bed ) 16 m in size . a total of 12 scenarios were made by 56 participants ( table 1 , figure 2 ) during the design improvisation sessions that were guided by creative facilitators ( 10 professional group facilitators and 9 assistant with a background in industrial design and creative techniques ) . all session facilitators and assistants had been briefed about the procedures and were provided with a manual and an instruction guide prior to the start of the sessions . the role for the session facilitator was to stimulate creativity , supporting the creative process , and to obtain a maximum of variety in responses and input . in the group rooms , session facilitators and assistants supported the various groups of participants . apart from these facilitators and assistants , jvh , ro , and ad managed the overall processes of the sessions , supported the supply of materials , and collected written documentation . the participants of this study had a mixed background in healthcare , design , housing , and technology ( table 1 ) . at the start of each collaborative design session , informed consent , covering the permission to use of all written , videographic , and photographic materials , was obtained from the participants at the start of the session . the participants were invited to consider the beds in the rooms as actual nursing home beds and to enhance the environment with materials presented to them in a shared material library , including paper and card board , sticky tape and ropes , balloons , plastic covers , and various other do - it - yourself materials . these materials allowed the participants with a high degree of freedom to shape the modifications to the bed and use their imagination . the participants were told that the new scenarios and concepts mattered , not the level of perfection of the results or the execution of the modifications . the 12 scenarios that the participants developed were based on a persona ( a fictional character ) of a nursing home resident [ 21 , 22 , 24 , 25 ] . . stated that fictional characters have been used as user representatives , either substituting actual users or supporting idea generation . their foremost objective is to facilitate the identification of user needs and goals and to support the development of detailed and comprehensive scenarios . context scenarios were defined as a short story , which represented the use of the bed environment by the resident . the scenarios were represented by making actual changes and adaptations to the beds and the direct surroundings using a wide range of materials , which symbolized actual environmental modifications ( figure 2 ) . at the end of the session , the personas were enacted by participants from each group who served as proxy residents ( figure 2 ) . as creativity can not be forced upon participants , it was important to create optimal conditions in which new ideas can come to the fore . for creativity to thrive , participants had to be comfortable , motivated , and stimulated through challenging goals . found that design alter egos technique liberated the majority of participants from the fear of straightforwardly exposing themselves , supported and enhanced their introspection , stimulated their creativity , and helped to establish an informal and constructive atmosphere throughout the design sessions . the scenarios of the sessions were each documented on paper by the participants and filmed by a professional camera crew for further analysis . this is in line with the narrative videotaping approach to storytelling laboratory described by bate and robert . the results of all 12 scenarios and the rationale behind each one of them ( combination of a context scenario and requirements analysis ) are presented as case studies containing potential design innovations that can be thought - through and developed in more detail for implementation in the nursing home setting . the transcripts of the scenarios were analysed in conjunction with the videos , in order to also include visual data . the data were analysed based on the six phases by braun and clarke and in conjunction with the steps of content analysis by krippendorff . then , they were read a second time to develop open codes identifying design solutions . open coding concerns the process of unravelling all of the collected data into fragments or codes . similar codes and quotes were clustered and labelled , and themes emerged from this process . together , the research team organised the codes and clustered them into smaller thematic groups . the final themes were grouped in amalgamation with graphical material from the films that reflected aspects of these themes , which is a form of axial coding . these themes are made up of design concepts and solutions , which in turn could also be clustered in major themes , that is , to which level of human functioning they relate . researcher triangulation was applied during the entire process ; for example , separate analyses of the scenarios were conducted by two of the authors ( j. van hoof , a. m. c. dooremalen ) . a method triangulation was applied due to the use of both written scenarios and video images . the 12 scenarios contained suggestions that can be used for improving the living situation of bed - ridden nursing home residents or for changing product design and are given in the appendix . the main themes which emerged from the analysis of the written and video materials are shown in table 2 . a high degree of convergence and data saturation has been found . the interdisciplinary character of the groups ( and the interaction between participants ) and the mixed location in both single - person and group rooms contributed to the richness of the scenarios and the data . some of the themes found in this study are overarching ( such as the use of tablet computers ) , whereas smaller themes are an elaboration of the functionalities one should use the tablet computer for ( for instance , opening curtains ) . the most frequently mentioned theme ( 11 out of 12 ) is the use of virtual and interactive walls for displaying pictures , television programs , videos , and materials from the past . this theme is followed by the possibility to have online contacts and to chat with relatives , friends , and fellow residents ( 8 out of 12 ) . seven out of twelve scenarios deal with the use of touch screens and tablet computers for the control of the space around them or to place orders for meals and plan the care tasks throughout the day and to control the environment such as curtains . lights and luminaires , sounds , and music are mentioned ( 7 out of 12 ) as important areas for development when designing nursing home environments , for creating a proper atmosphere , for functionality , in order to stimulate day and night rhythms , and in order to make people feel more at ease through music and sounds of nature . the so - called transformer beds , beds that can change their shape into a chair , wheelchair , or bathtub are mentioned by 7 out of 12 scenarios and reflect new products on the market place : beds which can bend and turn and become like real chairs in which residents / patients can sit upright . these developments go together with the desire for smart mattress and smart beds measuring vital signs ( 6 out of 12 ) and special mattresses that improve comfort and prevent decubitus ( 6 out of 12 ) . seven out of twelve scenarios give attention to the free choice of food and drinks and the need for minibars and coffee corners where visitors can get something to drink . autonomy is an overarching or major theme , which encompasses the freedom and being in control of residents , which is reflected in the choice for meals , planning care activities , being in control of people entering the room , and so on . robotics is a theme mentioned by 3 out of 12 scenarios , mainly in relation to transfers and delivering meals . homelikeness and atmosphere are another overarching theme , of which smaller themes as flowers , furniture for relatives , and picture frames could be an elaboration . the twelve scenarios resulting from this study contained numerous design ideas and solutions that can be used for improving the quality of life for bed - ridden nursing home residents . in this round of workshops , there was a lot of focus on technologies improving the autonomy of residents and for improving communication capabilities . for instance , tablet computers are things or items and are named as design solutions , whereas the autonomy relates to a personal experience or a human state . the definition of environmental enrichment used in this study , which is the enhancement of a person 's physical or social environment , allows for themes that relate to different levels . various technological solutions were identified , which , apart from the aforementioned tablet computers and touchscreens , include assistive and medical technology such as special beds containing sensors , architectural modifications as windows , and building services solutions including heating , ventilation , and air - conditioning systems and smart home technologies . although some of the design solutions proposed are futuristic , the vast majority is already available on the marketplace . these innovations are not yet implemented on a large scale in practice , and , therefore , they are still innovative . the goal was to come up with innovative and conceptual design concepts which can be developed into implementable solutions . this study welcomed five types of innovation as distinguished by delhoofen , namely , new products , product improvements , product differentiation , service innovations , and process innovations . the most important aspects of improvement to be achieved with innovations are savings , improvements of functionalities , and extensions of functionalities . whether the identified solutions can in fact contribute to improvements to healthcare is beyond the scope of the current work . the chosen approach to this study , participatory action research , has been used within the domain of public health care before . its combination with user - centred design approaches , however , is relatively new within the domain of healthcare . a small number of scientific publications applying these methodologies within the domain of healthcare and technology are being published ( for instance , [ 29 , 30 ] ) . the current method had been shown successful in identifying aspects of healthcare facilities which are necessary for the quality of care and life of bed - ridden nursing home residents and for obtaining convergence in design solutions . the creative methodology is very suitable for bringing together people with different backgrounds and quality of knowledge . participatory action research aims to obtain knowledge and action directly useful to people , and also to empower people through the process of constructing and using their own knowledge [ 31 , p. 225 the methodology could , therefore , also be applied within healthcare settings in order to think about improving products , environments , and services and , most of all , to involve nondesigning professionals in a design process . in this methodology , all participants have a role to play , whether it is a designer or engineer or a healthcare specialist . it is , therefore , a truly inclusive design process , but inclusive design in this sense does not pertain to inclusive design or design - for - all . in addition , the participants working in commerce can use the methodology to improve products being designed and produced within their own enterprises . the methodology can also be used for educational practices , for instance , in multi- or interdisciplinary classes [ 32 , 33 ] , for a large spectrum of design challenges , as is currently being done . in this study , older persons , nursing home residents , and actual bed - ridden persons were not included in the creative sessions . this means that any solution that comes to the fore does not necessarily reflect the actual needs of older , bed - ridden people . at the same time , the participating healthcare professionals could provide a foundation for the design solutions based on their practical knowledge . in future sessions , one could think of including informal or family carers or professional actors trained in playing nursing home residents , in order to obtain more accurate scenarios . the current cohort of participants , as was discussed in the methodology section , could have based their scenarios on stereotypes of bed - ridden residents or could have portrayed them as somewhat active and independently functioning older people , which does not match with the actual health status and abilities of bed - ridden nursing home residents . moreover , the methodology chosen may lead to scenarios that are enacted in too playful a manner . another potential weakness of the study is that participants are not aware of all innovations in the field , of those that appear on the market or are being researched in research and development ( r&d ) facilities . this may give a skewed perspective on the scenarios , which are either too optimistic or somewhat out - of - date . this also means that concepts are not being developed into real innovative products , which can , in turn , be tested in the nursing home . this means that no field - evidence is being gathered , although the scenarios themselves are based on practice - based evidence . the evidence - based and performance - based designs ( van hoof et al . ) are becoming important tools in optimizing the spatial layout and interior design of healthcare facilities . marquardt and motzek [ 35 , page 116 ] stated that because architects and designers are often not trained in research methods , they may feel somewhat overwhelmed by the terms used in the description of the methodology of scientific papers . therefore , when designing healthcare facilities , they may not feel confident discussing the evidence available from research studies with members of the medical professions , who often display a profound knowledge of research methods . when evidence of certain measures is absent , or when new innovations need to be designed , a qualitative approach based on performance ethnography and collaborative design may be a good step in exchanging ideas and come up with new innovative design solutions . the duration of an optimal session should be researched . in the current study , the 22.5-hour period was considered to be sufficient for writing the scenario , building the scene , and enacting the scenario . in conclusion , the chosen methodology may help practitioners identify potential solutions that exist to support the challenges in nursing home care . moreover , the results of this study can be used to adequately design the private rooms and bed environments of nursing home residents in accordance with personal preferences and needs and to stimulate innovations in product design which may become available on the marketplace in the future .

bed - ridden nursing home residents are in need of environments which are homelike and facilitate the provision of care . design guidance for this group of older people is limited . this study concerned the exploration and generation of innovative environmental enrichment scenarios for bed - ridden residents . this exploration was conducted through a combination of participatory action research with user - centred design involving 56 professional stakeholders in interactive work sessions . this study identified numerous design solutions , both concepts and products that are available on the marketplace and that on a higher level relate to improvements in resident autonomy and the supply of technological items and architectural features . the methodology chosen can be used to explore the creative potential of stakeholders from the domain of healthcare in product innovation .

1. Introduction 2. Methodology 3. Results 4. Discussion and Conclusions

PMC3748550

az senge 1990 declares , learning is a much more complicated phenomenon that can ever be limited to a classroom ( p. 63 ) . many of the students today are attempting to adjust their family life , job , and university life toward having more flexibility in their learning programs . current studies have suggested that a combination of face - to - face and e - learning methods would be an adjustable way of teaching that helps the students meet these challenges . continuous learning , saving time , and reducing transportation expenses are the most commonly quoted benefits of blended learning ( bl ) . in addition , learners are being educated in a holistic manner , in which their individual characteristics including attitudes , beliefs , view points , knowledge , skills , and mental power are regarded . some authors with a constructivist , student - centered , and person - centered standpoint have noted that holistic educational pedagogies have more likelihood of success than traditional ones . application of bl has been increased in both academic and business fields and many studies have been conducted in this area . some of these investigations have shown that students outcomes in bl method are better than or similar to those of face - to - face method . however , recent investigations have revealed the defeat of most web - based projects because of disregarding stakeholders opinion , and lacking appropriate technological and executive infrastructures . thus , conducting investigations which address these pre - requirements seems essential . on the other hand , there has been less attention to application of current electronic learning methods by universities and bl is relatively new to the educational systems of iran ; therefore , there would be a concern about the outcomes of applying such programs . to the best of our knowledge , outcomes of the implication of bl program within the educational , technological , and cultural context of iran have not been investigated . therefore , the aim of present study was to assess the outcomes of the bl program in the faculty of nursing and midwifery , tehran university of medical sciences ( tums ) , iran ; these outcomes included students grade point average ( gpa ) , participation in , and attitude and opinion about the program , and faculty instructors attitude and opinion about the program . this study was conducted in the form of participatory action research ( par ) which encompasses five steps including analysis , design , development , implementation , and evaluation ( addie ) ( chang et al . , 2008 ) to develop the bl program in the faculty of nursing and midwifery , evaluate its preliminary outcomes , and re - run it after potential modifications . in this paper , nursing and midwifery students who were studying bachelor 's , master 's , and doctoral degrees ( n = 467 ) and instructors who were fulltime faculty members in faculty of nursing and midwifery ( including 50 faculty members and 10 mentors ) participated in this study . students who had no theoretical courses ( those in 1 and 2 semesters of bachelor 's degree program ) were excluded from the study . this step included defining the need for such program , and evaluating the existence of the capacity for development and implementation of a bl program . as the need for such a program and the required capacity was recognized to exist ( after group discussions and collecting experts opinion inside and outside the university ) , decision was made to design a bl program for educating nursing and midwifery students in the faculty of nursing and midwifery ( tums ) . technological infrastructures a computer lab with sufficient number of computers connected to high - speed internet was established to facilitate students access to computer and the internet ( n = 104 ) . moreover , fulltime access to high - speed internet was provided for faculty members in their office . as instructors were not familiar enough with it and bl programs , a number of workshops were held a semester before execution of the program to improve instructors it knowledge and skills . the importance and necessity of using bl method and how to design course matters , course scenarios , specify individuals responsibilities were debated in these workshops . moreover , in a single session , students were informed about the way they should work with bl system . executive infrastructure in order to promote the feasibility of the program , some executive infrastructures were developed . most importantly , a distance learning committee was organized consisting of five specialists in the field of distance learning who were responsible for making decisions , rules , macro policies , and developing new strategies . moreover , members of either educational committee or distance learning committee of tums passed a number of educational regulations ( e.g. , each faculty instructor was permitted to hold 4/17 of his / her course credit in the form of e - learning ; some incentives were designed to be offered to instructors who had used bl method in teaching students , etc . ) . designing bl platform and bl courses bl platform was designed based on three major principles : user friendliness , stability of the system , and accessibility . twenty - two theoretical courses ( including basic , core , and specialized courses which were valued as 48 course credits ) were approved by distance learning committee for designing in the form of bl courses . preparing courses for bl program , each instructor should write and design course scenarios , designate particular activities for face - to - face and e - learning parts , specify individual activities and responsibilities , schedule for the course , develop online quizzes , and consider how he / she will give feedback to students and how teacher after they signed the agreement form , students were given a personal user account ( including a user name and a password ) in order to sign in to bl website at//http://dln.x.ac.ir/. then , the students were supposed to select their courses at bl website . initial instructions were provided for students by researcher about how to work with the bl system , get access to scientific resources , take online tests , upload tasks , download educational e - contents , and interact with teacher and other students before starting the course . bl program was implemented at the faculty of nursing and midwifery in tums for one semester . instructors delivered their courses which had been designed and approved a couple of months before running the program . checklists of students participation were filled for each student in face - to - face method and for bl method by the in - charge instructors . students individual characteristics , gpa , participation in courses , and opinion , and instructors demographics , attitude , and opinion toward teaching methods were assessed by the following researcher - made data collection tools : students individual characteristics : were recorded via a 9-item questionnaire . students participation in courses : we developed two checklists ( one for bl and one for face - to - face method ) to quantify each student 's participation throughout the course sessions . we considered the time and duration each student logged in to the bl website and the time he / she used the bl website 's chat room as indicators of participation with the bl courses . on the other hand , the time and duration the student was present in class and the time he / she involved in scientific discussions were considered as indicators of participation in face - to - face classes . both checklists were scored based on their values and the sums of scores were classified into three distinct levels : high participation , > 75% ; moderate participation , 50 - 75% ; and low participation , < 50% . students gpa : students gpa in bl method was compared to that of previous semester ( face - to - face method ) . instructors attitude : was measured via a 3-point , likert - scale questionnaire ( 1 = agree , 2 = agree to some extent , and 3 = disagree ) , including an open - ended question at the end to seek the instructors suggestions . participants opinion : focus group discussions were conducted separately for students and instructors to explore their opinion about bl and face - to - face methods . these qualitative data were analyzed with textual analysis . to develop the data collection tools , relevant literature reports were reviewed . those items were then evaluated for clarity and face and content validity by 10 experts in the field of education . cronbach 's alpha was applied to examine the internal consistency of the opinion and attitude questionnaires ( alpha = 0.83 and 0.81 , respectively ) . were used to compare students gpa between the two methods ( bl and face - to - face ) paired t - test and wilcoxon test were respectively used to compare students gpa and participation between bl and face - to - face methods . chi - square and fisher exact test were used to examine the association between categorical variables . this step included defining the need for such program , and evaluating the existence of the capacity for development and implementation of a bl program . as the need for such a program and the required capacity was recognized to exist ( after group discussions and collecting experts opinion inside and outside the university ) , decision was made to design a bl program for educating nursing and midwifery students in the faculty of nursing and midwifery ( tums ) . technological infrastructures a computer lab with sufficient number of computers connected to high - speed internet was established to facilitate students access to computer and the internet ( n = 104 ) . moreover , fulltime access to high - speed internet was provided for faculty members in their office . as instructors were not familiar enough with it and bl programs , a number of workshops were held a semester before execution of the program to improve instructors it knowledge and skills . the importance and necessity of using bl method and how to design course matters , course scenarios , moreover , in a single session , students were informed about the way they should work with bl system . executive infrastructure in order to promote the feasibility of the program , some executive infrastructures were developed . most importantly , a distance learning committee was organized consisting of five specialists in the field of distance learning who were responsible for making decisions , rules , macro policies , and developing new strategies . moreover , members of either educational committee or distance learning committee of tums passed a number of educational regulations ( e.g. , each faculty instructor was permitted to hold 4/17 of his / her course credit in the form of e - learning ; some incentives were designed to be offered to instructors who had used bl method in teaching students , etc . ) . designing bl platform and bl courses bl platform was designed based on three major principles : user friendliness , stability of the system , and accessibility . twenty - two theoretical courses ( including basic , core , and specialized courses which were valued as 48 course credits ) were approved by distance learning committee for designing in the form of bl courses . preparing courses for bl program , each instructor should write and design course scenarios , designate particular activities for face - to - face and e - learning parts , specify individual activities and responsibilities , schedule for the course , develop online quizzes , and consider how he / she will give feedback to students and how teacher after they signed the agreement form , students were given a personal user account ( including a user name and a password ) in order to sign in to bl website at//http://dln.x.ac.ir/. then , the students were supposed to select their courses at bl website . initial instructions were provided for students by researcher about how to work with the bl system , get access to scientific resources , take online tests , upload tasks , download educational e - contents , and interact with teacher and other students before starting the course . bl program was implemented at the faculty of nursing and midwifery in tums for one semester . instructors delivered their courses which had been designed and approved a couple of months before running the program . checklists of students participation were filled for each student in face - to - face method and for bl method by the in - charge instructors . this step included defining the need for such program , and evaluating the existence of the capacity for development and implementation of a bl program . as the need for such a program and the required capacity was recognized to exist ( after group discussions and collecting experts opinion inside and outside the university ) , decision was made to design a bl program for educating nursing and midwifery students in the faculty of nursing and midwifery ( tums ) . technological infrastructures a computer lab with sufficient number of computers connected to high - speed internet was established to facilitate students access to computer and the internet ( n = 104 ) . moreover , fulltime access to high - speed internet was provided for faculty members in their office . as instructors were not familiar enough with it and bl programs , a number of workshops were held a semester before execution of the program to improve instructors it knowledge and skills . the importance and necessity of using bl method and how to design course matters , course scenarios , specify individuals responsibilities were debated in these workshops . moreover , in a single session , students were informed about the way they should work with bl system . executive infrastructure in order to promote the feasibility of the program , some executive infrastructures were developed . most importantly , a distance learning committee was organized consisting of five specialists in the field of distance learning who were responsible for making decisions , rules , macro policies , and developing new strategies . moreover , members of either educational committee or distance learning committee of tums passed a number of educational regulations ( e.g. , each faculty instructor was permitted to hold 4/17 of his / her course credit in the form of e - learning ; some incentives were designed to be offered to instructors who had used bl method in teaching students , etc . ) . designing bl platform and bl courses bl platform was designed based on three major principles : user friendliness , stability of the system , and accessibility . twenty - two theoretical courses ( including basic , core , and specialized courses which were valued as 48 course credits ) were approved by distance learning committee for designing in the form of bl courses . preparing courses for bl program , each instructor should write and design course scenarios , designate particular activities for face - to - face and e - learning parts , specify individual activities and responsibilities , schedule for the course , develop online quizzes , and consider how he / she will give feedback to students and how teacher after they signed the agreement form , students were given a personal user account ( including a user name and a password ) in order to sign in to bl website at//http://dln.x.ac.ir/. then , the students were supposed to select their courses at bl website . initial instructions were provided for students by researcher about how to work with the bl system , get access to scientific resources , take online tests , upload tasks , download educational e - contents , and interact with teacher and other students before starting the course . bl program was implemented at the faculty of nursing and midwifery in tums for one semester . instructors delivered their courses which had been designed and approved a couple of months before running the program . checklists of students participation were filled for each student in face - to - face method and for bl method by the in - charge instructors . students individual characteristics , gpa , participation in courses , and opinion , and instructors demographics , attitude , and opinion toward teaching methods were assessed by the following researcher - made data collection tools : students individual characteristics : were recorded via a 9-item questionnaire . students participation in courses : we developed two checklists ( one for bl and one for face - to - face method ) to quantify each student 's participation throughout the course sessions . we considered the time and duration each student logged in to the bl website and the time he / she used the bl website 's chat room as indicators of participation with the bl courses . on the other hand , the time and duration the student was present in class and the time he / she involved in scientific discussions were considered as indicators of participation in face - to - face classes . both checklists were scored based on their values and the sums of scores were classified into three distinct levels : high participation , > 75% ; moderate participation , 50 - 75% ; and low participation , < 50% . students gpa : students gpa in bl method was compared to that of previous semester ( face - to - face method ) . instructors attitude : was measured via a 3-point , likert - scale questionnaire ( 1 = agree , 2 = agree to some extent , and 3 = disagree ) , including an open - ended question at the end to seek the instructors suggestions . participants opinion : focus group discussions were conducted separately for students and instructors to explore their opinion about bl and face - to - face methods . students individual characteristics , gpa , participation in courses , and opinion , and instructors demographics , attitude , and opinion toward teaching methods were assessed by the following researcher - made data collection tools : students individual characteristics : were recorded via a 9-item questionnaire . students participation in courses : we developed two checklists ( one for bl and one for face - to - face method ) to quantify each student 's participation throughout the course sessions . we considered the time and duration each student logged in to the bl website and the time he / she used the bl website 's chat room as indicators of participation with the bl courses . on the other hand , the time and duration the student was present in class and the time he / she involved in scientific discussions were considered as indicators of participation in face - to - face classes . both checklists were scored based on their values and the sums of scores were classified into three distinct levels : high participation , > 75% ; moderate participation , 50 - 75% ; and low participation , < 50% . students gpa : students gpa in bl method was compared to that of previous semester ( face - to - face method ) . instructors attitude : was measured via a 3-point , likert - scale questionnaire ( 1 = agree , 2 = agree to some extent , and 3 = disagree ) , including an open - ended question at the end to seek the instructors suggestions . participants opinion : focus group discussions were conducted separately for students and instructors to explore their opinion about bl and face - to - face methods . those items were then evaluated for clarity and face and content validity by 10 experts in the field of education . cronbach 's alpha was applied to examine the internal consistency of the opinion and attitude questionnaires ( alpha = 0.83 and 0.81 , respectively ) . were used to compare students gpa between the two methods ( bl and face - to - face ) paired t - test and wilcoxon test were respectively used to compare students gpa and participation between bl and face - to - face methods . chi - square and fisher exact test were used to examine the association between categorical variables . one hundred and eighty - one nursing and midwifery students and seventeen instructors consented to take part in the study . students individual characteristics : most students were 18 - 25 years old ( n = 148 , 81.8% ) , single ( n = 162 , 89.5% ) , and females ( n = 163 , 90.1% ) . details of students demographics and computer skills are presented in tables 1 and 2 , respectively . participants individual characteristics participants level of familiarity with computer skills students participation in courses : students participation during course sessions was significantly higher in bl compared to face - to - face method ( wilcoxon , p < 0.0001 ) [ table 3 ] . students participation and grade point average in blended learning and face - to - face methods students gpa : averages of students grade points were significantly higher in bl than in face - to - face method [ mean ( sd ) of gpas in bl method = 15.96 ( 1.43 ) , in face - to - face method = 15.44 ( 1.49 ) ; paired t - test , p < 0.0001 ] [ table 3 ] . instructors demographics : most instructors were married ( n = 14 , 82.4% ) and were interested in participating in bl training workshops ( n = 15 , 88.2% ) . instructors attitude : about 34% of respondents indicated completely positive attitude , 66% of them indicated positive attitude , and none of them indicated negative attitude toward bl method ; all participants appreciated the bl method as one of inevitable educational requirements in today 's world . a significant positive association was revealed between instructors attitude and their willingness to participate in workshops ( fisher 's exact , p < 0.0001 ) , and also their interest to bl course design ( fisher 's exact , p < 0.0001 ) . there was no significant difference between instructors attitude and their individual characteristics . participants opinion : textual analysis of the focus group discussions showed that most students admired the bl program as a teaching method in nursing and midwifery education and preferred it to traditional method . one student said : previously , getting the right course materials and contents was a big concern for me . now i can easily find the right content ; contents are also clearly classified into subtitles and make it easy to know which topics are included in each course . about content development issue there was a plenty of references for each course , it is good that we are offered a variety of resource addresses , in different types . i prefer to have written opinions rather than just hear them , because in the former , i can save them and refer to , whenever i need . another student indicated thus : i feel i can learn better because it is possible to get my instructor 's instant feedback about my task performance , which was not occurring in previous semesters . i could test myself whenever i wanted and surprisingly i could get the result of the online test in the moment ! i like the electronic parts of the course because i study them whenever i wish to . in terms of easy access to internet , one student said thus : although we have access to high speed internet in the faculty , i have problems about uploading my tasks when i am at home . another student pointed to her weakness of computer skills as a limitation and said : it is hard for me to work with the bl system , tasks are also too much , sometimes i get confused . some of them also complained about supportive aspects of the program . including one student : there are lots to do within a course . one instructor noted thus : i can schedule in a more flexible manner because i do parts of my course duties in my spare time . i think we can gradually gain a reach collection of course materials and questions for quizzes by working with bl system . instructors were also appreciated with incentives which had been provided to persuade them for using bl method . one of them said : if there were not an incentive for instructors who use this method in the faculty , the participation of instructors could n't be guaranteed . a couple of instructors complained about the time - consuming nature of bl course design . one of them said : answering to students questions , applying online tests and development of e - contents takes a lot of time . most of the faculty members believed that existence of a reward ( 67.90% ) and a supervisory system ( 80.40% ) for instructors who use this method is necessary . our results showed that students gpa and participation in courses were higher in the bl method . students had also a high regard for some aspects of the bl program , including lesson plan , and feedback ; however , they claimed about some technological aspects ( including lack of access to and skill of using the computer and the internet ) . our result about the improvement of students gpa in the bl method is congruent with that of some other investigations . however , some authors have reported similar learning outcomes for e - learning and face - to - face methods . in this regard , a longitudinal investigation that had included teacher 's routine feedback in e - learning method has shown a 19% boost in e - learning efficacy rather than in face - to - face method . thus , it seems logical to gain better learning outcomes in bl rather than e - learning and traditional method since in this way , students can benefit from both methods . thurmond ( 2003 ) concluded that the more frequent interactions are prepared ( whether in the form of email or online chat ) , the more participation rates are gained , which can itself lead to better learning outcomes . however , beard and harper ( 2002 ) suggested that interactions more frequently happen in face - to - face method rather than e - learning and considered that as a weak point of the latter method . high proportion of faculty members had positive attitude toward applying bl method and were eager to participate in bl workshops . development of distance learning has been a macro policy of tums since the year 2002 ; in addition , the university and faculty developed some obligation in which they offered some job promotion and financial incentives for faculty members who became volunteers to use bl method . it seems that all these situations made instructors aware of the new system and led to positive atmosphere in the university about bl ; these changes themselves seem to influence on instructors attitude in a positive manner . maneie ( 2003 ) and bagherian ( 2002 ) noted that efficacy and success of new educational technologies is highly related to users psychosocial background and attitude as well as options of the technology ; the positive correlation between faculty members attitude and their willingness to enroll in workshops also proves this matter . in our study , students were more satisfied with bl educational contents rather than those of face - to - face method students also admired supportive aspects of the program . thurmond ( 2003 ) concluded that since students devote a considerable amount of time on task fulfillment , preparing inadequate support makes them disappointed with their attempts . sherry et al . ( 1998 ) noted that teacher 's instant feedback to students in e - learning method positively influences learner 's motivation and provides a feeling of support . learner and teacher learner interaction , designing more supportive and interactive programs is suggested ; in this regard , further investigations should determine the effect of interaction option in bl program on participants satisfaction . learner - related matters could not have made students completely satisfied . since learners success in bl program depends on their computer skills , an average technological skill and lack of constant access to computer for half of the students may affect their satisfaction of the program . therefore , to ensure easy access and application of technologies required for working with bl system , better educational and technological infrastructures could help participants satisfaction and appropriate involvement with the program . ( 2008 ) found that there is a positive correlation between easy application of educational methods and learners satisfaction . development of appropriate technological and institutional infrastructures before running the program ( such as providing access to it center and high - speed internet , holding bl training workshops for educators , and removing some structural and organizational barriers ) and motivating participants to use bl program seem to be influential elements . philips ( 2002 ) conducted a comprehensive analysis on the causes of defeat in web - based educational approaches ; he explored three major causes : causes related to project output ( e.g. , weak site design , inappropriate or inadequate technical infrastructures ) , causes related to learners ( e.g. , learner 's unwillingness , unpreparedness , or lack of time ) , and causes related to organization ( e.g. , lack of organizational support or reward system ) . the nature of the way we assessed students participation in bl method was different from that of face to face method , however , we tried to make them more similar by considering all aspects which can be considered as participating in a course and asking experts opinion in this regard . we presented preliminary outcomes of bl program in educating nursing and midwifery students in this paper . the strengths and weaknesses of the program will be considered for design and implementation of subsequent steps of our par project . considering lack of evidence regarding effectiveness of bl programs in the field of medical education in iran , preliminary results gained by the present study might be helpful for decision makers who tend to develop similar programs in the country . in this regard , considering motivational and supportive aspects and providing appropriate technology basis besides proper development of knowledge and skills for users is recommended for developing successful programs .

background : we aimed to develop and evaluate outcomes of a blended learning ( bl ) program for educating nursing and midwifery students of tehran university of medical sciences ( tehran , iran).materials and methods : this was a participatory action research project . after designing bl website , providing technological infrastructures , and holding preparatory workshops , 22 blended courses were designed . bl method was implemented for one semester . students grade point average , participation with courses , and opinion about educational methods , and instructors attitude and opinion about educational methods were assessed.results:most students ( n = 181 ; 72.1% ) and 17 instructors ( 28.3% ) consented to participate in the study . students grade point average and participation was significantly higher in bl rather than in face - to - face method ( p < 0.0001 ) . most instructors ( n = 11 , 65% ) had positive attitude toward bl method . textual analysis of participants opinion showed that most students preferred bl method and felt more independent in this method . however , they complained about lack of easy access to internet and weakness in computer skills . instructors admired the flexibility and incentives that had been provided in the program . however , some of them complained about the time - consuming nature of bl course design.conclusion:the program showed positive effect on students learning outcomes and participation . the strengths and weaknesses of the program should be considered for development of next phase of the project . lessons learned in this phase might be helpful for decision makers who tend to develop similar programs in iran . motivational and communicational issues and users it skills should be addressed in every bl program .

I M The program Analysis (January-August 2008) Design (August-December 2008) Implementation (February-June 2009) Evaluation Data collection tools Validity and reliability Data analysis Ethical consideration R D C

PMC3483744

the potato commission did not effect much good , although it was said thirteen thousand pounds were expended upon the inquiry an extravagance which very much annoyed the doctors , who only got five shillings a day without meat or drink$\ldots $ for doctoring a hundred or two starving wretches in typhus fever . the chemists then took up the question , and found that there was too much phosphorus in one description of food , and too much sulphur in another , and too much or too little lime in a third ; and , in fine , that , with few exceptions , man could not live on bread alone . then the fact became evident to the chemists , which had long ago been practically demonstrated by the people , that the potato , bad as it was , contained more life - sustaining elements , added to more palatable qualities , and less deleterious constituents , when taken for any length of time into the system , than any other vegetable that could be procured . the potato commission did not effect much good , although it was said thirteen thousand pounds were expended upon the inquiry an extravagance which very much annoyed the doctors , who only got five shillings a day without meat or drink$\ldots $ for doctoring a hundred or two starving wretches in typhus fever . the chemists then took up the question , and found that there was too much phosphorus in one description of food , and too much sulphur in another , and too much or too little lime in a third ; and , in fine , that , with few exceptions , man could not live on bread alone . then the fact became evident to the chemists , which had long ago been practically demonstrated by the people , that the potato , bad as it was , contained more life - sustaining elements , added to more palatable qualities , and less deleterious constituents , when taken for any length of time into the system , than any other vegetable that could be procured . penned in 1854 by prestigious dublin surgeon william wilde , this critique suggests that ideas derived from contemporary nutritional science helped to structure how medico - scientific communities responded to the failure of the potato crop during the irish famine ( c.184552 ) . the day - to - day activities of medical men have been well documented within the voluminous famine historiography . yet wilde s statement also points to the existence of a pervasive web of ideas which shaped understandings of irish dietary habits , and informed medico - scientific efforts to comprehend the catastrophe . notably , wilde records the apparent failure of scientific knowledge to perform successfully and efficiently as a component of relief policy . even wilde a highly influential medical figure whose research into ophthalmology and ear diseases had earned him an international reputation retained scepticism towards the practical utility of nutritional theory within official relief mechanisms , a stance which reveals an inherent turbulence in the interaction between science , medicine and politics during the famine . according to wilde s account , scientific speculation had proven to be of limited practical value as a state tool , merely reiterating forms of knowledge in a language steeped in the technicalities of professional analysis already intuitively known by the irish peasantry through day - to - day experience . in fact , as his statement reveals , chemical analysis had been allocated a privileged position within state - organised schemes at the commencement of the famine , providing a conceptual backdrop to the activities of scientific men dispatched to investigate the blight . firstly , why was such a degree of faith and optimism attached to chemical science at this particular historical juncture in relation to the famine ? james vernon has persuasively argued that the hungry and starving began to be transformed into figures deemed worthy of humanitarian concern during the 1840s . yet vernon identifies scientific intervention into hunger as occurring in much later decades , whereas wilde s critique contrarily identifies scientific efforts to relieve potential starvation as significant in the 1840s . secondly , why did the application of this scientific knowledge seemingly fail to resolve famine - related problems , and what impact did this have upon irish public perceptions of nutritional science ? jan golinski has remarked upon the historicity of gulfs between the production of scientific knowledge and its interpretation by the public who , at worst , condemn science as a source of authority and a tool of powerful social interests , and this scenario is in many ways affirmed by wilde s statement . within this article i argue that strong links were forged between nutritional science and state - sponsored relief practices during the opening years of the famine . yet the relationship between science , the state and the public was intricate and precarious , so much so that scientific intervention sparked widespread debates which reached an apex during the soup kitchen controversies of 1847 . hence , nutritional science s stance during the famine became remarkably fluid and shifting as governmental policy evolved between 1845 and 1847 . in doing so , i aim to draw attention to the complexities of food reform in mid - century ireland . nineteenth - century irish historiography often depicts the dramatic dietary changes that followed the famine as having emerged as a natural response to the harrowing lessons learnt between 1845 and 1852 on over - relying upon one crop alone . this article , and the wider body of research from which it stems , reveals how the issue of dietary reform was far more intricate , and that discussion of it was embedded within wider international developments in medical science and nutritional thinking . food reveals itself as a subject that offers wider insights into the tense relationships between medicine , science , government agencies and the irish public in this period . governmental interest in harnessing nutritional knowledge also demonstrates an early experiment in investigating and regulating the diet of citizens , the likes of which tended not to be seen again until the early twentieth century . in 1845 , an estimated 40% of the irish population subsisted principally upon the potato , typically consuming the crop with buttermilk , water , fish or whiskey . prior to the famine , critical commentary on this mono - crop existence was rife , and tended to focus upon its detrimental impact upon ireland s socio - economic structure . condemnation of the crop s widespread usage permeated english perceptions of ireland , while the issue of food remained closely intertwined with wider debates regarding irish socio - economic underdevelopment . the potato became critically derided as a remarkably easy crop to produce ; this observation was applied to justify prevailing stereotypes of the irish peasantry as inherently idle . easy cultivation meant less time working on the fields , so influential political economists insisted , thereby promoting over - population and further poverty as irish peasants allegedly chose to spend their excess spare time procreating . ending reliance upon the potato thus became central to politico - economic debates regarding the need to stabilise irish society , rear the country s inhabitants into modernity and promote moral values of restraint ( dietary or otherwise ) . even outside the level of abstract economic theory , the potato was prone to frequent failures , a problem compounded by a lack of alternative foodstuffs being available to counter communal starvation . poor nationwide harvests were witnessed in 17401 , 18001 , 181618 , 1822 and 1831 , while partial , regional potato scarcities were common in intermittent years . yet this was a period when an established framework for considering diet from a nutritional perspective was in a nascent state , and it is unsurprising to find that the health advantages or disadvantages of the potato remained undocumented . nonetheless , a broad consensus existed which foresaw dietary transformation as a mechanism with which to assist in resolving immediate and long - term problems in ireland s social infrastructure . why , then , had confidence in dietary change being guided by scientific expertise gained currency by 1845 ? in part , this can be ascribed to the gradual development of refined , nuanced models of consumption during the early nineteenth century . during this period , important shifts in food analysis occurred which partially transformed diet into a medicalised issue . traditional approaches to consuming had typically posited the relationship between food and the individual as being determined by constitution . hence , discussion of dietetic health had tended to devote less attention to understanding the properties of individual foodstuffs , as the manner by which food was assimilated into bodily systems was seen as differing from person to person and being influenced by a diverse range of factors . yet , during the nineteenth century food intake became subject to closer analytical scrutiny . initially , attention remained focused on understanding how the inner body reacted to ingested food rather than on analysing food content . accordingly , the physiology of digestion captured the attention of a wide range of medical investigators . physicians , pathological anatomists and physiologists speculated widely on the manner by which food was taken into the bodily system , assimilated into the blood and passed into the tissues . numerous investigators explored the mastication of food , its admixture with saliva , its digestion by the stomach s muscular powers , how it was propelled into the bowels , and then carried into the blood or transformed into excrementitious matter . new norms of healthy digestion were established , with physiological deviation from these being increasingly referred to as indigestion or dyspepsia . methods of chewing , stomach sizes , the ideal time which the body spent digesting , and so on , all became closely analysed by emerging regimes of internal exploration . given the unique nature of irish dietary customs , it is unsurprising to find the irish agricultural labourer gradually falling under the gaze of digestive analysis . a burgeoning interest in irish dietary customs accumulated in the early 1840s when firm distinctions became mapped between normative models of digestion and irish dietary realities . a sense of alarm became attached to the large quantities of potatoes consumed in ireland , a tradition seen as clashing profoundly with the ideals of a burgeoning dietetic science that venerated dietary restraint . high levels of attention were awarded to this subject within derry - born physician james johnson s popular a tour of ireland ( 1844 ) , for instance . although best remembered for attending the duke of clarence and editing the medico - chirurgical review , johnson also published a series of influential works on digestion . it was perhaps johnson s interest in ascertaining the workings of the stomach , combined with his irish background , which fostered his interest in the relationship between physiology and ireland s mono - crop culture . within his a tour of ireland ( 1844 ) , johnson expressed bewilderment at the widespread habit of only half - boiling potatoes , so that the centre remained so solid that it became commonly referred to as the bone of the potatoe. this custom no doubt made sense to the irish peasant as it allayed hunger for longer , but it fitted uneasily with contemporary discourse on digestion . hence , in his text , johnson proclaimed that there is scarcely a more indigestible substance taken into the human stomach than a half - boiled potatoe ; and , to , a moderately dyspeptic englishman such diet would be less than poison. alarmingly , in a cultural milieu informed by industrial advancement and productivity , johnson implicated widespread reliance upon potato consumption as maintaining wide segments of the irish populace in a realm akin to primitive culture . by relying upon simplistic modes of food cultivation , the irish peasant was depicted as perpetuating an agricultural system that proved detrimental to ireland s progression into social modernity . johnson s unabashed views regarding irish cultural primitivism reached a notable climax when he attributed popular belief in fairies , goblins , daemons and kelpies to gastric phenomena . for him , the stomach and digestive organs constituted a rich epicentre of nerves from which , through gastric sympathy and internal nervous connections , abdominal problems might present themselves in other parts of the body in this instance in the mind . this model allowed johnson to ascribe the sightings of fantastical creatures to the potato diet , and to argue that indigestion , stimulated by excessive levels of poteen and potatoes , created vivid nightmares later recollected as actual sightings . johnson s arguments were imbued with a sense that the british stomach was a far healthier organ than its irish equivalent , while the deviant composition of irish digestive physiology provided him with an explanatory factor for irish cultural backwardness . in johnson s view , the impact of the country s economic system had become metaphorically , and literally , inscribed onto the peasant s internal organs . overall , inappropriate dietary arrangements among large sections of the irish populace became presented as bearing implications that extended far beyond the corporeal realm , contributing significantly to adverse socio - economic conditions . certainly , johnson s depictions served little purpose in terms of disseminating health advice . instead , his physiological models were intended to add weight to wider political and economic debates regarding how best to stabilise and modernise irish society , which is suggestive that medical opinion , in this instance , was willing to position itself as compatible with politico - economic agendas . literature such as johnson s undoubtedly influenced the perspectives of non - medical writers such as thomas campbell foster who claimed in the early 1840s that the irish populace had a natural craving for large quantities of food . potatoes , so foster asserted , contain very little nourishment meaning that large amounts needed to be consumed if the instinctive requirements of the bodily system were to be fulfilled . yet by perpetually subsisting on excessive food quantities , the irish peasant s stomach seemed to have become engorged , constantly craving quantity . in his text , foster even claimed that physiological investigation had proven the stomach of the potato - fed irish peasant to be double the size of the average stomach , although his source for this weighty claim remains elusive . in foster s example , as with johnson s , the concept of communal abnormal digestion provided a trope which in many ways affirmed contemporary impressions of a seemingly superior british culture by articulating profound difference between centralised cultures and peripheral subjects . foster drew heavily upon visions of a normative digestive system which rendered that of the irish peasant as essentially pathological an allegoric device intended to denote social primitivism and cultural backwardness . tellingly , when not penning travel literature , foster regularly contributed to the times , a newspaper which persistently promulgated unsympathetic attitudes to irish poverty by placing blame for it upon irish idleness . hence , foster s observations of the internal bodily workings of the irish peasant formed part of a wider agenda of promoting irish socio - economic reform . these writings reveal how physiological ideas might be adapted to suit political and economic ends while perpetuating pre - existing racial presumptions . yet , as a tool , digestive physiology was less useful for suggesting what crops might replace the potato in order to effect the elusive goal of irish dietary reform . however , the varieties of nutritional science which developed from the 1830s employed a radically different approach , taking food itself as an analytical starting point rather than physiological reactions to it . internationally renowned german chemist justus von liebig was particularly successful in impressing upon the public the premise that findings derived from chemistry contain social , economic and political utility if applied appropriately . by analysing the contents of a range of foodstuffs , liebig deciphered a comprehensive list of which chemicals enter the body during digestion , placing himself in an authoritative position to advise on consumption . liebig explained physiological phenomenon as chemical in nature , speculating that all organic nitrogenous constituents are derived from plant proteins . within his work on animal chemistry , for instance , he described blood as being composed in part of fibrin and albumin , while protein was determined as produced within vegetables which , when ingested , strengthened flesh and cellular tissue . the idea that chemical transformations occur following ingestion , and that foodstuffs are best chosen with this in mind , formed one of liebig s most inspiring contributions to an already rich international literature on diet . he constructed a model of consumption that proved attentive to food articles and their components while simultaneously producing research findings which were potentially socially applicable during the hungry 40s. and , as others have noted , the impact of liebig s research upon discussion of diet was profound in subsequent decades , stimulating new approaches to everyday dietary matters . hence , by the eve of the famine a strong intellectual framework had evolved which explained food intake and nutrition scientifically . meanwhile , chemistry was being presented as a socially beneficial force , and scientific cookery as a useful civilising agent . as adelman has persuasively argued , in ireland during the 1840s science became particularly valued as a discourse by a government eager to embrace and promote it as a means of effecting socio - economic reform . contemporaneously , as vernon has demonstrated , starvation became an increasingly emotive issue during this decade , and one which governments found increasingly harder to justify using malthusian logic . hence , starvation gradually transformed into an issue that fell under the aegis of governmental responsibility while organic chemistry increasingly presented itself as a socially transformative tool with the potential to assist in this process . it is in these contexts that proponents of food chemistry became well positioned to exert an intellectual and practical influence within state - managed relief practice , and to occupy a socially influential space in famine - period ireland . in 1845 , potato yields were 33% less than in previous years , and in 1846 75% less . by black 47 , potato acreage had fallen from over two million acres to only 284 000 . this opened up opportunities for inclined medico - scientific figures to apply understandings of dietary intake to resolve , or at best relieve , the ramifications of blight . importantly , given the prevalence of presumptions that the irish populace would seek alternative food sources once famine receded , ireland presented a remarkably suitable terrain upon which nutritional science s social claims could be tested , imposed and proven on a populace presumed to be eager to alter its dietary traditions . from the outset , chemical theories dominated understandings of the famine . in october 1845 , prime minister robert peel established a scientific commission to ascertain the cause of crop failure and to formulate solutions . historians have frequently observed how this group were mistaken in their conclusions on the cause of the blight , which was only established as fungal in the 1890s . murchadha , for instance , has recently argued that the commission s members were hampered by a lack of expertise in plant biology , and passes judgement upon them for ignoring suggestions of the blight s fungal origins . yet this perspective is retrospective , relying upon hindsight , and ultimately fails to assess why the commission chose the path which they did . to simply state that historical opinions were inaccurate does not explain why speculation on the blight s chemical origin became privileged over fungal explanations . fungal theories had certainly been developed , and remedies to exterminate fungi involving the application of acids , alkaline liquors and chlorine were well circulated . yet chemistry was a more developed discipline in the 1840s , and more socially active . certainly , the composition of the commission reveals the confidence placed in this branch of scientific activity by peel . the commission was made up of a group who subscribed to and promulgated contemporary confidence in organic chemistry . lyon playfair was a scottish professor of chemistry at the royal manchester institution ; john lindley was an accomplished english botanist ; and robert kane was the president of queen s college , cork . an often un - noted but significant point about the commission is that playfair was a former pupil of liebig . kane , meanwhile , had also studied organic chemistry in giessen under liebig , and had made important attempts to encourage the adoption of liebigian principles in ireland . given this weighty liebigian influence , it is unsurprising that the commission s activities focused so intently upon the chemistry of famine , and that its members were disinclined to explore suggestions running counter to their professional presuppositions . peel established the scientific commission shortly after the publication of george phillips the potato disease : its origin , nature and prevention . phillips had gained prominence as a highly active member of the london chemical society , being well respected in political circles for his scientific prowess . in 1842 , he had established a chemical laboratory to help enforce the pure tobacco act , an activity which underlined the official confidence placed in chemistry . phillips work presented the fullest elucidation of the potato as a target of scientific scrutiny published in the uk so far , and stimulated a chemical reimagining of the potato . within it , the potato emerged as an object analysed and divided into its constituent parts : water , starch , sugar , potateine , gum , albumen , ligneous fibre , silica , alumina , lime , potash , magnesia and so on . these highly specific elements were depicted as not only contributing to the overall constitution of the potato , but also as individually serving particular purposes within the human system . the potato s tuber , meanwhile , became reconstituted as a cellular tissue filled with granules of starch floating in water , gum , sugar and albumen . importantly , philips analysis of the potato suggested that the crop was in fact a highly useful food article containing all of the essential constituents of healthy digestion and nutrition : starch , gum , sugar and albumen , providing the body with a healthy supply of carbon , oxygen , hydrogen and nitrogen . in his work , phillips closely delineated a transformation from healthy to diseased potato . gradually decreasing levels of sugar , potateine , gum , albumen and ligneous fibre were hypothesised as resulting from blight , whilst a subtle transformation from healthiness to rottenness was mapped involving the decomposition of albumen , the colouring of the potato with oxygen , the browning of the diseased crop with alkalis , the consistency of the potato shifting from acid to alkali and ammonia being produced by putrid fermentation . phillips concluded that rain was the primary cause of these chemical alterations , explaining that excess moisture had encouraged the formation of an surplus of pendulums and tubers that had ceased to be supplied with nutriment once the rain had prostrated the crop s powers . newly formed pendulums and tubers were thus left unsupported by the plant , causing the living principle to cease to exist and chemical processes of decomposition to begin . overall , rain and moisture had stimulated the crop beyond its ability , overpowering it and producing putrefaction . the influence of phillips chemical paradigms became visibly clear in the commission s initial suggestions that nutritional elements could be salvaged from diseased potatoes . this plan was rendered possible by the pervasiveness of theoretical frameworks which conceptualised the potato as a crop divisible into constituent chemical elements . kane felt confident that the starch content of diseased potatoes could be salvaged but maintained that extracted starch could never substitute the potato itself , as the crop also contained other substances necessary to support human life . a practical , cost - efficient solution proposed involved educating the irish poor on how to produce starch at home , an approach resting on presumptions that the techniques of chemistry could be imitated in domestic settings . notably , this approach entailed minimal official intervention , thereby satisfying prevailing principles of laissez - faire intervention . the commission s instructions for this process were , however , complex to say the least . a rasp or grater was to be made by punctuating a sheet of tin or iron with holes . the pulp produced from grating should then be drenched with water , and the remaining pulp and starch separated . the pulp was then to be dried on a griddle over a fire , and put aside , whilst wet lumps of starch could be dried on a shelf for a number of days . the irish peasantry were informed through leaflets , posters and talks given in churches that wholesome bread could be made by mixing starch and pulp with peas - meal , bean - meal , oatmeal or flour . ultimately , however , the dispersion of such complex advice accentuated public perceptions of rifts between scientific theory and social practice , while the apparent collusion of state and science in producing inefficient advice did little to bolster nutritional science s public image in ireland . sporadic evidence exists of localised enthusiasm for the commission s remedies . in october 1845 , for instance , henry john porter , land agent to the duke of manchester of tanderagee , co. kildare , held meetings at the school houses on his estate to instigate the domestic conversion of potatoes . yet , to critical observers , such advice seemed to be of little value , a predicament which provoked widespread condemnation from various actors across the irish social spectrum . the commission persisted in disseminating advice despite an influx of letters sent to them and the famine relief commissioners lamenting the impracticality of starch production . reverend edward hoare of ballymore , co. kildare , for instance , wrote to the commission expressing doubts about persuading the peasantry to implement their recommendations . a paper read to the natural history society of dublin in december , meanwhile , envisaged problems with starch production as residing in the apathy and indifference of the peasantry who were reported to show a tendency to dislike innovations in methods of storing their potatoes . peasants , it was claimed , did not look much beyond the present hour and , in any case , were under the belief that public funds would become available to cover their loss . other critical voices saw problems with implementation as residing more in financial considerations than peasant conservatism or apathy . letters sent to the commission and the relief commissioners demonstrate the prevalence of opinions that the former were oblivious to the irish peasantry s financial situation . one of these stressed that cottiers could not afford the lime suggested as useful to dry potatoes with . similarly , reverend william fisher , rector of killmore , co. cork , estimated that 2500 peasants in his parish could not afford to purchase the oatmeal recommended as a mixer for starch , adding that financial aid would be more appropriate . others expressed indignation at irish corn exports not being prohibited , a seemingly more practical solution than encouraging the peasantry to apply complex processes of potato conversion . on a subtextual level , these conflicting accounts of peasant conservatism and calls for financial support represented far wider cultural debates regarding preferences for self - help or state intervention . underpinning discussion of financial aid were strong implications that ireland was deserving of state support , and that a system of scientifically guided starch production reliant upon self - initiative was impractical . the existence of intra - professional disputes also undermined public confidence in officially sanctioned scientific suggestions . edward carroll , agricultural superintendent on the estates of william wrexon beecher , mallow , co. cork and member of the royal agricultural society of ireland , wrote to the freeman s journal in october declaring official scientific advice on potato conversion as a collection of silly nostrums created by men who knew little of practice applicable to the circumstances of this country. accordingly , he condemned kane s idea of drying potatoes in the open air on the basis that ireland s weather , especially in autumn , was such that barely two days passed without rain . carroll also lamented the commission s advice that potatoes should be dried in uninhabited rooms , as most small irish labourers did not have the luxury of space in their huts . although carroll s statements indicate some degree of professional tensions between scientific groups , they were also resonant of wider concerns felt in ireland that a central government located in distant london was proving inattentive to irish needs . his articulation of this in a nationalist - minded newspaper ensured that he addressed a receptive audience who might adapt the ambiguity of science s function within relief practice into a trope with which to articulate wider apprehension regarding centralised approaches to ireland . such public critiques of the impracticalities of scientific advice placed nutritional science in danger of becoming perceived as synonymous with political inefficacy . tellingly , reverend richard walsh of headford , co. galway , returned recommendations dispatched to him as a measure not only of his lack of confidence in the commission but also of his horror at governmental indifference towards the want and distress of the irish nation . by november , the nation was calling for all official recommendations to be rejected as , if anything , the decomposition of the potato seemed to be accelerated by them . journalistic hostility to official activities accentuated in december 1845 when the commission circulated a nationwide questionnaire requesting information on the spread of the blight . it seemed to many critics that , although months had now passed , all kane and his colleagues had achieved so far was to meet and theorise , rather than undertaking more practical tasks such as collating information regarding the extent of loss of the potato crop , when scarcity was likely , the state of the food markets and levels of alternative foodstuffs available in ireland . journal s declaration that this negligence this apathy this carelessness of the obligations of humanity is so shameful , and we have no doubt that the government will yet be made to feel that this is so. the cork examiner , meanwhile , insisted that the government were mocking the irish with this unaccountable delay , pronouncing that our rulers would lavishly squander public money on itinerant commissioners who came here to abuse us , but are deaf to our cries for bread. at a meeting in mayo held in january 1846 , later recorded in the freeman s journal , one speaker announced that the learned pundits , if anything , had recommended a course of action which was the complete opposite of that which would have been wisest , and that such is the dependence to be placed on the theories of those self - sufficient gentry , who take upon themselves so dogmatically to point out the causes of and the remedies for the visitations of providence. in all these examples , nutritional science itself became deeply entangled within a wider web of simmering anti - union sentiment and depicted as an integral and illustrative facet of british political neglect . as the cost of the commission became apparent in the summer of 1846 , anger peaked , as did attacks on the utility of nutritional science . despite a costly financial outlay , it seemed to many critics that all the expense surrounding the commission had achieved was to confirm that the potato crop was failing . although lindley was acknowledged with possessing a profound knowledge of vegetable physiology , the nation stated we defy anyone to point out a single physiological reason advanced on the subject by him , or even the smallest exhibition of scientific knowledge , from the appearance of the celebrated manifestoes of the potato commissioners to the present day. the contributor also suggested that lindley has scarcely advanced a single opinion on the disease which he has not renounced at a subsequent period. by november , the tuam herald was describing the so - called potato commission as the greatest farcical humbug ever got up even in ireland and promptly renamed it the commission for the destruction of potatoes on the basis that it is a notorious fact that in every instance where the direction of the commissioners was followed , the potatoes were utterly destroyed ! clearly , some months into the operations of the commission , attitudes had evolved from scepticism to outright rejection . the approach of the scientific commission demonstrates the prominence of chemical and nutritional theory during the 1840s in official circles . the famine presented liebig s disciples with an opportunity to publicly demonstrate social prowess and to promulgate a sense of chemistry s social usefulness . the attempted conversion of diseased potatoes was an expression of the excitement and confidence placed in contemporary food investigations . the nutritional quality of the potato diet had been confirmed , to some surprise , and it could now be hypothesised that the irish peasantry could subsist once they had salvaged the nutritional goodness from their diseased crops . on one hand , reactions against the commission s scientific suggestions represented scepticism towards medical science s practical applicability . yet science s entanglement with political critique simultaneously undermined public perceptions of the discipline , whilst its solutions became publicly castigated as further evidence of governmental neglect , providing anti - union voices with a rich resource with which to articulate political critique . the most that officially sanctioned research into the chemical constitution of the potato seemed to many to have proven was what the irish had instinctively known all along : that they could subsist healthily on the potato . although this appeared as a momentous revelation to chemical scientists , from the perspective of the irish peasant and critics , the financial costs incurred discovering what was inside a potato and how to extract its elements might have been better spent by the state on providing food for an increasingly destitute population . the onset of widespread famine - related diseases during the winter of 18456 suggested direct connections between innutritious diet and epidemic disease . it also became increasingly apparent that diseased potatoes , even if peasants had managed to extract their nutritional content , were entirely unsuitable for consumption . throughout late 1845 , prominent dublin physician dominic corrigan had written to the scientific commission warning that the consumption of extracted starch encouraged epidemic disease by lowering nutritional standards , although his advice was ignored . his stance was intended to add weight to arguments for a more efficient relief policy involving supplying food . yet by winter , doctors were observing that patients who had consumed diseased potatoes were contracting symptoms including rigors , hot skin , abdominal pain , diarrhoea and swollen muscles . initially , these observations seemed to suggest the existence of an entirely new form of gastro - enteritis . however , in april 1846 , the dublin hospital gazette noted that symptoms shared commonalities with the land scurvy recently witnessed in indian famines . such discoveries became utilised as evidence that the government needed to assist the irish population in locating an alternative food supply . in the meantime , corrigan had been aggressively reiterating his views , published in the lancet fifteen years earlier , on the correlation between innutritious diets and epidemic disease , a connection not universally agreed upon . alternative explanations for famine fever instead emphasised associations between poverty and fever , and the role of atmospheric or electrical phenomena in disease generation . corrigan republished his findings in pamphlet format in 1846 to warn of the strong likelihood of fever striking again . to solidify his point , within his on famine and fever corrigan detailed the experiences of over one hundred years of irish famines , concluding that regardless of season or climate , each time a fever epidemic had struck ireland , famine had been present . cynically , he suggested that those who persisted in drawing linkage between filth and fever did so to provide justification for withholding the most suitable form of aid food to the starving . corrigan s ideas were invariably dismissed as conjecture , and responses insisted that fever also recurred in times of plenty . yet corrigan became notably prominent during the famine , being appointed as a key member of the central board of health , which was , due to changes in patterns of famine fever , established in march 1846 , disbanded in august and reappointed in february 1847 . hence , he became well positioned to project his interpretation of the relationship between fever and famine , and eagerly employed ideas drawn from nutritional science to emphasise his views . the board also consisted of esteemed dublin medical practitioner philip crampton , robert kane and two civil servants , randolph routh and edward twistleton . this group attempted to instigate a more practical application of scientific theory than the scientific commission had achieved . both the commission and the board made use of liebigian science as a shared frame of reference , formulating advice upon notions that the potato contained all of the essential nutritional elements which now had to be obtained separately in combinations of meats , vegetables and grains . yet for the board only a scientifically based varied diet could relieve the impact of famine , a perspective expressed in their arguments that ignorance of this tenet had led voluntary relief providers including the quakers to make erroneous decisions . rice , for instance , had been dispersed on the basis that it acted as a sole article of food in oriental nations . yet the board publicly insisted that rice was not consumed alone anywhere , always being instinctively prepared with vegetables , oil , butter , meat or fish to add important nutritional elements which it did not itself contain . supposition that bulks of rice would supply the missing quantity required by the irish poor were thus presented as mistaken . rice , the board maintained , would certainly swell into a visible mass , yet this mass contained a smaller proportionate quantity of nutriment than potatoes . this critique of unofficial food relief programmes was a clear attempt at articulating the superior nature of dietetic knowledge derived from laboratory analysis and experimentation , and at carving out an important social function for nutritional knowledge in ireland . importantly , the stance of the board challenged older assumptions regarding irish diet based upon physiological differentiation . unlike johnson and foster s earlier accounts , discussion of food focused less upon judging the quantity of potatoes consumed in ireland and more upon ensuring that nutritional quality would underpin wider processes of dietary reform and modernisation it is in this context that the board looked favourably towards indian meal and oatmeal as substitutes as these foodstuffs were seen to have an immediate influence on dietetic health as well as the potential to permanently modernise irish food systems if popularised , a stance which further reveals the forging of synergies between scientific and politico - economic agendas . certainly , this perspective was remarkably evident when the board optimistically claimed in an official report that the introduction of meal had transformed how the irish thought about food . on this matter , they claimed : it has often been desired , that the people of ireland could be induced to turn from the potato to grain as their food , as tending to produce improvement in their habits , and as rendering them less liable to suffer from periodic famines . however , the knowledge that they have now acquired of the very superior nutritious qualities of oatmeal , and its price continuing to bear such a relation to the cost of potatoes , as to render its consumption often more economical than that of the potato , will , it would seem to us , eventually and certainly lead to the desired end . it has often been desired , that the people of ireland could be induced to turn from the potato to grain as their food , as tending to produce improvement in their habits , and as rendering them less liable to suffer from periodic famines . . however , the knowledge that they have now acquired of the very superior nutritious qualities of oatmeal , and its price continuing to bear such a relation to the cost of potatoes , as to render its consumption often more economical than that of the potato , will , it would seem to us , eventually and certainly lead to the desired end . this statement clearly demonstrates a faith in the good that could result once the short - term implications of the potato blight had passed : a change in crop culture would simultaneously protect from future famines and promote changes in social and individual behaviour . in autumn 1845 , robert peel purchased 100 000 worth of indian meal from america and sales commenced from march 1846 . not only was it described as highly nutritious for humans , but it was also noted to be an admirable food for fattening pigs and oxen , whilst dogs were reported to relish it . in reality , however , supplying the public with scientifically defined food supplies was a process punctuated with practical difficulties , and members of the board became increasingly critical of indoor relief policies . poor law unions were allowed to substitute potatoes for alternative foodstuffs from 1846 . by 1848 only three out of 140 workhouses were reported to be serving neither type of meal , whilst fifty - four served a mixture of oatmeal and indian meal , the remainder serving one or the other . however , these statistics fail to account for problems in preparing dishes containing meal . when meal was introduced into the south dublin union workhouse in april 1846 , for instance , the baker had no comprehension of how it could be mixed with flour to produce bread , a scenario which implied the pitfalls of a policy that simply provided food without instructions on how to cook it in a country primarily accustomed to preparing potatoes . these issues challenged idealised images of the harmonious compatibility of culinary practice and nutritional theory . revealingly , in may , members of the south dublin union relief committee publicly asserted that allocated portions of a pound of bread made from meal and three pints of porridge fed only two people rather than six . on this subject , we do not think that sixteen ounces of food , the allowance given by the south and by the north union committees , has been recognised by physiologists as sufficient for the healthy sustenance of an adult man , adding that the economic savings were not enough to justify keeping pauper populations in a precarious balance between healthy and diseased existence . a fortnight later , the lord mayor of dublin toured the city s workhouses , tasting the dishes on offer . paupers complained to him that they would prefer bread to porridge , as the latter played havoc with their stomachs . hence , nutritional theory became presented as failing to account for individual bodily reactions to new dietary articles , a discourse which formed an important part of critical public coverage of this aspect of relief practice . in response to concerns regarding indoor relief , the board intensified its arguments regarding the provision of cooked food , a step which suggests increasing divergence from official relief policies amongst medico - scientific advisors in ireland . unlike the commission , the board increasingly distanced itself from official relief strategies as the realities of famine became ever more apparent , and as nutritional science became increasingly critiqued in public domains . with workhouses becoming ever more swamped and epidemic disease rapidly spreading , the board increasingly articulated discontent with the limitations of legislation intended to meet the demands of famine , in one instance asserting that institutionalisation was a greater evil then allowing the poor to fend for themselves in the community . this was probably an exaggeration intended to justify arguments for a more interventionist form of state relief , but it did raise pertinent questions regarding whether peasants were better off in institutions or in the community . meal supplies in the latter were often so poor that the consumption of diseased potatoes continued . a limited understanding of how to prepare and cook meal was just as prevalent in the community as it was in workhouse kitchens . initially , indian meal had been imported un - milled , and was not always subsequently ground sufficiently by millers , resulting in widespread illness . the board insisted that if meal was insufficiently cooked then a full supply of nutriment failed to be provided , predisposing the community to dysentery and diarrhoea , an argument which further justified arguments regarding linkage between dietary deficiency and disease . as the dissemination of recipes appeared to be proving ineffective , the board argued that closer regulation of the irish poor s nutritional health was necessary if the tide of epidemic disease was to be halted , a process requiring scientific guidance . hence , the board began to encourage an official relief policy involving the provision of cooked food as part of outdoor relief . only by doing overall , the central board of health strove to bridge the chasm between nutritional theory and nutritional practice . as well as promulgating awareness of connections between disease and innutritious diet , the board stressed the importance of food quality as a means of enabling irish modernisation , an approach which displaced discourse on the physiological ramifications of a bulky potato diet and which asserted the authority of new forms of nutritional science . the board came under less public criticism than the commission had done as , if anything , its members increasingly challenged the inefficacies of government policy . nonetheless , its medical representatives corrigan and crampton simultaneously publicly participated in wider politico - economic visions of promoting permanent societal change in ireland through dietary transformation . drawing upon narratives of irish improvement , they defined a move away from a mono - crop existence as compatible with nutritional science although their efforts to achieve this were impeded by the intensity of famine . the board s aspirations for adequate relief policies were thwarted when the government did embark on dispersing cooked food . throughout 1846 , overwhelming applications for indoor relief encouraged the government to open nationwide soup kitchens , despite this policy being contrary to their pervasive laissez - faire ideologies . the temporary relief act instigated the setting up of these establishments . in 1847 , the government consulted alexis soyer , famed french chef of the reform club , and quickly dispatched him to dublin where he began constructing a network of kitchens . cooked food provision was approved of by the board s members who were becoming ever more exasperated by observations of people devouring raw meal . yet the scheme was immediately stricken with controversy , not least because the very idea of the soup kitchen was closely associated with the stigma and social degradation of pauperism , as had become evident during international attempts to establish them on count romford s model during earlier decades . from the onset , the grandiose ceremonial opening of the scheme caused consternation . the freeman s journal expressed nationalist fury at the opening of dublin s first soup kitchen as the union jack was flaunted from the top of the soup kitchen$\ldots $ proclaiming to the heavens the degradation of our provincialism , but unfolding a tale , as it flaps in the listless air , of a union with england brought about by force , stained by corruption , cemented in blood , and now consummated in famine. here , the soup kitchen became perceived as emblematic of the extension of an unwelcome foreign power into ireland , symbolising a lack of sensitivity to the needs of a vulnerable , subjugated populace . for the freeman s journal , the public feeding of members of the poor at the scheme s opening ceremony was an inappropriate , but telling , articulation of english attitudes towards the irish poor . five shillings each to see paupers feed ! , exclaimed the newspaper , five shillings each to watch the burning blush of shame chasing pallidness from poverty s wan cheek ! five shillings each ! when the animals at the zoological gardens may be inspected at feeding time for sixpence ! hence , despite their ideals , soup kitchens became condemned as emblems of imperial expansion by nationalist voices . the very act of eating and digesting food in these establishments carried meanings of subjugation to an imperial power , of colonial unjustness and inhumane treatment . soup kitchens were presented not as benevolent structures , but instead as sites established to ease the conscience of those in power ; buildings masquerading as expressions of british kindness but in reality serving concoctions devoid of nutritional or palatable value . the physical act of consuming food there carried symbolic meanings relating to subjugation and a shunning of responsibility to care for starving citizens . these issues permeated the objections of medical authorities who took issue with the manner by which nutritional science was being publicly presented in such schemes , an approach which also allowed them to adopt a more detached stance from official relief policies . allegations of the dishonesty and public spectacle of soyer s soups coloured professional critiques of the kitchens . while his dublin kitchen was under construction , soyer had penned a booklet entitled the poor man s regenerator which recorded his economical recipes intended to assist the irish poor . soyer proudly proclaimed to have personally discovered that existing soup providers were ignorant of the nutritional quality of their ingredients . proper seasoning , soyer maintained , was necessary to restore and strengthen the digestive organs . however , soyer would have been better positioned to publish such strong claims had he actually been equipped with an understanding of scientific cookery . certainly , for many medical men the digestive organs of famine - stricken peasants required more than the addition of herbs to their soups , as soyer recommended . critical medical professionals in both ireland and britain spotted soyer s precarious comprehension of the principles of food science , and angrily denounced his ideas as fraudulent and his soups as deficient . prestigious edinburgh doctor james simpson wrote to henry labouchere , chief secretary of ireland , condemning soyer . drawing upon liebigian animal chemistry , simpson asserted that most of soyer s soups contained no meat , despite meat having been scientifically proven to contain more nutriment than vegetable products . reciting liebigian theory , he recommended that soup should not be provided unless prepared using valid scientific principles . in similar fashion , soup quackery and as soups of pretence$\ldots $ taken by the rich as a salve for their consciences as part of a spasmodic feeling of benevolence. the journal noted that other clubs , committees and relief associations had dispatched food without any boasting or ostentation , in sharp contrast to the government s public spectacle . the lancet then undertook a thorough chemical analysis of the content of each soup , dismissing some of its contents turnip parings , celery tops and salt as items not even describable as food . soyer s soups were determined to contain less than three ounces of solid aliment per quart , despite nutritional chemistry stating that a healthy individual required between twelve to fourteen ounces . on this , the lancet sardonically warned that organic chemistry will not , we fear , bend to the receipts of the most miraculous cookery book. continuing its scathing attack , the lancet complained that both soyer and the government were inaccurate in assuming that a bulk of water would compensate for a deficiency of solid aliment , as : no culinary digestion , or stewing , or boiling , can convert four ounces into twelve , unless , indeed , the laws of animal physiology can be unwritten , and some magical power be made to reside in the cap and apron of the cook for substituting fluids in place of solids , and aqua pura for solid aliment , in the animal economy . no culinary digestion , or stewing , or boiling , can convert four ounces into twelve , unless , indeed , the laws of animal physiology can be unwritten , and some magical power be made to reside in the cap and apron of the cook for substituting fluids in place of solids , and aqua pura for solid aliment , in the animal economy . the journal concluded by declaring that although the public mind had been satisfied by developments in relief provision , this was unlikely to extend to the public stomach . marquises , and lords and ladies , may taste the meagre liquid , and pronounce it agreeable to their gustative inclinations , claimed the journal , but something more than an agreeable titillation of the palate is required to keep up the manufactory of blood , bone and muscle , which constitutes the strong healthy man. what angered the lancet the most was an apparent misappropriation of accumulated scientific knowledge , the misrepresentation of which was feared to be strengthening public associations of food chemistry with deficient relief policies . at stake in the debates regarding the nutritional quality of soyer s soups were important questions of medical respectability and professionalism , as well as ownership of knowledge . certainly , the lancet s accusation of quackery was not accidental . in a period when medicine was professionalising , public articulations of inaccurate scientific knowledge were potentially damaging , especially when seemingly sanctioned by a london - based government . furthermore , the interest aroused by the famine meant that relief practices were now publicly discussed internationally . the medical profession had been notably quiet when the scientific commission had dispersed publicly challenged advice at the commencement of the famine . then , state policy was at least attempting to make good use of analytical knowledge , even if in a misguided fashion . yet , two years later , the government appeared to be relying upon nutritional knowledge whom they castigated as a medical charlatan a development with potential ramifications to public images of professional respectability . hence , the idea that nutritional science had in any way informed soup kitchen schemes was staunchly dismissed , while the forms of knowledge underpinning soup provision were denigrated as false . after all , this was the sort of quack activity which medical reformers were eager to distance themselves from . proper nutritional science , although it is notably remarkable that the dublin medical press , then the irish equivalent of the lancet , remained mostly silent on such issues despite normally being outspoken . even queen victoria s doctor sir henry marsh suggested within a widely distributed pamphlet that soup was an unhealthy , dangerous relief mechanism , maintaining that semi - liquid diets could only maintain the health of children and sedentary adults . the labourer s food , marsh insisted , should be partly solid as digestion requires mastication and insalivation . liquid soups , on the contrary , were naturally digested too quickly , producing hunger and debility . to confirm this point , marsh argued that the habit of eating the bone of the potatoe had in fact long fulfilled these bodily requirements as it had necessitated a slow digestion of a solid substance . for marsh , the consideration of the diet best calculated to uphold his [ the labourer s ] strength and maintain his health is at all times one of the highest national importance at the present time , and in the existing disastrous state of the country , it is one which claims the utmost amount of attention , scientific and practical , which can be devoted to it. yet , for marsh , official responses dangerously violated established scientific laws , as expressed through statements which reasserted the validity of the final publication related to the soup kitchen controversy was a report commissioned by the royal dublin society to examine soyer s soups , compiled and penned by prominent irish chemist john aldridge . aldridge analysed the nutritive values of the soups on offer in soyer s soup kitchen , comparing these with analytical chemistry s dietary laws . within his report yet aldridge concluded by asserting that , in order to sustain healthy animal life , it was necessary to combine flesh , seeds and vegetables in a bulky preparation of food proportionate to the capacity of the digestive organs , rather than by the methods employed by soyer . notably , like many contemporaries , aldridge perceived the onset of blight as a suitable opportunity to generate irish industriousness , which suggests that such visions were not discarded despite the immediate urgency of providing short - term solutions to famine by 1847 . revealingly , within his report , aldridge argued that professional forms of scientific knowledge , rather than soyer s interpretation , had much to offer the irish situation in both the short term and long term . he noted that carnivorous animals living on highly concentrated food tended to be energetic , whilst herbivorous animals , tending to eat more gradually , were sluggish and idle . aldridge insisted that if ireland s population was fed in a similar manner to cows and sheep , then industrial exertion could never result . yet the irish poor were considered by him to have been even worse fed than cattle for decades , as hay was a far more concentrated food than potatoes . thus , in contrast to the dominant iconography of the famine which focused upon the skeletal , starved rural peasant , medico - scientific communities continued to depict a potentially empowered peasant invigorated by a precise comprehension of nutritional science , although this was achievable only if he was shielded from false forms of nutritional knowledge . hence , aldridge , like many contemporaries , remained eager to position nutritional science as a field with potential long - term benefits to ireland , provided that professional forms of scientific knowledge based upon analysis and deduction were applied . despite this , however , a general distancing of the medical profession from state relief policies occurred in both britain and ireland which contrasted sharply with an earlier willingness to collude with state activity . by 1847 , nutritional science s positioning within official policy had become contested and complicated , a scenario which structured shifts in how scientists interested in nutrition publicly presented themselves . this article has demonstrated that medico - scientific ideas relating to nutritional health played a visible role in state - sponsored relief policy , a consequence of a strong socially active dietetic science having been developed . during the famine , various scientists acted as authoritative voices on substitute foodstuffs and the nutritive properties of soups . by doing so , these scientists were able to engage with wider debates about the reform of irish society , a development that suited the social agenda of organic chemistry more generally . yet , associations were fostered between nutritional science and the shortcomings of central policy among irish lay communities . towards the end of the famine , even leading irish medical figures including william wilde were forced to admit that medico - scientific intervention had failed to live up to its aspirations , having been received with public hostility , and its ideas having been misappropriated within official relief strategies .

the activities of irish medical practitioners in relieving the impact of the irish famine ( c.184552 ) have been well documented . however , analysis of the function of contemporary medico - scientific ideas relating to food has remained mostly absent from famine historiography . this is surprising , given the burgeoning influence of liebigian chemistry and the rising social prominence of nutritional science in the 1840s . within this article , i argue that the famine opened up avenues for advocates of the social value of nutritional science to engage with politico - economic discussion regarding irish dietary , social and economic transformation . nutritional science was prominent within the activities of the scientific commission , the central board of health and in debates regarding soup kitchen schemes . however , the practical inefficacy of many scientific suggestions resulted in public associations being forged between nutritional science and the inefficiencies of state relief policy , whilst emergent tensions between the state , science and the public encouraged scientists in ireland to gradually distance themselves from state - sponsored relief practices .

Article Ireland, the Potato and Science Salvaging Potatoes Replacing the Potato Soup Controversies Conclusions

PMC3348677

since the first simulation of an enzyme using molecular dynamics ( md ) was reported by mccammon et al . in 1977 , md simulations have evolved to become important tools in rationalizing the behavior of biomolecules . the field has grown from that first 10-ps - long simulation of a mere 500 atoms to the point where small enzymes can be simulated on the microsecond time scale and simulations containing millions of atoms can be considered routine . however , such simulations are numerically very intensive , and using traditional cpu - centric hardware requires access to large - scale supercomputers or well - designed clusters with expensive interconnects that are beyond the reach of many research groups . numerous attempts have been made over the years to accelerate classical md simulations by exploiting alternative hardware technologies . some notable examples include atoms by at&t bell laboratories , fastrun by columbia university and brookhaven national laboratory , mdgrape by riken , and most recently anton by de shaw research llc . all of these approaches have , however , failed to make an impact on mainstream research because of their excessive cost . additionally , these technologies have been based on custom hardware and do not form part of what would be considered a standard workstation specification . this has made it difficult to experiment with such technologies , leading to a lack of sustained development or innovation and ultimately their failure to mature into ubiquitous community - maintained research tools . graphics processing units ( gpus ) , on the other hand , have been an integral part of personal computers for decades , and a strong demand from the consumer electronics industry has resulted in significant sustained industrial investment in the stable , long - term development of gpu technology . in addition to low prices for gpus , this has led to a continuous increase in the computational power and memory bandwidth of gpus , significantly outstripping the improvements in cpus . as a consequence , high - end gpus can be considered standard equipment in scientific workstations , which means that they either already exist in many research laboratories or can be purchased easily with new equipment . this makes them readily available to researchers and thus attractive targets for acceleration of many scientific applications including md simulations . the nature of gpu hardware , however , has until recently made their use in general purpose computing challenging to all but those with extensive three - dimensional ( 3d ) graphics programming experience . however , the development of application programming interfaces ( apis ) targeted at general purpose scientific computing has reduced this complexity substantially such thatgpus are now accepted as serious tools for the economically efficient acceleration of an extensive range of scientific problems . the computational complexity and fine grained parallelism of md simulations of macromolecules makes them an ideal candidate for implementation on gpus . indeed , as we illustrate here for implicit solvent and in a subsequent paper for explicit solvent , the careful implementation of modern md algorithms on gpus can provide capability , in terms of performance , that exceeds that achieveable with any current cpu - based supercomputer . several previous studies have investigated the use of gpus to accelerate md simulations . for a detailed review of the use of gpus for acceleration of condensed phase biomolecular md simulations we present our high - performance gpu implementation of implicit solvent generalized born ( gb ) md for the amber and charmm pairwise additive force fields on cuda - enabled nvidia gpus . we have implemented this within the amber pmemd dynamics engine in a manner that is designed to be as transparent to the user as possible , and we give an overview of what the code currently supports , as well as our plans for future developments . we discuss the specifics by which we exploit the processing power of gpus , both in serial and using multiple gpus , and show the performance that can be achieved in comparison to conventional cpu clusters . we also discuss our implementation and validation of three specific precision models that we developed and their impact on the numerical results of implicit solvent md simulations . as illustrated by figure 1 , gpus offer a tremendous amount of computing power in a compact package . this , however , comes at the cost of reduced flexibility and increased programming complexity as compared to cpus . in order to develop software that runs efficiently on gpus , it is necessary to have a thorough understanding of the characteristics of the gpu hardware architecture . a number of manuscripts have already discussed this in detail in the context of md . for this reason , we provide simply a brief overview of the complexities involved in programming gpus as they relate to our implementation , focusing on nvidia hardware . for a more detailed description , peak floating - point operations per second ( flop / s ; left ) and memory bandwidth ( right ) for intel cpus and nvidia gpus . a gpu is an example of a massively parallel stream - processing architecture which uses the single - instruction multiple data ( simd ) vector processing model . unlike a regular cpu , which typically operates on one to four threads in parallel , gpus typically process threads in blocks ( termed warps within the cuda programming language ) containing between 16 and 64 threads . these thread blocks logically map to the underlying hardware , which consists of streaming multiprocessors . at the time of writing , high - end gpus typically have between 16 and 32 multiprocessors . for example , an nvidia m2090 gpu consists of 16 multiprocessors , each containing 32 cores for a total of 512 cores . all threads in a single block must execute the same instruction on the same clock cycle . this necessarily implies that , for optimum performance , codes must be vectorized to match the size of a thread block . branching must therefore be used with extreme care since if any two threads in the same warp have to follow different code paths of the branch , then threads in the warp will stall while each side of the branch is executed sequentially . the memory hierarchy of gpus has its origins in their graphics lineage , and the high density of arithmetic units comes at the expense of cache memory and control units . all of the cores making up a multiprocessor have a small number of registers that they can access , a few kilobytes ( 64 kb on an m2090 ) of shared memory [ this can be split into directly accessible memory and l1 cache ; in the case of an m2090 , it can be split 48/16 kb or 16/48 kb ; in the case of amber , the configuration is switched at runtime for optimal performance of a given kernel ] which is private to each multiprocessor and a small amount ( typically 48 kb ) of high - speed but read - only texture memory . the majority of the memory ( 6 gb on an m2090 ) , termed global device memory , is available to all multiprocessors . while being fast compared to the main memory accessible by cpus , access to the device memory by gpus is still relatively slow compared to the local cache memory . the nature by which the multiprocessors are connected to this memory also means that there is a significant performance penalty for nonstride-1 access . finally , it should be noted that currently the cpu and gpu memories are in different address spaces and this requires careful consideration . the unique nature of this memory model leads to several considerations for optimizing gpu performance , including optimizing device memory access for contiguous data , utilizing the multiprocessor shared memory to store intermediate results or to reorganize data that would otherwise require nonstride-1 memory accesses , and using the texture memory to store read - only information , such as various force field parameters , in a fashion that allows very rapid access . as mentioned above , the cpu and gpu memories are , at the time of writing , in different address spaces . this means it is up to the programmer to ensure that the memories are synchronized as necessary to avoid race conditions . however , there is a big performance penalty for such synchronizations which have to occur via the peripheral component interconnect express ( pcie ) bus , and thus they should be avoided unless absolutely necessary . the traditional method for programming scientific algorithms in paralleluses the message passing interface ( mpi ) in which each thread runs in a separate address space . when running gpus in parallel under an mpi paradigm , additional complexity is introduced since sending data between two gpus involves copying the data from the memory of the sending gpu to the cpu memory of the corresponding mpi thread over the pcie bus , an mpisend by this cpu thread , and corresponding mpireceive by the receiving cpu thread , which copies the data between the memories of the cpus , and finally copying the data to the memory of the receiving gpu . clearly , this introduces additional considerations for maximizing parallel performance as compared to traditional cpu programming . at the time of writing , there are efforts to streamline gpu to gpu communication , particularly within a single node but also for infiniband connections between nodes . one such approach under development by nvidia and mellanox is termed gpudirect , which ultimately seeks to unify address spaces between multiple cpus and gpus . currently , the degree to which this can be utilized is heavily dependent on the underlying hardware design . therefore , at present , the added complexity of using the advanced features of gpudirect , beyond the pinned memory mpi optimizations offered by gpudirect version 1 , on the large number of possible different hardware combinations is not worth the effort for a widely used production code . early versions of gpus in nvidia s lineup ( prior to the gt200 model ) only supported single precision ( sp ) floating point arithmetic . this was due to the fact that graphics rendering did not require double precision ( dp ) . scientific algorithms , however , typically require dp arithmetic ( for a discussion in the context of quantum chemistry , see for example the work by kniziaet al . ) . the generation of gpus at the time of our initial implementation ( 2008 ) supported dp in hardware , but only at 1/8 the performance of sp . in the latest generation of cards , at the time of writing , termed the fermi lineup by nvidia , the dp to sp performance ratio is 1/2 and thus equivalent to that in cpus . this , however , only holds for the professional ( termed tesla ) series of cards . the significantly cheaper gaming cards ( termed geforce ) still only support dp at a fraction of the speed of sp . it is therefore important to optimize the use of dp such that it is only used when necessary to maintain numerical accuracy . early use of gpus for scientific computing was hampered by the lack of an application programming interface ( api ) for general purpose calculations . the problems to be solved had to be described in terms of a graphics pipeline employing either opengl or directx , which made the software development time - consuming and hardware - specific . the barrier to utilizing gpu hardware for general purpose computation has since been reduced by the introduction of gpu programming models such as the brook stream programming language , opencl , and nvidia s compute unified device architecture ( cuda ) and the availability of corresponding software development toolkits ( sdks ) . the amber implementation uses cuda , which is a relatively simple extension of the standard c programming language that allows one to code in an inherently parallel fashion and perform all necessary operations to access and manipulate data on a gpu device . realizing the full potential of gpus , however , still requires considerable effort as indicated above and outlined below to take advantage of the particular gpu architecture , and not all algorithms are suitable to achieve good performance on these massively parallel processors . the nature of md simulations requires what in computer science is referred to as strong scaling , that is , reduction of the solution time with an increasing number of processors for a fixed total problem size . this enables access to simulations at longer time scales , which is required for a proper convergence of results . this becomes more important as one moves to larger system sizes since the number of degrees of freedom increases . weak scaling , that is , the solution time with the number of processors for a fixed problem size per processor , is only of secondary importance , since this merely enables simulating larger molecules at currently attainable time scales . our implementation therefore has focused on accelerating problem sizes that correspond to those typically studied by amber users . in the case of gb simulations , the initial driving force in accelerating amber implicit solvent gb calculations with gpus was to provide the scientific community with a computational tool that would allow an individual researcher to obtain performance on a simple desktop workstation equivalent to that of a small cpu cluster . such a tool alleviates the costs , both capital and recurring , involved in purchasing , maintaining , and using individual research compute clusters . to this end , our goal was that a single state - of - the - art gpu should provide a performance equivalent to that of four to six high - end cpu cluster nodes . such an approach also removes the need to purchase and maintain expensive interconnects that are required to achieve scaling even on a modest number of nodes . beyond this initial serial development , which was first released as an update to amber 10 in august 2009 , we have also developed a parallel implementation based on the mpi-2 message passing protocol , released as an update to amber 11 in october 2010 , that allows a single job to span multiple gpus . as shown below , it is possible with this implementation to achieve a performance improvement that goes beyond simply making a desktop workstation faster , ultimately providing a performance capability that surpasses what is achievable on all current conventional supercomputers . achieving this level of performance required implementing the entire implicit solvent md algorithm including energy and force evaluations , restraints , constraints , thermostats , and time step integration on the gpu . as described in section 3.2 , cpu to gpu communication only occurs during i / o or to some extent when data is sent between gpus during parallel runs . while we have designed our gpu implementation to achieve substantial acceleration of implicit solvent md simulations over that achievable with amber s cpu implementation , our overriding goal has always been to maintain the precision of the calculations . to this end , we have focused on ensuring that gpu simulations will match cpu simulations . all approximations made in order to achieve performance on gpu hardware have been rigorously tested as highlighted in the following sections . an additional design goal has been to attempt to preserve forward compatibility of our implementation . using the cuda programming language provides this by abstracting the program from the underlying hardware . the gpu accelerated version of amber can be used on all nvidia cards that support double precision in hardware , that is , those with hardware revision 1.3 or 2.0 or higher . our choice of cuda and nvidia graphics cards was largely guided by the fact that , at the time we began this work , opencl was not mature enough to offer the same performance and stability benefits that cuda did . a port to opencl is certainly possible , and this would support amd hardware . however , with the public release of the cuda api by nvidia and the release of cuda compilers for x86 platforms by pgi , it is possible that an amber implementation will soon be available on a variety of accelerator hardware . we have attempted to make our gpu implementation all - inclusive of the features available in the pmemd program . at the time of writing , the majority of features applicable to implicit solvent simulations are available as described below . support is provided for all gb models currently implemented within amber as well as the analytical linearized poisson in addition to constant energy simulations , thermostats have been implemented to perform constant temperature simulations . this includes all three thermostats available in pmemd , that is , the berendsen weak coupling algorithm , the andersen temperature coupling scheme , and the langevin dynamics thermostat . constraints for hydrogen bond distances use a gpu version of the standard shake algorithm employed in pmemd , and harmonic restraints to a reference structure are supported . to the best of our knowledge , no gb formalism currently exists that corrects for the errors introduced by the use of cutoffs for long - range nonbonded interactions . the use of cutoffs in gb simulations as implemented in pmemd does not conserve energy , and their use involves an approximation with an unknown effect on accuracy . for this reason , we chose not to implement van der waals ( vdw ) and electrostatic cutoffs in the gpu version of this code . [ cutoffs for the nonbonded interactions are implemented for explicit solvent simulations with periodic boundary conditions using the particle mesh ewald ( pme ) method , as described in a later paper . a design feature of the gpu code that goes beyond the cpu implementation is the deterministic nature of the implementation on a given hardware configuration . serial cpu calculations for a given set of input parameters on identical hardware are perfectly reproducible . this does not hold for the parallel cpu implementation since the need to load balance aggressively to achieve good parallel scaling means that the order of numerical operations is not defined , and therefore two simulations started from identical conditions will always diverge due to rounding differences . this poses a problem when transitioning to microsecond or greater simulation time scales since it can be of advantage to store trajectory information less frequently than what is optimal in order to conserve available storage space and produce data files of manageable size . it is thus not possible to go back to a given point of the simulation and analyze the trajectory in finer detail by restarting and sampling more frequently unless the implementation is deterministic . the deterministic nature of the gpu code coupled with machine precision binary restart files ( currently under development ) makes this mode of simulation possible . another key feature and a primary design goal of our gpu acccelerated implementation is that its use is completely transparent to the user . as far as the user is concerned , our gpu implementation is indistinguishable from the cpu implementation , and using the gpu version of the code is simply a case of switching the executable name from pmemd to pmemd.cuda or from pmemd.mpi to pmemd.cuda.mpi for the mpi parallelized implementation . all other items such as input and output files and regression tests within the code remain identical . the only difference to be noticed by the user is an increase of performance . this guarantees effective uptake of our gpu implementation by the scientific commmunity because there is no learning curve for the use of the code , and all tools and scripts that have been developed for the cpu version of pmemd can be utilized without modifications . the maximum system size that can be treated with the gpu implementation is a function of both the gpu hardware and the md simulation parameters . in particular , langevin temperature regulation and the use of larger cutoffs for the effective born radii calculations increase the memory requirements . the physical gpu hardware also affects memory usage since the optimizations used are nonidentical for different gpu types . table 1 gives an overview of the approximate maximum atom counts that can be treated with the present version of the code . the dominant sources of gpu memory usage are the output buffers used for the nonbonded interactions as described in section 3.2 . the memory used by those buffers is proportional to the square of the number of atoms . currently , the atom count limitations imposed by gpu memory usage are roughly identical in serial and parallel . all simulations use shake ( amber input ntf=2 , ntc=2 ) ; a time step of 2 fs ; the hawkins , cramer , truhlar gb model ( amber input igb=1 ) ; the default cutoff value of 25 for gb radii ( amber input rgbmax=25 ) ; and temperature control with the langevin thermostat ( amber input ntt=3 ) , if applicable . error - correction code ( ecc ) was switched off on the tesla cards . in classical md , the majority of the computational effort is spent evaluating the potential energy and gradients , which has to be repeated each time step . in the case of the amber pairwise additive force fields , the potential takes the form1where the bond and angle terms are represented by a simple harmonic expression with force constants bi and ai and equilibrium bond distances / angles ri , eq and i , eq , respectively . torsional potentials for the dihedral angles are represented using a truncated fourier expansion in which the individual terms have a potential vi , n with periodicity n and phase shift i , n . the last two terms are the vdw interaction represented by a lennard - jones potential with diatomic parameters aij and bij and the electrostatic interaction between atom - centered point charges qi and qj separated by the distance rij . the prime on the summation of the nonbonded interactions indicates that vdw and electrostatic interactions are only calculated for atoms in different molecules or for atoms in the same molecule separated by at least three bonds . those nonbonded interactions separated by exactly three bonds ( 14 interactions ) are reduced by the application of independent vdw and electrostatic scale factors , termed scnb and scee in amber , which are dependent on the specific version of the force field ( 2.0 and 1.2 , respectively , for the ff99sb version of the amber force field ) . the charmm force field takes a similar form but includes three additional bonded terms:2the two - body urey bradley terms account for angle bending between atoms that are separated by two bonds using a harmonic potential with force constant ui and equilibrium distance ri , eq . the improper dihedrals with force constant ki describe out - of - plane bending and are used to maintain planarity and prevent undesired chiral inversions . within the amber force field , the third additional term is a cross term between two sequential protein backbone dihedral angles , termed cmap . additionally , the charmm and amber force fields handle 14 nonbonded interactions in a different manner . the single prime on the electrostatic summation has the same meaning as described above for the amber force field with the exception that 14 interactions are not scaled . the double prime on the vdw summation implies the same exclusions as the single prime but the use of different values rijmin and ij for 14 interactions . in the gb implicit solvent model , the effect of a surrounding solvent is described via a continuum electrostatics model that uses a pairwise descreening approximation and in general also includes a debye hckel term to account for salt effects at low salt concentrations . the general form of the correction to the energy of the solute is given as3where qi and qj are the atomic partial charges , r is the relative permittivity of the solvent , and is the debye the function fijgb interpolates between an effective born radius ri when the distance rij between atoms is short and rij at large distances according to4the effective born radius ri reflects how deeply buried a charge is in the low - dielectric medium ( solute ) , and it depends on the intrinsic radius i of an atom and the relative positions and intrinsic radii of all other atoms in the solute . the models implemented in pmemd differ in the intrinsic radii and the function that is used to determine the effective born radii . the remainder of this section discusses key aspects of the gpu implementation of these equations . this includes design features to maximize performance while preserving accuracy as well as addressing issues of reproducibility which are critical when moving to longer time scale simulations . one of the initial ideas we considered when implementing gpu acceleration for amber was to write an entirely new code from scratch in a combination of c++ and cuda . however , this was quickly dismissed for a number of reasons . in particular , we wanted to1.keep the maintenance of the amber code base simple2.minimize the amount of coding required3.simplify the way in which features are ported to the gpu4.maintain backward compatibility with existing input files and regression tests . keep the maintenance of the amber code base simple minimize the amount of coding required simplify the way in which features are ported to the gpu maintain backward compatibility with existing input files and regression tests . the approach we ultimately chose was to utilize the existing fortran code base in pmemd and extend it with calls to specific cuda kernels for the gpu acceleration with the cuda code protected with # ifdef cuda preprocessor directives . a series of gpu synchronization routines provide an abstract way to copy relevant data to and from the gpu memory , for example , gpu_upload_vel ( ) and gpu_download_vel ( ) for atomic velocities . a complete list of synchronization routines is provided in the supporting information . for performance , we have implemented the entire md algorithm on the gpu , which means uploads ( to gpu memory ) are only needed at the beginning of a run and downloads ( to cpu memory ) are only needed when i / o is required , for example , downloading the coordinates to write to the trajectory file . it remains nevertheless simple to add new features to the code in a way that will work for both the cpu and gpu ( see the supporting information for an example ) . while this limits the performance for the new feature due to repeated up- and downloads , it does provide a mechanism by which new features can be easily added and tested with the gpu code before writing an additional gpu kernel . in order to achieve performance in parallel on cpus , it has always been necessary to include complex dynamic load balancing algorithms and extensive use of asynchronous communication within the implementation . this arises from the fact that the cpus are expected to perform a number of tasks above and beyond running the underlying simulation . for example , the operating systems run multiple daemons controlling i / o and various other os related tasks . in addition , the latency between any two cpus in a large parallel run can span an order of magnitude or more . as a result a downside of this is that the order of operations is not well - defined in the cpu implementation . this results in rounding differences between otherwise identical runs . due to the chaotic nature of numerically integrating newton s equations of motion , this means that two initially identical simulations will always decorrelate on time scales of a few nanoseconds or less . there is nothing inherently wrong with this ; the two simulations are just exploring two different regions of phase space . as mentioned previously , this does , however , pose a problem when one starts to routinely run simulations on the microsecond time scale since it can be necessary to return to a given point in a simulation and repeat it exactly while saving the output more frequently . . attempted to address with a bit - wise reversible integrator based on scaled integers . the architectural design of the gpu with many floating point units controlled by a single instruction unit , and the fact that the gpu is not required to time slice with os related tasks , means that work can be divided between gpu threads and indeed entire gpus in a predetermined fashion without detrimentally impacting performance . this can be exploited by careful programming and bookkeeping to ensure that the order of floating point operations is always predefined at each given time step . the gpu implementation of pmemd has been designed such that any two simulations with identical starting conditions run on the same gpu model will always be bit - wise identical . this is extremely useful for debugging purposes , for example , for detecting shared memory race conditions , and has also been used to determine that the use of ecc on gpus has little to no impact on the reliability of amber simulations . for anderson and langevin thermostatted simulations , it is necessary for the random number generator ( rng ) to also be perfectly deterministic in order for any two initially identical simulations to be reproducible . to this end , we use the parallelized rng that is implemented in the curand library and available with the cuda toolkits . in amber , all of the traditional cpu codes are written entirely using double precision ( dp ) floating point arithmetic . uses single precision ( sp ) floating point numbers throughout the calculations with the exception of a single double - precision accumulator in the reduction phase of the force accumulation . use of sp in all places within the code , however , can cause substantial instabilities in the md simulations . while this error can be hidden using tightly coupled thermostats , the true effects of such approximations have not been well characterized . we distinguish three different precision models in our gpu implementation in which the contributions to the nonbonded forces are calculated in single precision arithmetic , but bonded terms and force accumulation are in double precision ( spdp ) , or everything is computed and accumulated in single precision ( spsp ) or double precision ( dpdp ) . the exception to this is the shake algorithm ( see below ) , which is implemented in dp for all precision models since it involves calculating relative differences in distance on the order of 10 . attempting to use sp for the shake algorithm as implemented leads to numerically unstable simulations . the aim in developing our spdp precision model for the gpu implementation of pmemd was to achieve numerical stability during md simulations equivalent to that of traditional dp implementations but with performance as close to the spsp model as possible . as highlighted in the subsequent sections , the spdp model achieves these aims and for this reason is the default precision model used in the gpu implementation of pmemd , although the other precision models can be chosen if desired . our approach to the calculation of nonbonded interactions is similar to that described in friedrichs et al . having been developed at the same time and with overlapping authors but with several differences and additional optimizations , including the way in which accumulations are handled . the algorithm used for the calculation of the nonbond forces is identical for all three precision models . the pairwise interactions between atoms i and j , which can schematically be represented by a matrix as in figure 2 , are grouped together into tiles of dimension w w. the evaluation of the nonbond forces for each tile is dynamically assigned to independent warps across all of the streaming multiprocessors ( sms ) of a given gpu , which are each running a sufficient number of threads to bury operational latency . the reason for doing this is that the gpu schedules work in terms of warps which effectively perform the same mathematical operation on w values at once . in the case of nvidia , all current gpus process warps as 32-threads - wide ; in this case , optimum performance is achieved by making w = 32 . since the interaction between two atoms i and j is symmetric , only the blue diagonal tiles and the green off - diagonal tiles need to be considered for the calculation as described below . unlike the cpu code , which can just simply loop over atoms i and j , it is crucial to divide up the calculation on the gpu into as many warp size blocks as possible . however , this presents a problem when it comes to accumulating the forces for each atom since the warps can be scheduled for computation in any order . a nave accumulation of the resulting forces on each atom can thus lead to race conditions and incorrect results . the use of atomic operations within the nonbond routine to avoid this problem , however , represents a serial bottleneck that would be unacceptable in terms of performance . the approach we use is thus to allocate ( n / w ) + 1 output buffers per atom where n is the total number of atoms and w is the warp size . in the case of the nonbond force calculation , each output buffer is three double - precision - values - wide for the spdp and dpdp precision models and three single - precision - values - wide for the spsp precision model corresponding to the force in the x , y , and z directions of cartesian coordinate space . forces calculated within a tile can then be summed for each atom without fear of overwriting memory effectively providing a separation in space between tiles . at the conclusion of the force calculation , we assign one thread per atom in linear order and cycle through the output buffers , summing them to obtain the total force on each atom . schematic representation of the work - load distribution for the calculation of nonbond forces with n atoms . each square represents the interactions between two atoms i and j for which the resulting forces need to be evaluated . these are grouped together in tiles of size w w that are each assigned to an independent warp . due to symmetry , only the blue diagonal tiles and the green off - diagonal tiles need to be considered for the calculation . for details , see the text . while this approach provides a separation in space between tiles , it is still necessary to deal with race conditions within a tile . we have to distinguish on - diagonal tiles ( blue ) and off - diagonal tiles ( green ) , see figure 2 . the on - diagonal tiles include the interaction of atoms i to i + w 1 with themselves and thus include the self - interaction from the gb term . in contrast , off - diagonal tiles include the interaction of atoms i to i + w 1 with j to j + w 1 , where i j. to avoid race conditions , it is necessary to handle these two types of tile in different ways . on - diagonal tiles store two copies of the coordinates and associated parameters for each atom , one set in shared memory ( index atom i in figure 2 ) and the second set in the registers ( index atom j in figure 2 ) . each thread in the warp then runs linearly through shared memory ( atom j ) , accumulating only the force acting on the register atoms i in the corresponding force output buffer . as illustrated in figure 2 , on the first iteration , the force on atom i resulting from interacting with itself is obtained and stored in the corresponding register for i , while simultaneously the force on atom i + 1 resulting from interacting with atom i is obtained and stored in the register for atom i + 1 . similarly the forces on atoms i + 2 to i + w 1 resulting from interacting with atom i are obtained and stored in the corresponding output buffer . all threads then step to the next column to obtain the forces on atoms i to i + w 1 due to interactions with atom i + 1 etc . this approach does some excessive computational work since , for any given atom pair ij , the force on atom j is just the negative of the force on atom i , and thus only the upper or lower triangle of the tile would have to be considered . however , this is far outweighed by the advantage of avoiding race conditions that would result from several threads updating their force contribution to the same atom . in the case of off - diagonal tiles , a different approach is taken such that the symmetry in the interactions is exploited while avoiding race conditions . again , the coordinates and associated parameters of atoms labeled j to j + w 1 are placed in the registers , while the coordinates and associated parameters of atoms i to i + w 1 are placed in shared memory . if the off - diagonal tiles were handled in the same fashion as the on - diagonal tiles , on the first iteration , the force on atom j due to interacting with atom i would be calculated and from this the corresponding force on atom i obtained by negation . at the same time , the force on atom j + 1 due to interacting with atom i would be calculated and from this the corresponding force on atom i obtained by negation . thus , the forces on atom i due to all atoms j of the tile would be calculated at the same time , which would require atomic operations to accumulate correctly . the solution to this problem that is implemented in amber is to partition the off - diagonal tiles in time as illustrated in figure 2 . by starting each thread offset by its thread i d , we avoid race conditions in the accumulation and eliminate the need for performance destroying atomic operations . the gb terms are calculated in an identical fashion to the nonbond terms described above . the born radii are first calculated within their own kernel and reduced to per atom radii in an analogous fashion to how the nonbond forces are handled . the bonded and 14 terms represent a very small fraction of the computational workload ( typically < 1% of an iteration ) , and thus optimization of their calculation is not critical for performance compared with , for example , the nonbond interactions . however , efficient calculation on a gpu still requires some consideration in how these terms are calculated in order to exploit the massive parallelism within the gpu while avoiding race conditions and memory overwrites . our implementation simply creates a list of the interactions , sorted by type , and then divides up the bonded and 14 terms across sms on a per interaction basis . since the resulting forces need to be summed for each atom , there is the potential for a race condition to occur during this reduction if two or more gpu threads attempt to accumulate forces for the same atom at the same time in global memory . our initial attempts to avoid this used atomic operations for reduction of the resulting forces to individual atoms but showed very poor performance . our solution to this is to make use of the tile reduction buffers from the nonbond calculation as described above and then sum these forces in the reduction step used in the nonbond calculation . at the time of writing , these are handled in an identical fashion to the bonded interactions being calculated as a bond between an atom and a fixed virtual particle representing the reference structure . the shake implementation is currently restricted to hydrogen atoms since this represents > 99% of the types of simulation amber users run . this restriction has the benefit that each heavy atom and its attached hydrogen atoms can thus be handled independently . our approach is to simply spawn a thread for each heavy atom during the shake calculation . the use of double precision entirely within the shake routine provides sufficient precision to handle shake tolerances on the order of 10 and thus avoids complexities involved in attempting to implement shake constraints in an internal coordinate representation as would be required if shake was to be carried out in single precision . integration is carried out on the gpu in a manner analogous to that on the cpu since it is intrinsically parallel given that each atom can be handled independently . the integration is done on the gpu rather than on the cpu to negate the need for expensive copy operations of the coordinates back and forth between cpu and gpu . as mentioned above , the berendsen and langevin thermostats are applied in the same way during the coordinate update step , while the anderson thermostat is implemented as a separate kernel that randomizes the velocities at regular intervals . the reason for including the berendsen and langevin thermostats within the integration kernel is that they operate on every time step , while the anderson thermostat is typically called every few hundred time steps . in the case of the langevin thermostat , a total of 3n random numbers are required on each step , which are extracted from a pool of gpu generated random numbers , stored in global memory , which is refilled as needed using the parallel random number generator implemented within the curand library from the cuda toolkit . the same pool is used for the anderson thermostat as needed . in the interest of performance and conserving limited gpu resources , the energy is only calculated alongside the forces when needed . calculation of the energies causes a small degree of register spillage to global memory as well as additional code execution , and since one only periodically needs these values , typically only every 1 to 2 ps when writing to the output file , it is not necessary to calculate these on every iteration . additionally , this is one aspect of the algorithm that is allowed to be nondeterministic at the level of double - precision round - off error . we allow this because the energy values have no effect on the trajectory of the simulation . however , it was disturbing to observe this in action in the spsp mode where nondeterminism was initially allowed at the level of single - precision round - off error . this was harmless as the forces remained consistent , but in the interest of avoiding confusion among end - users , with regression tests showing differences for repeat runs in spsp mode , all energy summation was promoted to double - precision where differences in round - off of the energies are seen only once in approximately 10 printed interactions . the reasons for this were to maximize portability of the code and avoid hardware - induced complexities such as the fact that inter - gpu communication as implemented in cuda 4.0 requires all gpus to be on the same pcie controller . support for gpu to gpu copies in different nodes is also not yet sufficiently mature to be exploited in a widely used software package . for these reasons , the current parallel gpu scaling should be considered as a lower bound on performance that will likely improve considerably in subsequent versions of amber running on next generation hardware . unlike the cpu mpi implementation , the gpu mpi implementation is fully deterministic for a given number of nodes and gpus . at present , our gpu implementation performs significant load - balancing between the sms within each gpu rather than between gpus , which makes it possible to produce a deterministic implementation . achieving good parallel scaling on cpu clusters has always required the use of extensive load - balancing due to the noise introduced from the operating system sharing the cpu resources on a node . gpus on the other hand provide significantly more stable performance , and thus load balancing between gpus is not as critical . in the current version of the software ( amber v11 ) , the gpu parallel support for gb calculations is implemented by dividing the force calculation evenly and linearly across all gpus . for m gpus running a simulation consisting of n atoms , gpu i calculates all forces for atoms ( i 1 ) n / m + 1 to i n / m . since gb is a full o(n ) calculation , this introduces a small degree of redundancy at the gpu level with a worst - case outcome of doing twice the overall calculation , but usually much less than this as long as n m. as currently implemented , there are three stages to the calculation in parallel , each in need of synchronizing force data across all gpus . the procedure used consists of three sequential mpi_allgather operations per iteration to merge born radii , born force , and general force data . as the hardware evolves and gpus can reliably communicate directly with each other within a node , we intend to scrap the internode mpi communication and instead replace it with direct peer to peer copies between gpus . to assess both the serial and parallel performance that can be achieved with gpu accelerated amber , we ran a series of md simulations representative of typical research scenarios using the gpu and cpu implementations on a variety of hardware . the systems used consisted of partially folded trpcage ( 304 atoms ) , ubiquitin ( 1231 atoms , pdb code 1ubq ) , apo - myoglobin ( 2492 atoms ) , and nucleosome ( 25 095 atoms , pdb code 1kx5 ) . in all three simulations , the ff99sb version of the amber force field was used with a time step of 2 fs and bonds to hydrogen atoms constrained using the shake algorithm . the hawkins , cramer , and truhlar gb model ( amber input igb=1 ) was used with no cutoff applied to the nonbonded interactions and a cutoff of 15 for the calculation of the effective gb radii . the output and trajectory files were written to every 1000 steps ( 2 ps ) . the software base used for all simulations was amber version 11 , including patches 115 for ambertools and patches 117 for amber , which were released on august 18 , 2011 . the executables were built under the redhat enterprise linux 5 operating system with intel compiler version 11.1.069 , the intel mkl library version 10.1.1.019 , and the nvidia cuda compiler version 4.0 . mvapich2 version 1.5 was used for the parallel runs for both the cpu and gpu versions of the code . while the fortran compiler and use of mkl has little impact on the gpu code performance , this compiler / library combination gives the best performance for the cpu code and thus forms a fair basis when assessing the capabilities of the gpu implementation . both the cpu and gpu simulations were run on machines equipped with a supermicro x8dtg - df motherboard with dual hex - core intel x5670 processors clocked at 2.93 ghz and mellanox quadruple data rate ( qdr ) infiniband interconnects . each compute node was equipped with one gpu and one infiniband card , which were each connected to a pcie x16 slot . error - correction code ( ecc ) was switched off on the tesla cards , and the 64-bit nvidia linux driver version 275.21 was used . additionally , cpu simulations were run on the xsede trestles supercomputer at the san diego supercomputer center , whose nodes are equipped with four oct - core amd opteron 6136 processors clocked at 2.4 ghz and also interconnected with qdr infiniband . the results of the simulation timings for single gpu runs are summarized in table 2 . for small simulations such as trpcage with 304 atoms where the gpu s arithmetic hardware can not be fully utilized , the serial gpu implementation is approximately twice as fast as a current state of the art cpu node . the real advantage of the amber gpu implementation , however , becomes apparent for implicit solvent gb simulations of medium to large systems with 2500 to 25 000 atoms . for apo - myoglobin ( 2492 atoms ) , the serial gpu version of the code is significantly faster on a single gpu than a state of the art cpu node . for nucleosome , this performance gap increases dramatically such that a simulation that takes two weeks on a single desktop machine with one gpu would take nine months using the same desktop machine without gpu acceleration . for details on the hardware and software stack , the cpu code requires > 10 atoms per core , and thus the trpcage simulation was run on 24 cpu cores . the parallel performance of the gpu and cpu implementations is compared in table 3 . a throughput of up to 135.1 ns / day for apo - myoglobin and up to 3.95 ns / day for nucleosome can be achieved with eight nvidia m2090 gpus , which is a factor of 4 to 5 faster than the maximum throughput that can be achieved on a typical supercomputer given that the cpu scaling plateaus long before it reaches the performance achieved by the gpu implementation . even a single gpu is a factor of 2 to 3 faster than the cpu scaling limit . for details on the hardware and software stack , as mentioned above , the amber gpu implementation supports three different precision models . the dpdp model is logically equivalent to the traditional dp approach used on the cpu . however , the use of dp on gpus can be more detrimental to performance than its use on cpus . therefore , the desire to achieve high performance prompts for use of sp where possible . it is critical though to use sp carefully in order not to detrimentally affect the accuracy of the md . for this reason , we designed our hybrid precision spdp model to achieve performance very close to that achievable with sp but in a manner that does not impact accuracy . while we recommend using the spdp precision model , it is instructive to compare the performance of the different precision models available in the amber gpu implementation . as shown in table 4 , the single precision ( spsp ) model achieves the highest performance as expected but , as highlighted in section 5 , at the cost of accuracy . our hybrid spdp precision model , however , achieves a performance of greater than 75% of the spsp precision model with accuracy comparable to that of the full double precision ( dpdp ) model , which is between 4 to 7 times slower . for details on the hardware and software stack , a critical and often overlooked aspect of any major change to a widely used scientific software package is a detailed validation of any and all new approximations made . in the case of the amber gpu implementation the first is a detailed testing of the code itself to ensure that it correctly simulates the existing systems and does not introduce any new bugs or contain critical logic errors . this is the key reason why we implemented a full double precision ( dpdp ) model within the gpu code . this precision model matches the cpu code to machine precision and thus can be used to check that the gpu code is indeed giving the correct answers . this allowed us to take the cpu - based regression tests that we have used for many years to validate an amber installation and run them using the gpu code and obtain answers to the limits of machine precision . more complex , however , is the second category for validation which is the careful testing of our hybrid ( spdp ) precision model . this is significantly more complex since it requires a careful evaluation of any subtle differences in the dynamics as well as ensemble properties from converged simulations run in dpdp and spdp precision . in this section , we attempt to extensively validate our spdp model comparing a number of key observables across all three , dpdp , spdp , and spsp , precision models . the first metric tested was the effect of the numerical precision model on the force calculations as compared to a reference precision model , in this case , the result from the cpu implementation . we are using the starting structures of the test systems described in section 4 above . the dpdp model matches the reference forces very closely with maximum deviations not exceeding 10 kcal/(mol ) and rms deviations not exceeding 10 kcal/(mol ) , even for systems as large as nucleosome . these deviations are entirely due to the different order of execution of the floating point operations in the cpu and gpu implementation . the dpdp gpu implementation of pmemd will thus generate trajectories of precision equivalent to the cpu implementation . the forces obtained from the spsp model , however , show very large deviations from the reference values on the order of up to 10 kcal/(mol ) for the largest system studied . such large deviations introduce significant numerical noise into the simulations , and it is reasonable to expect that this may render the results of md simulations questionable . calculating the force contributions in sp and accumulating them in dp , however , leads to an improvement of more than 1 order of magnitude in all cases for the spdp model as compared to the spsp model . in the following section , we will indeed show that the forces obtained from the spsp model are not precise enough to conserve energy in biomolecular md simulations and can lead to instabilities in long time scale md simulations , while the spdp model is sufficient for this purpose . one of the most important gauges for judging the precision of md software is its numerical stability , which is reflected in its ability to conserve the constants of motion , in particular the energy . to this end , we have performed constant energy md simulations of the first three test systems described in section 4 above . we collected data for 100 ns ( trpcage ) , 50 ns ( ubiquitin ) , and 20 ns ( apo - myoglobin ) after an initial equilibration for 1 ns at 300 k using langevin dynamics . in addition to simulations using a time step of 2 fs with bonds to hydrogen atoms constrained using the shake algorithm with a relative geometrical tolerance of 10 , we have run simulations using time steps of 0.5 and 1.0 fs without constraints . the energy drifts along the trajectories are summarized in table 6 , while figure 3 shows a plot of the total energy for the trajectories of trpcage and ubiquitin for the different precision models at a time step of 0.5 fs . the corresponding plot for apo - myoglobin is very similar to that for ubiquitin and can be found in the supporting information along with plots for the larger time steps . the plots underline that it is important to validate the precision models for trajectories that are long enough to uncover numerical instabilities . while the spsp model seems to perform reasonably well for a trajectory of 1 ns length , in particular for smaller systems like trpcage , it becomes apparent that the errors introduced lead to unacceptably large energy drifts in the long term . apart from the magnitude in the energy drift , the plots for the other time steps show a similar behavior ( see supporting information ) . table 6 shows that the spdp model is able to conserve energy to the same degree as the full double precision model ( dpdp ) and the reference cpu implementation . the spsp model , on the other hand , shows energy drifts that are 1 to 2 orders of magnitude larger and , on the time scales investigated , leads to completely unphysical results . while the cpu implementation and the gpu implementations with the full double precision model ( dpdp ) and mixed precision model ( spdp ) achieve excellent energy conservation on the order of 10 kt / ns / dof , where dof means degree of freedom , for simulations with a time step of 0.5 fs , the energy drift for the spsp model remains as large as with a time step of 1.0 fs . in addition , the energy drift for the spsp model is not constant and in general increases significantly along the trajectory as the temperature increases , which eventually leads to unstable simulations . total energy ( kcal / mol ) along constant energy trajectories using a time step of 0.5 fs without constraints . shown are results for trpcage ( left ) and ubiquitin ( right ) for different precision models . the shake algorithm to constrain bond lengths to hydrogen atoms was used for a time step of 2.0 fs ; no constraints were used for smaller time steps . a dash indicates that the system heated up extremely during the simulation to the point that it is meaningless to report an energy drift . an asterisk indicates that the energy drift increases dramatically for longer time scales . by way of comparison to other gpu implementations of md , the closest published constant energy gb simulation is that of the repressor ( 1254 atoms ) using openmm . simulations of this system at a time step of 1.0 fs over 1 ns of simulation time without constraints using the gpu implementation of openmm gave energy drifts of 0.0054 kt / ns / dof ( ati hardware ) ( table 3 of friedrichs et al . ) compared with our values of 0.000006 kt / ns / dof ( dpdp ) , 0.000030 kt / ns / dof ( spdp ) , and 0.000305 kt / ns / dof ( spsp ) for the similarly sized ubiquitin system ( 1231 atoms ) . it is also interesting to compare the energy conservation achieved with the amber gpu implementation using the typical simulation settings that one would use in a production md simulation with those published for the openmm implementation . most amber simulations would use shake constraints and a 2 fs time step with the spdp precision model , which for ubiquitin gives an absolute energy drift of 0.000165 kt / ns / dof . this compares extremely favorably with the drift of 0.0060 ( nvidia hardware ) and 0.0558 ( ati hardware ) for openmm . the energy conservation of the amber gpu implementation looks even better when one considers that the openmm constraint numbers were obtained with a smaller time step of 1 fs . on the molecular level , protein dynamics are at the heart of biological function . md simulations that are able to accurately describe protein motions aid in understanding the multitude of functions that proteins carry out and can be used to interpret and predict experimental results that are related to its structural and dynamic properties , for example , nmr spectroscopic parameters such as spin relaxation or residual dipolar couplings . in order to scrutinize the reliability of our implementation for protein dynamics , we present results of md simulations of ubiquitin with the different accuracy models of our gpu implementation and compare these to trajectories obtained with the cpu implementation which serves as a reference . we focus on root - mean - square deviations ( rmsds ) and root - mean - square fluctuations ( rmsfs ) of the c backbone carbon atoms with respect to the crystal structure ( pdb code 1ubq(53,54 ) ) . the highly flexible end tail of ubiquitin ( residues 71 to 76 ) was excluded from our analysis . results for the radius of gyration rg of ubiquitin can be found in the supporting information . although the use of a thermostat will to some extent cover up numerical noise introduced by numerical inaccuracies in the implementation , we are analyzing constant temperature simulations here since typical biomolecular amber simulations are generally run with some form of temperature control . in order to obtain statistically meaningful results , 50 independent md trajectories each of 100 ns length were generated at 300 k both for the cpu implementation and for each of the precision models of the gpu implementation . the ff99sb force field was used for all simulations with a time step of 2 fs and bonds to hydrogen atoms constrained using the shake algorithm with a relative geometrical tolerance of 10 . ( amber input igb=5 ) was used with no cutoff applied to the nonbonded interactions and a cutoff of 15 for the calculation of the effective gb radii . the output and trajectory files were written to every 1000 steps ( 2 ps ) . temperature control during production runs was achieved with the berendsen weak coupling algorithm using a time constant of t = 10.0 ps for the heat bath coupling . the initial coordinates and velocities that form the starting point of the 50 trajectories were generated using the cpu implementation as follows . after energy minimization for 2000 steps followed by heating and an initial equilibration for 1 ns at 300 k using langevin dynamics with a collision frequency of = 1.0 ps , snapshots were extracted at time intervals of 4 ns from a constant temperature md simulation at 300 k. each of these snapshots was then assigned random velocities corresponding to a temperature of 10 k followed by heating and equilibration to 300 k using langevin dynamics for 50 ps and removal of any center of mass motion that may have been introduced . using the cpu generated restart files in all cases guarantees that any numerical differences observed between the various implementations can be traced back to the numerical precision of the implementation and is not an artifact of different initial conditions . the plot of the rmsd values vs time ( figure 4 ) clearly shows the stability of the md simulations using the cpu implementation and the gpu implementation with the full double precision model ( dpdp ) and the mixed single / double precision model ( spdp ) . the value of rg also remains constant throughout all simulations of ubiquitin using the cpu and gpu dpdp and spdp versions of the code ( see supporting information ) , indicating that the relative compactness of the protein is maintained . the rg value stays around 11 to 12 , typical for proteins of this size , assuming a spherical conformation , and are in good agreement with the rg reported for other native - state simulations of ubiquitin . the rmsf values for each residue for the 50 native - state simulations are shown in figure 5 . the data show an excellent agreement among the cpu implementation and the gpu implementation using both the dpdp and the spdp precision model with the majority of the residues remaining within 2 to 3 of their positions in the experimental structure and somewhat larger fluctuations around residue numbers 33 , 48 , and 63 . on the time scale investigated , no major structural change is taking place during the simulation , confirming that the protein remains within a native - state ensemble . root - mean - square deviations ( rmsds ) of the c backbone carbon atoms of ubiquitin ( excluding the flexible tail , residues 7176 ) with respect to the crystal structure for 50 independent trajectories as obtained with the cpu implementation and the gpu implementation of pmemd using different precision models . root - mean - square fluctuations ( rmsfs ) of the c backbone carbon atoms of ubiquitin residues 7176 with respect to the crystal structure for 50 independent trajectories of 100 ns length as obtained with the cpu implementation and the gpu implementation of pmemd using different precision models . the situation is very different for the spsp precision model of the gpu implementation . while approximately half of the trajectories show a similar behavior and stability as the trajectories obtained with the other precision models and the reference cpu implementation of pmemd , the remaining trajectories experience a very large increase in the rmsd values ( figure 4 ) and a corresponding increase in the rmsf values ( figure 5 ) . the reason for this behavior is that the numerical noise introduced in the simulations due to rounding errors can lead to an accumulation of errors such that ubiquitin starts to unfold . this is accompanied by an increase in rg ( see supporting information ) and is also reflected in the large rmsf values of the residues close to the termini of the protein . since it can not be predicted whether a simulation using the spsp precision model will be stable or not , no judgment can be made whether the results obtained are meaningful . the gpu implementation of pmemd should thus only be used with the dpdp and the spdp precision models . on the basis of the results shown here , we recommend the use of the mixed - precison spdp model since the numerical results obtained are comparable with those obtained with the full double precision dpdp model but at significantly reduced computational cost . we presented a framework for the acceleration of classical molecular dynamics simulations on graphical processing units within the pmemd program of the software package amber . in this contribution , we described the efficient and highly precise implementation of implicit solvent generalized born molecular dynamics simulations realizing performance that exceeds that achievable on any current conventional supercomputer . the implementation supports serial runs on a single nvidia gpu or parallel runs across multiple gpus using mpi . performance for moderately sized systems can exceed 100 ns per day , making microsecond or longer simulations routine on deskside workstations . for large systems , the performance differential between our gpu code and the cpu implementation increases significantly . a gb simulation of the nucleosome running on just one nvidia m2090 gpu completes in two weeks what a current high - end workstation would take nine months to run using cpus . this massive performance increase for minimal investment in hardware will transform the traditional molecular dynamics workflow , providing a platform for sustained innovation . the implementation is transparent to the end user and does not require any specialized knowledge beyond that required to run the traditional cpu code . our gpu implementation supports the majority of the features provided by the cpu implementation including the amber and charmm families of biomolecular force fields . we have developed a hybrid precision model for floating point arithmetic termed spdp that provides performance close to full single - precision calculations but retains energy drifts in constant energy md simulations and structural properties of proteins for long time scale md simulations equivalent to what is achievable with full double precision . energy conservation with our hybrid spdp model is approximately 1 to 2 orders of magnitude better than what has been published for other similar gpu implementations of molecular dynamics . simulation size is limited by the memory available on gpu cards , which at the time of writing is at maximum 6 gb corresponding to approximately 54 000 atoms . the current cost benefits of gpus are enticing , and this is driving both soft- and hardware development at a rapid pace . in a few short years , gpu - based md codes have evolved from proof - of - concept prototypes to the production level amber gpu implementation we described here . with gpus becoming ubiquitous in workstations and also as accelerators in high - performance computing ( hpc ) platforms , the impact of our implementation on the field of molecular dynamics is broad and transformative . a gpu accelerated implementation of the particle mesh ewald ( pme ) algorithm for explicit solvent calculations is also available in amber 11 as described in another publication . this , along with future support for advanced features including accelerated md ( amd ) , umbrella sampling , replica exchange md ( remd ) , thermodynamic integration ( ti ) , polarizable force fields , and constant ph simulations means the use of gpus is a viable alternative to traditional supercomputers for classical molecular dynamics simulations .

we present an implementation of generalized born implicit solvent all - atom classical molecular dynamics ( md ) within the amber program package that runs entirely on cuda enabled nvidia graphics processing units ( gpus ) . we discuss the algorithms that are used to exploit the processing power of the gpus and show the performance that can be achieved in comparison to simulations on conventional cpu clusters . the implementation supports three different precision models in which the contributions to the forces are calculated in single precision floating point arithmetic but accumulated in double precision ( spdp ) , or everything is computed in single precision ( spsp ) or double precision ( dpdp ) . in addition to performance , we have focused on understanding the implications of the different precision models on the outcome of implicit solvent md simulations . we show results for a range of tests including the accuracy of single point force evaluations and energy conservation as well as structural properties pertainining to protein dynamics . the numerical noise due to rounding errors within the spsp precision model is sufficiently large to lead to an accumulation of errors which can result in unphysical trajectories for long time scale simulations . we recommend the use of the mixed - precision spdp model since the numerical results obtained are comparable with those of the full double precision dpdp model and the reference double precision cpu implementation but at significantly reduced computational cost . our implementation provides performance for gb simulations on a single desktop that is on par with , and in some cases exceeds , that of traditional supercomputers .

Introduction GPU Programming Complexities Overview of the AMBER Implicit Solvent GPU Implementation Performance Validation Conclusions and outlook

PMC3560507

whole body vibration ( wbv ) has received a great deal of attention with reported increases in muscular strength [ 15 ] , improvements in body composition , changes in muscle performance and balance [ 710 ] , increases in lower back and hamstring flexibility , improvements in human athletic performance such as improvement in jumping and running parameters , and changes in peripheral circulation . standing on a platform , vibrating at 26 hz for 9 minutes , increased relative moving blood volume of the quadriceps and gastrocnemius muscles and popliteal artery mean blood flow with a decrease of flow resistance . ( 2007 ) documented significant transient increases in skin blood flow due to a short duration ( 3 minutes ) of high - amplitude ( 56 mm ) , high - frequency vibration ( 30 hz ) mechanically delivered to the posterior calf muscles with the subjects in a relaxed supine position . a follow up study focusing on passive vibration applied to the forearm muscles demonstrated that both 30 hz and 50 hz vibration significantly increased skin blood flow ; however , 50 hz was more efficient and gains lasted during the entire 15 minute recovery period . the authors found that blood flow peaked at 5 minutes ; with no added benefits from an additional 5 minutes of vibration application . ( 2008 ) reported that increases in skin temperature occur after 8 minutes of lower extremity vibration exercise . significant increases in skin blood flow were also reported after 12 minutes of vibration exercise . both skin temperature and skin blood flow remained elevated during the 10 minute recovery period . the vast majority of wbv studies have involved vertically directed mechanical stimuli ( 25 to 50hz ) in standing or combined with vibration exercise . for this study , we have coined the term active vibration for this form of vibration intervention . one vascular study applied vertical vibration in the supine position ( head - to - toe whole body acceleration ) using a lower frequency ( 2 hz ) . to date , there have been a small number of studies that have examined the effects of wbv on circulation with the subject resting and the target area receiving horizontal vibration . the mechanical sinusoidal vibration was delivered at a right angle to the long bones of the extremity , blood vessels , and muscle fibers . for this study , we have coined the term passive vibration for horizontally delivered vibration to specific body regions in the relaxed subject because the subject is not receiving vibration to the whole body nor are they active in the process . the active process is when the subject stands on the platform , holding the safety handles , and is not relaxed . the active process involves whole body weight bearing while the passive process is not whole body weight bearing . impaired autonomic and endothelial function is attributed to the aging and disease processes such as diabetes mellitus . circulation plays an essential role in maintaining healthy integumentary system function and promotion of tissue healing . local heating , such as moist heat hydrocollator packs ( hot packs ) and warm water immersions increase circulation in healthy individuals . increased blood flow has been shown to increase tissue nutrients , decrease susceptibility to infection , improve wound healing , and facilitate soft tissue repair [ 2123 ] . it is well established that when heat is applied to the skin there is an increase in skin blood flow [ 2426 ] . initially , after the application of local heat , tactile neurons in the skin release substance p and calcitonin gene related peptide which causes an increase in potassium permeability in vascular smooth muscle surrounding the endothelial cell [ 24,2729 ] . but this response only lasts a few minutes . the sustained response to increasing temperature in the skin is mediated by transient receptor potential vanilloid-4 ( trpv-4 ) voltage gated calcium channels in the vascular endothelial cells . above a temperature of 35c , these cells cause an exponential increase in calcium influx into the endothelial cell from the interstitial space . nitric oxide , a potent vasodilator , diffuses into the surrounding smooth muscle activating cyclic gmp which in turn increases potassium permeability and relaxes vascular smooth muscle [ 28,29,3234 ] . moist heat is more effective than dry heat at raising tissue temperature , but moist heat does have a greater risk of causing a skin burn . this is especially true in individuals with compromised circulation who would benefit most from modalities that increase skin circulation . to achieve these therapeutic benefits , moist heat must be applied for a minimum of 10 minutes . according to prentice ( 2002 ) , it takes approximately 10 minutes of superficial therapeutic moist heat to reach a maximum heating depth of up to 1 cm below the skin . the purpose of our study is to determine the effects of short - term exposure to vibration versus moist heat ( alone and combined ) on lower extremity skin blood flow and skin temperature . our hypothesis is that skin blood flow will increase following short - term , low frequency vibration under the treatment site for all interventions except for active vibration ( vibration exercise ) as compared to the control intervention . we hypothesize skin blood flow and skin temperature at the calf will rank as following ( most to least ) : 1 ) passive vibration combined with moist heat , 2 ) moist heat only , 3 ) passive vibration only , 4 ) commercial massaging ( vibrating ) heating pad , 5 ) active vibration , and 6 ) control . furthermore , it is hypothesized that with the combination of moist heat and vibration , local skin temperature will increase at a slower rate than with moist heat alone due to increased circulation and decreased skin impedance provided by passive vibration . the subjects included 10 healthy adult volunteers aged 2030 years ( mean = 25.7 years ) from the loma linda university community . five females ( age = 25.84.1 years ; weight = 53.8 6.1 kg . ; height = 160.63.2 cm ) and five males ( age = 25.61.5 years ; weight = 67.811.3 kg ; height = 168.98.7 cm ) participated in the study . the subjects received the following interventions on three separate days to the posterior calf region : intervention 1 active vibration ( vibration exercise ) only , intervention 2 passive vibration only , intervention 3 moist heat only , intervention 4 passive vibration combined with moist heat , intervention 5 a commercially marketed , over the counter massaging heating pad , and intervention 6 the control intervention ( no intervention ) . the institutional review board at loma linda university approved all of the procedures and all subjects signed a statement of informed consent . all subjects were free of neurological , orthopedic , and circulatory disorders ( e.g. , deep vein thrombophlebitis , bleeding disorders ) that which would have limited their participation in the study . pregnant subjects and subjects with pacemakers or skin ulcers were also excluded from this study . skin blood flow was measured by a laser doppler flow meter ( figures 1 and 2 ) produced by biopac , inc . the device was secured with hypo - allergic tape to the middle of the gastrocnemius muscle belly . the units of blood flow stated in the results are in flux units , the measure of flow generated by laser doppler systems . the thermoster was placed within 2 cm of the doppler probe ( figures 1 and 2 ) . the mechanical stimulation of wbv was provided by a power plate for five of the subjects . the power plate is a vibration platform ( power plate north america , llc , culver city , california , usa ) , as shown in figure 3 , with frequency settings ranging from 3050 hz . the power plate has an option for either low amplitude ( 23 mm ) or high amplitude ( 56 mm ) plate oscillations . a frequency of 4050 hz with low or high amplitude is recommended for massage and non - weight bearing activities . for this study , the vibration parameters were set at a frequency of 50 hz , an amplitude of a0=56 mm ( high amplitude ) , and therefore a peak acceleration of approximately 62.40 m / s ( 6.24 g ) . the other five subjects received vibration interventions from a similar vibration platform , the globus physioplate , using the same settings . a 2 cm rubber pad was used to reduce friction to the skin during massage related vibration techniques . this also reduces the amplitude by approximately 12 mm ; however , frequency is unchanged . moist heat was supplied by a commercial hydrocollator heat pack from an approximately 160f water tank with a heating unit . the hydrocollator pack is a canvas - covered pouch , filled with hydrophilic substances such as petroleum distillate or bentonite that provides superficial therapeutic moist heat up to 1 cm below the skin . it takes approximately 10 minutes for the maximum depth of 1 cm tissue heating to occur . according to prentice ( 2002 ) , the therapeutic temperature range is approximately 86 to 104f . the canvas cover is used with 68 layers of dry towels between the subject s skin and the hydrocollator pack to avoid skin burns . , the weight of the leg is relatively low so the patient s calf was placed on top of the hydrocollator pack . as an example , a 90 kg - male - subject s lower leg will produce approximately 5.44 kg of force over a surface area of approximately 24 cm or 0.23 kg / cm . this commercial heating pad product , powered by 120-volt ac , provides a massaging ( vibration ) heat with either a dry or moist heat option . to create the moist heat option for this study , the product sponge was saturated in warm water and then gently wrung out before attaching the sponge under the cloth heating pad cover . both the heat and massage ( vibration ) settings were set to the vibration characteristics are that the unit runs at 35 watts and at 0.3 amps at 120 volts . the temperature at the surface of this product ranged from 90.7 to 93.6f after 10 minutes of use at the high settings of both massage and heat . this temperature range is at the lower half of the therapeutic temperature range of 86 to 104f . subjects who meet the inclusion / exclusion criteria were given an invitation to participate in this study and signed an informed consent document prior to participation . the subjects were led into an environmental room where room temperature was regulated between 72 to 75f . the room was pre - warmed to 7275f for at least 30 minutes so that all tables and walls temperatures were constant . the skin was marked with skin - safe ink on the muscle belly of the gastrocnemius muscles to insure that the doppler flow readings were measured at the same location on all three test days . the subjects rested comfortably for a ten - minute rest period prior to doppler testing . following the initial rest period and without repositioning the subject , a homan s sign test was performed to help rule out the possibility of a deep vein thrombosis . the subject s ankle was passively dorsiflexed while assessing for pain in the posterior aspect of the calf . pallor , swelling , tenderness , and warmth to palpation of the posterior lower limb were also assessed bilaterally . these signs and symptoms are indicative of a possible deep vein thrombosis , thromboembolism , or superficial thrombophlebitis ; which are contraindications for wbv . a positive finding of any of the above procedures served as exclusion from this study . the subjects were then repositioned to the supine position with their calf muscles elevated approximately 10 cm . a baseline doppler skin blood flow and skin temperature readings were taken in this position . the subjects received two of the following interventions for 10 minutes each , per day , over a three day period : 1 ) standing on power plate ( active vibration ) , 2 ) a passive calf massage with the calf resting on the power plate or globus physioplate ( passive vibration ) , 3 ) moist heat , 4 ) a combination of passive vibration and moist heat , 5 ) sunbeam massaging heating pad , and 6 ) no intervention , resting in supine ( control ) . an intervention was applied to the right and left posterior calf each day ( at approximately the same time of day ) to allow for all six interventions to be tested in a three day period without retesting the same extremity on any one day . refer to table 1 for a description of the six interventions . during the heat interventions , if the subject became too warm anytime during the 10-minute intervention or if the skin became excessively blotchy at the 5-minute inspection , an additional layer of toweling was added . once the therapeutic intervention was completed , the subject s skin blood flow and temperatures were recorded for an additional nine minutes . during this time period the posterior calf was elevated approximately 2 cm above the platform ( a small bolster under the subject s heel ) to prevent skin contact with the residual warmth from the heating modality on the surface of the platform . this procedure assured avoidance of continued heating of the skin during the nine post - intervention assessments . friedman s anova was used to test for changes in temperature and blood flow between the different modalities . the subjects included 10 healthy adult volunteers aged 2030 years ( mean = 25.7 years ) from the loma linda university community . five females ( age = 25.84.1 years ; weight = 53.8 6.1 kg . ; height = 160.63.2 cm ) and five males ( age = 25.61.5 years ; weight = 67.811.3 kg ; height = 168.98.7 cm ) participated in the study . the subjects received the following interventions on three separate days to the posterior calf region : intervention 1 active vibration ( vibration exercise ) only , intervention 2 passive vibration only , intervention 3 moist heat only , intervention 4 passive vibration combined with moist heat , intervention 5 a commercially marketed , over the counter massaging heating pad , and intervention 6 the control intervention ( no intervention ) . the institutional review board at loma linda university approved all of the procedures and all subjects signed a statement of informed consent . all subjects were free of neurological , orthopedic , and circulatory disorders ( e.g. , deep vein thrombophlebitis , bleeding disorders ) that which would have limited their participation in the study . pregnant subjects and subjects with pacemakers or skin ulcers were also excluded from this study . skin blood flow was measured by a laser doppler flow meter ( figures 1 and 2 ) produced by biopac , inc . ( goleta , california ) . the device was secured with hypo - allergic tape to the middle of the gastrocnemius muscle belly . the units of blood flow stated in the results are in flux units , the measure of flow generated by laser doppler systems . the thermoster was placed within 2 cm of the doppler probe ( figures 1 and 2 ) . the mechanical stimulation of wbv was provided by a power plate for five of the subjects . the power plate is a vibration platform ( power plate north america , llc , culver city , california , usa ) , as shown in figure 3 , with frequency settings ranging from 3050 hz . the power plate has an option for either low amplitude ( 23 mm ) or high amplitude ( 56 mm ) plate oscillations . a frequency of 4050 hz with low or high amplitude is recommended for massage and non - weight bearing activities . for this study , the vibration parameters were set at a frequency of 50 hz , an amplitude of a0=56 mm ( high amplitude ) , and therefore a peak acceleration of approximately 62.40 m / s ( 6.24 g ) . the other five subjects received vibration interventions from a similar vibration platform , the globus physioplate , using the same settings . a 2 cm rubber pad was used to reduce friction to the skin during massage related vibration techniques . this also reduces the amplitude by approximately 12 mm ; however , frequency is unchanged . moist heat was supplied by a commercial hydrocollator heat pack from an approximately 160f water tank with a heating unit . the hydrocollator pack is a canvas - covered pouch , filled with hydrophilic substances such as petroleum distillate or bentonite that provides superficial therapeutic moist heat up to 1 cm below the skin . it takes approximately 10 minutes for the maximum depth of 1 cm tissue heating to occur . according to prentice ( 2002 ) , the therapeutic temperature range is approximately 86 to 104f . the canvas cover is used with 68 layers of dry towels between the subject s skin and the hydrocollator pack to avoid skin burns . , the weight of the leg is relatively low so the patient s calf was placed on top of the hydrocollator pack . as an example , a 90 kg - male - subject s lower leg will produce approximately 5.44 kg of force over a surface area of approximately 24 cm or 0.23 kg / cm . this commercial heating pad product , powered by 120-volt ac , provides a massaging ( vibration ) heat with either a dry or moist heat option . to create the moist heat option for this study , the product sponge was saturated in warm water and then gently wrung out before attaching the sponge under the cloth heating pad cover . both the heat and massage ( vibration ) settings were set to the vibration characteristics are that the unit runs at 35 watts and at 0.3 amps at 120 volts . the temperature at the surface of this product ranged from 90.7 to 93.6f after 10 minutes of use at the high settings of both massage and heat . this temperature range is at the lower half of the therapeutic temperature range of 86 to 104f . subjects who meet the inclusion / exclusion criteria were given an invitation to participate in this study and signed an informed consent document prior to participation . the subjects were led into an environmental room where room temperature was regulated between 72 to 75f . the room was pre - warmed to 7275f for at least 30 minutes so that all tables and walls temperatures were constant . the skin was marked with skin - safe ink on the muscle belly of the gastrocnemius muscles to insure that the doppler flow readings were measured at the same location on all three test days . the subjects rested comfortably for a ten - minute rest period prior to doppler testing . following the initial rest period and without repositioning the subject , a homan s sign test was performed to help rule out the possibility of a deep vein thrombosis . the subject s ankle was passively dorsiflexed while assessing for pain in the posterior aspect of the calf . pallor , swelling , tenderness , and warmth to palpation of the posterior lower limb were also assessed bilaterally . these signs and symptoms are indicative of a possible deep vein thrombosis , thromboembolism , or superficial thrombophlebitis ; which are contraindications for wbv . a positive finding of any of the above procedures served as exclusion from this study . the subjects were then repositioned to the supine position with their calf muscles elevated approximately 10 cm . a baseline doppler skin blood flow and skin temperature readings were taken in this position . the subjects received two of the following interventions for 10 minutes each , per day , over a three day period : 1 ) standing on power plate ( active vibration ) , 2 ) a passive calf massage with the calf resting on the power plate or globus physioplate ( passive vibration ) , 3 ) moist heat , 4 ) a combination of passive vibration and moist heat , 5 ) sunbeam massaging heating pad , and 6 ) no intervention , resting in supine ( control ) . an intervention was applied to the right and left posterior calf each day ( at approximately the same time of day ) to allow for all six interventions to be tested in a three day period without retesting the same extremity on any one day . refer to table 1 for a description of the six interventions . during the heat interventions , if the subject became too warm anytime during the 10-minute intervention or if the skin became excessively blotchy at the 5-minute inspection , an additional layer of toweling was added . once the therapeutic intervention was completed , the subject s skin blood flow and temperatures were recorded for an additional nine minutes . during this time period the posterior calf was elevated approximately 2 cm above the platform ( a small bolster under the subject s heel ) to prevent skin contact with the residual warmth from the heating modality on the surface of the platform . this procedure assured avoidance of continued heating of the skin during the nine post - intervention assessments . friedman s anova was used to test for changes in temperature and blood flow between the different modalities . changes in mean skin blood flow across time during the different modalities are represented in figure 4 and tables 24 . for the passive vibration modality , there was a significant increase in mean skin blood flow from baseline to 9 minutes post - intervention ( 88.316.9 flux vs. 150.540.4 flux ) ( p=0.01 ) . this increase in skin blood flow was also significant from the start of the intervention to 9 minutes post - intervention ( 96.652.5 flux vs. 150.540.4 flux ) ( p=0.03 ) . at the end of the intervention , mean skin blood flow was significantly higher in the moist heat and passive vibration combination intervention than compared to the sunbeam ( commercial massaging heating pad ) intervention ( 163.9 flux 71.8 vs. 85.028.6 flux);(p=.01 ) , control ( 163.9 flux 71.8 vs. 90.056.8 flux ) ; ( p=.04 ) , and active vibration ( 163.9 flux 71.8 vs. 84.340.8 flux ) ; ( p=.04 ) . the moist heat and passive vibration combination intervention had significantly higher skin blood flows than both the sunbeam intervention ( 133.746.1 flux vs. 73.018.7 flux ) ; ( p=.01 ) and the control intervention ( 133.746.1 flux vs. 96.739.1 flux ) ; ( p=.04 ) . in addition , at 9 minutes post - intervention , mean skin blood flow was significantly higher in the passive vibration intervention than the moist heat intervention ( 150.540.4 flux vs. 135.295.1 flux);(p=.04 ) and sunbeam intervention ( 150.540.4 flux vs. 73.018.7 flux ) ; ( p=.01 ) . changes in mean skin temperature across time during the different interventions are represented in figure 5 and tables 57 . in the passive vibration intervention , there was a significant increase in mean skin temperature from baseline to the end of the intervention ( 95.42.7 deg f vs. 97.02.8 deg f ) ; ( p=.02 ) and from baseline to 9 minutes post intervention ( 95.42.7 deg f vs. 96.63.3 deg f ) ; ( p=.05 ) . this increase in skin temperature was also significant from start of the intervention to the end of the intervention ( 95.73.1 deg f vs. 97.02.8 deg f ) ; ( p=.01 ) . in the moist heat & passive vibration combination intervention , mean skin temperature at the end of the intervention ( 96.44.7 deg f ) and 9 minutes post - intervention ( 95.94.6 deg f ) were significantly higher than mean baseline skin temperature ( 94.04.6 deg f ) ; ( p=.01 ; p=.01 respectively ) . mean skin temperature was significantly higher in the moist heat intervention at the end of the intervention compared to sunbeam intervention ( 97.45.3 deg f vs. 95.24.6 deg f ) ; ( p=0.05 ) and active vibration interventions ( 97.45.3 deg f vs. 95.13.8 deg f ) ; ( p=.03 ) . these differences remained significant at 9 minutes post - intervention with the moist heat intervention s mean skin temperature being significantly higher than sunbeam intervention ( 97.25.5 deg f vs. 95.05.2 deg f ) ; ( p=.02 ) and active vibration intervention ( 97.25.5 deg f vs. 95.04.2 deg f ) ; ( p=.02 ) . changes in mean skin blood flow across time during the different modalities are represented in figure 4 and tables 24 . for the passive vibration modality , there was a significant increase in mean skin blood flow from baseline to 9 minutes post - intervention ( 88.316.9 flux vs. 150.540.4 flux ) ( p=0.01 ) . this increase in skin blood flow was also significant from the start of the intervention to 9 minutes post - intervention ( 96.652.5 flux vs. 150.540.4 flux ) ( p=0.03 ) . at the end of the intervention , mean skin blood flow was significantly higher in the moist heat and passive vibration combination intervention than compared to the sunbeam ( commercial massaging heating pad ) intervention ( 163.9 flux 71.8 vs. 85.028.6 flux);(p=.01 ) , control ( 163.9 flux 71.8 vs. 90.056.8 flux ) ; ( p=.04 ) , and active vibration ( 163.9 flux 71.8 vs. 84.340.8 flux ) ; ( p=.04 ) . the moist heat and passive vibration combination intervention had significantly higher skin blood flows than both the sunbeam intervention ( 133.746.1 flux vs. 73.018.7 flux ) ; ( p=.01 ) and the control intervention ( 133.746.1 flux vs. 96.739.1 flux ) ; ( p=.04 ) . in addition , at 9 minutes post - intervention , mean skin blood flow was significantly higher in the passive vibration intervention than the moist heat intervention ( 150.540.4 flux vs. 135.295.1 flux);(p=.04 ) and sunbeam intervention ( 150.540.4 flux vs. 73.018.7 flux ) ; ( p=.01 ) . changes in mean skin temperature across time during the different interventions are represented in figure 5 and tables 57 . in the passive vibration intervention , there was a significant increase in mean skin temperature from baseline to the end of the intervention ( 95.42.7 deg f vs. 97.02.8 deg f ) ; ( p=.02 ) and from baseline to 9 minutes post intervention ( 95.42.7 deg f vs. 96.63.3 deg f ) ; ( p=.05 ) . this increase in skin temperature was also significant from start of the intervention to the end of the intervention ( 95.73.1 deg f vs. 97.02.8 deg f ) ; ( p=.01 ) . in the moist heat & passive vibration combination intervention , mean skin temperature at the end of the intervention ( 96.44.7 deg f ) and 9 minutes post - intervention ( 95.94.6 deg f ) were significantly higher than mean baseline skin temperature ( 94.04.6 deg f ) ; ( p=.01 ; p=.01 respectively ) . mean skin temperature was significantly higher in the moist heat intervention at the end of the intervention compared to sunbeam intervention ( 97.45.3 deg f vs. 95.24.6 deg f ) ; ( p=0.05 ) and active vibration interventions ( 97.45.3 deg f vs. 95.13.8 deg f ) ; ( p=.03 ) . these differences remained significant at 9 minutes post - intervention with the moist heat intervention s mean skin temperature being significantly higher than sunbeam intervention ( 97.25.5 deg f vs. 95.05.2 deg f ) ; ( p=.02 ) and active vibration intervention ( 97.25.5 deg f vs. 95.04.2 deg f ) ; ( p=.02 ) . we hypothesized that local skin blood flow would increase while skin temperature would increase less with the combination of moist heat and passive vibration as compared to moist heat alone . after the ten - minute interventions , skin blood flow was significantly greater with the combination of passive vibration and moist heat as compared to active vibration , sunbeam massaging heating pad , and the control intervention . although the combination of passive vibration and moist heat had a greater mean skin blood flow after a 10-minute intervention , it was not significantly different from either moist heat or passive vibration alone . nine minutes after the 10-minute intervention , passive vibration alone had the highest mean skin blood ( 150.5 flux ) , followed by passive vibration plus moist heat ( 133.7 flux ) , and moist heat alone ( 111.3 flux ) . the sunbeam massaging heating pad was turned on at the start of the intervention and began immediately to massage and also began warming . since the skin blood flow measurements were recorded within the first minute ( start ) and immediately after the tenth minute of the intervention , this device may not have reached either its maximum heating potential at these data collection points . in fairness to the product , if this product is used in a follow up study , the product should be allowed to reach the maximum warmth prior to application to the subject or collecting data . after the ten - minute interventions , mean skin temperature was highest with moist heat alone ( 97.4f ) , followed closely by passive vibration alone ( 97.0f ) . mean skin temperature following ten - minute interventions of both passive vibration alone and moist heat alone was significantly different than active vibration . the control ( 95.8f ) , sunbeam massaging heating pad ( 95.2f ) , and active vibration ( 95.1f ) intervention had similar low skin temperatures at the end of the ten - minute intervention . the combination of moist heat and passive vibration ( 96.4f ) skin temperature was between these other interventions . nine minutes after the cessation of the interventions , mean skin temperature remained relatively unchanged for all interventions with the combination of passive vibration and moist heat having the greatest drop in skin temperature of only.5f . the authors conclude that this lowered skin temperature is due to the high skin blood from the combination of moist heat and passive vibration . the most plausible explanation for why skin blood flow was reduced during active vibration is that during high intensity exercise activities , the body directs blood away from the organs such as the skin and is redirected to the skeletal muscles . adrenal medulla regulated epinephrine release and sympathetic regulation results in vasodilation of skeletal muscle vessels and vasoconstriction of the vessels of the epidermis and dermis . the muscle contractions necessary for skeletal movement and support during exercise are executed by muscle fibers that contain cells that must be supplied with nutrients ( e.g. , glucose ) and oxygen by the capillaries . as nutrients and oxygen are supplied to the muscle cells during exercise , waste products ( e.g. , carbon dioxide ) are also removed by the capillary system . at rest , approximately 20% of cardiac output is directed to skeletal muscle at a rate of 3 ml / minute per 100 mg of muscle mass . at rest , only approximately 20% of capillaries in skeletal muscles are open . at the start of exercise , this rate of skeletal blood flow approaches 20 times the resting rate . during high intensity , endurance exercises ( e.g. , cross country skiing ) , skeletal muscle blood flow approaches 80% of cardiac output and 100% of capillaries in skeletal muscles are open . this initial sympathetic regulated reduction of skin blood flow at the start of exercise is replaced by hypothalamus regulated skin vasodilation as body temperature increases . the exercise regimen performed in this study during active vibration was that of short duration activities . in addition , active exercises were performed in standing with the vibratory forces being applied vertically through the soles of the shoes . during passive vibration , the vibratory forces were applied directly to the skin of the calf in a perpendicular direction . the muscles are relaxed and the skin is mechanically stressed resulting in shear stress - induced nitric oxide production . one of the concerns with heat modality applications is the risk of increasing skin temperature to the point that a skin burn occurs . skin temperature , with the combination of passive vibration and moist heat to the calf , was similar to and not significantly different from the control intervention at both the end of the intervention and nine minutes post intervention . although not a thermal modality , passive vibration to the posterior calf increased skin temperature almost as much as moist heat which had the highest skin temperature at both the end of the application and nine minutes post application . the three interventions in this study that produced significant increases in skin blood flow ( sbf ) , ranked highest to lowest , include : 1 ) passive vibration combined with moist heat , 2 ) passive vibration alone , and 3 ) moist heat alone . of these top three sbf interventions , the rank order for skin temperature ( st ) , ranked highest to lowest , include : 1 ) moist heat alone , 2 ) passive vibration alone , and 3 ) passive vibration combined with moist heat . these relatively low skin temperature increases with the combined intervention occurred at both the end of the intervention as well as nine minutes post - intervention . when considering an intervention that has the ability to increase skin blood flow to dissipate heat from the skin to the blood stream while having a relatively low increase in skin temperature , the combination of passive vibration and moist heat appears to be the superior intervention . there are no reports in literature to suggest any risk of a skin burn from the non - thermal modality of passive vibration . anecdotally , prolonged passive vibration could potentially cause a friction rub that could cause skin irritation ; however , this theoretical risk can be further reduced through the use of the 2 cm rubber pad ( skin - platform interface ) provided by the manufacture . since passive vibration alone does not possess the risk of a burn , this modality provides a safe alternative to moist heat to increase skin blood flow in individuals at risk for burns such as individuals with compromised skin blood flow . increased blood flow has been shown to increase tissue nutrients , decrease susceptibility to infection , improve wound healing , and facilitate soft tissue repair [ 2123 ] . in our study , moist heat , passive vibration , and a combination of the two all provided increased skin blood flow . of these three , moist heat alone increases skin temperature more than the other two modalities , suggesting that this modality may place individuals at a greater risk of a burn . consequently , this study does not provide direct applicability to the elderly and diabetic populations who have a greater prevalence of skin wounds due to decreased extremity circulation . a follow up study applying these modalities and methods to these vulnerable populations would be beneficial . follow - up studies to compare passive vibration to warm water immersion on skin blood flow and temperature are also suggested . a study to determine the benefit of the combination of both of these modalities may also prove beneficial . the combination of passive vibration and moist heat produced the greatest increase in skin blood flow and the second highest skin blood flow at nine minutes post - intervention . these therapeutic benefits combined with relatively low increases in skin temperature , may make the combination of these two modalities an intervention of choice .

summarybackgroundcirculation plays an essential role in tissue healing . moist heat and warm water immersion have been shown to increase skin circulation ; however , these heating modalities can cause burns . recent research has shown that passive vibration can also increase circulation but without the risk of burns.material/methodsthe aim of this study is to compare the effects of short - duration vibration , moist heat , and a combination of the two on skin blood flow ( sbf ) and skin temperature ( st ) . ten ( 10 ) subjects , 5 female and 5 male , aged 2030 years of age , received two interventions a day for 3 consecutive days : intervention 1 active vibration only ( vibration exercise ) , intervention 2 passive vibration only , intervention 3 moist heat only , intervention 4 passive vibration combined with moist heat , intervention 5 a commercial massaging heating pad , and intervention 6 no intervention , resting in supine only ( control ) . sbf and st were measured using a laser doppler imager during the 10 minute intervention and then throughout the nine minute recovery period.resultsthe mean skin blood flow following a ten - minute intervention of the combination of passive vibration and moist heat was significantly different from the control , active vibration , and the commercial massaging heating pad . skin temperature following the ten - minute interventions of moist heat alone and passive vibration alone were both significantly different from the commercial massaging heating pad and active vibration interventions.conclusionsthe combination of passive vibration and moist heat produced the greatest increase in skin blood flow and the second highest increase in skin blood flow nine minutes post application .

Background Material and Methods Subjects Laser Doppler Skin temperature Whole body vibration Moist heat Sunbeam Procedures Data analysis Results Skin blood flow Skin temperature Disscussion Conclusions

PMC3430050

isfahan province is one of the biggest and the most important area that is located in center of iran . it is a population based cancer registry based on iran ministry of health guideline.6 data was collected from all hospitals , pathology lab , physician office , radiography centers , and radiotherapy centers . histopathologic diagnosis were coded based on international classification of diseases for oncology ( icd - o).7 data entry was performed by special software named canreg which was designed by cancer department of ministry of health . the history of cancer registry in iran was described previously by mohagheghi and mosavi - jarrahi.8 the population based data of isfahan cancer registry has been completed since 2005 . it was tried to cover all centers in isfahan university of medical sciences district area . for checking sensitivity of data gathering , ten percent of data we decided to enter data from 2005 to 2010 because of confidence in gathering information to prevent sampling bias . we calculated crude incidence rate and the average annual adjusted specific incidence rate ( asr ) per 100000 persons . we obtained population of isfahan province and then adjusted it based on who standard population tables . afterwards , the incidence rates regarding calculated standard populations were calculated.9 trend of yearly cancer incidences and asrs were compared by poisson regression analysis and were presented as incidence rate ratios ( irr ) with % 95 confidence interval ( ci ) . the poisson regression model is a method for multiple regression analysis of data with a dichotomous outcome and one or more categorically define predictors . the asrs were defined as dependent variable while age sex and years were considered as independent variable and irr was calculated by stata 10 software . during 5-year period from 2005 to 2010 , 24771 new cases of cancer were registered in isfahan cancer registry center . of these , 54.6% of patients were male and 45.4% were female aged 56.1 18.03 years ( mean sd ) . the mean age of cancer patients in men and women frequency of cancers based on their topography is shown in figure 2 . the most prevalent cancers were gastrointestinal system , skin , breast and urinary system neoplasms . the most frequent cancer in men was gastrointestinal but in women it was breast cancer . frequency of cancers by organs after direct standardization , it was demonstrated that the incidence rate of cancer is increasing in isfahan province . poisson regression analysis determined the rate of cancer increased approximately 4 per 1000 population ( irr = 1.004 95%ci : 1.002 - 1.005 ) . table 1 shows the incidence rate ratio ( irr ) of cancer in each year adjusted for sex and age . trend of cancer incidence from 2005 to 2010 annual incidence rate ratio of cancer from 2005 to 2010 the findings of this study showed that incidence of cancer in isfahan province has been increasing in recent years ( 4 per 1000 population ) . it is important for policy makers and health manager to seriously consider cancer in future plan in this district area -isfahan . there was a gender difference in cancer incidence and most people were involved in middle ages with mean age of 56 years . the most prevalent cancer was gastrointestinal and breast cancers in male and female , respectively . the mean age of cancer patients in other reports in iran was around 56 - 57 years old.15 the world population data shows the most frequent cancer in men is respiratory system cancers and gi cancers are after that . the serial studies by moore and colleagues showed the contribution of cancer in asia - pacific area . our recent data was comparable with incidence rates from different population of the world.1320 one of differences in iran in contrast to other countries was respiratory system cancers . in iran , the smoking pattern in iran differs from other country and it seems to be lower than other countries , although it has been progressing . more important reason was an information defect regarding respiratory cancers in our cancer registry data . many cases with lung cancer are undiagnosed and some of them did not have any biopsy . they have been diagnosed only by radiographic procedures . colorectal cancer in our data was the most frequent and after that stomach cancer was in the second order . however , it is notable that iran is located in esophageal cancer belt of asia.10 in women , breast cancer was on the top of list . our rates was the same as asian countries but it was lower than north american and european countries.21 enhancement in breast cancer incidence should be taken into consideration . existence of early detection programs of breast cancer plays the leading role in reducing mortality and improving the patient 's prognosis . the comparison between our data and countrywide data which published by ministry of health determined the incidence of bladder cancer was high in isfahan province . we presented 5-year analysis and will try to report ten - year or more in future . we did not have data from 2 cities of isfahan province -kashan and aran - bidgol- because they are covered by kashan medical sciences university . however , we have been conducting some research project to find cancer risk factors and distribution in the district area . as discussed in aforementioned paragraphs , our cancer registry had defect in lung cancer . we have tried to solve this problem to call on all medical center like oncology , radiology , surgery and respiratory disease centers . fm designed and edited manuscript , mar designed , analyzed and wrote manuscript , kh and mt supported the project and edited manuscript and ht helped in data collection and entry and managed data .

background : cancer incidence rate is increasing in the world particularly in developing countries . the awareness regarding cancer incidence and distribution helps policy makers and researchers to design comprehensive plan for controlling cancer . the aim of this study was to determine the incidence rate and trend of cancer in isfahan area , one of the most importance provinces of iran.methods:data of isfahan cancer registry were derived from 2005 to 2010 . direct standardization through world standard population produced by the world health organization was used and adjusted standard rate ( asr ) was calculated . the poisson regression analysis was employed to estimate cancer incidence trend during 5 years.results:the new cases of cancer were 24771 patients from 2005 to 2010 . mean age of these patients was 56.1 18.0 years and 54.6% were male . male patients were approximately 7 years older than females . the most frequent cancer was gastrointestinal in men and breast cancer in women . the rate of cancer increased approximately 4 per 1000 population and incidence rate ratio ( irr ) was 1.004 ( 95%ci : 1.002 - 1.005).conclusions : the rate of cancer is increasing rapidly in isfahan province . cancer control and comprehensive prevention plan for isfahan is necessary .

Method Result Discussion Conclusion Authors Contributions

PMC5080157

females commonly wear high heels to give an appearance of being taller and to accentuate the appearance of their legs1 . in addition , as they have social and cultural meaning , as well as psychosexual significance , high heels have become a consistent component of fashion trends for females2 . in fact , it has been reported that fashion - conscious young females can wear high - heel shoes for up to 8 hours per day while performing activities of daily living , such as standing and walking3 . prolonged wearing of high heels may be a predisposing risk factor for impairments in body alignment , lower limb proprioception , and musculoskeletal injury4 , 5 . it has been suggested that an interaction between high heels and hormonal factors could explain the 2-fold higher incidence of knee osteoarthritis in females , compared to males6 . moreover , as they increase the magnitude of torque about the knee during standing and walking , high heels could increase the risk for knee joint injury and degenerative changes due to accumulated fatigue7 . such degenerative changes could alter knee joint position sense , negatively affecting balance control and leading to lowered capacity for activities of daily living and mobility8 , 9 . balance control largely refers to the ability to maintain alignment of the body along the line of gravity and lower the amplitude and velocity of sway of the body center of gravity10 , 11 , and is strongly influenced by knee joint position sense . high heels narrow the base of support and restrict the area within which body sway must be controlled to avoid triggering a fall . compounding this problem of balance control is the negative effect of high heels on knee joint position sense , which reduces the efficiency of the system to use proprioceptive information to strictly control body sway within the available base of support5 . the chronic demand on lower limb muscles and soft tissues to manage these changes in proprioception and base of support can eventually lead to inefficient contraction of leg muscles and abnormal muscle lengths12 . however , the effects of high heels on muscle activity , proprioception , and lower limb alignment have only been evaluated within the context of standing4 , 13 , without consideration of the potentiating effects of walking in high heels . therefore , the aim of our study was to examine the effects of increased heel height and gait velocity on balance control and knee joint position sense . all participants were right - foot dominant , naive to high - heel use , and had no previous history of neuromuscular disease , fractures , spine - related diseases , or lower limb surgery . after being provided with a full explanation of the study procedure , without disclosure of the study purpose , participants gave informed consent . participants were randomly allocated to the low - heel , low - speed group ( 3 cm , 2 km / h , ll ) , the low - heel , high - speed group ( 3 cm , 4 km / h , lh ) , the high - heel , low - speed group ( 9 cm , 2 km / h , hl ) , or the high - heel , high - speed group ( 9 cm , 4 km / h , hh ) , with 10 subjects per group . the distribution of age , height , and weight within each group is reported in table 1table 1.general characteristics of the study group ( n=40)grouplllhhlhh(n=10)(n=10)(n=10)(n=10)age ( years)21.6 1.921.9 2.222.0 1.922.8 2.0height ( cm)158.4 5.5157.2 5.0158.4 8.0158.5 5.4weight ( kg)56.2 5.057.6 4.356.5 4.257.4 ll(a ) : low - heel low - speed group ; lh(b ) : low - heel high - speed group ; hl(c ) : high - heel low - speed group ; hh(d ) : high - heel high - speed group . baseline measurements of static and dynamic balance and joint position sense were obtained prior to the experimental session . participants in the ll and hl groups wore shoes with a 3-cm heel , while participants in the lh and hh groups wore shoes with a 9-cm heel . all walking trials were performed on a treadmill ( t25 , motus , korea ) , at 2 speeds for 20 min : 2 km / h for the low - speed group and 4 km / h for the high - speed group . following the walking trial , participants removed their shoes and completed posttest assessments of static and dynamic balance , and knee joint position sense . all participants were evaluated under the same experimental conditions , and provided with an adaptation time of approximately 10 min before and after the measurements . ll(a ) : low - heel low - speed group ; lh(b ) : low - heel high - speed group ; hl(c ) : high - heel low - speed group ; hh(d ) : high - heel high - speed group static and dynamic balance tests were performed using an automatic balance calibration system ( model i balance s , cybermedic co. , iksan , korea ) . the i - balance system provides a measure of the sway velocity of the center of gravity of the body and the total amplitude of sway , using a force platform equipped with 4 load cells . for static balance testing , participants stood on the force platform , with feet shoulder - width apart and hands folded across the chest . participants were asked to look directly in front at a monitor providing feedback of the location of their center of gravity and to maintain the position within the spatial area provided by the program . static balance was quantified by the length of time ( s ) the reference position of the center of gravity was maintained within the reference spatial area . for dynamic balance testing , participants assumed the same posture as for static balance testing but were asked to change the position of the center of gravity to match locations presented by the program : anterior , posterior , right , left , and along diagonals relative to their neutral position . dynamic balance was quantified by the response time along each direction ( s ) . diagnostic tests using smartphone applications have recently been conducted for clinical and scientific diagnosis , and the reliability of the drgoniometer application has been verified in previous studies15 , 16 . participants were placed in the prone position , with eyes blindfolded and the torso and thighs stabilized using a belt . the knee being assessed was passively flexed to 90 and held for 5 s to provide participants with a sensory representation of the reference angle . participants were then asked to actively flex their knee further to 120 , holding the new end - point position again for 5 s. the learning trial was repeated 3 times to ensure that participants had obtained an accurate sensory representation of 90 and 120 of knee flexion . for testing , the knee was again passively flexed to 90 , and participants were then asked to actively flex their knee further to 120 , telling the experimenter when they felt they had reached the target knee angle . a total of 6 measurements were completed and the difference between the target angle of 120 and the measured angle calculated . the assessments were performed under identical experimental conditions for the pre- and posttest , and all tests were performed by the same experimenter . the normality of the distribution of variables was evaluated using the shapiro - wilk test , with data transformation used as needed to achieve a normal distribution . effects of heel height and walking speed were evaluated using a two - way analysis of variance ( anova ) , and statistical significance was set at an -level of 0.05 . changes in static balance as a function of shoe heel height and gait velocity are reported for each group in table 2table 2.changes of static balance according to shoe heel height and gait velocity ( sec)groupmean sdprepostll0.34 0.10.39 0.1***lh0.36 0.10.41 0.1***hl0.34 0.10.45 0.2***hh0.34 0.10.53 0.1****p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . p<0.05 , p<0.01 , p<0.001 , comparison between group .. overall , we identified an increase in sway amplitude as a function of increased heel height and gait velocity , with an increase in sway amplitude of 0.05 in the ll group , 0.05 in the lh group , 0.11 in the hl group , and 0.19 in the hh group . a significant group time interaction was identified ( p<0.05 ) , with the condition of high heels and high velocity causing the largest increase in sway amplitude . * p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . changes in dynamic balance as a function of shoe heel height and gait velocity are reported for each group in table 3table 3.change of dynamic balance according to shoe heel height and gait velocity ( sec)groupmean sdprepostll1.69 0.62.23 0.9***lh1.66 0.42.33 0.8***hl1.50 0.42.76 0.9***hh1.60 0.43.89 0.9****p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . p<0.05 , p<0.01 , p<0.001 , comparison between group .. sway amplitude increased by 0.54 in the ll group , 0.67 in the lh group , 1.26 in the hl group , and 2.29 in the hh group , again indicating that amplitude of sway increased as shoe heel height and gait velocity increased . a significant group time interaction was identified ( p<0.001 ) , with the condition of high heels and high velocity causing the largest increase in sway amplitude . * p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . assessment of knee position sense provided evidence of an increased magnitude of error with increasing heel height and gait velocity . the magnitude of error in joint position sense across the 4 groups , using the reference position of 60 , was as follows : 0.68 for the ll group ; 1 for the lh group ; 0.86 for the hl group , and 4.62 for the hl group ( table 4table 4.change of position sense of knee joint according to shoe heel height and gait velocity ( )groupmean sdprepostll0.64 3.31.32 2.8lh0.16 5.71.16 2.2hl0.76 3.31.62 3.0hh0.48 3.05.10 4.2*p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . again , a significant group time interaction was identified ( p<0.05 ) , with the condition . * p<0.05 , * * p<0.01 , * * * p<0.001 , comparison between pre and post . an improper fitting of the shoe can negatively affect the body alignment and balance19 . in particular , high - heel shoes can modify the position sense of the knee joint , which itself modifies the responsiveness of the muscles responsible for aligning the trunk and lower limbs along the line of gravity . these modifications in sensory - motor control can functionally reduce balance capacity and increase the risk for falls20,21,22 , 24 . balance capacity has been shown to be closely correlated with proprioception , with knee joint position sense being an important component of proprioception influencing balance and walking performance23 . single - leg balance training on a trampoline has been shown to enhance knee joint position sense , resulting in increased balance capacity , measured by a significant reduction in the amplitude of body sway . our study contributes novel knowledge on the effects of wearing high heels on balance control . specifically , we demonstrated that a trial of 20 min of treadmill walking with high heels was sufficient to lower the acuity of knee position sense and to increase the amplitude of body sway under both conditions of static and dynamic balance . our findings are comparable to previous reports of the effect of high heels in increasing the amplitude and area of sway during static standing4 and reduced dynamic stability during fore - aft walking22 . similar negative effects of high heels on sensory input were identified by a reduction in two - point discrimination and sensitivity to light tactile touch on the soles of the feet , and lower position sense of the great toe in young females who wore high heels for more than 20 h per week13 . other studies have reported a decrease in knee joint position sense following high - intensity isokinetic knee exercises26 . the effects of high heels on knee joint position sense could be mediated by an increase in ankle plantar flexion angle and an associated larger range in vertical displacement of the center of gravity during walking21 , 25 . another important component of decreased knee joint position sense might be muscle fatigue induced by increased gait velocity , which increases the number of strides per minute26,27,28 . overall , our results show that higher gait velocity and heel height induce a change in knee joint position sense , which negatively affect static and dynamic posture control . future studies regarding the effects of heel height on lower limb proprioception and balance capacity are needed to evaluate age effects that would be prognostic of an increased risk for falls .

[ purpose ] the aim of this study was to examine the effects of increased heel height and gait velocity on balance control and knee joint position sense . [ subjects and methods ] forty healthy adults were randomly allocated to 4 groups : low - heel , low - speed group ( 3 cm , 2 km / h ) , low - heel , high - speed group ( 3 cm , 4 km / h ) , high - heel , low - speed group ( 9 cm , 2 km / h ) , high - heel , and high - speed group ( 9 cm , 4 km / h ) , with 10 subjects per group . static and dynamic balance was evaluated using the i - balance system and knee joint position sense using a goniometer . measurements were compared using a pre- and posttest design . [ results ] increasing heel height and gait velocity decreased knee joint position sense and significantly increased the amplitude of body sway under conditions of static and dynamic balance , with highest sway amplitude induced by the high - heel , high - speed condition . [ conclusion ] increased walking speed in high heels produced significant negative effects on knee joint sense and balance control .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3881858

the oral candidiasis known as denture stomatitis is related to the use of removable dentures and is considered the most common oral lesion observed ( 65% ) in patients wearing removable dentures . local factors associated with the denture are also related to this pathology , such as : presence of biofilm , local trauma caused by dentures12 , xerostomia , continuous use of the dentures and alteration in salivary ph . different treatments for denture stomatitis are available and may include topical antifungal and systemic therapy , care with oral hygiene , denture cleaning and disinfection procedures , replacement of old dentures , elimination of anatomic irregularities , re - establishment of atraumatic occlusion , and nutritional restitution . furthermore , in order to protect and preserve the integrity of the mucosal epithelium , patients should sleep without the dentures . the choice of a treatment or association of more than one treatment is an aspect to be individually considered . re - infection of the treated oral mucosa may occur in up to two weeks post - treatment , and is attributed to the survival of candida spp . therefore , it is crucial to adopt methods that reduce or preferably eliminate the microorganisms from denture surfaces . in addition , resilient materials have been routinely used with the purpose of recovering tissues that are in contact with the denture base . these materials partially absorb chewing load on the denture during function , thus reducing the energy transmitted to the associated paraprosthetic tissues . however , these materials are easily degradable and susceptible to microbial colonization , which may cause different degrees of denture stomatitis . to prolong the clinical longevity of resilient materials and reduce plaque accumulation , incorporation of antimicrobial agents into these materials this combination may be a logical therapy in the treatment of denture stomatitis because of several factors : 1 . reducing the trauma caused by the internal surface of removable dentures ; 2 . eliminating contact of the contaminated surface with the oral tissues and consequently , interrupting the cycle of re - infection , and 3 . action of antimicrobial agents incorporated into the material on the infected tissues . in this context , denture stomatitis may be treated before fabricating new dentures , in a relatively short period . the reason is attributed to their gradual degradation and hardening , so it should not take longer than two weeks , which is a period similar to the one required for the treatment with conventional topical antifungal drugs . the incorporation of antimicrobial agents into resilient materials has shown to be effective and feasible both in in vitro and in vivo studies . despite these therapeutic advantages , the incorporation of drugs into polymeric materials , including tissue conditioners and resilient liners , may affect their properties . for the resilient liner to adequately perform its function of recovering the tissues injured by trauma , it should remain bonded to the acrylic base of the removable denture5 . peeling of the resilient material from the denture base has been reported as the cause of clinical failure and the bond between the resilient materials and the denture base acrylic resins has been the object of previous investigations . thus , the aim of this study was to evaluate the effect of the addition of antimicrobial agents ( nystatin , miconazole , ketoconazole , and chlorhexidine diacetate ) to a resilient liner on its peel bond strength to a denture base acrylic resin . the hypothesis investigated in this study was that the addition of antimicrobial agents to a resilient liner would result in changes in the peel bond strength to a denture base acrylic resin . the acrylic materials , manufacturers , batch numbers , compositions , powder / liquid ratios , and polymerization conditions selected for this study are listed in figure 1 . the selected antimicrobial agents were nystatin , miconazole , ketoconazole ( alonatu farmcia de manipulao e cosmticos / farmcia dermatus , macei , al , brazil - req . 119704 - 1 ) , and 98% chlorhexidine diacetate ( acros organics , morris plains , nj , usa ) . materials selected for this study specimens ( n=9 ) measuring 75x10x3 mm of heat - curing acrylic resin qc 20 ( dentsply ind . for this purpose , stainless steel matrixes measuring 75x10x3 mm were molded using laboratory silicone ( zetalabor , rovigo , veneto , italy ) between two glass plates . the mold / matrix set was invested in conventional metal dental flasks in type iii dental stone ( herodent , vigodent , rio de janeiro , rj , brazil ) . the dental flasks were closed and remained under pressure ( 500 kgf ) in a hydraulic press during stone setting time . after this period , the dental flasks were opened and the stainless steel matrixes were removed . qc 20 was proportioned , mixed according to the manufacturer 's instructions ( figure 1 ) , and was inserted into the silicone matrix mold . the dental flask was closed and kept under pressure at room temperature ( 232c ) for 30 min . after this period , the test specimens were submitted to the polymerization cycle " b " recommended by resin manufacturer ( figure 1 ) . when the polymerization cycle ended , the dental flasks were bench cooled for 30 min and then under running water for 15 min . the specimens were removed from the molds and stored in distilled water at 37c for 48 h. after this period , specimens were submitted to surface preparation to receive the modified resilient liner . one of the specimen surfaces was abraded automatically in a polishing machine using # 600 silica carbide abrasive paper ( norton abrasivos , so paulo , sp , brazil ) . the abraded surface was cleaned with detergent for 20 s , washed under running water , and dried . the specimen was then placed in a hollow stainless steel mold with internal measurements of 75x10x6 mm . the specimen area ( 650 mm ) to not be bonded to the resilient material was covered with a polyester strip . the antimicrobial powders in each experimental group ( figure 2 ) were manually mixed with resilient lining powder with a spatula , until a homogenous mixture was obtained . the resilient lining liquid was added to this mixture and the material was mixed in accordance to the manufacturer 's instructions ( figure 1 ) . the modified material was inserted into the hollow mold containing the test specimen of the heat - curing acrylic resin prepared for the relining procedure . this set was covered with glass slide and kept under finger pressure during the resilient liner polymerization time recommended by the manufacturer ( figure 1 ) . the excesses of the modified resilient liner were eliminated and the specimen was removed from the mold . the relined specimens were then stored in distilled water at 37c for 24 h prior to the peel test . drug dosages incorporated into the resilient liner powder in all experimental groups a universal testing machine ( versat 2000 , panambra ind . tech . sa , so paulo , sp , brazil ) was used to perform the peeling bond strength test of the relined test specimens at an angle of 180. a portion of modified resilient material not bonded to the resin base ( 65 mm ) was folded upwards and fixed onto the top hook of the equipment at 20 mm from the adhesive bond area of the test specimen . the other un - relined portion of the heat - curing acrylic resin was fixed onto the bottom hook of the equipment at the same distance from the adhesive bond area . each test specimen was submitted to tension to promote peeling of the modified resilient liner from the heat - curing acrylic resin base at a speed of 10 mm / min until failure occurred . bond failures were visually observed and classified into three categories : adhesive , when peeling occurred between the modified resilient liner and the denture base acrylic resin ; cohesive , when there was tearing ( rupture of the resilient liner within the area bonded to the denture base ) or snapping ( resilient material had stretched and then ruptured away from the bonded area ) within the modified resilient liner ; and mixed , when regions with two types of failure were observed on the surface of the denture base material . the results of rupture force were initially obtained in n and transformed into peeling bond strength in mpa and then submitted to one - way anova at a significant level of 5% . specimens ( n=9 ) measuring 75x10x3 mm of heat - curing acrylic resin qc 20 ( dentsply ind for this purpose , stainless steel matrixes measuring 75x10x3 mm were molded using laboratory silicone ( zetalabor , rovigo , veneto , italy ) between two glass plates . the mold / matrix set was invested in conventional metal dental flasks in type iii dental stone ( herodent , vigodent , rio de janeiro , rj , brazil ) . the dental flasks were closed and remained under pressure ( 500 kgf ) in a hydraulic press during stone setting time . after this period , the dental flasks were opened and the stainless steel matrixes were removed . qc 20 was proportioned , mixed according to the manufacturer 's instructions ( figure 1 ) , and was inserted into the silicone matrix mold . the dental flask was closed and kept under pressure at room temperature ( 232c ) for 30 min . after this period , the test specimens were submitted to the polymerization cycle " b " recommended by resin manufacturer ( figure 1 ) . when the polymerization cycle ended , the dental flasks were bench cooled for 30 min and then under running water for 15 min . the specimens were removed from the molds and stored in distilled water at 37c for 48 h. after this period , specimens were submitted to surface preparation to receive the modified resilient liner . one of the specimen surfaces was abraded automatically in a polishing machine using # 600 silica carbide abrasive paper ( norton abrasivos , so paulo , sp , brazil ) . the abraded surface was cleaned with detergent for 20 s , washed under running water , and dried . the specimen was then placed in a hollow stainless steel mold with internal measurements of 75x10x6 mm . the specimen area ( 650 mm ) to not be bonded to the resilient material was covered with a polyester strip . the antimicrobial powders in each experimental group ( figure 2 ) were manually mixed with resilient lining powder with a spatula , until a homogenous mixture was obtained . the resilient lining liquid was added to this mixture and the material was mixed in accordance to the manufacturer 's instructions ( figure 1 ) . the modified material was inserted into the hollow mold containing the test specimen of the heat - curing acrylic resin prepared for the relining procedure . this set was covered with glass slide and kept under finger pressure during the resilient liner polymerization time recommended by the manufacturer ( figure 1 ) . the excesses of the modified resilient liner were eliminated and the specimen was removed from the mold . the relined specimens were then stored in distilled water at 37c for 24 h prior to the peel test . sa , so paulo , sp , brazil ) was used to perform the peeling bond strength test of the relined test specimens at an angle of 180. a portion of modified resilient material not bonded to the resin base ( 65 mm ) was folded upwards and fixed onto the top hook of the equipment at 20 mm from the adhesive bond area of the test specimen . the other un - relined portion of the heat - curing acrylic resin was fixed onto the bottom hook of the equipment at the same distance from the adhesive bond area . each test specimen was submitted to tension to promote peeling of the modified resilient liner from the heat - curing acrylic resin base at a speed of 10 mm / min until failure occurred . bond failures were visually observed and classified into three categories : adhesive , when peeling occurred between the modified resilient liner and the denture base acrylic resin ; cohesive , when there was tearing ( rupture of the resilient liner within the area bonded to the denture base ) or snapping ( resilient material had stretched and then ruptured away from the bonded area ) within the modified resilient liner ; and mixed , when regions with two types of failure were observed on the surface of the denture base material . the results of rupture force were initially obtained in n and transformed into peeling bond strength in mpa and then submitted to one - way anova at a significant level of 5% . therefore , the incorporation of antimicrobial agents in the concentrations assessed did not affect the peeling bond strength between the resilient liner and the denture base resin after 24 h of immersion in distilled water . peel strength ( mpa ) at 24 h there was no statistical difference ( p>0.05 ) among the experimental groups the failure modes obtained after performing the tests are shown in table 1 . the majority of bond failures were cohesive ( tearing and/or snapping ) within the resilient liner . for the experimental groups g2 ( nystatin at 500,000 u ) , g5 ( miconazole at 250 mg ) , and g8 ( 5% chlorhexidine diacetate ) , a mixture of tearing and snapping was observed . peeling away from the denture base was only observed for groups g6 ( ketoconazole at 100 mg ) and g9 ( 10% chlorhexidine diacetate ) . for the other groups , the hypothesis investigated in this study that " the addition of antimicrobial agents to the resilient liner would result in alterations in the peeling bond strength to denture base resin " was rejected because there was no difference between the experimental groups assessed in comparison with the control group without the addition of drugs . during clinical use , the resilient materials are highly subjected to degradation and susceptible to the colonization by microorganisms . if these materials are not regularly replaced , they may act as microorganism reservoirs , causing systemic complications . an example of this is the presence in the oral cavity of staphylococcus aureus , a microorganism responsible for respiratory infections . the combination between resilient materials and antimicrobial agents seems to be a logical therapeutic modality for denture stomatitis . this method results in a reduction of the trauma caused by the old denture and tissue reconditioning associated with antimicrobial therapy ; important etiologic factors in triggering infection by candida spp . are simultaneously eliminated . in addition , this method favors a relined denture that can more easily be kept clean by the patient . several drugs have shown reduced water solubility , so maximum dose is required to have the effectiveness required for a certain medication . among the antimicrobial agents assessed , chlorhexidine shows higher solubility in water , followed by nystatin , miconazole , and ketoconazole . although these medications are soluble in water , they are insoluble in monomers and plasticizers.thus , they could not interfere with the polymerization or plasticization process of these materials . however , their physical presence within the polymer matrix could interrupt the structure of the polymerized materials . resilient materials containing nystatin showed increased water sorption , and for these materials , this resulted in breaking their morphological structure . according to addy and handley ( 1981 ) , change in material properties may be consistent with the incorporation pattern of the medication into the polymer matrix . a previous study assessed the incorporation pattern of antimicrobial agents into a tissue conditioner with the same concentrations to those investigated in this study , by scanning electronic microscopy ( sem ) and energy dispersive spectroscopy x - ray ( eds ) . the test specimens containing nystatin and miconazole exhibited particles with irregular shapes and sizes distributed uniformly within the tissue conditioner matrix while specimens with chlorhexidine exhibited more irregular particles distributed randomly within the material . however , these alterations would not prevent the incorporation of drugs for release in the oral cavity if they were added to materials to reline already existent dentures , without necessarily reducing their strength . although some soft materials are submitted only to compression and shear , tensile strength is used to measure the quality of the material . the ability of the material to resist tearing is of practical importance . in clinical use , including the cleaning and disinfection procedures , the soft materials are submitted to conditions that start the tearing process . . clinical failure of these materials is frequently attributed to the rupture of this bond , and the measurements of this bond are clinically relevant . reduced bond between the soft liner and the denture base resin effectively negates any other property considered adequate for this material . in the peel bond strength test , the stress is confined to a line restricted to the end of the bond , and is considered the most clinically representative of the failure modes . this is the only method in which the failure proceeds at controlled speed and it is a direct measure of peeling , while it also represents the elastic deformation of the material . the peeling test simulates the lining procedure more precisely , with a uniform and constant distribution of force throughout the bond area . the results of this study demonstrated that the addition of antimicrobial agents in all the assessed concentrations did not affect the peeling bond strength of the resilient liner to denture base resin . however , the bond strength values were considered low , since they were approximately 10 times lower than the acceptable value for the clinical use of resilient liners ( 0.44 mpa ) . while the methodology in this study was performed , some modifications were made , such as the reduction in bond area and surface roughness of the denture base , to ensure that the methodology evaluated the bonding between materials rather than the cohesive strength of the liner material . if the bond failures observed in this study were predominantly cohesive within the liner material , the peeling bond strength would not be measured . the failure mode of the cohesive type provides information related to the material itself and not to the bond between materials . ( 1995 ) suggested the term " strength failure " instead of " bond failure " when cohesive failures occur . predominant cohesive failures , such as those that occurred in this study , indicate poor resistance to tearing of the resilient material . however , mixed and adhesive failures were observed in some samples , indicating that the cohesive strength values of the resilient liner and bond strength values to base resin were similar . a previous study observed that a tissue conditioner ( duraconditioner , reliance manufacturing nystatin showed cohesive strength values similar to those of the control group . these values were close , if not similar , to the ones obtained in this study . therefore , the cohesive strength of the resilient material tested in this study is equivalent to its bond strength to the denture base material . thus , the material will snap or tear at the bond interface at forces lower than those necessary to cause bond failures . one of the limitations of this study was that only one brand of the resilient liner was assessed . moreover , the peeling bond strength could have been assessed after other storage periods . this assessment is also important to observe a possible reduction in bond strength of the modified liner to the denture base material , since it has been reported that plasticizers and alcohols are released from resilient materials after periods of storage in water and this release is responsible for the decrease in the bond strength values between the materials . within the limitations of this in vitro study , it can be concluded that it is possible to incorporate any of the antimicrobial agents assessed in the selected concentrations into a resilient liner without changing the bond strength of this material to denture base resin . a clinical study is still needed to determine the therapeutic validity of this alternative treatment modality .

in order to prolong the clinical longevity of resilient denture relining materials and reduce plaque accumulation , incorporation of antimicrobial agents into these materials has been proposed . however , this addition may affect their properties.objectivethis study evaluated the effect of the addition of antimicrobial agents into one soft liner ( soft confort , dencril ) on its peel bond strength to one denture base ( qc 20 , dentsply ) . material and methodsacrylic specimens ( n=9 ) were made ( 75x10x3 mm ) and stored in distilled water at 37c for 48 h. the drug powder concentrations ( nystatin 500,000u - g2 ; nystatin 1,000,000u - g3 ; miconazole 125 mg - g4 ; miconazole 250 mg - g5 ; ketoconazole 100 mg - g6 ; ketoconazole 200 mg - g7 ; chlorhexidine diacetate 5% - g8 ; and 10% chlorhexidine diacetate - g9 ) were blended with the soft liner powder before the addition of the soft liner liquid . a group ( g1 ) without any drug incorporation was used as control . specimens ( n=9 ) ( 75x10x6 mm ) were plasticized according to the manufacturers ' instructions and stored in distilled water at 37c for 24 h. relined specimens were then submitted to a 180-degree peel test at a crosshead speed of 10 mm / min . data ( mpa ) were analyzed by analysis of variance ( =0.05 ) and the failure modes were visually classified . resultsno significant difference was found among experimental groups ( p=0.148 ) . cohesive failure located within the resilient material was predominantly observed in all tested groups . conclusionspeel bond strength between the denture base and the modified soft liner was not affected by the addition of antimicrobial agents .

INTRODUCTION MATERIAL AND METHODS Specimen preparation Peel test RESULTS DISCUSSION CONCLUSIONS

PMC4181422

in common with many non - communicable diseases , alcohol - related health harms result from a broad range of social , economic and political determinants that are affected by policies made in a number of non - health sectors . recognizing this , the health in all policies movement calls for health professionals to work synergistically with non - health sectors to negotiate policy changes that enhance health and well - being . a range of non - health sectors share concerns regarding alcohol - related harms in particular crime , disorder and lost economic productivity presenting an opportunity for incorporating health goals across alcohol control policies . yet broadly similar aims belie differences in how sectors prioritize alcohol control interventions , in particular the relative policy importance of targeting acute intoxication or chronic overconsumption . while many determinants impact on both acute and chronic harms for example alcohol outlet density is associated with violence , assault and health the relative magnitude of this impact may differ . whether a government body is concerned with the immediate or long - term effects of alcohol consumption not only influences its policy preferences , but also leads to distinct operating principles and practices that may be at odds with population health considerations . alcohol consumption is regulated by a complex mix of government institutions that act within country - specific legal and political contexts . in england and wales , local governments are directly responsible for controlling alcohol provision through licensing , planning and trading standards . however , local government powers are limited to activity within a national framework of prescribed legal considerations and policy objectives . this legal framework focuses predominantly on balancing individual liberties and economic considerations with immediate societal harms resulting from acute alcohol intoxication . systematic reviews , national and international guidelines consistently emphasize the health importance of reducing the affordability and physical availability of alcohol . the most effective interventions from a health perspective include reducing licensed alcohol outlet density , their opening days and times , increasing taxation and minimum unit pricing . conversely , standalone server or design interventions for on - premises are less consistently effective and less likely to impact on chronic consumption . in keeping with this evidence , the uk government 's 2012 alcohol strategy included pledges to introduce national minimum unit pricing and to consult on proposals to ban off - trade multi - buy promotions and introduce a cumulative impact health licensing objective for local alcohol policy . however , despite positive progress in scotland , recent government statements suggest these policies are no closer to becoming a reality in england . public health practitioners tackling alcohol - related health harms are therefore faced with a paradox : interventions with the most evidence supporting their effectiveness often appear the least feasible to implement . individual - level interventions , despite good evidence for the effectiveness of brief interventions in reducing alcohol consumption , are unlikely to be sufficient in isolation to reduce the 31% of women and 44% of men in england who drink more than recommended weekly alcohol limits . localism , the best opportunities to intervene on the physical availability and affordability of alcohol currently appear to be at the local government level . this article analyses the implications for local alcohol control of recent changes in alcohol licensing laws and practice in england and wales . we present uk case studies to show how alcohol - related health harms can be tackled through licensing , planning and local partnerships . while this legal framework is specific to england and wales , the challenge of reconciling population health needs with the legal and political principles governing alcohol regulation is internationally relevant . a focused search was conducted in april 2013 to identify laws , legal rulings and government policy documents relevant to current english local government alcohol control policies , processes or practices . lexis and westlaw uk database searches identified the legislative framework for local alcohol control in england and wales , drawing on legal commentary and secondary sources including halsbury 's laws of england . policy articles were identified from medline , web of science and google scholar database searches ; hand - searching relevant non - governmental and local government websites and contacting key experts from local government and non - governmental organizations . documents were reviewed and analysed for their implications for the ability of local government practitioners to enact effective policies to address alcohol - related health harms . policy implementation processes were analysed by drawing on the authors ' prior legal , public health and policy experiences , combined with contacting key informants known to the researchers for non - audio recorded discussions of policy implementation to ensure the accuracy of our process descriptions . powers to control local alcohol supply and consumption are established by the licensing act 2003 , which transferred authority for granting and reviewing licenses from magistrate courts to local authorities . implemented in 2005 , the act sets out the licensing process ( box 1 ) and defines the statutory licensing objectives that legally underpin all licensing activities ( box 2 ) . the police reform and social responsibility act 2011 granted health leads a statutory role in the licensing process for the first time and gave local authorities additional powers to address the cumulative impact of alcohol sales . the recent return of public health to local authorities presents additional opportunities for cross - sector collaboration on shared objectives . in contrast with scotland , potential health opportunities arising from this new legislation in england and wales must contend with the absence of a statutory health licensing objective . box 1 overview of the process and legal framework governing licensing decisions in england and wales box 2 licensing objectives in england and wales as defined by the licensing act ( 2003 ) prevention of crime and disorder : based on police advice concerning , preventing crime and maintaining order . public safety : physical safety of people using a premises , immediate harms , e.g. accidents , injuries , unconsciousness . prevention of public nuisance : noise nuisance , light pollution , noxious smells , litter and where an effect is prejudicial to health. protection of children from harm : moral , psychological and physical harm , including underage sale of alcohol . local health leads in england and wales have , as responsible authorities , a recognized role in commenting on all licensing applications , yet evidence they present must legally be framed in terms of non - health objectives . the licensing process ( box 1 ) is primarily a method for controlling immediate harms associated with alcohol sales at a particular premises . all license decisions must relate specifically to the premises in question and the promotion of the four statutory licensing objectives ( box 2 ) . government guidance explicitly states that public health should not be the primary consideration for a licensing decision , although health considerations can support concerns regarding a statutory licensing objective . alcohol - related injury rates , for example , are considered relevant to public safety . rates of chronic conditions , on the other hand , are harder to link directly with any of the four licensing objectives , despite accounting for 75% of alcohol - related hospital admissions in england . licensing authorities can only consider health - related evidence that directly links the premises in question to a threat to one of the named licensing objectives ( box 2 ) . the more specifically evidence relates to the premises or location of concern , the greater its legal weight and the less vulnerable it is to appeal . routine health data , rarely collected in a way that can be linked to individual premises , are unlikely to be considered relevant . an area where pub or supermarket chains hold an advantage , since evidence of good operating practice by their company elsewhere is considered relevant to new applications . the practical challenges of acquiring sufficiently detailed health data to support licensing decisions must be weighed against the potential consequences of submitting weakly justified health representations . repeated submissions based on health evidence that is unrelated to the licensing objectives , or not deemed relevant to the applicant , may weaken the credibility of future representations to the licensing sub - committee . conversely , and somewhat paradoxically , not submitting a health representation for an application may be interpreted as evidence that the application holds no health threat . a 2008 high court decision suggests that the absence of expert representation in this case by the police signified that there were no serious concerns about the impact on licensing objectives within that responsible authority 's domain of expertise . despite these limitations , licensing policy statements can be powerful tools to support and coordinate local government actions against alcohol - related harms . licensing statements , open to challenge on appeal or by way of judicial review , must still be consistent with promoting the four licensing objectives . they do , however , allow for broader responses to alcohol consumption above the individual premises level by designating special policies or establishing the local relevance of particular licensing approaches . special policies can be implemented to address area - wide impacts of alcohol consumption , namely : cumulative impact zones ( cizs ) , early morning restriction orders and late night levies . policy statements can also document population - level data , including health impacts , at a higher spatial scale than individual license decisions allow . using policy statements to clearly set out a licensing authority 's reasoning and justification for a licensing approach , backed up by appropriate local evidence , can make individual representations more legally robust while reducing the time needed to write them . this could include , for example , establishing a health rationale for limiting the density of alcohol premises using rates of local alcohol - related hospital admissions . local authorities can designate cizs to control new on- or off - premises alcohol outlets in areas where the cumulative stress caused by existing overprovision of alcohol outlets demonstrably threatens the licensing objectives . licensing decisions are normally made with a presumption in favour of the applicant . under a ciz , the burden of proof is reversed and it is the applicant who must demonstrate how they will avoid threatening the licensing objectives . cizs do not , however , affect existing license holders and decisions are still made on a case - by - case basis . for example , an initial refusal to grant a license within a ciz in leeds was overturned on appeal due to the applicant 's short opening hours , clientele and history of running trouble - free premises . although yet to be implemented by any local authority , early morning restriction orders ( emro ) and late night levies ( lnl ) offer additional mechanisms for controlling alcohol sales from both on- and off - premises between 12 and 6 a.m. emros are designed to address recurrent licensing objective infringements that are not attributable to a single premises , such as night time anti - social behaviour . lnls recoup financial costs associated with late night alcohol provision by levying a charge on any premises licensed to sell alcohol between specified night hours . acting outside of the licensing process , emros and lnls are decided on an area rather than individual - premises basis . lnls must apply to the whole of a licensing authority 's area , while emros can apply to selected parts of this area . the absence of a health licensing objective does not preclude addressing health needs where they concern other licensing objectives . producing specific evidence linking local alcohol consumption practices with licensing objectives does , however , require a change of approach from traditional local health data analysis , demonstrated by the ongoing difficulties scottish licensing boards face in reflecting health evidence in licensing decisions ( box 3 ) even since the introduction of an explicit public health licensing objective . more successfully , the cardiff model has pioneered the production of detailed local health data for use by non - health sectors . linking anonymized data on alcohol - related injuries with the precise location of where the injury occurred provides evidence that is highly relevant to the public safety objective . box 3 case study : scotland 's experience of a public health licensing objective licensing policy in scotland in contrast to england and wales , scottish licensing decisions have been required to promote a fifth licensing objectiveprotecting and improving public healthas set out in the licensing ( scotland ) act 2005 , implemented in 2009 . two years after implementation , a report by alcohol focus scotland concluded that the potential to tackle alcohol - related health harms through this objective had not been met . a major barrier identified was the discrepancy between the population perspective of public health considerations and the case - by - case perspective of licensing decisions . in practice , the public health objective in most scottish licensing statements has most commonly equated to the provision of health information in licensed premises . the few licensing boards who did recognize population - level health determinants , including the overprovision of alcohol , justified their policy positions using systematically gathered and analysed evidence that they documented in policy statements . what international lessons can be learnt from scotland 's experience of a health licensing objective ? first , broadening the scope of alcohol control frameworks to explicitly address health concerns does not change the underlying legal principles governing individual licensing decisions . , population - level evidence can nevertheless be used to justify local policy positions and , to a degree , mitigate legal uncertainty regarding individual licensing decisions . even without a health licensing objective , there are examples of licensing authorities in england and wales establishing the relevance of certain health indicators to current licensing objectives within their policy statements , including child protection cases , domestic violence , alcohol - related injuries and under-18 health attendances associated with alcohol . while not fully reflecting the health harms caused by alcohol consumption , such indicators if linked specifically enough with a particular area or premises can be used to justify licensing decisions even without further national legislative changes . innovations by a number of local authorities demonstrate that the existing licensing objectives in england and wales can promote health needs even in the absence of a legal public health objective . while isolated licensing rulings on individual premises are unlikely to impact considerably on health , case studies give an indication of what the licensing process allows and examples of how local authorities can implement innovative policy ahead of national regulation . arguably the greatest strengths of recent legislative and organizational changes are the new opportunities for collaboration within local government to address a broad range of alcohol - related harms . box 4 gives an example of where a commitment across newcastle city council to tackle alcohol - related harms has led , among other initiatives , to the introduction of minimum unit pricing license conditions for certain premises . planning processes and strategic partnerships are two opportunities to align strategic alcohol goals across sectors including planning , trading standards , police and community safety as well as public health . box 4 case studies : local authority interventions addressing alcohol affordability in newcastle and westminster as part of a pro - active council - wide approach to tackling alcohol harms , newcastle city council have recently granted new licenses for a small number of premises on the condition that they agree to a minimum unit pricing policy . the mup condition is one of a number of cross - sectoral initiatives addressing alcohol overprovision and overconsumption . set at 1.25/unit , significantly higher than the mup level proposed nationally , this condition has been introduced for bars applying for licenses in one street covered by one of a number of cumulative impact policies . so far , this condition has only been applied to high - end on - premises licensees who would probably have priced their drinks above the minimum level even without the condition the impetus behind these conditions was precautionary to protect against the future transfer of the license to owners with business models based around large volume sales of heavily price - promoted drinks . nevertheless , this example joins cases elsewhere of licensing authorities implementing health - relevant policies despite the absence of a health objective . for example , westminster council have granted a supermarket license with a condition banning drinks promotions . these demonstrate the potential for health leads to use existing statutory powers available to local governments to address population - level determinants of alcohol - related health harms . in contrast to licensing , the legal framework governing planning is broad enough to include the goal of health promotion . furthermore , there is precedence for using spatial planning to improve health through regulating the concentration and proximity of takeaway food outlets . where licensing and planning conditions differ , a premises must comply with both , for example by observing whichever specified closing time is earliest . there is therefore scope for addressing long - term population health impacts by controlling local alcohol availability through local development frameworks and development plans . partnership working has formed an important part of local alcohol control policy for many years and is encouraged in the government 's alcohol strategy . where individual partners ' interests conflict with the partnership 's overall aims , however , such collaborations may not be effective . alcohol industry partnerships , including community alcohol partnerships , may prioritize individual - focused interventions such as health information campaigns over more effective population - level regulation . joint strategic needs assessments or strategic partnerships led by local government , for example the safer newham crime and disorder reduction partnership , may offer more effective ways of simultaneously addressing a broad range of alcohol harms . powers to control local alcohol supply and consumption are established by the licensing act 2003 , which transferred authority for granting and reviewing licenses from magistrate courts to local authorities . implemented in 2005 , the act sets out the licensing process ( box 1 ) and defines the statutory licensing objectives that legally underpin all licensing activities ( box 2 ) . the police reform and social responsibility act 2011 granted health leads a statutory role in the licensing process for the first time and gave local authorities additional powers to address the cumulative impact of alcohol sales . the recent return of public health to local authorities presents additional opportunities for cross - sector collaboration on shared objectives . in contrast with scotland , potential health opportunities arising from this new legislation in england and wales must contend with the absence of a statutory health licensing objective . box 1 overview of the process and legal framework governing licensing decisions in england and wales box 2 licensing objectives in england and wales as defined by the licensing act ( 2003 ) prevention of crime and disorder : based on police advice concerning , preventing crime and maintaining order . public safety : physical safety of people using a premises , immediate harms , e.g. accidents , injuries , unconsciousness . prevention of public nuisance : noise nuisance , light pollution , noxious smells , litter and where an effect is prejudicial to health. protection of children from harm : moral , psychological and physical harm , including underage sale of alcohol . local health leads in england and wales have , as responsible authorities , a recognized role in commenting on all licensing applications , yet evidence they present must legally be framed in terms of non - health objectives . the licensing process ( box 1 ) is primarily a method for controlling immediate harms associated with alcohol sales at a particular premises . all license decisions must relate specifically to the premises in question and the promotion of the four statutory licensing objectives ( box 2 ) . government guidance explicitly states that public health should not be the primary consideration for a licensing decision , although health considerations can support concerns regarding a statutory licensing objective . alcohol - related injury rates , for example , are considered relevant to public safety . rates of chronic conditions , on the other hand , are harder to link directly with any of the four licensing objectives , despite accounting for 75% of alcohol - related hospital admissions in england . licensing authorities can only consider health - related evidence that directly links the premises in question to a threat to one of the named licensing objectives ( box 2 ) . the more specifically evidence relates to the premises or location of concern , the greater its legal weight and the less vulnerable it is to appeal . routine health data , rarely collected in a way that can be linked to individual premises , are unlikely to be considered relevant . an area where pub or supermarket chains hold an advantage , since evidence of good operating practice by their company elsewhere is considered relevant to new applications . the practical challenges of acquiring sufficiently detailed health data to support licensing decisions must be weighed against the potential consequences of submitting weakly justified health representations . repeated submissions based on health evidence that is unrelated to the licensing objectives , or not deemed relevant to the applicant , may weaken the credibility of future representations to the licensing sub - committee . conversely , and somewhat paradoxically , not submitting a health representation for an application may be interpreted as evidence that the application holds no health threat . a 2008 high court decision suggests that the absence of expert representation in this case by the police signified that there were no serious concerns about the impact on licensing objectives within that responsible authority 's domain of expertise . despite these limitations , licensing policy statements can be powerful tools to support and coordinate local government actions against alcohol - related harms . licensing statements , open to challenge on appeal or by way of judicial review , must still be consistent with promoting the four licensing objectives . they do , however , allow for broader responses to alcohol consumption above the individual premises level by designating special policies or establishing the local relevance of particular licensing approaches . special policies can be implemented to address area - wide impacts of alcohol consumption , namely : cumulative impact zones ( cizs ) , early morning restriction orders and late night levies . policy statements can also document population - level data , including health impacts , at a higher spatial scale than individual license decisions allow . using policy statements to clearly set out a licensing authority 's reasoning and justification for a licensing approach , backed up by appropriate local evidence , can make individual representations more legally robust while reducing the time needed to write them . this could include , for example , establishing a health rationale for limiting the density of alcohol premises using rates of local alcohol - related hospital admissions . local authorities can designate cizs to control new on- or off - premises alcohol outlets in areas where the cumulative stress caused by existing overprovision of alcohol outlets demonstrably threatens the licensing objectives . licensing decisions are normally made with a presumption in favour of the applicant . under a ciz , the burden of proof is reversed and it is the applicant who must demonstrate how they will avoid threatening the licensing objectives . cizs do not , however , affect existing license holders and decisions are still made on a case - by - case basis . for example , an initial refusal to grant a license within a ciz in leeds was overturned on appeal due to the applicant 's short opening hours , clientele and history of running trouble - free premises . although yet to be implemented by any local authority , early morning restriction orders ( emro ) and late night levies ( lnl ) offer additional mechanisms for controlling alcohol sales from both on- and off - premises between 12 and 6 a.m. emros are designed to address recurrent licensing objective infringements that are not attributable to a single premises , such as night time anti - social behaviour . lnls recoup financial costs associated with late night alcohol provision by levying a charge on any premises licensed to sell alcohol between specified night hours . acting outside of the licensing process , emros and lnls are decided on an area rather than individual - premises basis . lnls must apply to the whole of a licensing authority 's area , while emros can apply to selected parts of this area . the absence of a health licensing objective does not preclude addressing health needs where they concern other licensing objectives . producing specific evidence linking local alcohol consumption practices with licensing objectives does , however , require a change of approach from traditional local health data analysis , demonstrated by the ongoing difficulties scottish licensing boards face in reflecting health evidence in licensing decisions ( box 3 ) even since the introduction of an explicit public health licensing objective . more successfully , the cardiff model has pioneered the production of detailed local health data for use by non - health sectors . linking anonymized data on alcohol - related injuries with the precise location of where the injury occurred provides evidence that is highly relevant to the public safety objective . box 3 case study : scotland 's experience of a public health licensing objective licensing policy in scotland in contrast to england and wales , scottish licensing decisions have been required to promote a fifth licensing objectiveprotecting and improving public healthas set out in the licensing ( scotland ) act 2005 , implemented in 2009 . two years after implementation , a report by alcohol focus scotland concluded that the potential to tackle alcohol - related health harms through this objective had not been met . a major barrier identified was the discrepancy between the population perspective of public health considerations and the case - by - case perspective of licensing decisions . in practice , the public health objective in most scottish licensing statements has most commonly equated to the provision of health information in licensed premises . the few licensing boards who did recognize population - level health determinants , including the overprovision of alcohol , justified their policy positions using systematically gathered and analysed evidence that they documented in policy statements . what international lessons can be learnt from scotland 's experience of a health licensing objective ? first , broadening the scope of alcohol control frameworks to explicitly address health concerns does not change the underlying legal principles governing individual licensing decisions . , population - level evidence can nevertheless be used to justify local policy positions and , to a degree , mitigate legal uncertainty regarding individual licensing decisions . even without a health licensing objective , there are examples of licensing authorities in england and wales establishing the relevance of certain health indicators to current licensing objectives within their policy statements , including child protection cases , domestic violence , alcohol - related injuries and under-18 health attendances associated with alcohol . while not fully reflecting the health harms caused by alcohol consumption , such indicators if linked specifically enough with a particular area or premises can be used to justify licensing decisions even without further national legislative changes . innovations by a number of local authorities demonstrate that the existing licensing objectives in england and wales can promote health needs even in the absence of a legal public health objective . while isolated licensing rulings on individual premises are unlikely to impact considerably on health , case studies give an indication of what the licensing process allows and examples of how local authorities can implement innovative policy ahead of national regulation . arguably the greatest strengths of recent legislative and organizational changes are the new opportunities for collaboration within local government to address a broad range of alcohol - related harms . box 4 gives an example of where a commitment across newcastle city council to tackle alcohol - related harms has led , among other initiatives , to the introduction of minimum unit pricing license conditions for certain premises . planning processes and strategic partnerships are two opportunities to align strategic alcohol goals across sectors including planning , trading standards , police and community safety as well as public health . box 4 case studies : local authority interventions addressing alcohol affordability in newcastle and westminster as part of a pro - active council - wide approach to tackling alcohol harms , newcastle city council have recently granted new licenses for a small number of premises on the condition that they agree to a minimum unit pricing policy . the mup condition is one of a number of cross - sectoral initiatives addressing alcohol overprovision and overconsumption . set at 1.25/unit , significantly higher than the mup level proposed nationally , this condition has been introduced for bars applying for licenses in one street covered by one of a number of cumulative impact policies . so far , this condition has only been applied to high - end on - premises licensees who would probably have priced their drinks above the minimum level even without the condition the impetus behind these conditions was precautionary to protect against the future transfer of the license to owners with business models based around large volume sales of heavily price - promoted drinks . nevertheless , this example joins cases elsewhere of licensing authorities implementing health - relevant policies despite the absence of a health objective . for example , westminster council have granted a supermarket license with a condition banning drinks promotions . these demonstrate the potential for health leads to use existing statutory powers available to local governments to address population - level determinants of alcohol - related health harms . in contrast to licensing , the legal framework governing planning is broad enough to include the goal of health promotion . furthermore , there is precedence for using spatial planning to improve health through regulating the concentration and proximity of takeaway food outlets . where licensing and planning conditions differ , a premises must comply with both , for example by observing whichever specified closing time is earliest . there is therefore scope for addressing long - term population health impacts by controlling local alcohol availability through local development frameworks and development plans . partnership working has formed an important part of local alcohol control policy for many years and is encouraged in the government 's alcohol strategy . where individual partners ' interests conflict with the partnership 's overall aims , however , such collaborations may not be effective . alcohol industry partnerships , including community alcohol partnerships , may prioritize individual - focused interventions such as health information campaigns over more effective population - level regulation . joint strategic needs assessments or strategic partnerships led by local government , for example the safer newham crime and disorder reduction partnership , may offer more effective ways of simultaneously addressing a broad range of alcohol harms . this article describes the mechanisms currently available for addressing alcohol - related health harms at a local level in england and wales . while decisions regarding specific policy interventions depend on the particular needs and context of the target population , our analysis clarifies which interventions local practitioners can feasibly implement . although the current licensing framework imposes a number of constraints on public health , local government interventions continue to be one of the most important ways of addressing alcohol - related health harms . at present , licensing decisions must be framed around non - health arguments and processes . local alcohol policy currently focuses on criminal justice and the immediate management of drunkenness . in contrast to the routine public health data and evidence reviews that public health practitioners are more commonly familiar with , licensing committees need data and arguments specific to the individual geography of the premises or circumstances of the applicant . although it is important to recognize and respect individual rights within the licensing legal framework , public health needs to find ways to highlight the considerable burden of alcohol harms in licensing processes . local public health efforts can be supported nationally in a number of ways . developing resources including evidence reviews , evaluation tools and case studies of best practice will strengthen local representations . providing evidence linking local contexts to alcohol harm will support appropriate licensing policy statements by , for example , justifying how alcohol outlet density influences drinking behaviour . advocating for the addition of a health licensing objective would allow licensing decisions to reflect the growing evidence for alcohol - related health harms and make health representations less burdensome on already overstretched health leads . without a public health licensing objective , health leads face the prospect of being responsible for addressing the potential health harms of granting a license without the clear legal authority to do so . licensing has largely confined its focus to on - premises and immediate harms associated with alcohol consumption . the uk government 's 2012 alcohol strategy marks a shift towards public health - oriented alcohol policy ; however , the majority of this strategy 's content has yet to be enacted . the challenges faced by local governments responsible for local alcohol control without appropriate powers to address important health determinants have been described in new zealand and australia . this article adds to the international literature by analysing legal as well as policy frameworks for local alcohol control within england and wales . alcohol health concerns can be addressed within the current alcohol control framework by utilizing the potential for cross - sector collaboration within local government . pre - emptive data collection to support representations and identify priority areas can improve effectiveness while reducing costs . aligning local planning policy with licensing can improve the long - term control of alcohol availability . finally , local partnerships can harness mechanisms from across local government to address shared concerns related to alcohol consumption . joint strategic needs assessments and joint health and well - being strategies are two mechanisms for public health to collaborate with non - health sectors , but it is important to work with other powerful allies including the police and community safety . our legal analysis is specific to england and wales ; details of our findings can not be assumed to apply directly to other legal contexts . however , the challenges of overcoming the tensions between tackling acute and chronic , social and health harms caused by alcohol consumption while respecting individual freedoms are likely to apply to alcohol policy - making in all contexts . our findings should be interpreted alongside other country - specific policy analyses and international alcohol policy comparisons . this article describes the mechanisms currently available for addressing alcohol - related health harms at a local level in england and wales . while decisions regarding specific policy interventions depend on the particular needs and context of the target population , our analysis clarifies which interventions local practitioners can feasibly implement . although the current licensing framework imposes a number of constraints on public health , local government interventions continue to be one of the most important ways of addressing alcohol - related health harms . at present , licensing decisions must be framed around non - health arguments and processes . local alcohol policy currently focuses on criminal justice and the immediate management of drunkenness . in contrast to the routine public health data and evidence reviews that public health practitioners are more commonly familiar with , licensing committees need data and arguments specific to the individual geography of the premises or circumstances of the applicant . although it is important to recognize and respect individual rights within the licensing legal framework , public health needs to find ways to highlight the considerable burden of alcohol harms in licensing processes . local public health efforts can be supported nationally in a number of ways . developing resources including evidence reviews , evaluation tools and case studies of best practice will strengthen local representations . providing evidence linking local contexts to alcohol harm will support appropriate licensing policy statements by , for example , justifying how alcohol outlet density influences drinking behaviour . advocating for the addition of a health licensing objective would allow licensing decisions to reflect the growing evidence for alcohol - related health harms and make health representations less burdensome on already overstretched health leads . without a public health licensing objective , health leads face the prospect of being responsible for addressing the potential health harms of granting a license without the clear legal authority to do so . licensing has largely confined its focus to on - premises and immediate harms associated with alcohol consumption . the uk government 's 2012 alcohol strategy marks a shift towards public health - oriented alcohol policy ; however , the majority of this strategy 's content has yet to be enacted . the challenges faced by local governments responsible for local alcohol control without appropriate powers to address important health determinants have been described in new zealand and australia . this article adds to the international literature by analysing legal as well as policy frameworks for local alcohol control within england and wales . alcohol health concerns can be addressed within the current alcohol control framework by utilizing the potential for cross - sector collaboration within local government . pre - emptive data collection to support representations and identify priority areas can improve effectiveness while reducing costs . such data can justify cumulative impact policies and support license policy statements . aligning local planning policy with licensing can improve the long - term control of alcohol availability . finally , local partnerships can harness mechanisms from across local government to address shared concerns related to alcohol consumption . joint strategic needs assessments and joint health and well - being strategies are two mechanisms for public health to collaborate with non - health sectors , but it is important to work with other powerful allies including the police and community safety . our legal analysis is specific to england and wales ; details of our findings can not be assumed to apply directly to other legal contexts . however , the challenges of overcoming the tensions between tackling acute and chronic , social and health harms caused by alcohol consumption while respecting individual freedoms are likely to apply to alcohol policy - making in all contexts . our findings should be interpreted alongside other country - specific policy analyses and international alcohol policy comparisons . despite health not being a legally recognized licensing objective , our analysis demonstrates how public health practitioners can address local health consequences of alcohol consumption in england through non - health sector policies . important barriers to working collaboratively across sectors include differences in prioritizing interventions and the types of evidence that can be used to justify policy decisions . collaborations with non - health sectors are more likely to succeed if these differences are understood and addressed . there is , however , a limit to what can be achieved at a local level . this includes using the greater fiscal control available to the national government to upholding pledges to reduce alcohol affordability , for example by introducing minimum unit pricing and multi - buy promotion bans . successful local implementation of minimum unit pricing as a licensing condition or across a province could act as test cases for the introduction of similar national policies , thus embedding health objectives more robustly in alcohol policies . this work was supported by the national institute for health research ( nihr ) 's school for public health research ( sphr ) . the views expressed are those of the author(s ) and not necessarily those of the nhs , the nihr or the department of health . funding to pay for the open access charges was provided by the nihr school for public health research .

backgroundthe power to influence many social determinants of health lies within local government sectors that are outside public health 's traditional remit . we analyse the challenges of achieving health gains through local government alcohol control policies , where legal and professional practice frameworks appear to conflict with public health action.methodscurrent legislation governing local alcohol control in england and wales is reviewed and analysed for barriers and opportunities to implement effective population - level health interventions . case studies of local government alcohol control practices are described.resultsaddressing alcohol - related health harms is constrained by the absence of a specific legal health licensing objective and differences between public health and legal assessments of the relevance of health evidence to a specific place . local governments can , however , implement health - relevant policies by developing local evidence for alcohol - related health harms ; addressing cumulative impact in licensing policy statements and through other non - legislative approaches such as health and non - health sector partnerships . innovative local initiatives for example , minimum unit pricing licensing conditions can serve as test cases for wider national implementation.conclusionsby combining the powers available to the many local government sectors involved in alcohol control , alcohol - related health and social harms can be tackled through existing local mechanisms .

Introduction Methods Results Current English National Legislation Health and alcohol licensing Statements of licensing policy Addressing cumulative impact Developing relevant local health evidence Local government collaboration Discussion Main findings of this study What is already known on this topic What this study adds Limitations Conclusion Funding

PMC3953463

cr allergy has been recognized as important ige - mediated type i hypersensitivity since 1964 . it is associated with the development of asthma and recognized as a risk factor for emergency room admission of asthmatic patients , especially among inner city children living in low - income houses infested with cockroaches . in china , totally 25.7% of allergy patients are skin prick test ( spt ) positive to the american cr ( periplaneta americana , per a ) and 18.7% spt positive to the german cr ( blattella germanica , bla g ) . american cr , german cr , and smoky brown cr ( periplaneta fuliginosa ) are the dominant indoor cr species which cause allergy among human population worldwide . twenty - two immunoglobulin e ( ige ) binding components including the proteins of 23 , 28 , 35 , 38 , 40 , 49 , 72 , 78 , and 97 kda were identified as major allergens in american cr , but only per a 1 , per a 2 , per a 3 , per a 4 , per a 7 , per a 9 , and per a 10 are characterized . for example , per a 1 is an isoallergen with 5 isoforms reported so far , that is , per a 1.0101per a 1.0105 . per a 1 is a major american cr allergen as it binds to ige in the sera of 90100% of cr allergic subjects . per a 2 . per a 5 belongs to gsts , but the allergenicity of the gst homologues in american cr has not been reported . since the gst of german cr ( bla g 5 ) is a major cockroach allergen , which induces specific ige expression in 30 to 71% of cr allergy [ 1214 ] , we anticipate that per a 5 is likely a major allergen of american cr and investigate the potential allergenicity of it in the present study . two gst homologue ( per a 5 ) genes are available in the genbank ( accession : ay792949 and aev23867 ) , which provides the possibility of producing recombinant per a 5 ( rper a 5 ) in large amounts to study its role in allergic reactions . many , but not all allergens expressed from cdna have shown a considerable ige binding reactivity that seems to be comparable to their natural counterparts . the majority of these recombinant allergens are produced in e. coli , but , unfortunately , the amount and/or reactivity is sometimes reduced when the allergen is purified and subjected to immunological and biochemical assays . to overcome some of these problems , the aim of the present study is to generate rper a 5 by using eukaryotic ( baculovirus - infected insect cells ) and prokaryotic ( e. coli ) expression systems and characterize its biochemical and immunologic properties . the study protocol was approved by the ethical committee of the first affiliated hospital of nanjing medical university . written informed consent for the use of blood samples was obtained from all participants before study entry according to the declaration of helsinki . a total of 16 allergic rhinitis patients with positive spt ( allergens were supplied by alk - abell , inc . , denmark ) and positive serum ige test to american cr extract ( by using immuno - cap assay ( pharmacia diagnostics ab , uppsala , sweden ) ) and 6 healthy controls ( hc ) were recruited in the study . serum ( 4 ml ) from peripheral venous blood was collected from each patient and hc for western blot analysis . total rna was isolated from adult female cr reared at our institute by using trizol reagent ( invitrogen , carlsbad , ca , usa ) . the cdna was prepared by reverse transcription using a commercial rna - pcr kit according to the manufacturer 's instruction ( takara biotech co. ltd . , dalian , china ) . for each reaction , 1 g of total rna was reversely transcribed using oligo - d ( t ) . the cdnas encoding per a 5 were amplified by pcr using primers based nucleotide sequence of per a 5 gene ( ay792949 and aev23867 ; forward : 5-atgaccatcgacttctacta-3 ; reverse : 5-tcacttcttggcgaggttat-3 ) . pcr condition was 95c/5 min ( one cycle ) , 95c/1 min , 52c/1 min and 72c/1 min ( 30 cycles ) , and 72c/5 min ( one cycle ) . the purified pcr product was cloned into apmd18-t vector ( takara biotech co. ltd . , dalian , china ) before being transformed into escherichia coli strain dh5. the inserts were sequenced on an abiprism 377 dna sequencer ( applied biosystems , foster , ca , usa ) . dna sequence data were translated to amino acid sequence by show translation tool in sms software package ( http://www.bioinformatics.org/sms/ ) . the glycosylation motifs of per a 5 were predicted by using netnglyc 1.0 server ( http://www.cbs.dtu.dk/services/netnglyc ) . the per a 5 gene was subcloned into pfastbac1 vector ( novagen , madison , wi , usa ) using ecor i and sal i sites and the resulted construct was transformed into e. coli strain dh10bac to generate recombinant bacmid . the recombinant bacmid was transfected into sf-9 cells by using cellfectin ( invitrogen corporation , carlsbad , usa ) and incubated in sf-900ii liquid medium ( invitrogen corporation , carlsbad , usa ) for 5 days at 27c until the cells got swollen . a total of 500 ml of sf-9 cells were infected by p2 viruses and harvested at 72 h. the cells were lysed against 50 mm tris - hcl with 300 mm nacl and 5% glycerol . the supernatant was loaded on ni - nta column ( genscript , nanjing , china ) , washed with running buffer containing 50 mm tris - hcl , 300 mm nacl , and 5% glycerol ( ph 8.0 ) and eluted with elution buffer containing 50 mm tris - hcl , 300 mm nacl , 250 mm imidazole , and 5% glycerol ( ph 8.0 ) . the eluted fractions were obtained and identified as per a 5 ( iper a 5 ) . the purified iper a 5 was dialyzed in carbonate - bicarbonate buffer ( 0.05 m , ph 9.6 ) for further investigation . the concentration of iper a 5 was determined by using a coomassie plus assay kit with bsa as standard ( thermo scientific pierce , rockford , il , usa ) . the per a 5 gene was subcloned into pcold ii vector ( takara biotech co. ltd . , dalian , china ) using nde i and xba i sites and verified by dna sequencing . the recombinant pcold ii - per a 5 plasmid was transformed into e. coli origami host strain . a colony of the selected transformed e. coli origami on an overnight lb - ampicillin agar plate was inoculated into 5 ml of lb - ampicillin broth and incubated at 37c overnight . for iptg optimization , the overnight culture was added into fresh lb media in a ratio of 1 : 100 . once cell density reached the optical density at a600 nm ( od600 ) , the cells were incubated with 0.1 , 0.5 , and 1 mm iptg , respectively , at 15c overnight . expression of the target protein was analyzed by sds - page . for scale - up expression , 40 ml of the culture was inoculated into 2 l of fresh lb - ampicillin broth and incubated at 37c until od600 reached 0.6 . iptg was added to the final concentration of 0.5 mm and the culture was incubated overnight . the bacterial cells were harvested by centrifugation at 4,000 g at 4c for 20 min and were lysed in a lysis buffer by sonication at 20 khz , 2 min pulse - on , 3 min pulse - off . cell debris was removed by centrifugation at 12,000 g at 4c for 20 min . the supernatant was loaded on the nickel column ( genscript , nanjing , china ) as described above , and the eluted fractions were obtained and identified as per a 5 ( bper a 5 ) . the purified bper a 5 was dialyzed in carbonate - bicarbonate buffer ( 0.05 m , ph 9.6 ) for further investigation . the concentration of bper a 5 was determined by using a coomassie plus assay kit with bsa as standard . far uv cd spectra of bper a 5 and iper a 5 were collected on a jasco j-810 spectropolarimeter ( japan spectroscopic co. , tokyo , japan ) using a 1 mm path length quartz cuvette at protein concentrations of 0.1 mg / ml . spectra were measured from 240 to 190 nm , with a 0.5 nm resolution at a scanning speed of 50 nm / min , and resulted from averaging of three scans . results were expressed as the mean residue ellipticity ( y ) at a given wavelength . the secondary structure content of bper a 5 and iper a 5 was calculated by using the secondary structure estimation program k2d2 . a 96-well plate was coated with purified bper a 5 and iper a 5 at 10 g / ml in carbonate - bicarbonate buffer ( 0.05 m , ph 9.6 ) overnight at 4c , 100 l per well . human serum samples ( 1 : 20 dilution in pbs - tween with 2% bsa ) were then added to the plates for 2 h at room temperature . after ige binding , plates were incubated with horseradish peroxidase - labeled goat anti - human ige ( 1 : 2500 dilution ) ( kpl , inc . , md , usa ) , and the color was developed with tetramethylbenzidine peroxidase substrate ( thermo scientific pierce , rockford , il , usa ) . the cutoff of the elisa was calculated as the mean of the negative controls plus 2 sds . for competitive elisa test , a 96-well plate was coated with american cockroach extract ( 10 g / ml ) in carbonate - bicarbonate buffer ( 0.05 m , ph 9.6 ) overnight at 4c . the 1 : 20-diluted pooled serum alone or preincubated with various quantities of the crude extract , iper a 5 and bper a 5 , for 2 h was added to the plates . crude extract of cockroaches were prepared according to the methods described previously with few modifications . the sample was defatted in 200 ml of ethyl ether and ethyl acetate ( 1 : 1 , by volume ) and extracted with slow overhead stirring in carbonate - bicarbonate buffer ( 0.05 m , ph 9.6 ) , containing 6 mm 2-mercaptoethanol and 1/1,000 volume of protease inhibitor cocktail ( shenggong , shanghai , china ) at 4c overnight . the extract was then centrifuged at 10,000 g for 30 min at 4c , and the supernatant was filtered through a 0.22 m - pore - size filter ( millipore , bedford , usa ) before use . immunoblots for detection of serum specific ige were performed with bper a 5 and iper a 5 as described previously [ 19 , 20 ] . bper a 5 and iper a 5 ( 5 g ) were added to a sds - page ( gel concentration of 15% ) under reducing conditions and then transferred to nitrocellulose membranes . the nitrocellulose membranes were incubated with the sera from the patients with american cr allergy ( 1 : 5 in pbs - tween with 1% bsa , 10% normal goat serum ) for 90 min . following rinsing with pbs , the membranes were incubated with peroxidase - labeled anti - human ige monoclonal antibody . expression of cd63 and ccr3 on basophil surface has been considered as the indicator of basophil activation [ 21 , 22 ] . briefly , peripheral blood mononucleated cells ( pbmc ) from 20 ml blood donated by 4 healthy volunteers were separated by ficoll - paque density gradient and treated with 10 ml ls ( a solution containing 1.3 m nacl , 0.005 m kcl , and 0.01 lactic acid , at ph 3.9 ) for 2 min at 8c . after neutralization with 12% tris ( ph 10.9 ) , nonspecific ige on basophils was stripped off and cells were passively sensitized with sera of the patients with american cr allergy or hc ( n = 4 , 1 in 10 dilution , 2 h at 37c ) as described previously . the cells were then challenged with various concentrations of bper a 5 and iper a 5 for 15 min at 37c . a goat anti - human ige antibody ( serotec , kidlington , uk ) san diego , ca , usa ) and anti - human cd63-fitc antibody ( invitrogen corporation , camarillo , ca , usa ) were added to cells for 15 min at 37c in the dark . flow cytometry analysis of surface markers was performed at 488 nm on a facsaria flow cytometer ( becton dickinson , franklin lakes , nj , usa ) and analyzed by facsdiva software . basophils were gated in the lymphocyte region of the ssc or fsc pattern , and identified as a single population of cells that stained positively for ccr3-pe antibody . upregulation of cd63 expression was determined by an increase in fluorescence in the fl-1 channel . responses were quantified as percentages of cd63 expressing basophils in a higher fl-1 region , which had been adjusted to contain 4% of basophils . data are expressed as mean sem for the indicated number of independently performed duplicated experiments . statistical significance between means was analyzed by one - way anova or the student 's t - test utilizing the spss 13.0 version . the cdnas encoding per a 5 were amplified by pcr using primers based on the nucleotide sequence of per a 5 gene . it is a 645 bp gene and encoded 215 amino acids protein ( figure 1 ) . the sequence identity of per a 5 to cdnas deposited in genbank ( accession no . . per a 5 shows 81 , 15 , and 13% sequence similarity to german cr allergen bggstd1 , der p 8 , and bla g 5 ( figure 2 ) . one motif ( 198nhsg201 ) was predicted to be the glycosylation motif of per a 5 . the per a 5 gene was subcloned into pcold ii vector and transformed into e. coli origami host strain . the optimal induction condition for per a 5 was 0.5 mm iptg ( figure 3(a ) ) , the concentration chosen as the final condition throughout the study . more than 6 mg bper a 5 was obtained from 2 l cell culture medium . it showed single band with an apparent molecular weight of 25 kda ( figure 3(b ) ) . the per a 5 encoding gene was subcloned into pfastbac1 vector and transformed into e. coli strain dh10bac to generate recombinant bacmid . the per a 5 protein was expressed in sf-9 cells and purified by ni column . about 16 mg of iper a 5 was obtained from 2 l cell culture medium . the purity of the purified per a 5 was identified by sds - page , which showed major band with an apparent molecular weight of 25 kda ( figure 4 ) . the far uv cd spectra of iper a 5 and bper a 5 showed similar curves with two minima at 220 and 209.5 nm and a large maximum at 192 nm , which represent characteristics of proteins with predominantly -helical structure ( figure 5 ) . calculation of the secondary structure using the program k2d2 resulted in predicted 45.93 and 8.69% of -helix and -sheets in iper a 5 and 42.54 and 8.49% of -helix and -sheets in bper a 5 , respectively . in order to determine the allergenicity of per a 5 , we examined the ability of per a 5 to bind ige in the sera of american cr allergy by a direct elisa technique . patients serum including patients 5 , 9 , 10 , and 14 showed positive ige reactivity to both bper a 5 and iper a 5 . the results showed that 4 out of 16 ( 25% ) sera from these patients reacted to both bper a 5 and iper a 5 ( figure 6(a ) ) . the ige reactivity of bper a 5 and iper a 5 in the sera from the per a 5 positive patients was increased by 5.0- and 7.9-fold , respectively in comparison with the sera from hc . moreover , competitive elisa showed that bper a 5 and iper a 5 inhibited the ige reactivity to the cockroach extract by approximately 25.4 and 35.5% , respectively ( figure 6(b ) ) . ige binding activity of per a 5 in a representative group of 3 patients and two hc was assessed by western blot and was illustrated in figure 6(c ) . ige binding bands appeared clearer with iper a 5 than with bper a 5 . both iper a 5 and bper a 5 iper a 5 and bper a 5 at 1.0 g / ml induced approximately up to 4.5- and 3.2-fold increase in the expression of cd63 and ccr3 in cd63 and ccr3 double positive cells when incubating with passively sensitized basophils ( by sera from american cr allergy ) . both iper a 5 and bper a 5 had no effect on the basophils sensitized by the sera from hc ( figure 7 ) . aerosolized proteins derived from saliva , fecal material , secretions , cast skins , debris , and dead bodies of cockroaches induce ige - mediated hypersensitivity . to better understand the per a 5 mediated cr allergies and promise to improve diagnosis and treatment of cr allergies , we prepared biologically active and highly pure american cr allergen per a 5 in relatively large amount in the present study . we have identified per a 5 as a novel american cr allergen , which is recognized by 25% of the subjects with american cr allergy . several allergens from german and american cr have been identified and their ige cross - reactivity has been described . these include bla g 1 and per a 1 ( food intake and digestion ) ; bla g 2 and per a 2 ( inactive aspartic protease ) ; bla g 3 and per a 3 ( arylphorin - like storage protein ) ; bla g 4 and per a 4 ( male pheromone transport lipocalin ) ; and bla g 7 and per a 7 ( tropomyosin ) . among cr allergens , 3 gsts were identified from male adults of german cr by glutathione - agarose affinity chromatography including one in which n - terminal amino acid sequence is identical to that of bla g 5 . two ige - reactive gsts were detected from 25 ige - reactive spots by proteomic analysis , and one of them was found to be bla g 5 . recently , a delta class gst ( bggstd1 ) , which has 15% amino acid sequence identity with bla g 5 , was purified from the german cr . however , the allergenicity and ige cross - reactivity of the gst homologues in american cr have not been reported . as a member of the delta class gsts , per a 5 shows 81% sequence similarity to bggstd1 , but only 13% similarity to bla g 5 , a sigma class gst identified from german cr . in the present study , only 25% ige reactivity to rper a 5 was observed when sera from cockroach - sensitive patients was incubated with rper a 5 , suggesting that per a 5 is not likely a major allergen in american cr . the ige reactivity rate of per a 5 seems lower than that of bla g 5 ( 30 to 71% ) [ 1214 ] , but higher than that of bggstd1 ( 17.9% ) . it has previously been reported that american cr extracts could not inhibit ige antibody binding to bla g 5 using an ria assay , suggesting that there was no significant ige cross - reactivity between gsts from these two species . however , huang et al . found that gst(s ) in american cr is(are ) allergenic and is(are ) pan - allergens in cr and mites . the finding that per a 5 reacts with specific ige of american cr allergy in the present study suggests that gst in american cr is allergenic . we prepared rper a 5 by using two different expression systems in the present study and found that as little as 2 l of e. coli and sf-9 cell culture medium was able to produce 8 and 16 mg of highly pure rper a 5 , respectively , which is enough for functional study of per a 5 . the e. coli system is a well - established system offering many advantages : easy handling of the bacteria cells and selection of a large variety of vectors using different promoters . among the disadvantages , overexpressed proteins can be incorrectly folded and may require chemical refolding procedures to obtain the protein in a native , fully active , biological form . in the present study , we chose pcold ii vector and e. coli origami host strain to produce bper a 5 in a soluble form without any reconstitution process . the eukaryotic baculovirus expression system is characterized by an extensive array of posttranslational processing , typical for higher eukaryotic cells . the production of recombinant proteins in this system offers the advantage that secreted proteins are often glycosylated and disulphide - bonded correctly leading to a biologically active conformation . because of the advantage of baculovirus - infected insect cell expression system , we employed it to express iper a 5 and managed to obtain substantial quantity of iper a 5 in the present study . numerous insect allergens such as api m 1 , api m 2 from honeybee venom , sol i 3 from ant venom , dol m 5 from bald - faced hornet ( dolichovespula maculate ) , cul s 1 from the north american midge ( culicoides sonorensis ) , der f 1 from house dust mite , blo t 11 from dust mite ( blomia tropicalis ) , lep d 2 from dust mite ( lepidoglyphus destructor ) , and aed a 1 and aed a 2 from mosquito ( aedes aegypti ) have been successfully expressed in insect cells using a baculovirus expression system . they are reported to possess similar structural and biological activities to their natural forms . regarding the immunoreactivity , rper a 5 produced in the two systems showed ige - binding activities towards the sera from the american cr allergic patients . iper a 5 seems to have better binding activity than bper a 5 , implicating that the ige - binding activity of rper a 5 is likely to associate with its glycosylation sites and tertiary conformation . one motif ( 198nhsg201 ) was predicted to be the glycosylation motif in per a 5 , but we are not sure of the numbers of -gals in it , as most of the carbohydrate epitopes are known to lead false - positive reactions except -gal . the far uv cd spectra of recombinant iper a 5 and bper a 5 showed similar secondary structures , which consist of predominantly -helical structure . ige - binding epitopes recognized by ige antibodies are either linear or conformational and are located on the surface of the molecule accessible to antibodies . conformational epitope comprises amino acids that are close in space in the folded molecule , despite being noncontiguous in the amino acid sequence . for globular inhaled allergens , conformational epitopes play a very important role in allergenicity [ 41 , 42 ] . therefore , it seems likely that extra conformational ige - binding epitopes may exist in iper a 5 , as a result of more -helical structures in its molecular structure . the basophil activation test we employed herein is a more advanced technique for determination of allergenicity of a given compound . we confirm that per a 5 is an active allergen of cr as it is able to activate cr - sensitized basophils . similar to ige - binding activity , iper a 5 seems more potent in activation of basophils compared with bper a 5 , suggesting that the basophil - activating activity of rper a 5 is likely to associate with its glycosylation sites and tertiary conformation . the availability of recombinant allergens has increased our understanding of ige - mediated allergies and promises to improve diagnosis and treatment of these diseases . in our case , these observations also confirm that baculovirus - infected insect cell expression system is more suitable for the production of more active per a 5 allergen than e. coli expression system . in conclusion , we have cloned and prepared two rper a 5 allergens by using a eukaryotic and a prokaryotic expression system . we confirm that there is gst allergen ( per a 5 ) in american cr , though it is not a major allergen . rper a 5 should be a useful tool for studying and understanding the role of per a 5 in cr allergy .

glutathione s - transferase ( gst ) from various arthropods can elicit allergic reactions . in the present study , per a 5 , a gst , was cloned from american cockroach ( cr ) and expressed in both baculovirus - infected insect cell ( iper a 5 ) and e. coli expression ( bper a 5 ) systems . the secondary structures were predicted to be 45.93 and 8.69% of -helix -sheets in iper a 5 and 42.54 and 8.49% of -helix and -sheets in bper a 5 , respectively . it is found that 4 out of 16 ( 25% ) sera from american cr allergy patients reacted to both bper a 9 and iper a 9 as assessed by elisa and western blotting analysis , confirming that per a 5 is not a major allergen of american cr . induction of upregulated expression of cd63 and ccr3 on passively sensitized human basophils ( sera from american cr allergy patients ) by approximately up to 4.5- and 3.2-fold indicates that iper a 5 and bper a 5 are functionally active . recombinant per a 5 ( rper a 5 ) should be a useful tool for studying and understanding the role of per a 5 in cr allergy .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4756205

the hypothalamic suprachiasmatic nucleus , the primary circadian oscillator in humans , is the site for the master circadian clock [ 1 , 2 ] . light phase shifts the endogenous oscillator in the hypothalamic suprachiasmatic nucleus , synchronizing it with the day - night cycle [ 1 , 2 ] . some years ago , a study of human eyes culminated in the breakthrough discovery of intrinsically photosensitive retinal ganglion cells ( iprgcs ) , which constitute a subtype of retinal ganglion cells ( rgcs ) that express melanopsin , a photopigment that is most sensitive to short - wavelength blue light ( 480 nm ) [ 3 , 4 ] . in addition to being intrinsically light - sensitive [ 3 , 57 ] , iprgcs are associated with circadian , neuroendocrine , and neurobehavioral functions as well as having influence on some image - forming functions [ 69 ] . glaucoma , an optic neuropathy characterized by the progressive loss of rgcs , is associated with morphological changes in the optic nerve and the retinal nerve fiber layer ( rnfl ) . several investigators have explored whether light transmission to the hypothalamic suprachiasmatic nucleus or the postillumination pupillary light response ( plr ) is compromised as a result of iprgc damage in glaucoma [ 1114 ] . the activity of iprgcs has been evaluated in patients with glaucoma using either the light - induced reduction of nocturnal pineal melatonin secretion or the plr test as a functional indicator of melanopsin - based phototransduction [ 1214 ] . most of these previous studies have shown significant reductions in iprgc function in affected eyes compared with the eyes of healthy subjects [ 1114 ] . in addition , previous studies using the plr test have demonstrated significant correlations between decreased iprgc function and functional [ 11 , 13 ] and structural damage in patients with glaucoma . few studies have tried to find an association between abnormal iprgc function and the severity of sleep disorders in glaucoma patients [ 16 , 17 ] . wang et al . used the self - rated pittsburgh sleep quality index ( psqi ) to evaluate whether glaucoma affects sleep quality . these authors showed that the prevalence of sleep disorders was higher among patients with glaucoma than among healthy participants . although these studies indicate that patients with glaucoma exhibit significantly altered sleep patterns that are also correlated with the plr test , the relationships between objective polysomnographic measures and self - reported diurnal symptoms ( e.g. , excessive daytime sleepiness ) have yet to be established . the degree of daytime sleepiness can be assessed using the epworth sleepiness scale ( ess ) , which can be helpful in diagnosing sleep disorders . the ess score has never been determined in a glaucomatous population , nor has it been associated with the objective polysomnography measures that are considered as the gold - standard test for assessing sleep disorders . the study uses the plr ( as a parameter for iprgc dysfunction ) and correlated these measurements with ess and polysomnography to evaluated correlations between sleep disorder and iprgc function . the protocol for this cross - sectional study followed the tenets of the declaration of helsinki and was approved by the institutional review board of the federal university of so paulo ( cep 262.470 ) . written informed consent thirty of these participants had primary open - angle glaucoma , and 10 others were included for the control group . the inclusion and exclusion criteria , as well as the ophthalmological assessments , have been previously used and are described in detail as follows [ 15 , 17 ] . all subjects underwent a complete ophthalmological examination , including a medical history review , best - corrected visual acuity measurement , slit - lamp biomicroscopy , gonioscopy , intraocular pressure ( iop ) measurement , dilated fundoscopic examination using a 78-diopter ( d ) lens , refraction , and standard automated perimetry ( sap ) using the swedish interactive threshold algorithm ( sita standard 24 - 2 ; carl zeiss meditec , inc . , subjects were excluded if they were younger than 40 years old or older than 80 years old ; had a best - corrected visual acuity worse than 0.2 logmar ( logarithm of the minimum angle of resolution ) ; had a previous history of ophthalmic surgery ; had lens opacity greater than 0.5 ( cortical opacity , nuclear opalescence , posterior subcapsular opacity , or changes in nuclear color ) according to the lens opacity classification system iii ( locs iii ) ; had any corneal , retinal , or orbital diseases ; presented with an absolute spherical refractive error > 5 d or an absolute cylindrical error > 3 d ; or were using alpha - adrenergic agonist eye drop medication or any systemic medication that might affect plr . in addition , none of our patients reported any travel or shift work during the examination period . only patients with an open angle at gonioscopy the participants were considered as having glaucoma if they had at least 3 repeatable , consecutive , abnormal visual field test results , which were defined as a pattern standard deviation outside the 95% confidence interval for the normal range or a glaucoma hemifield test result outside the normal limits and a corresponding alteration in the appearance of the optic disc . patients were also considered as having glaucoma if they showed signs of glaucomatous optic neuropathy at clinical examination by a glaucoma expert , confirmed by stereophotography . glaucomatous damage to the optic disc nerve was defined as the presence of rnfl defects or localized or diffuse neuroretinal rim loss . the control participants showed normal results at ophthalmological examination , an iop of < 21 mmhg , normal visual field test results , and absence of glaucomatous optic neuropathy based on fundoscopic and stereoscopic optic disc photograph evaluations . the entire cohort ( i.e. , the 30 patients with primary open - angle glaucoma and the 10 controls ) belongs to the same sample of a study that was published elsewhere [ 15 , 17 ] . no additional participant was recruited to augment that previously published sample [ 15 , 17 ] . sleep disorders were evaluated using the ess , which has been widely used as a subjective measurement of daytime sleepiness . this test measures the probability of falling asleep across eight daily situations ; each question is scored as 0 ( e.g. , would never doze ) , 1 ( slight change of dozing ) , 2 ( moderate chance of dozing ) , or 3 ( high chance of dozing ) , for a total possible score of 24 . the clinically normal score of this scale ranges from 2 to 10 . a score higher than 10 indicates a borderline or increased level of chronic sleepiness , and a score higher than 15 is defined as excessive daytime sleepiness . measurement of the plr was based on a method developed by park et al . ; our previous studies [ 15 , 17 ] and other research groups have used this technique . to preferentially stimulate iprgc function , we used 470 nm ( blue ) flashes with luminance of 250 cd / m ( 14.1 log photons / cm / s melanopic excitation ) of 1 s in duration . alternatively , to preferentially stimulate different retinal photoreceptors ( e.g. , cones and rods ) without directly stimulating the iprgcs , we used 640 nm ( red ) flashes of 1 s in duration with luminance of 250 cd / m ( 10.7 log photons / cm / s melanopic excitation ) . table 1 summarizes full equations for calculating illuminance values for iprgc , cones , and rods functions . for each stimulus , a red flash was presented first , followed by a blue flash 60 s after the offset of the red flash . the interval between stimuli was used to allow the pupil size to return to baseline before the presentation of the subsequent stimulus . the patients were dark - adapted for 10 min ; then , alternating 1 s red and blue flashes were presented at luminance of 250 cd / m ( 10.7 and 14.1 log photons / cm / s melanopic excitation , resp . ) . stimuli were generated by corresponding light - emitting diodes ( leds ) in a ganzfeld system ( retiport ; roland consult , brandenburg , germany ) , and responses were recorded using an eye - tracking camera system with an infrared led ( ir333-a ; arrington research , scottsdale , az , usa ) . the peak wavelength of the infrared leds was 940 nm [ 20 , 22 , 23 ] . pupil size was directly measured ; the largest diameter was considered as the primary measure . the peak plr amplitude was calculated as the maximal pupillary constriction and expressed relative to the baseline value ( peak plr amplitude = maximal constriction diameter / baseline diameter ) . in addition , the sustained plr was expressed as the pupil diameter 6 s after the flash offset relative to baseline . the absolute values of the peak and sustained responses to blue and red flashes at 250 cd / m for a similar cohort were reported in our previous studies [ 15 , 17 ] . to obtain sleep parameter data , polysomnographic recordings were performed over a full night in a temperature - controlled and sound - attenuated room ( embla_s7000 , embla systems inc . , the following parameters were included in the polysomnographic assessment : total sleep time ( tst ; the amount of actual sleep time over a sleep period ) ; sleep efficiency ( tst per sleep period ) ; sleep latency ( i.e. , the amount of time before starting the effective sleep period ) ; rem sleep latency ( i.e. , the amount of time before starting rem sleep ) ; and the s1 to s3 and rem sleep stages ( as a percentage of tst ) . arousal was defined as a 3 s increase in the electroencephalographic frequency preceded by 10 s of stable sleep . the following data were collected for the arousal assessment : total arousal duration after falling asleep ( in minutes ) , total number of arousals ( n ) , and arousal index ( number of events per hour ) . the apnea - hypopnea index ( ahi , event count per hour of tst ) was calculated as the total number of episodes of apnea and hypopnea per hour of electroencephalographically confirmed sleep . apnea was defined as the cessation of airflow for at least 10 s combined with the effort to breathe . hypopnea was classified as a minimum 30% reduction in thoracoabdominal movement or airflow compared with baseline lasting at least 10 s together with 4% oxygen desaturation . our previous study reported different polysomnographic parameters from a similar cohort [ 15 , 17 ] . a board - certified physician who was unaware of the study design ( and was therefore blind to patients ' ophthalmic examination results ) analyzed the polysomnographic tests . the polysomnographic tests were performed within 6 months of the administration of the ess questionnaire , the ophthalmological exams , and the plr tests . both eyes of each participant were included in this analysis . to correct for the bias introduced by the expected correlation between the two eyes of each participant , a generalized estimating equation ( gee ) was used to adjust for intereye correlations . after adjusting for within - patient intereye correlations , the gee was used to examine the relationship between the ess score and the plr across the entire cohort . in addition , a linear regression was used to determine the association between the ess score and the polysomnographic parameters for both groups . to obtain the correlation coefficients regarding the association between the ess score and the plr as well as the polysomnographic parameters , a linear regression was performed . whenever both eyes were eligible , the right eye was arbitrarily selected for this specific analysis . statistical analyses were performed using commercially available stata software ( version 13 , statacorp lp , college station , tx , usa ) . the level ( type i error ) this study examined both eyes of 40 subjects : 30 with glaucoma and 10 healthy ( control group ) . the mean ( standard deviation ) ages of the control and glaucoma groups were 56.10 8.08 ( range = 45 to 74 ) and 62.00 9.39 ( range = 42 to 75 ) years , respectively ( p = 0.084 ) . the demographic and clinical characteristics of the participants are summarized in table 2 . relevant variation was present with regard to the average md in the glaucomatous eyes , ranging from 32.28 to 0.53 db . the mean ess score of the glaucoma patients was significantly higher than that of the control group ( 13.10 5.14 and 9.10 3.73 , resp . , nineteen glaucoma patients ( 63.33% ) and three control patients ( 30.00% ) reported an ess score > 10 , which is considered as borderline or indicative of an increased level of chronic sleepiness ; however , this difference was not significant ( p = 0.234 ; fisher 's exact test ) . the ess score was greater than 15 for 10 of the participants in the glaucoma group ( 33.33% ) , suggesting a high level of daytime sleepiness ; however , none of the participants in the control group had an ess score greater than 15 . figure 1 illustrates the distribution of the ess scores among the controls and glaucoma patients . a significant association was found between the ess score and glaucoma severity based on sap md ( p < 0.001 ) ; specifically , higher ess scores were related to worse damage ( figure 2(a ) ) . table 3 shows the associations between the ess scores and the polysomnographic parameters for the entire cohort . a significant inverse correlation was observed between the ess score and sleep efficiency ( p = 0.002 ) , indicating that higher ess scores were associated with lower sleep efficiencies . a significant association was also found between the ess score and arousal duration after falling asleep ( p < 0.001 ) ; specifically , higher ess scores were related to longer arousal durations after falling asleep . the ess score was also associated with the number of arousals and the arousal index ( p = 0.039 and p = 0.013 , resp . ) . figure 2(b ) illustrates the association between the ess score and the number of arousals at polysomnography for the entire cohort . age was included in the multivariate model but did not exert a significant effect ( p > 0.05 for all polysomnographic parameters ) . a significant inverse correlation was observed between the ess score and the peak response to the blue flash with a luminance of 250 cd / m ( p = 0.017 , r = 0.080 ) . furthermore , a significant correlation was found between the ess score and the sustained response to the blue flash with luminance of 250 cd / m ( p = 0.009 , r = 0.068 ) . a higher ess score was associated with lower peak and sustained responses to the blue flash at 250 cd / m . figure 2(c ) shows the association between the ess score and the sustained response to the blue flash with luminance of 250 cd / m for the entire cohort . an inverse association was also found between the ess score and the sustained response to the red flash at 250 cd / m ( 10.7 log photons / cm / s ) ( p = 0.010 ; r = 0.080 ) ; specifically , a higher ess score was associated with a lower sustained response to the red flash with luminance of 250 cd / m ( 10.7 log photons / cm / s ) . however , no correlation was observed between the ess score and the peak response to the red flash at 250 cd / m ( 10.7 log photons / cm / s ; p = 0.199 ; r = 0.021 ) . in addition , age was examined in the multivariate model ; however , it was found not to exert a significant effect on the associations between the ess score and either the peak or sustained response to the blue flash with luminance of 250 cd / m for either the control or glaucoma group ( p = 0.403 and p = 0.290 , resp . ) . in addition , age did not significantly affect the associations between the ess score and either the peak or sustained response to the red flash with luminance of 250 cd / m for either the control or glaucoma group ( p = 0.223 and p = 0.375 , resp . ) . this study used the ess to evaluate sleep disorders , specifically daytime sleepiness , in glaucoma patients . in addition , we addressed the relationship between the ess score and both polysomnographic measures and plr in glaucoma patients . increased daytime sleepiness , as measured by the ess , was associated with arousal duration after falling asleep , sleep efficiency , number of arousals , and the arousal index at polysomnography . in addition , increased ess scores were associated with reduced peak and sustained plrs to a blue flash with luminance of 250 cd / m . to the best of our knowledge , this study is the first to identify the associations between daytime sleepiness and polysomnography and plr parameters . sleep disorders ( evaluated using ess and polysomnography ) and plr might be related to certain types of iprgcs . the results of this study are in agreement with those of previous studies showing that glaucoma is associated with a decrease in sleep quality [ 16 , 30 , 31 ] . wang et al . used the psqi to investigate whether glaucoma affects sleep quality and to evaluate the differences between disparate types of glaucoma ( i.e. , primary open - angle glaucoma and primary closed - angle glaucoma ) . these authors found that the prevalence of sleep disorders was higher among patients with glaucoma and that the patients with primary closed - angle glaucoma had a greater incidence of sleep disorders than those with primary open - angle glaucoma . using the ess the ess is a self - report questionnaire that is considered as the gold standard for assessing daytime sleepiness ; however , to date , the ess score has never been used to investigate glaucoma patients . we hypothesized that ocular damage in patients with glaucoma affects all types of rgcs ( including iprgcs ) and that this damage is linked to the dysregulation of the circadian system , thereby leading to decreased sleep quality and resulting in excessive daytime sleepiness . the finding in our study supported these hypotheses , showing that patients with glaucoma have higher daytime sleepiness associated with worse parameters in polysomnography . the excessive daytime sleepiness observed among glaucoma patients was likely because of the increased number or total duration of arousals after falling asleep , decreased sleep efficiency , and briefer tst . daytime sleepiness ( as measured using the ess ) was also associated with different polysomnographic parameters such as sleep efficiency , arousal duration after falling asleep , arousals , and the arousal index . a previous study performed by our group demonstrated that patients with glaucoma have significantly lower average tsts and sleep efficiencies as well as longer arousal durations after falling asleep . daytime sleepiness was also associated with plr , a well - known indicator of the afferent input from the retina and the optic nerve . briefly , the plr shows a transient phase of pupillary constriction , which is attributed to rod and cone input to the iprgcs , followed by sustained pupil constriction , which is primarily driven by the melanopsin response in the iprgcs [ 20 , 37 ] . previous studies have shown an inverse correlation between sustained pupillary constriction and glaucoma severity [ 11 , 13 , 15 ] . for example , gracitelli et al . showed that the sustained response to the blue flash at 250 cd / m ( which preferentially stimulates iprgcs [ 3 , 4 ] ) was reduced in patients with advanced glaucoma ( i.e. , patients with sap md worse than 12 db ) . the present study adds new information by showing that , in addition to the decreased sustained pupillary response in glaucoma patients , the degree of daytime sleepiness is associated with a reduced sustained response to a blue flash at 250 cd / m . one possible explanation for this finding is that iprgcs are among the cells in the total rgc population that are damaged in glaucoma patients ; thus , they affect several functions of iprgcs , including the plr and circadian rhythms . importantly , although a significant association was observed between daytime sleepiness and the plr in this study , the correlation coefficient was not substantial ( r = 0.080 for the correlation between the ess score and peak plr and r = 0.068 for the correlation between the ess score and the sustained plr ) . for example , the subjectivity of all self - report questionnaires might affect the final score . showed that self - reported questionnaire of sleepiness can often be inaccurate when viewed alongside quantitative measures of alertness and sometimes is often viewed as a common and natural aspect of aging that can lead to a misinterpretation of the results [ 38 , 39 ] . in addition , daytime sleepiness , in glaucoma group , was associated with a sustained plr to the red flash . the peak and sustained plrs to a red flash are well known to be correlated with the number and function of the rods and cones , not iprgcs . one hypothesis explaining this finding is that rod and cones photoreceptors might also be affected in patients with chronic or advanced glaucoma , resulting in decreased signaling from these cells [ 4042 ] . however , this topic remains controversial in the literature [ 4043 ] , and additional studies must be conducted to clarify the actual connection between the functions of iprgcs and rod and cones photoreceptors . another interesting issue is the controversy concerning the relative preservation of iprgcs among the diversity of rgcs [ 21 , 4447 ] . showed that although the conventional rgc number was decreased in rats with increased iop , the number of iprgcs did not change . this result suggests that iprgcs were resistant to the deleterious effects caused by increased iop . however , another study in mice demonstrated that iprgcs appeared to be resistant to damage resulting from iop elevation at an early age ( approximately 5 months ) but became vulnerable at a later age ( approximately 11 months ) . the authors of that study suggested that the magnitude of iop elevation required to damage iprgcs must be greater than that required to induce rgc damage [ 48 , 49 ] . in addition , drouyer et al . and de zavala et al . showed that glaucomatous rats and other rodents exhibited a delayed phase angle with respect to darkness [ 46 , 47 ] . these results are in agreement with ours , suggesting that glaucoma induces a circadian system dysfunction . one explanation for the differences between certain animal models and studies of patients with glaucoma is the endpoint evaluated , that is , morphological versusfunctional endpoints . the primary focus of human studies has typically been iprgc function , whereas the number and morphology of these cells have usually been considered in animal studies . the resistance mechanism that underlies although this question persists , our study analyzed two different functions involving iprgcs in the same group of patients , and we found a strong association between them . thus , our results strongly suggest that both non - image - forming functions of iprgcs were affected . recently , chen et al . demonstrated that there is not one specific type of iprgc ; rather , different subpopulations of iprgcs exist with similar properties that innervate disparate brain regions to execute specific functions , such as the plr and circadian photoentrainment . in this study , pupil constriction , circadian oscillation ( wheel running activity ) , the adjustment of the circadian clock to different light stimuli ( i.e. , circadian photoentrainment , jet - lag paradigms , phase shifting , and skeleton photoperiod ) , and the direct effect of constant and ultradian light on activity were measured . the results showed that iprgcs comprise functionally distinct subpopulations that differentially express a specific transcription factor . iprgcs that do not express this transcription factor innervate the suprachiasmatic nucleus of the hypothalamus , whereas iprgcs that express this transcription factor innervate other known brain targets such as the olivary pretectal nucleus . thus , the iprgcs that express this transcription factor are associated with the plr but not with circadian photoentrainment . because we did not evaluate this issue in the present study , future investigations should be conducted to elucidate the subpopulations of iprgcs to determine how they are involved in non - image - forming functions . the primary clinical finding of this study was that glaucoma patients had increased daytime sleepiness compared with healthy participants , and these results were associated with worse polysomnographic parameters and lower sustained plrs to a blue flash with luminance of 250 cd / m . therefore , circadian rhythm regulation , which is linked with iprgcs ' functions , should be evaluated in certain patients with glaucoma . excessive daytime sleepiness is well known to affect quality of life , daytime function , and mortality ; furthermore , previous studies of geriatric populations have revealed that excessive daytime sleepiness is associated with age , disability , dementia , and vision and hearing impairments . although future studies with greater numbers of patients should be undertaken to better understand the role of iprgcs in sleep disorders , the present study did show strong agreement between the subjective and objective measures of sleep quality among patients with glaucoma . second , sleep patterns are most likely affected by factors other than the hypothesized iprgc loss associated with glaucoma . however , the subjective and objective measures of sleep quality ( i.e. , the ess questionnaire and the polysomnographic parameters ) strongly suggested that the damage to iprgcs associated with glaucoma results in certain types of sleep disorders . furthermore , regarding the primary endpoint of this study , we relied on self - reported histories of daytime sleepiness ; it is possible that inaccuracies were present in patient recollection and reporting . however , we also used polysomnographic parameters ( i.e. , the gold standard test of sleep quality ) , and these parameters were in agreement with the ess scores . in addition , because patient age is associated with a decrease in sleep quality and can affect plr , this variable might have affected our final results [ 16 , 51 ] . although the influence of age - related factors could not be completely excluded , our study did not reveal any significant differences in age between the two groups , and the multivariate analysis found that age was not a significant covariate . in addition , previous studies have shown that the postillumination response did not significantly decrease with age and that only pupil diameter was reduced among the elderly population . in conclusion , the results of this study demonstrated that patients with glaucoma had increased daytime sleepiness relative to healthy controls as measured by a self - report questionnaire . in addition , daytime sleepiness was associated with a reduced sustained pupillary response and polysomnographic parameters . these iprgc functions might be impaired in patients with glaucoma , thereby affecting their quality of life .

patients with glaucoma showed to have higher daytime sleepiness measured by epworth sleepiness scale . in addition , this symptom was associated with pupillary reflex and polysomnography parameters . these iprgc functions might be impaired in patients with glaucoma , leading to worse quality of life .

1. Introduction 2. Methods 3. Results 4. Discussion

PMC4108101

optimal and timely antibiotic treatment of critically ill , infected patients is paramount to maximizing survival [ 5 , 6 ] . given the epidemiological trends of gram - negative pathogens and the increased incidence of resistance , many treatment guidelines recommend the use of empiric dual gram - negative coverage , which frequently includes the use of an aminoglycoside [ 79 ] . the surviving sepsis campaign guidelines further recommend that adequate initial doses of antibiotics should be given to ensure that serum concentrations are attained to maximize efficacy and minimize toxicity ; nevertheless , these antibiotic doses are infrequently evidence based in critically ill patients . infected patients may develop a spectrum of biologic response , ranging from systemic inflammatory response syndrome to septic shock and death . acute renal failure occurs proportionally to the extent of the biologic response to infection , ranging from 19% in patients with sepsis to 51% in patients with septic shock [ 11 , 12 ] . among critically ill patients with acute kidney injury requiring renal replacement therapy , continuous renal replacement therapy ( crrt ) understanding the pharmacokinetic ( pk ) characteristics of aminoglycoside during crrt warrants further investigation , given the importance of attaining adequate antibiotic serum concentrations and the increasing need for this class of antimicrobials in critically ill patients . among the aminoglycosides , amikacin is useful for gentamicin - resistant gram - negative pathogen infections or as empiric treatment in institutions with a local epidemiological pattern suggesting the need to use this medication . despite its crucial role in therapy , a survey of the literature reveals a relative paucity of amikacin pk data among critically ill patients . in particular , there are fewer than 50 reports of amikacin pk parameters during crrt [ 1522 ] . despite the availability of these reports , drug clearance for a particular medication may be affected by the mode of crrt used , inter- and intra - patient variation in dialytic dose , and institutional variations in crrt machines and filters . the majority of the reports on amikacin pk characteristics during crrt were from a period of time where crrt was performed with relatively lower dialysate or replacement fluid flow rates ( 0.61.2 l / h ) compared to current crrt prescriptions ( 24 l / h ) , or with hemofilters no longer used in clinical practice [ 1518 ] . in addition , few of the reports provided the characteristics of the dialysis machine , the mode of crrt , and filter details . lastly , only one report describes the pk characteristics of amikacin in patients undergoing continuous veno - venous hemodialysis ( cvvhd ) . there are several reports of amikacin pk with novel crrt parameters ; however , they comprise fewer than 30 cases in total . furthermore , some novel reports of amikacin pk characteristics involved five or fewer patients in their analysis [ 21 , 22 ] and one report focused on patients with burn injury , which may have confounding pk implications . given the paucity of data and the continued need for broad - spectrum antibiotics targeting gram - negative pathogens in an era of newer crrt machines and filters with drastically higher flow rates , the pk characteristics of amikacin warrant further investigation . as such , we performed a prospective observational study of patients who received amikacin therapy while on cvvhd to further characterize the pk parameters of the medication . this was a prospective observational study of a convenient sample of patients admitted to a medical icu of a tertiary care academic medical center , who received amikacin therapy while on cvvhd . patient characteristics , amikacin dosing , and cvvhd parameters , including machine , filter , effluent , and dialysate flow rates , were collected from an intensive care database that was approved by the cleveland clinic institutional review board ( irb ) . the database was approved by the local irb as part of a registry for the evaluation of intensive care pharmacotherapy - related outcomes . the current study was performed by querying the existing data within the registry with no additional information collected through chart review or patient contact . a waiver of informed consent was granted by the local irb . the decision to administer amikacin and the prescribed dose / frequency were determined by the primary icu service , and not prescribed by the study protocol . patients with at least two amikacin serum sample concentrations measured after the first dose of amikacin were included in the study . serum amikacin concentration measurements were drawn as part of routine patient monitoring and levels were generally determined more than 8 h apart . amikacin levels were measured by our local institutional laboratory using the advia 1200 system ( siemens medical solutions , malvern , pa , united states ) chemistry analyzer with an enzyme immunoassay technique . the assay measures total amikacin level and has a quantification range of 2.550 g / ml , with a detection limit of 1 g / ml and a coefficient of variation of approximately 10% . first - order pharmacokinetics with a single compartment were assumed and estimations of the peak concentration ( cmax ) , volume of distribution ( vd ) , elimination constant ( kel ) , clearance ( cl ) , and terminal half - life ( t ) were performed . the equations used to calculate the various pk criteria can be found in table 1 . for all calculations , the ideal body weight ( ibw ) was used unless patients were more than 30% above their ibw . if patients were more than 30% above their ibw , then a dosing weight ( dw ) was used [ dw = ibw + 0.4 ( actual weight in kg ibw ) ] .table 1pharmacokineticformulaspharmacokinetic parameterequationelimination constant ( k el ) , h ln(c 2/c 1)/(t 2 t 1)half - life ( t ) , h0.693/k el projected peak ( c max ) , g / ml \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { c_{1 } } \mathord{\left/ { \vphantom { { c_{1 } } { { \text{ln(}}e^ { { - k_{\text{el } } { \kern 1pt } \times { \kern 1pt } \updelta t } } ) } } } \right . \kern-0pt } { { \text{ln(}}e^ { { - k_{\text{el } } { \kern 1pt } \times { \kern 1pt } \updelta t } } ) } } $ $ \end{document}c1/c1ln(ekelt)ln(ekelt ) volume of distribution ( v d ) , l d / c max clearance ( cl ) , ml / min v d k el t time between first concentration drawn and 30 min after infusion completion , c 1 first measured concentration , c 2 second measured concentration , d dose , t 1 time when first concentration was drawn , t 2 time when second concentration was drawn pharmacokineticformulas t time between first concentration drawn and 30 min after infusion completion , c 1 first measured concentration , c 2 second measured concentration , d dose , t 1 time when first concentration was drawn , t 2 time when second concentration was drawn the decision to administer crrt was made as per recommendations from the nephrology icu consult service . selection of the machine for dialysis and filter choice were based upon chance equipment availability at the time of cvvhd initiation . however , in accordance with our local practice , cvvhd was performed using a prismaflex system ( gambro , lakewood , co , usa ) or system one dialysis system ( nxstage , lawrence , ma , usa ) with either a polyacrylonitrile [ ( an69)prismaflex m100 , 0.9 m membrane surface area ] or a polysulfone hemofilter ( nxstage cartridge express , 1.5 m membrane surface area ) , respectively . the cvvhd parameters , including blood flow rate , dialysate flow rate , ultrafiltration rate , or the need for filter anticoagulation , were determined by the nephrology icu consult service based on individual patient needs . in general , an ultrafiltration rate ranging from 50 to 150 ml / h was added to the cvvhd dialysate rate to optimize machine running time and facilitate volume removal ( as determined by the nephrology and primary icu services ) . because this ultrafiltration rate was relatively small compared to the dialysate rate ( about 5% ) , the dialysis modality was still considered cvvhd , as opposed to continuous veno - venous hemodiafiltration , or cvvhdf . continuous data are presented as median ( interquartile range , iqr ) , unless otherwise specified . pearson correlation was utilized to assess the relationship between amikacin pk parameters and cvvhd characteristics . linear regression was performed to evaluate the relationship between the dose administered and the projected peak amikacin concentration , as well as the relationship between dialysate flow rate and amikacin clearance . chicago , illinois ) , and a p value < 0.05 was considered statistically significant . continuous data are presented as median ( interquartile range , iqr ) , unless otherwise specified . pearson correlation was utilized to assess the relationship between amikacin pk parameters and cvvhd characteristics . linear regression was performed to evaluate the relationship between the dose administered and the projected peak amikacin concentration , as well as the relationship between dialysate flow rate and amikacin clearance . statistics were computed using spss software , version 15.0 ( spss inc . , chicago , illinois ) , and a p value < 0.05 was considered statistically significant . their median ( iqr ) age was 56 ( 4567 ) years with a median ( iqr ) weight of 84 ( 64117 ) kg . only two of the patients had end - stage renal disease , while the remainder required crrt due to acute kidney injury . patients had minimal residual renal function with a median ( iqr ) urine output of 10 ( 052 ) ml in the 24 h after amikacin administration . the patients were all critically ill with a median ( iqr ) apache ii score of 25 ( 2230 ) , with 14 ( 93% ) requiring mechanical ventilation . four patients ( 26.7% ) were dialyzed using the nxstage machine with nxstagecartridge express polysulfone filter , while 11 ( 73.3% ) patients were dialyzed using the prismaflex machine with the m100 acrylonitrile filter . the median ( iqr ) age of the dialysis filter at the time of amikacin administration was 10 ( 328 ) h. minimal interruption in continuous dialysis was observed during the amikacin sampling period , with a median ( iqr ) interruption time of 15 ( 0300 ) min . the median ( iqr ) dialysate , weight - adjusted dialysate , ultrafiltration , and blood flow rates were 2,000 ( 1,8252,450 ) ml / h , 23.9 ( 19.029.5 ) ml / kg / h , 50 ( 50100 ) ml / h , and 200 ( 150200 ) ml / min , respectively.table 2individual characteristics of continuous veno - venous hemodialysis parameterspatient numbermachineblood flow ( ml / min)dialysate rate ( ml / h)effluent rate ( ml / h)age of filter ( h)1prismaflex2002,5005040.02prismaflex1502,00010023.53prismaflex1602,350509.04prismaflex2003,00010010.05nxstage1502,800503.06prismaflex2002,00015043.07prismaflex1502,400500.58prismaflex1502,000501.59nxstage1501,200500.510prismaflex2001,8005028.011nxstage2001,600508.012prismaflex2002,5001003.813nxstage2002,00010022.514prismaflex1601,8505047.015prismaflex2001,8005010.0 individual characteristics of continuous veno - venous hemodialysis parameters the median ( iqr ) dose of amikacin , based on adjusted body weight ( dw ) , was 14.1 ( 11.717.3 ) mg / kg . the individual amikacin dose and pk parameters are presented in table 3 . the amikacin dose administered corresponded with a median ( iqr ) projected cmax of 28.5 ( 20.939.0 ) g / ml . the vd , cl , and t were 0.39 ( 0.280.57 ) l / kg , 36.7 ( 22.844.5 ) ml / min , and 12.7 ( 8.716.7 ) h , respectively . correlation analyses found a significant correlation between clearance and dialytic dose . using simple linear regression , for every 1 l / h increase in dialysate flow rate , the clearance rate increased by 23.6 ml / min ( 95% ci 1.745.4 ml / min ; p = 0.037 ) . in addition , the dose administered corresponded significantly with the projected peak amikacin serum concentration ( fig . 1).table 3amikacin pharmacokinetic parameterspatient numberdose ( mg)dose ( mg / kg ) * c max ( g / ml ) v d ( l / kg)*clearance ( ml / min ) t ( h)time to serum level < 5 g / ml11,30012.428.50.4361.08.621.7275011.737.70.3137.76.117.831,00012.989.50.2312.416.769.741,00012.219.80.6136.715.931.651,25014.727.60.5395.15.513.661,25021.160.10.3518.912.745.671,00010.421.00.5044.512.425.6875010.524.30.4320.117.840.5954010.817.40.6222.815.828.4101,00014.231.30.4537.29.926.31183014.620.90.7022.820.241.6121,50017.319.50.8943.120.640.5131,25017.757.60.3148.95.118.1141,00018.339.00.6830.114.242.21580015.331.40.4933.18.923.5 * per adjusted body weightfig . 1association between cmax and dose amikacin pharmacokinetic parameters * per adjusted body weight association between cmax and dose in this study of a convenient sample of patients who received amikacin while on cvvhd , a significant positive correlation was found between amikacin clearance rate and dialysate flow rates . all patients in this study were treated with cvvhd utilizing synthetic dialysis filters and relatively high dialysate flow rates . the dialytic dose used in this study was complementary to those described by a recent survey of the management of critically ill patients with acute renal failure . despite the correlation between amikacin clearance and dialysate flow rates , the wide range of projected cmax and t seen in this study indicate that the exact amikacin dosing regimen can not be accurately predicted based on the dialytic dose or other factors available at the bedside . as such , it would appear to be most appropriate to perform first - dose pk calculations to determine the appropriate dosing regimen for each patient . among many gram - negative species across the world , the minimum inhibitory concentration to inhibit 90% of bacterial isolates ( mic90 ) for amikacin is 8 g / ml ; optimal antibacterial activity is achieved when the amikacin cmax is eight to ten times greater than the mic . based on the projected pk from this analysis , to achieve a peak of 64 g / ml ( 8-times an mic of 8 g / ml ) , a projected dose of about 25 mg / kg ( based on dw ) is needed . this is consistent with a recent report by taccone and colleagues , who studied pk parameters after a dose of 25 mg / kg of total body weight was administered to patients with severe sepsis and septic shock . among patients with renal dysfunction ( defined as creatinine cl < 50 ml / min ) in this study , a dose of 25 mg / kg achieved a cmax , vd , cl , and t of 71.5 g / ml , 0.42 l / kg , 1.29 ml / min / kg , and 7.6 h , respectively . remarkable similarities were seen between the vd in the study by taccone and colleagues and that in the present study . in a subgroup of the patients from the taccone study undergoing cvvhdf , the t and cl were 6.5 h and 1.26 ml / kg / min ( about 5.3 l / h for a 70-kg patient ) , respectively . these values are drastically higher than those found in our study , which could be explained by the considerably higher dialytic dose used in that study ( median dialysate and ultrafiltration rates of 29 ml / kg / h and 33 ml / kg / h , respectively , for an approximate total crrt dose of 62 ml / kg / h ) . similarly , in a recent study of five patients undergoing cvvhdf , darcy and colleagues demonstrated an amikacin t and cl of 6.7 h and 56.6 ml / min , respectively . this study utilized an ultrafiltration rate of 2 l / h and a dialysate rate of 12 l / h . in contrast to the studies listed above , other studies have found considerably lower clearance rates than our study . armendariz and colleagues presented a case report of a patient undergoing cvvh and found that total body clearance of amikacin was 10.5 ml / min and cvvh clearance was 10.11 ml / min . they found an elimination constant of 0.023 h , which corresponds to a t of 29.7 h. this study found clearance rates from crrt to be similar to those reported for patients in renal failure without the use of dialysis . the median clearance rate of amikacin in our study ( 36.7 ml / min ) was drastically higher than that reported by armendariz and colleagues . of note , the dialysate flow rates described in the current report are approximately twice those reported by armendariz and colleagues . given the high sieving coefficient of 0.93 for amikacin , it is conceivable that the flow rates during crrt would dictate the amount of drug removal . this premise is supported by other studies that utilized higher dialysate or ultrafiltration rates with subsequent findings of higher rates of amikacin clearance . roberts and colleagues reported data from five patients on cvvh , with average flow rates of 19.2 ml / min ( 1.2 l / h ) and found a mean hemofiltration clearance rate of 16.4 ml / min . taken together , it appears that across studies , the overall dialytic dose may affect amikacin clearance . this is consistent with the findings of our current study , which suggest that dialytic dose correlates with amikacin clearance . however , there are still many other factors that would ultimately determine the pk profile of amikacin . these may include inter - patient variability in non - dialytic measures , such as volume status , non - renal intrinsic clearance , the age of the filter , and interruptions to cvvhd . of interest , a study by cotera and colleagues that evaluated amikacin clearance in five patients with acute oliguric renal failure undergoing cvvhd found that the amikacin clearance rates were only 3.57 and 4.18 ml / min with 1 and 2 l / h dialysate rates , respectively . even though the 2 l / h dialysate rate was only slightly lower than that reported in the current study , the authors noted drastically lower clearance rates than in our study . notably , all the previous studies discussed and the current study utilized synthetic hemodialyzer membranes composed of either acrylonitrile or polysulfone . in contrast , the study by cotera and colleagues utilized a cuprofen ( cellulose ) dialysis membrane . a decrease in drug clearance with the use of cellulose dialysis membranes compared to polysulfone has been well documented [ 2730 ] . this may partially be explained by significant adsorption of aminoglycosides to synthetic membranes , which may contribute to an increase in overall clearance [ 3133 ] . as such , all pk evaluations of aminoglycosides should readily report the type of filter , its age at the time of drug administration , and any potential filter changes during the pk sampling period . similar to previous studies , the external validity of this study may be limited , given that all patients received cvvhd using either the prismaflex or nxstage machine . of note , only 4 of the 15 patients received dialysis via the nxstage machine ; therefore , the data presented here may be more applicable to patients receiving dialysis via the prismaflex machine . likewise , the considerable institutional differences in the practice of crrt , including the mode , filter material , and dialysate and ultrafiltration rates , may limit the external applicability of this study . in addition , the methods used in the current study do not allow for differentiation between extracorporeal clearance and intrinsic clearance . the patients in our study had minimal residual kidney function , but in patients with some remaining renal function , clearance of amikacin may be higher . lastly , the pk profiles evaluated in this study were obtained after the first dose of amikacin . therefore , no conclusions could be made regarding the pk characteristics of amikacin beyond the initial dose . the strengths of our study include the largest number of patients evaluated to date and explicit notation of dialytic characteristics ( which could affect pk parameters ) that reflect more current practices with crrt . in conclusion , our study found a significant correlation between dialysate flow rate and amikacin clearance . institutions should evaluate their usual dialytic practice to examine the flow rates routinely prescribed , which may provide a good starting estimate for amikacin clearance . however , given the considerable inter - individual variability observed in this study , an a priori prediction of pk parameters and optimal amikacin dose to be administered to patients on cvvhd may be challenging . therefore , determination of the optimal dose of amikacin and dosing interval should be achieved by serum concentration monitoring and subsequent dose adjustments . furthermore , the exact amikacin dosing regimen needs to be individualized based on the presumed mic of the pathogen , site of infection , and other host factors . due to the large number of potential confounders , which may include dialysate rate , ultrafiltration rate , hemodialyzer properties , patient residual intrinsic clearance , and host volume status , first - dose pk evaluations would be prudent in all critically ill patients on crrt who are administered amikacin . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

introductionlittle is known about the pharmacokinetics of amikacin during continuous renal replacement therapy.methodsthis prospective observational study included patients admitted to an academic medical center who received amikacin therapy while on continuous veno - venous hemodialysis ( cvvhd ) and had at least two serum sample concentrations measured after first - dose administration . first - order pharmacokinetic parameters , patient characteristics , and cvvhd parameters were recorded.resultsfifteen patients were included in the analysis . the median ( interquartile range ) dose of amikacin and dialysate flow rate , based on adjusted body weight , were 14.1 mg / kg ( 11.717.3 mg / kg ) and 23.9 ml / kg / h ( 19.029.5 ml / kg / h ) , respectively . this corresponded with a median cmax of 28.5 g / ml ( 20.939.0 g / ml ) . there was a significant correlation between clearance and dialytic dose ( for every 1 l / h increase in dialysate flow rate , clearance rate increased by 23.6 ml / min [ 95% confidence interval 1.745.4 ml / min ; p = 0.037]).conclusionthe results of this study suggest that amikacin dose and interval should be individualized for each patient on cvvhd based on first - dose pharmacokinetic assessment .

Introduction Materials and Methods Statistical Analysis Results Discussion Conclusion Conflict of interest Compliance with ethics guidelines Open Access

PMC3991318

the role of emotions within social work practice may at first glance appear to be intuitively obvious and incontestable . indeed , howe ( 2008 , p. 13 ) described the day of a social worker as suffused with emotional content. munro ( 2011 ) highlighted the centrality of the social work relationship and acknowledged the importance of workers being able to identify their own emotional responses and those of service users in achieving positive relationships . the role of emotions is at the core of literature regarding relationship - based practice and the separation of feelings from professionalism can be seen as an anathema in an interpersonal profession ( hennessey , 2011 ) . it is possibly around the concept of professionalism that we begin to get into less certain territory when speaking about the role of emotions in social work practice . munro ( 2011 ) recognised the tensions between proceduralist approaches to practice and the desirability that social workers should be able to exercise greater autonomy in their practice and decision making . the attractiveness of seeking a conceptualisation of the profession as one being underpinned by concrete knowledge and explanation is understandable . hennessey ( 2011 ) suggested , however , that if one does separate off one 's emotions from practice , then one is essentially separating the relationship with the service user from the practice . remove emotions from the presentation and recording of their practice and , in turn , act in ways that do not make use of their emotions and feelings relating to their practice . it is the debate around the presence , use , suppression and/or removal of emotions that underpins this paper and a model for emotionally inclusive supervision is proposed . this paper intends to view the emotional elements of social work practice as being something which has a significant impact on the content , direction and experience of practice . the concept of emotional intelligence sits comfortably with a conscious and proactive awareness and use of emotions within a social work relationship . alongside this conceptual framework of emotions , i will also take cognisance of the unconscious and potentially repressed aspects of emotions that if left unexplored can impact on the actions and behaviours of social workers in practice ( jopling , 2000 ; ferguson , 2005 ) . the concept of emotional intelligence places emphasis on the ability of an individual to identify , understand and manage the emotional content of their interactions and experiences ( goleman , 1995 ) . similarly , it has been proposed that emotional intelligence involves an individual 's ability to be aware of their own emotional reactions in differing situations and their abilities to manage their responses accordingly ( mayer et al . , they suggested that this balance of awareness and control allows individuals to make more appropriate and confident decisions . in addition to this self - regulation and awareness , they suggested that an ability to identify emotional responses in others is a key aspect of emotional intelligence . these abilities are further linked to an individual 's ability for empathic understanding and their skills in communication . ingram ( 2012 ) forged links between the aforementioned qualities associated with emotional intelligence and the ability to establish and maintain positive relationships with service users in social work practice . the core argument from ingram ( 2012 ) being that emotional attunement and empathy are the foundations of establishing an open and trusting relationship , and are also congruent with the service user literature in terms of the qualities most valued in social workers ( harding & beresford , 1996 ) . lazarus and lazarus ( 1994 , p. 151 ) stated that an emotion is a personal life drama , which has to do with the fate of our goals in a particular encounter and our beliefs about ourselves and the world we live in . it is aroused by an appraisal of the personal significance or meaning of what is happening in that encounter . an emotion is a personal life drama , which has to do with the fate of our goals in a particular encounter and our beliefs about ourselves and the world we live in . it is aroused by an appraisal of the personal significance or meaning of what is happening in that encounter . this fundamental centrality to human experience suggests that emotions are purposeful and operant ( davidson , 1994 ; lazarus & lazarus , 1994 ) . emotions are aroused when a person perceives that something they desire comes to fruition or is compromised . the emotional response will , in turn , be dependent on how we assess the potential impact of a circumstance on our perceived goals . this goal - orientated view of emotions is echoed by goleman ( 1995 ) , who makes explicit links between goal attainment and emotional awareness and regulation . to purely focus on goals runs the risk of overlooking key processes involved . for example , if a social worker is running late when travelling to an initial home visit to a family , it is the process of a delayed journey that arouses an emotional reaction rather than the goal which is the home visit . this illustrates that there may be implicit and explicit variables at play when emotional reactions are experienced . lazarus and lazarus ( 1994 ) stated that our ego or self - identity is developed through experience and the associated emotional reactions to these experiences . they described ego as being at the core of an individual 's personal view of the world , and , as such , emotional responses arise from events and circumstances that impact upon it . clearly , this suggests there is a cognitive process at play which helps us determine what elements of an event are important and , in turn , what is potentially at stake . this appraisal of events will , in turn , feed into decisions about subsequent actions . to return to the example of the social work home visit , the worker may hold a strong belief about the importance of reliability and the development of trust with service users ( lishman , 2009 ) . it is this aspect of their professional identity and their appraisal of its importance that may guide their emotional response and subsequent actions . the personal meaning that we then attach to the circumstances will drive our decision making at this point . he argued that emotions are adaptive responses to a current event , while moods are a longer - term cognitive state . it is argued that moods impact on the aforementioned appraisal of events and dictate what aspects we are likely to focus on . for example , the worker may angrily phone their office to complain about the impact their workload is having on their ability to keep to timescales or , perhaps , they will contact the service user to apologise for the delay and negotiate a change to the meeting . the selection of response to the emotional reaction to being late will be in part controlled by an existing mood state . if we accept that emotions are associated with actions and consequences , then it could be argued that the former response may provide short - term relief by letting off steam while the latter response may have a longer - lasting impact on the quality of the worker 's relationship with the family . this apparently simple example will be influenced by a succession of previous experiences and responses and may have an ongoing impact on behaviour during the home visit . a key issue that underpins such decision making is the degree of self - knowledge at play ( hennessey , 2011 ) . where this is lacking , unconscious emotional drivers may have a significant impact on the course of events . for example , if we consider that the avoidant behaviours of social workers apparent in the victoria climbie case ( ferguson , 2005 ) were associated with repressed and/or unexplored feelings of fear and anxiety , we can begin to add to the complexity of what might be happening in the example of lateness above . rustin ( 2005 ) further explored the victoria climbie case and suggested that the repression of gut feelings and emotion on the part of social workers led to confusion and indecisiveness about what actions to take and what information was pertinent to them . applied to our example , it is possible to envisage the worker 's lateness and reactions to it being influenced by unconscious factors such as fear and anxiety , rather than purely the conscious practical issues such as poor timekeeping . the crucial issue here is that without opportunities and permissions to explore such emotional depth , such drivers are likely to remain less visible but no less significant . the role of emotions and the impact that they may have on practice is a contentious one . popular aphorisms such as do n't let your heart rule your head can be traced back to the work of plato ( forgas , 2001 ) . howe ( 2009 ) suggested that emotions have been viewed as wayward and crude by western philosophers such as kant and descartes , and that the clarity of rational thought is compromised by their existence . this view was countered by enlightenment philosophers such as david hume who stated that emotions and reason should work in harmony ( howe , 2009 ) . 2000 ) suggested that emergent themes of social justice , anti - war movements and equality from the 1960s onwards reflect a challenge to the aforementioned paradigm . they go on to suggest that the humanist movement within psychology began to reflect this . for example , maslow ( 1943 ) emphasised the importance of individuals feeling satisfied and that consideration of emotions was central to this . it is useful to consider what we mean by rationality in order to clarify where emotions may sit in relation to it . lazarus and lazarus ( 1994 ) suggested that the common view of rationality is concerned with maximising chances of success and minimising chances of loss , and the focus is on logic and factual evidence . this would appear to overlook other factors such as kindness , justice , equality and fairness . they suggested that this then leads to the potential for considerable disagreement about what the rationally this has direct links with the aforementioned debate about whether practice can be purely evidence based . this they suggested opens the door for needing to explore why people make the decisions they do . figure 1 is based on this model and is intended to illustrate a cyclical process which links well with the concept of emotional intelligence and , in turn , has a resonance for social work practice . figure 1 illustrates that there is a relationship between an event , the associated emotions and the processes of decision making that ensue . a key element is that if we accept the aforementioned definition of rationalism , then the choices made through examination of the emotional response will be , by definition , rational . by this i mean that they will be goal orientated and bespoke to our individual wants and beliefs . another important element here is that if an individual undertakes this process , their ability to understand and reflect upon emotional cues and processes will help them interpret how others may feel . it is clear that this model sits comfortably within the concept of emotional intelligence in terms of the notion of choice , regulation and empathy . damasio ( 1994 ) undertook a study examining the decision making of individuals with damage to their amygdala which provides a useful and persuasive underpinning to the claim that emotions contribute to and aid decision making . damasio proposed that the amygdala is the central emotional hub of the brain , based on the observation that individuals who experience damage to this part of the brain display significantly impaired emotional responses . damasio suggested that damage to the amygdala led to individuals losing prior emotional learning ( i.e. impact of behaviour on others ) and , in turn , making decisions in their lives that led to negative outcomes ( i.e. relationship breakdown ) . this suggests that decisions made by workers in practice will similarly be infused and informed by emotional learning and that these emotions are a prerequisite for making sense of the complex information and circumstances that a social worker is required to make sense of . this emotional information stream is recognised by munro ( 2011 ) as being a crucial and valued part of a social worker 's toolkit . this view that emotions and thought are compatible and intrinsically linked is developed further within the context of social work practice by morrison ( 2007 ) . hence , the creation of an emotional response is in itself a process of thought . this is echoed by lazarus and lazarus ( 1994 ) who suggested that if emotions arise from our own personal meanings and identity , then emotions can not exist without thought . these choices may at times appear to be unreasonable or ill - advised , but are the product of a cognitive process . goleman ( 199s ) concurred with this view and suggested that if an individual lacks emotional intelligence , they will find it harder to assign values to the choices they are presented with , and , in turn , will find decision making more difficult . in a sense , this is the polar opposite to the familiar paradigm that emotions cloud thought . if we accept that there is a role , whether positive or at least unavoidable , for emotions in decision making , then we can begin to establish strong links with the notion of relationship - based practice ( hennessey , 2011 ) . if we then consider that service users highlight empathy , genuineness and warmth as key qualities in social workers , we can begin also to see a positive use of emotions . the notion that emotions and the thought process are compatible and have a function is not confined to the literature regarding emotions and emotional intelligence . the concept of social competence has much in common with emotional intelligence ( topping et al . , 2000 ) . the ability to integrate thinking , feeling and behaviour to achieve social tasks and outcomes valued in the host context and culture ( ibid . this concept has much greater emphasis on the influence of collective and cultural factors which determine how an individual behaves . the key element in common with emotional intelligence would appear to be that there is a thought process involved that recognises the role of feelings and that serves a function . averill ( 1994 ) pushed this idea further by suggesting that if we accept that emotions have a function , then to suppress or sideline emotions is dysfunctional . hennessey ( 2011 ) concurred with this view by emphasising that to have an emotional response to any given situation does not preclude an informed response . he goes on to rank emotions as an equal partner alongside theory and skill , and notes that there is an interaction between these elements . for example , it is suggested that the selection of which theoretical models to apply and/or prioritise in social work practice is affected by the emotional response to both the presenting situation and the knowledge base itself . myers ( 2008 ) echoed this view when he acknowledged the attractiveness of seeking a diagnosis in social work and highlighted the role that emotions and values may play in the choices we make in this process . houston ( 2011 ) builds on the care ethics work of sevenhuijsen ( 1998 ) by marrying the rational / cerebral ( head ) aspects of relationship - based ethics with the emotional and empathetic ( heart ) elements . in addition to these two elements , he adds the practical approaches adopted by social workers ( hands ) and the motivation that workers have to care ( feet ) . in a recent interview i conducted with a criminal justice social worker as part of a wider study of emotions and practice , the following example was provided that helps to illustrate the active awareness , management and use of emotion in practice . the worker provided an account of the internal ethical struggle he manages when working with a sex offender who does not take responsibility for their offences . he acknowledged feelings of anger and the potential this can have for him to misuse his power . this tension is resolved for him by adopting an approach that recognises the service user holistically and considers the motivations for defensiveness on the part of the service user as being based on the past and present experiences of social work interventions . this led to an active management of his emotions and drove his decision to draw on his knowledge of motivational interviewing and role modelling to positively engage with the service user . the worker reported the importance of supervision in allowing him space to develop his awareness and ability to deconstruct the emotional triggers in a way that not only manages them but also funnels them into an active part of his practice . head , heart and hands in practice table 1 helpfully pulls together the active goal - orientated aspects of emotional intelligence with potentially unconscious or repressed emotions such as anger to produce a piece of practice that reflects the management and use of emotions . the uneasy relationship between the recognition of emotions and the pursuit of rational decision making is further played out when we consider the emergence of the professionalisation of social work . social work has struggled to lay claim to a coherent vision of itself as a profession for a range of reasons . one issue is that the underpinning knowledge base of the profession is disparate ( dominelli , 2009 ) and draws from a range of sources such as psychology , sociology , moral philosophy and law . munro ( 2011 ) highlighted the need for social work to grapple with the organisational and individual contexts of social work to achieve a coherent and realistic vision of the profession . the notion of relationship - based social work suggested by hennessey ( 2011 ) is persuasive but is often pitted against more bureaucratic professional narratives rather than being seen to co - exist . there is an existing tension with the notion of social work as a profession that has its roots in the radical social work movement . the radical social work movement emerged in the late 1960s and expressed a view that social work should not seek to be viewed as a profession . this was due to the proposition that social work was a political activity and one which should focus on challenging the social structures and the system which impinge on service users ( bailey & brake , 1976 ) . the notion of professionalism was seen to reflect an elitist and paternalistic view of social work . an enduring outcome associated with radical social work was the emergence of anti - discriminatory practice ( pease & fook , 1999 ) and its synthesis with the psychosocial aspects of social work practice . one of the key aspects of this historical debate around professionalism is the constant presence of the relational nature of social work practice . a pragmatic and responsive view of the profession of social work emerges in the changing lives document ( scottish executive , 2006 ) which incorporates the need for a robust knowledge base , clear professional value base and an emphasis on partnership and collaboration with service users and wider multidisciplinary colleagues . with the registration of social workers and the associated responsibilities and accountabilities , the issue of professionalism has never been more cogent . myers ( 2008 ) noted that a non - emotional approach to social work could be seen to sit comfortably with the notion that social workers should be non - judgemental in their interactions and assessments of service users . myers countered this view by questioning whether a purely non - judgemental is achievable , and whether the best way to approach it is to allow for an awareness and acknowledgement of emotions and values in order to reach non - judgemental outcomes . for example , if a worker is to become involved in working with a service user who is aggressive and uncooperative , it is important that the emotional responses of the worker are considered and explored so that a non - judgemental approach to the work can be achieved . to avoid such a process may well lead to an illusion of non - judgmentalism but runs the risk of producing uncritical and repressed practice . this was echoed by nspcc ( 2008 ) with their suggestion that social workers often feel that their role is to focus on the strengths of service users and carers and to adopt an optimistic approach to their practice . they go on to argue that this approach appears to avoid significant reflection and analysis and may be a barrier to required action in child protection cases . mattison ( 2000 ) suggested that there is an increasing emphasis on social workers to act correctly. this inevitably leads workers to seek reliable and legitimate procedures and guidance to ensure their practice is correct. mattison noted that this approach overlooks another associated aspect of defensible decision making which is that workers must be able to understand and articulate the process of decision making as well as the actual decision itself . munro ( 2011 ) and howarth ( 2007 ) both warned against relying on a proceduralist approach to practice . the argument being that no matter how thorough procedures are , they are ultimately liable to the subjective choices and decisions of a worker . given the preceding discussion about potential unconscious and unexplored emotions , the pressure to present one 's practice as howarth ( 2007 ) discussed the limitations of holistic assessment models which are intended to give workers a clear framework to ensure decisions are well founded . howarth ( 2007 ) suggested that such tools do not encourage or provide signposts for practitioners to consider their own role in the choices and priorities that they make in relation to such tools . . it might be useful to describe this as the interpretative gap between procedures and their actual use in practice . it is within this gap that workers bring their values , emotions , experience , knowledge and context to bear on their decision making . holland ( 1999 ) found that many workers preferred to present themselves as neutral observers who could use quantifiable scientific measurements to explain their decision making in practice . if we accept that emotions and values impact on our decision making and that genuinely holistic practice involves a head , hands and heart approach ( petrie et al , 2009 ) , then the need for emotional awareness and intelligence would seem to be a logical aspect of this . mattison ( 2000 , p. 207 ) provided a persuasive argument for this when she states that there is general agreement in the literature that the ultimate decision for resolving an ethical dilemma lies in the circumstances and the value system or preferences of the decision maker. barlow and hall ( 2007 ) reported that social work students felt anxious when there was a disparity between their this could reasonably be applied to qualified practitioners and , again , suggests that workers would benefit from a forum to explore these issues . this clearly has links to the above example and suggests that social workers may need a forum to consider emotional responses . the role that supervision may play in this process will be discussed in the supervision and the exploration of emotions section . these issues are very pertinent when one considers the messages in the munro report ( munro , 2011 ) relating to professional autonomy and highlighting the need for an infrastructure that facilitates and supports social workers to achieve this . the preceding discussion makes a strong argument for social workers engaging in a significant degree of reflection about their practice and that this process could and should encompass emotions and feelings . this is coupled with the case that has been made to incorporate emotional intelligence in social work practice and decision making and the need to allow space and permissions to explore the unconscious emotional drivers and responses within practice . there are a range of forums where the emotional content of social work could be expressed . . the ability of workers to express the emotional content of their work in these forums will be dependent on workers feeling that it is valid and desirable to do so . this provides workers with a forum to discuss their practice with another practitioner ( usually a senior colleague ) and explore the functional aspects of their practice but also potentially to critically reflect upon the content of the practice . fook and gardner ( 2007 ) made explicit reference to the emotional aspects of critical reflection and suggested that this can have a therapeutic aspect to it while also directly feeding into ongoing practice and decision making . the importance of supervision is noted by england ( 1986 ) who suggested that social workers must be engaged in deep analysis of their practice and be clear about their own perceptions and those of others . this was echoed by the social work task force ( department of children , schools & families , 2009 ) with the recognition that supervision and management needed to resist restrictive and prescriptive conceptions of practice , and to aspire to a system that recognises the fluid and unpredictable relationship - based aspects of practice . the important role that supervision may play in promoting safe and positive social work practice was a key recommendation by nspcc ( 2008 ) in their evidence submitted as part of lord laming 's review of child protection . the central plank of the argument was that working with manipulative , violent , and possibly sadistic adults is emotionally draining not to mention frightening . to defend oneself it is common to put up protective emotional barriers ( nspcc , 2008 , p. 8) . it has been reported by nspcc ( 2008 ) that in the face of such challenging work that supervision was often of low quality . the quality that has been reported to be missing was the opportunity for workers to reflect upon their feelings and their practice . this was echoed in a study of decision making in child protection by holland ( 1999 ) who found that social workers had an underdeveloped language when it came to describing feelings and felt more comfortable when discussing their work in a technicist manner . it was argued that this was in part due to there being a lack of legitimacy in discussing practice in terms of feelings . supervision could provide a vehicle for this perception to be addressed and , in turn , the development of a language to support it . hawkins and shohet ( 2000 , p. 3 ) provided a useful description of the emotional element within a social work supervisory relationship : the supervisors role is not just to reassure the worker , but to allow the emotional disturbance to be felt within the safer setting of the supervisory relationship , where it can be survived , reflected and learned from . the supervisors role is not just to reassure the worker , but to allow the emotional disturbance to be felt within the safer setting of the supervisory relationship , where it can be survived , reflected and learned from . they go on to note the importance of allowing social workers to stand back from their practice so that they do not internalise all their emotional responses . fineman ( 198s ) noted the relationship between lack of supervision and stress among workers . there is an acknowledgement that supervision operates within a wider context and there are other factors which may impact on the nature and focus of the supervisory relationship . hawkins and shohet ( 2000 ) recognised that there is a potential tension between manager and educator roles that a supervisor may inhabit . they suggested that both partners in the supervisory relationship should construct a contract that acknowledges and clarifies the parameters of supervision . hughes and pengelly ( 1997 ) emphasised the need for clarity about confidentiality when constructing supervisory contracts . hawkins and shohet ( 2000 ) also noted that the procedural focus that is evident in many social work supervisory relationships is often driven by resources and the need to cover the practical elements of a caseload . collins ( 2007 ) approached this point from a different angle and noted that the emotional labour of social work continues regardless of the emphasis on procedures . collins goes on to note that positive emotions are often overlooked in the literature around emotions in social work . this is an important point in that supervision should allow for the exploration of feelings such as joy and contentment with particular cases . this can act as an emotional buffer to cope with negative emotions as well as allowing for reflection on why positive emotions are being elicited . cadman and brewer ( 2001 ) considered the need for role models in nursing who demonstrate emotionally intelligent approaches to practice . this is a helpful contribution to potential roles for a social work supervisor where emotional intelligence ( and emotion more broadly ) could be modelled within the supervisory relationship and be promoted from a top down perspective ( from supervisor to worker ) as well as a bottom up perspective ( the worker engaging in reflection within supervision ) . ( 2006 ) who explored the impact of supervisory styles in industry and found that supervisors who actively explored emotions within supervision produced positive emotions in their workers and , in turn , heightened performance . tsang ( 2006 ) suggested that a social work educator ( supervisor ) should swing between an emphasis on cognition and emotion to allow social workers to consider the often contradictory demands on them . for example , the tension between potential controlling aspects of the social work role and therapeutic roles requires consideration of the emotional impact of this tension , not just a cognitive awareness of it . tsang talks vividly about the need for supervision to have a nourishing and replenishing function . this links helpfully with the aforementioned emphasis that collins ( 2007 ) gives to exploration of the positive aspects of practice . barlow and hall ( 2007 ) studied the views of social work students on practice learning placements . they found that students reported feeling under stress when their individual emotional experiences in practice were incongruent with what they perceived to be the required the conclusion was a call for supervision to provide a forum for discussing emotional responses in practice . it could be argued that this style of supervision would also promote informed and reflective practice . the preceding discussion about the important role that supervision can play in relation to the exploration , management and use of the emotional elements of practice presents social work with an organisational challenge . this challenge is noted by the social work task force ( department of children , schools & families , 2009 ) but , at an operational level , may be subject to competing pressures and agendas which may lead to the perpetuation of performance indicator - led approaches ( ruch , 2011 ) . the model proposed here intends to place the supervisory relationship itself at the heart of the realisation of the aforementioned challenge . jindal - snape and ingram ( 2012 ) developed a model of supervision for international doctoral students that sought to encourage and facilitate a partnership approach to supervision which allowed both parties to clarify the balance and nature of the expected supervision relationship and , in turn , adjust and modify it accordingly . i wish to take this model as a starting point and develop it further within the context of social work supervision , and give particular focus to the place that the articulation of emotions has within it . figure 2 is a visual illustration of where the balance of supervision can be negotiated and also as a tool for co - constructing and reviewing the balance and content of supervision . this dual function of the model is crucial in terms of its application , as it acknowledges the dynamic and fluid nature of supervisory relationships . it should also be noted at this point that the model is adaptable across a range of supervisory debates . i have chosen the balance between the practical and emotional aspects of casework , but equally it could be about a balance between reflective approaches and a technical / rational approach . one can plot the degree to which a facet of supervision is relevant at any point on both axes . this allows for both parties to plot their own expectations and aspirations in terms of supervision . the use of this model would allow for each party to plot where they would hope the balance of supervision would lie.negotiation . having plotted the aspirational balance , any divergence in view can be explored and discussed.agreement . the model can then be used to reach an agreed balance , which can be linked to organisational and national perspectives.review . the model can be revisited at any stage to consider whether the agreement is still valid or requires adjustment . the use of this model would allow for each party to plot where they would hope the balance of supervision would lie . having plotted the aspirational balance , any divergence in view can be explored and discussed . the model can then be used to reach an agreed balance , which can be linked to organisational and national perspectives . the model can be revisited at any stage to consider whether the agreement is still valid or requires adjustment . , we can see that quadrant a depicts a supervisory relationship that focuses on the emotional aspects of practice but with less of a focus on practical casework . quadrant b represents a balance between the emotional and practical elements of casework and would reflect a supervisory relationship where both elements are seen as valid and appropriate . quadrant c depicts a supervisory relationship where the practical elements of casework take centre stage , as reflected in much of the discussion above . finally , quadrant d shows a supervisory relationship which does not focus on the practical nor on the emotional aspects of practice . it is difficult to envisage such a supervisory approach , although this would reflect a lack of supervisory support . figure 3 clarifies the potential use of this model further by illustrating where a supervisor and a supervisee may differ in their expectations . it is clear from figure 3 that the supervisee seeks an integration of the emotional and practical elements of the caseload , while the supervisor favours a practical focus . what is also useful about this model is the flexibility allowed in terms of plotting at any point on an axis to indicate the intensity anticipated . in the case of the supervisor , their lack of willingness to engage in the emotional aspects of practice is less than their enthusiasm for the practical elements . uneasy alliance at the heart of this paper , namely the balance between the rational and technical aspects of practice and how they sit next to the relationship - based aspects of practice and the emotional responses ( both conscious and unconscious ) of the social worker . the model requires the participants in the supervisory relationship to be explicit ( rather than passive ) about the content and focus supervision . next steps element of the diagram that directs the participant 's to supportive sources of guidance such as professional codes and national documents such as the aforementioned report from the social work task force ( department of children , schools & families , 2009 ) . this places the uneasy balance in a wider context ( rather than being left to the subjectivities of the participants ) and is intended to reduce the apparent disjuncture between the procedural and emotional aspects of practice . if we return to our previous practice example of the social worker who used his emotional response of anger as the starting point to engage in useful reflection and , in turn , as a key information stream that linked to subsequent actions , it is clear to see how this model could be utilised . if this had not been available , then the use of this model would allow for an active discussion about where the balance of supervision lay and , in turn , allow the worker to seek the quality of supervision to enable them to engage in positively and emotionally informed practice . it is important to note that this model encourages a co - created approach to supervision , and it provides the supervisor with a means to explore emotionally repressed practice and reflection and provide a basis for countering the potential for negative projection of anxieties within the supervision relationship that arise from anxieties relating to practice ( ruch , 2011 ) . in this paper i have considered the complex position of emotions within social work practice and supervision . if we consider emotions through the lens of relationship - based social work and recognise that emotions are inextricably linked to social work decision making , then we can establish a conception of the social work profession that must embrace the emotional elements of practice . it is recognised that relationship - based conceptions of practice sit within a wider context of processes , statutory responsibilities , professional knowledge and power . i would argue that the perceived tension between technicist approaches and relationship - based approaches to practice can be ameliorated by establishing a cultural shift in terms of the role of supervision . the proposed model allows for transparency and partnership in the construction of the essential supervisory relationship which seeks to place the emotional elements of practice at the core of practice rather than leaving them potentially marginalised . what is particularly heartening is that recent narratives concerning the profession ( scottish executive , 2006 ; department of children , schools & families , 2009 ; munro , 2011 ) recognise the tensions discussed above , and this paper is intended to contribute to the understanding of these tensions and a supervisory model to support the cultural shift required .

this paper examines the place of emotions within social work practice . the perceived tensions between emotions and rational decision making are explored and it is argued that their relationship is compatible and necessary . a model for the co - creation of emotionally intelligent supervision is developed to support this vision of practice .

Introduction The place of emotions setting the scene Emotions and decision making: exploring their relationship Professionalism, proceduralism and accountability Supervision and the exploration of emotions A partnership model and tool for supervision Conclusion recognising and managing the uneasy alliance

PMC3170810

alzheimer 's disease ( ad ) is a highly prevalent neurodegenerative disease accompanied by gradual and irreversible behavioral and cognitive impairments . brain lesions observed during the course of ad involve two main aspects : extr - acellular amyloid - beta ( a ) deposition in the senile plaques and intracellular tau accumulation forming neurofibrillary tangles and promoting cytoskeletal disorganization . current research on ad is largely guided by a dominant pathogenic theory , the so - called amyloid cascade hypothesis . regularly commented on and amended , this model posits accumulation of a in the brain as a key primary event that determines the onset of other brain alterations ( e.g. , synaptic and neuronal death ) , finally leading to the dementia . early onset familial forms of ad are indeed associated with mutations in different genes ( amyloid precursor protein ( app ) and presenilins 1&2 , ( ps1&2 ) ) involved in the biosynthesis of a. dysfunction of these genes is logically thought to alter the rate of app cleavage , resulting in exaggerated a production . high levels of brain a and associated parenchymal amyloid plaques are key phenotypes described in transgenic mice overexpressing one or more of these mutated genes ( see for review ) . these mice subsequently develop neuropathological alterations and behavioral impairments mimicking ad phenotype [ 4 , 5 ] . the exact impact of brain a accumulation on clinical symptoms remains to date difficult to decipher , both in ad patients and in animal models of the disease . clinicopathological correlative analyses have led to mitigated conclusions [ 610 ] , and it is now considered that preplaques a assemblies are the most deleterious species while aggregated insoluble deposits have a reduced pathogenicity . in addition white matter anomalies are described in ad patients , presumably in association with cerebrovascular impairments , and in transgenic mice modeling brain amyloidosis . these lesions can be detected through conventional postmortem neuropathological examination , but also in vivo by means of dedicated techniques such as diffusion tensor imaging [ 15 , 16 ] , and it has even been proposed that white matter defects are potent diagnostic biomarkers for ad [ 17 , 18 ] . the integrity of axonal bundles constituting white matter fiber tracts is particularly compromised during the course of ad , even at the very early stages of the disease [ 19 , 20 ] . alterations of fiber tracts have obvious functional consequences : disconnection of neural networks occurs following fibers loss , leading to diaschisis and cortical disorganized activity [ 21 , 22 ] . also , disruption of myelin in axonal bundles might have deleterious effects on neuronal communication by altering propagation of action potentials and increasing brain energy expenditure ( see [ 23 , 24 ] for reviews ) . white matter alterations in fiber tracts are related to demyelination and also to other axonal morphological defects that can ultimately lead to degeneration and loss of fibers . it has been demonstrated that ad patients show concurrent decreased axonal densities and myelin breakdown . interestingly , experimental studies in ad animal models have shown that axonal pathology can be driven by a deposition in the brain [ 2628 ] . the aim of the present study was to further evaluate fiber tracts changes in a double appxps1 mouse transgenic model with aggressive a-related pathology [ 2931 ] . two commissural fibers tracts ( corpus callosum and anterior commissure ) that show significant alterations in ad patients [ 32 , 33 ] were selected and analyzed : axonal integrity was quantified by assessing antineurofilament immunostainings . myelin density was evaluated by histochemistry using a gold chloride staining that provides high contrast and spatial resolution [ 34 , 35 ] and that has previously been used to underline myelin alterations in ad transgenics . female transgenic app / ps1 mice ( thy1 app751 sl ( swedish mutation km670/671nl , london mutation v717i introduced in human sequence app751 ) hmg ps1 m146l ) modeling early onset and progressive cerebral amyloid deposition were used [ 29 , 30 , 3739 ] . heterozygous app / ps1 mice were obtained by crossing app(+/wt)/ps1(wt / wt ) with app(wt / wt)/ps1(+/+ ) mice established on a c57bl6/cba background . note that , due to the use of homozygous ps1(+/+ ) mice , no wild - type mice could be generated from the breeding scheme . ps1 mice have been described in previous studies to be devoid of neuropathological alterations [ 30 , 37 ] and hence constitute good controls ( age - matched littermates with no overt phenotypes ) for analyzing appxps1 transgenics . two age groups were analysed : young ( 2 months : 8 app / ps1 and 7 ps1 mice ) and old animals ( 24months : 6 app / ps1 and 7 ps1 mice ) . following decapitation , the brains were extracted , fixed in 10% buffered formalin , and stored overnight in a solution of 20% glycerin and 2% dimethylsulfoxide in 0.1 m phosphate buffer for cryoprotection . brains were subsequently sectioned into 40 m thick coronal sections on a freezing microtome . twelve series of sections ranging from the frontal to the occipital poles were collected and stored at 20c in cryoprotectant before use . myelin staining was performed on a batch of serial sections using a protocol derived from with some modifications . brain sections were rinsed with 0.1 m phosphate buffer ( pb ) mounted on superfrost+ slides and dried overnight at 50c . the slides were then rinsed in 0.02 m phosphate saline buffer ( pbs ) . two milliliters of freshly prepared 0.2% gold chloride ( carlroth , karlsruhe , germany ) dissolved in 0.02 m pbs was added on each glass slides to completely cover the section . incubation was carried out at room temperature ( rt ) for 2 hours in obscure humid chambers . the tissue was briefly rinsed in water , fixed in 2.5% sodium thiosulfate for 5 minutes , and finally rinsed in running tap water for 30 minutes . sections were coverslipped with eukitt after dehydration in graded alcohols and clearing in xylene . free - floating sections were rinsed in pb and pretreated with 0.3% h2o2 in methanol for 10 minutes to block endogenous peroxidase activity . nonspecific antigenic sites were blocked by 5% goat serum in pbs - triton x-100 ( 1 hour at rt ) . sections were then incubated overnight at rt with a c - terminal antineurofilament m ( 145kd ) primary antibody ( ab1987 ; chemicon , temecula , calif ) diluted 1 : 1000 in serum . the following day sections were incubated in a secondary biotinylated antirabbit antibody solution ( sigma , st . louis , mo ; 1 : 200 , 1.5 hours at rt ) and then in an avidin - biotin peroxidase complex ( abc ) reagent ( vectastain elite abc - kit , vector laboratories , usa , 1 : 800 , 1.5 hours at rt ) . the reaction was stopped when the signal - to - noise ratio was considered optimal . the sections were finally mounted on superfrost slides , dried , and processed in alcohols / xylene before being coverslipped . one series of section was stained by congo red according to the standard technique ( 30 minutes in a 80% ethanol solution saturated with congo red and sodium chloride ) [ 30 , 40 ] . another series of sections was immunostained with the primary 4g8 monoclonal anti - a antibody ( signet laboratories , dedham , mass ; 1 : 10000 , incubation overnight at rt ) . a high dilution of the biotinylated 4g8 antibody was used to avoid cross - reaction with app and to solely immunodetect a . thanks to biotin complexion , no secondary antibodies were used and tissue was directly incubated in the abc kit solution before being developed in dab . staining of degenerating neurons was performed using the fluoro - jade b dye with a slightly modified protocol . glass - mounted sections were passed through absolute ethanol and 75% ethanol followed by a 1-minute rinse in distilled water . tissue was then incubated in 0.06% potassium permanganate solution for 15 minutes with slight agitation and rinsed before staining in fluoro - jade b ( histo - chem . , jefferson , ark ; 0.001% solution prepared in 0.1% acetic acid ; 30 minutes at rt ) . after extensive rinsing in distilled water , sections were dehydrated , cleared in xylene , and coverslipped . all slides were digitized using a super coolscan 8000 ed scanner ( nikon , champigny sur marne , france ) with a 4000 dpi in - plane digitization resolution ( pixel size 6.35 m ) . quantification of gold chloride staining was performed , in two regions of interest ( corpus callosum and anterior commissure ) , by relative optical density ( rod ) analysis , a method that has already been used to evaluate myelin stainings . in the rostral brain , 3 sections were selected to analyze myelin at the level of the anterior commissure ( bilateral countings , i.e. , n = 6 samples / mouse ) . for the analysis of myelination of the corpus callosum , 7 sections per animal were selected , spanning the rostrocaudal extent of the region ; the rod analysis was performed on the total corpus callosum volume and also separately in its anterior and posterior parts ( see below ) . measure of mean grey value was semiautomatically performed in the two regions of interest using photoshop cs2 software ( adobe , paris , france ) . rod was obtained after transformation of mean grey values using the formula rod = log10 ( 256/(mean grey value ) ) . values of background staining were taken in adjacent tissues with no myelin staining ( upper cortical layers or striatum ) and subtracted to get a final densitometric evaluation of myelin stain . while 2-month - old appxps1 mice were devoid of amyloid plaques , a deposition was very extensive in aged app / ps1 transgenics . notably many plaques were observed in the corpus callosum and in the anterior commissure . obviously the gold chloride negatively stained areas corresponding to plaque occupancy might artificially decrease the local mean grey values . to correct for this bias in old mice , plaques surfaces were manually removed from analyzed regions of interest before processing rod calculations . the sizes ( surface area from coronal sections ) of both the corpus callosum and the anterior commissure were evaluated by manually outlining the structures on digitized scans of myelin - stained brain sections ( see figure 1 ) . three measures of fibers tracts size were taken : ( 1 ) in the anterior corpus callosum ( sampling between 4.9 and 3.22 mm / interaural reference in the paxinos and franklin atlas , n = 4 sections / mice ) , ( 2 ) in the posterior corpus callosum ( 3 sections taken between 1.5 and 2.58 mm / interaural reference ) , and ( 3 ) in the anterior commissure ( sampling between 4.18 mm and 4.9 mm / interaural reference , n = 3 sections analyzed bilaterally / mice ) . for each region of interest , area size measurement was calculated by summing outlined areas across serial sections . assessment of neurofilament immunoreactivity was performed in the same two regions of interest ( corpus callosum , n = 7 sections / mouse ; anterior commissure , n = 3 sections / mouse ) by means of rod analysis . evaluation of the extracellular amyloid load was only performed in the 24-month - old app / ps1 mice as almost no congo red positive deposits were observed in young double transgenic mice . images were then processed with imagej freeware ( rasband , w.s . , imagej , us national institutes of health , bethesda , md , usa , http://rsb.info.nih.gov/ij/ , 19972006 ) using a dedicated macrocommand that extracts amyloid deposits from background tissue with no user inputs . regional amyloid loads were expressed as percent of tissue surface stained by the congo red dye that corresponds to the proportion of plaques volume . amyloid loads were evaluated ( 1 ) in the whole brain , ( 2 ) in the rostral isocortex that is richly innervated by axons passing through the anterior corpus callosum , and ( 3 ) directly in the two fiber tracts that were investigated in the present study ( anterior commissure , corpus callosum ) . intr - aneuronal a immunoreactivity was not found in aged mice ( as already reported for this mouse line ) and was quantified only in the young appxps1 mice . two sections / animals were selected at the level of the frontal cortex , and a semiquantitative analysis , based on a four - point scale , was performed on both hemispheres to evaluate levels of neuronal immunostainings in the cortex : 0 : no obvious positive staining ; ( 1 ) : weak intracellular staining ; ( 2 ) : moderate staining ; ( 3 ) : strong staining . for each animal semi - quantitative analyses were hence performed in four fields ( 2 slices ; bilateral countings ) . to evaluate the connection properties of a-containing neurons and their possible participation to commissural tracts we performed an axonal tracing study in two wild - type mice ( 3 month - old ) : biotinylated dextran amine ( bda ) , an anterograde tracer , was stereotactically injected in the cortical regions where a-positive neurons were concurrently observed in appxps1 transgenics , allowing fine visualization of cell bodies and of neurites . surgery and immunolabeling of bda stained axons were performed using standard protocols as previously described [ 47 , 48 ] . student 's t - tests were performed using statistica 7 ( statsoft , inc . , gold chloride myelin staining , as compared to standard stains ( e.g. , he or nissl stains ) , allowed to precisely outline the area of the corpus callosum . in particular , delineating the corpus callosum from adjacent white matter tracts ( e.g. , cingulate bundle , dorsal fornix , and dorsal hippocampal commissure ) was greatly facilitated by the myelin staining ( figure 1 ) . lateral limits of the corpus callosum and borders of the external capsule were identified by a horizontal - to - vertical shift in fiber orientation . also , the anterior commissure was easily identified and outlined in the gold chloride - stained sections ( see figure 5(b ) ) . the size of the anterior commissure was similar in 2-month - old ps1 mice and in age - matched appxps1 transgenics ( t(13 ) = 0.58 ; figures 2(b ) and 5(b ) ) . on the contrary , the corpus callosum size was significantly decreased in young appxps1 as compared to controls ( t(13 ) = 3.501 , p < .005 ; figures 2(a ) and 5(a ) ) . subregional analysis indicated a significant reduction in the size of the rostral corpus callosum of young appxps1 mice ( t(13 ) = 3.743 , p < .005 ) while there was no difference between genotypes in the size of the posterior corpus callosum ( t(13 ) = 0.136 , ns ) . with aging , a significant increase in the size of axonal bundles was observed in ps1 control mice ( corpus callosum : t(12 ) = 3.858 , p < .005 ; anterior commissure : t(12 ) = 4.275 , p we have observed the same developmental traits in wild - type c57bl6 mice ( increased volumes of the corpus callosum and of the anterior commissure with aging ; data not shown ) precluding the possibility that the ps1 transgene by itself triggered abnormal ( increased ) fiber tract growth in ps1 transgenic mice . on the other side , age - dependent enlargement of commissural bundles was clearly not evidenced in the double appxps1 transgenics : in this genotype , the size of the corpus callosum remained constant between 2 and 24 months ( t(12 ) = 1.850 , ns ) and the surface area of the anterior commissure even undergoes slight atrophy with aging ( t(12 ) = 2.284 , p < .05 ) . hence , strong differences between genotypes were observed in 24-month - old mice with appxps1 transgenics showing , in comparison to ps1 controls , decreased white matter surface areas . this was observed at the level of the anterior commissure ( t(11 ) = 6.388 , p < .0001 ) and of the corpus callosum ( total corpus callosum : t(11 ) = 4.653 , p < .001 ; anterior part : t(11 ) = 5.404 , p < .0005 ) . the only posterior region of the corpus callosum did not show significant atrophy in old appxps1 mice ( t(11 ) = 1.492 , ns ) . densities of axonal neurofilaments in the corpus callosum and in the anterior commissure were quantified using rod analysis of immunostainings . in 2-month - old mice axonal densities were similar in both genotypes whatever the fiber tract considered ( all p 's > .35 ) . with aging , a severe decrease in neurofilament density was observed , both in appxps1 and ps1 mice , in the corpus callosum ( figures 2(c ) and 3(a ) ) and in the anterior commissure ( figures 2(d ) and 3(b ) ) ( all p 's < .0001 ) . age - related reduction of neurofilament density was , however , largely more prominent in old appxps1 mice than in age - matched ps1 controls . this accelerated loss of axonal neurofilaments in old appxps1 mice was further confirmed by statistical analysis in the different subregions of the corpus callosum ( appxps1 < ps1 : all p 's < .0001 ) and 2 ) and also at the level of the anterior commissure ( t(10 ) = 4.16 ; p < .005 ) . rod analysis of gold chloride stainings in 2-month - old mice indicated comparable myelin densities in ps1 and appxps1 transgenics ( corpus callosum : t(13 ) = 0.318 , ns , figures 2(e ) and 5(a ) ; anterior commissure : t(13 ) = 1.277 , ns , figures 2(f ) and 5(b ) ) . the myelin density of the anterior commissure was not affected by aging ( ps1 : t(12 ) = 1.292 , ns ; appxps1 : t(12 ) = 0.556 , ns ) , and myelin densities in this fiber tract were similar in 24-month - old ps1 and in age - matched appxps1 mice ( t(11 ) = 0.679 , ns ) . on the other hand , an increase of the myelination of the corpus callosum was observed when comparing 2-month- and 24-month - old ps1 mice ( t(12 ) = 2.823 , p < .05 ) . noticeably , this increase in myelin density was significant in the rostral corpus callosum ( t(12 ) = 4.171 , p < .005 ) but not in its posterior part ( t(12 ) = 0.461 , ns ) . contrarily to ps1 mice , such an age - related increase of callosal myelination was not observed in appxps1 mice ( t(12 ) = 0.7 , ns ) . consequently decreased myelin staining was evidenced in 24-month - old appxps1 mice when compared to old ps1 controls ( total corpus callosum : t(11 ) = 3.332 , p < .01 ) . differences between genotypes were further confirmed at the level of the anterior corpus callosum ( t(11 ) = 3.512 , p < .005 ) , while no difference between ps1 and appxps1 mice were evidenced in more caudal regions ( t(11 ) = 1.9 ; ns ) . no evidence of myelin breakdown ( debris ) was found in the large myelinated bundles of the corpus callosum . however , in comparison to control animals , myelin appeared to be fragmented in the isocortex and hippocampus of appxps1 mice ( figure 4 ) . myelin material was often detected under the form of small tortuous segments with bead - like varicosities . these morphological anomalies , absent in young appxps1 mice , were found at the vicinity of a deposits but also in the parenchyma in areas distant from plaques . congo red positive aggregates were detected and quantified in the anterior commissure ( mean load= 2.8% ; min = 1.6% ; max = 6% ) and in the corpus callosum ( mean load = 2.2% ; min = 1.6% ; max = 2.7% ) of old appxps1 mice ( figures 6(a ) and 6(b ) ) . correlative analysis did not reveal significant associations between local amyloid loads in fiber tracts and decreased neurofilament immunoreactivity / myelin densities ( all p 's > .111 ) . also there were no correlations between the sizes of the corpus callosum / anterior commissure and total or cortical amyloid loads ( all p 's > .196 ) . in addition to amyloid plaques loads , intraneuronal a was semiquantitatively assessed in the isocortex of young appxps1 mice . as expected from previous observations , positive labeling was detected using the 4g8 antibody in a subset of cells . staining was mainly observed in deep cortical layers ( v ) involving a distinctive band of large pyramidal cells ( figures 6(c ) and 6(d ) ) . there were no direct correlations between levels of intracellular a that may significantly vary from one animal to the other ( mean = 7.8 ; min = 4.5 ; max = 11.5 ) and neurofilament and myelin densities ( all p 's > .119 ) . interestingly , tracing experiments underlined that the deep layers of the cortex , where specifically a-positive neurons were observed in the double transgenics , are the source of dense callosal projections ( figure 6(e ) ) . therefore , although no tight correlations were stressed between the density of intracellular a and the magnitude of axonal anomalies , we evidenced that callosal fibers still originate from a subpopulation of neurons overproducing toxic a species . in particular no degenerating neurons were observed in the cortical layers with high densities of a-positive neurons ( see above ) . only the core of amyloid deposits and the surrounding degenerating dystrophic neurites as well as reactive astrocytes were detected with fluoro - jade b in old appxps1 mice ( see [ 49 , 50 ] for similar observations ) . evidence from diffusion tensor imaging of fiber tracts suggests for instance that during the course of the disease there is a loss of barriers that restrict water motion and tissue anisotropy of white matter . these observations have obviously a neuropathological counterpart : anomalies of diffusion could reflect either loss of axons , demyelination , and/or oligodendroglial pathology . to date only a few studies have investigated the source of white matter / fiber tracts impairments in ad patients and in aged nondemented subjects [ 51 , 52 ] . recent attempts to decipher the origin and mechanisms of such morphological alterations have been performed in animal models of the disease [ 36 , 53 , 54 ] but a systematic screening of fiber tracts anomalies related to ad pathology is still lacking in these models . the goal of our work was therefore twofold : ( 1 ) to assess in an ad mouse model the severity of fiber tracts alteration across aging and ( 2 ) to refine the understanding of their histological substratum and in particular to evaluate the relation between a peptide deposition and the occurrence of the white matter anomalies . our quantitative analysis underlines the decreased size of forebrain fiber bundles in old appxps1 mice displaying concurrent severe brain amyloidosis . volume reductions were observed in the corpus callosum , as already reported in ad transgenics [ 14 , 30 , 55 ] but also , as a novel finding , at the level of the anterior commissure . these anomalies do not purely mimic an age - related atrophy and might also reflect a lack of normal development of fiber tracts in appxps1 mice ( see also for similar conclusions ) . as mentioned above , atrophy of the corpus callosum has previously been reported in ad transgenic mouse models but more interestingly is also classically depicted in ad patients ( e.g. , ) . we found that atrophy of the corpus callosum , in our appxps1 transgenics , largely predominates in its anterior part . although not constantly reported , similar findings concerning regional atrophies of the corpus callosum have been described in human patients [ 21 , 56 ] . in addition to callosal atrophy we also evidenced , in old appxps1 mice , a reduction in size of the anterior commissure that might find a parallel with recent observations in ad patients . supporting size reduction of fiber tracts we evidenced a very strong decrease of axonal neurofilament densities in the double transgenics , outclassing the normal loss of neurofilament we observed in aged control ps1 mice ( and in wild - type mice ; data not shown ) . the loss of myelin in aged appxps1 mice appears however to be limited in comparison to the high and accelerated decrease of neurofilament densities these mice undergo . as evidenced by correlative analysis , there were no strict linear relationships between fiber tracts atrophies and neurofilament or myelin markers . for instance , we found a reduced size of the corpus callosum in young appxps1 mice while these animals showed the same neurofilament and myelin densities as control ps1 mice such observations emphasizing similar densities of axonal markers in an overall reduced volume of tissue might indeed testify for an early net loss of callosal fibers in young appxps1 mice . also we found that fiber tracts continue to grow and pursue their myelinisation in normal mice between 2 and 24 months of age while neurofilament densities in the same mice concurrently display a 50% drop this observation can be explained ( 1 ) by a progressive enlargement of myelin sheets with aging that will maximize space around individual axons and consequently decrease their density and ( 2 ) by a concurrent diminution of the expression of axonal neurofilament proteins that has previously been described in aged animals . we have investigated various sources of neuropathological alterations that could explain fiber tracts atrophies in our appxps1 model . it has been claimed that a demyelination process is observed during the course of ad and is a trigger in the physiopathogeny of this disease [ 23 , 24 ] . it has even been proposed that myelin breakdown in ad initiates a vicious circle as release of iron after demyelination might promote a production and toxicity which in turn affects myelin integrity . in the present study we indeed observed qualitative anomalies of axonal myelination in old appxps1 mice accumulating a. similar observations , that might deserve confirmation and further characterization at the ultrastructural level using electron microscopy , have been recently reported in ad tg2576 and appxps1 mice but only in the neuropil in close association with amyloid plaques . in our study myelin alterations , including fragmentation and spheroid - like pathological enlargements , were observed in axons passing through the gray matter , even at distance of amyloid deposits , but not easily in dense fiber tracts under light microscopy . at the quantitative level , the overall myelination of the corpus callosum was found to be significantly decreased in old appxps1 mice . however no differences were observed between genotypes at the level of the anterior commissure , suggesting that loss of myelin is heterogeneous and does not affect similarly all axonal bundles . actually , myelination kinetics during ontogenesis might considerably vary from one brain region to the other [ 44 , 59 ] . it is hence possible that brain areas and fiber tracts with late or early myelination are differentially impeded during ( pathological ) aging [ 60 , 61 ] . in addition , it is known that the corpus callosum , in terms of phylogeny , is a neural pathway that is only found in placental mammals while the anterior commissure is a more ancestral axonal tract , adding supplementary differences between the two fiber bundles that could explain regional discrepancies in age - dependent ( de)myelination processes . interestingly , it has been shown that oligodendrocytes have a reduced myelin turnover in regions lately myelinated during ontogenesis . this would imply a diminished capacity for myelin repair in these regions , especially under challenging conditions ( oxidative stress , excitotoxicity , inflammation , etc . ) because oligodendrocytes are a strikingly vulnerable population of cells . toxicity of a on oligodendrocytes has been evidenced in several reports [ 36 , 64 ] with effects ranging from mitochondrial dysfunction to apoptosis and cell death . the involvement of oligodendrocytes in the myelin anomalies depicted in our appxps1 transgenics would deserve further analysis . in particular , it would be interesting to document possible anomalies at the level of myelin basic proteins . decreased sizes of the corpus callosum and of the anterior commissure in appxps1 mice might be related to defects in fiber myelination but , according to our observations , can also be linked to an accelerated loss of axonal neurofilaments , possibly reflecting loss of fibers . we have previously reported vascular alterations ( vessel voids and reduction of vessel lengths ) by means of in vivo magnetic resonance angiography in the appxps1 transgenic mouse model used in the present study . these lesions , closely associated with amyloid angiopathy , could contribute to the described axonopathies . anomalies in regulatory proteins involved in axonal structural plasticity during ad could also be causative but remain to be firmly established in our transgenic mouse model . axonopathy has previously been reported in the appxps1 mouse line but was originally evidenced at the spinal level . interestingly , it has been demonstrated that ad patients sustain axon loss [ 25 , 68 ] but the magnitude of the effects we reported ( large drop in neurofilament densities , first in aged ps1 control mice and second , greatly accentuated , in aged double appxps1 transgenics ) has not been reported up to now to the best of our knowledge . bussires and collaborators have demonstrated that neurons from ad brains expressing neurofilament proteins with medium- and heavy- molecular - weight subunits ( smi-32 immunoreactive neurons ) constitute an exquisite vulnerable subpopulation of cells during disease process . these neurons have a specific laminar distribution in the cortex compatible with their participation to corticocortical connections relying on fiber tracts and axonal fasciculi . back to our experimental data , it would be appealing to speculate that loss of axonal neurofilaments in the anterior commissure and corpus callosum indeed implies pathological events at the level of efferent neurons in specific brain regions ( e.g. , isocortex area for callosal neurons ) . we did not find however any evidence of cell degeneration in old appxps1 mice ( see also for similar report ) . on the other side we can not totally preclude that appxps1 mice have decreased neuronal densities in projections regions resulting from earlier insults , with no overt signs of active degeneration in the oldest animals . a substantial ( 30% ) loss of neurons has been described in the hippocampus of old appxps1 mice [ 37 , 70 ] . it is not yet known if a similar loss occurs in isocortical areas and further analysis will be required to ascertain this point . in accordance with previous studies ( e.g. , [ 37 , 71 ] ) , we did not find evidence of a clear pathogenicity of aggregated ( congo red positive ) insoluble a deposits . the density of amyloid plaques did not predict , in old appxps1 mice , the magnitude of fiber tracts anomalies these mice sustained . in a previous study we evidenced that axon terminals of cortico - cortical fibers endorse degeneration when contacting amyloid plaques but this local synaptotoxicity was not associated with clear retrograde degeneration . in another study of jantaratnotai axonal damage and demyelination however these effects , obtained in a context of sudden and potentiated toxicity , can not be considered as conclusive . furthermore , stokin and collaborators in a recent study have confirmed that axonal defects induced by app / ps1 mutations are not directly caused by a overproduction and amyloid accumulation . while amorphous insoluble amyloid deposits do not appear to be deleterious per se , our results support the hypothesis of a pathogenicity of a when it accumulates intraneuronally [ 73 , 74 ] . as described by others , young appxps1 mice display intracellular a accumulation , several weeks before the occurrence of amyloid plaques . in the present study , a-positive neurons were observed , in 2-month - old appxps1 mice , in specific layers of the cortex ( mostly layer v ) . in primates , including humans , it is known that pyramidal neurons projecting through the corpus callosum belong to lamina iii and v of the association cortex [ 7678 ] . in mice also the origins of callosal afferents have been traced back to cell bodies in layer v . by means of anterograde tracings we confirmed that the a-containing neurons in the deep layers of the cortex participate to commissural fibers passing the midline through the corpus callosum . one may infer from these data that , early during aging , appxps1 mice accumulate a in a subset of cortical neurons that later become dysfunctional , develop axonal pathology ( loss of neurofilament immunoreactivity and of myelin ) , possibly through a dying - back mechanism , and eventually die , leading to a definite loss of axons in brain commissures . to summarize , we found , in appxps1 mutant mice modeling ad brain amyloidosis , evidence of fiber tracts atrophies . these anomalies are potentiated in aged mice but might partly be explained by some neurodevelopmental defect . axonal pathology developed by appxps1 mice does not appear to be caused by amorphous insoluble amyloid deposits but intracellular a that accumulates in projecting neurons could be a causative factor . the atrophies in fiber bundles we depicted in appxps1 mice resemble those described in ad patients and are concomitant with a loss of axonal neurofilaments and a myelin breakdown . these results renew the importance of white matter impairments in alzheimer 's disease as these anomalies , with clear functional impact , can be reproduced in mice overexpressing a , a molecular actor that is the principal target for current therapeutical assays in this devastating disease .

amyloid beta ( a ) peptides are known to accumulate in the brain of patients with alzheimer 's disease ( ad ) . however , the link between brain amyloidosis and clinical symptoms has not been elucidated and could be mediated by secondary neuropathological alterations such as fiber tracts anomalies . in the present study , we have investigated the impact of a overproduction in appxps1 transgenic mice on the integrity of forebrain axonal bundles ( corpus callosum and anterior commissure ) . we found evidence of fiber tract volume reductions in appxps1 mice that were associated with an accelerated age - related loss of axonal neurofilaments and a myelin breakdown . the severity of these defects was neither correlated with the density of amyloid plaques nor associated with cell neurodegeneration . our data suggest that commissural fiber tract alterations are present in a-overproducing transgenic mice and that intracellular a accumulation preceding extracellular deposits may act as a trigger of such morphological anomalies .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC3518994

there is absence of penis and urethral opening is located in the anterior rectal wall in the majority of cases . phallic reconstruction using the free radial forearm flap ( rff ) or the pedicled anterolateral thigh flap ( altf ) has been routinely used in penile inadequacy or in female - to - male transsexuals . however , even if any reconstruction in infancy is carried out successfully , it is likely to cause encumberance during childhood and a further source of social embarrassment . we describe a novel technique as a temporizing procedure which would see the patient through childhood . we describe 2 patients who presented at the age of 9 months and 1 year with penile agenesis . both were passing urine and stools per rectum . the physical examination revealed complete absence of a phallus , a well - developed scrotum and normal sized testes . the urethral opening was located on the anterior rectal wall , about 2 cm . from the anal verge . the voiding cystourethrography revealed a short urethra with urethro - rectal communication a short distance beyond the bladder neck . while mri confirmed the above findings , it also revealed a normal looking urinary bladder and absence of corpora cavernosa and the corpus spongiosum . urinary bladder was seen to open lower down on the anterior rectal wall through the short posterior urethra ending approximately 2.0 cm from the anal verge . bowel preparation is done by peglec solution ( polyethylene glycol with electrolytes ) administered orally for 48 hours before surgery . under general anesthesia , the child urethral lumen is catheterized easily by inserting a # 8f feeding tube through the anal verge . we use a pre - anal , anterior coronal approach , to detach and mobilize the urethral opening from the anterior rectal wall [ figure 1a ] . a semilunar incision is given on the perineum , anterior to the anal verge , from 3 oclock to 9 oclock . the urethra - easily identified over the feeding tube - is mobilized and detached from the anterior surface of rectum . the rectal end of the opening is closed by a single layer of seromuscular , running , pds suture . ( a ) pre - anal anterior coronal approach : semilunar incision on the perineium , anterior to the anal verge , from 3 oclock to 9 oclock . urethra easily identified with prior insertion of feeding tube , mobilized from anterior surface of rectum and the rectal end of opening closed in two layers ( b ) quadrangular incision on the anterior surface of scrotum for phalloplasty . note the caudal projection for configuring the prepuce a quadrangular flap is taken from the anterior scrotal skin and raised with its dartos layer . the two edges of the flaps are folded together and the edges sutured together to create a neophallus [ figure 1b ] . the previously mobilized urethral opening is re - located in the midline , between the two scrotal halves and fixed to the root of the neophallus [ figures 2a and b ] . there is no tissue loss and the adjoining tissues on the abdominal wall remain undisturbed for any possible future use . six months later , a buccal mucosal graft can be laid on the undersurface of the neophallus [ figure 3a ] and later tubularized . neo - phallus. please note the urethral opening in scrotal location ( b ) post - operative appearance after scrotal phalloplasty ( a ) appearance later with buccal mucosal graft being laid on the undersurface of neo - phallus ( b ) one year later bowel preparation is done by peglec solution ( polyethylene glycol with electrolytes ) administered orally for 48 hours before surgery . under general anesthesia , the child urethral lumen is catheterized easily by inserting a # 8f feeding tube through the anal verge . we use a pre - anal , anterior coronal approach , to detach and mobilize the urethral opening from the anterior rectal wall [ figure 1a ] . a semilunar incision is given on the perineum , anterior to the anal verge , from 3 oclock to 9 oclock . the urethra - easily identified over the feeding tube - is mobilized and detached from the anterior surface of rectum . the rectal end of the opening is closed by a single layer of seromuscular , running , pds suture . ( a ) pre - anal anterior coronal approach : semilunar incision on the perineium , anterior to the anal verge , from 3 oclock to 9 oclock . urethra easily identified with prior insertion of feeding tube , mobilized from anterior surface of rectum and the rectal end of opening closed in two layers ( b ) quadrangular incision on the anterior surface of scrotum for phalloplasty . a quadrangular flap is taken from the anterior scrotal skin and raised with its dartos layer . the two edges of the flaps are folded together and the edges sutured together to create a neophallus [ figure 1b ] . the previously mobilized urethral opening is re - located in the midline , between the two scrotal halves and fixed to the root of the neophallus [ figures 2a and b ] . there is no tissue loss and the adjoining tissues on the abdominal wall remain undisturbed for any possible future use . six months later , a buccal mucosal graft can be laid on the undersurface of the neophallus [ figure 3a ] and later tubularized . neo - phallus. please note the urethral opening in scrotal location ( b ) post - operative appearance after scrotal phalloplasty ( a ) appearance later with buccal mucosal graft being laid on the undersurface of neo - phallus ( b ) one year later aphallia is a rare birth defect seen in 1 in 30 million births . in the past , however , pre- and postnatal effects of androgens on brain , cause a male - typical shift in terms of psychosocial and psychosexual development , resulting in gender dysphoria later in life . as a result , urethral opening is typically present lower down in the rectum in the post - sphincteric location ( 87% ) and less commonly in the presphincteric or prostatourethral location . both our cases had the former , typical presentation . cystography and mri are useful investigations to look for any associated anomaly as well as defining the anatomy . cystography , through suprapubic route , is often difficult to perform in un - cooperative children . mri should be the preferred modality of management as has been first demonstrated by us . the development of microsurgical free - flap techniques made the first microsurgical phalloplasty possible using a free radial forearm flap . however , there are several problems with early phalloplasty , such as , lack of further growth and the large neo - phallus being an encumberance to the young child . the microvascular techniques are more demanding at a young age and any tissue utilized earlier would hinder , any later age appropriate surgery . they are , anatomical phallic configurations in which , at best , penile implants could be inserted . at the same time , postponing surgery till post - pubertal period adds to parental anxiety , affects psychosocial development of the child who continues to pass urine per rectum . therefore , a temporizing procedure is necessary which may see the child through childhood [ figure 3b ] . scrotal phalloplasty , using scrotal skin to reconstruct a temporary phallus , can be performed easily . urethro - rectal disconnection is easily performed by our pre - anal anterior coronal approach . it provides an age - appropriate phallic appearance and does not disturb the tissues which may be required for post - pubertal phalloplasty . therefore , scrotal phalloplasty serves as a temporizing procedure which may see the child through early school going years . adding buccal mucosal graft allows urethral reconstruction and thus enables the child to micturate while standing . as he approaches teenage the other already available procedures could be introduced with ease , as , scrotal phalloplasty does not encroach on tissues used for any future surgeries .

all the currently known reconstructions for aphallia are carried out around puberty . we describe a novel technique as a temporizing procedure which would see the patient through childhood . this would not cause any hindrance while undertaking any of the other established procedures later on after puberty .

INTRODUCTION CASE REPORT TECHNIQUE Urethral re-location Scrotal flap-phallic reconstruction DISCUSSION

PMC2288682

the incidence rate of this malignancy is steadily rising in developing countries . it is expected to account for 31% of all new cancer cases among women in usa . in tunisia , the incidence of breast cancer is approximately 19 new cases per 100 000 women per year . invasive carcinomas where the basement membrane is totally or partially destroyed represent 7080% of all breast cancer and include more than 10 different types . among these , infiltrating ductal carcinomas ( idca ) are the most aggressive forms and have a poor prognosis . nowadays , improvement of breast - cancer detection at its early stage through the identification of selective diagnostic biomarkers and targets for therapeutic intervention is of critical importance . alterations that occur in disease versus normal tissues at either the gene or protein level have been regarded as an appropriate way to identify such markers of pathologies . in biomedicine , proteomics , with the recent advances in mass spectrometry , is considered as a powerful analytical method for deciphering protein expression alterations as a function of disease progression . recently , proteomics - based analyses of breast tissue lysates have resulted in the finding of a number of potential tumor biomarkers providing , therefore , a basis for a better understanding of the intracellular signalling pathways leading to breast - cancer development and progression , and eventually serving as diagnostic and prognostic markers . probably the most widely used proteomic technology is the identification of alterations in protein expression between two different samples through comparative two - dimensional gel electrophoresis ( 2-de ) which provides high - resolution separation of proteins and offers a powerful method for their identification and characterization . actually , there is no universal 2-de method that can be applied for all types of samples investigated , and protocols must be optimized and adapted according to the types of tissues and proteins of interest . it is also crucial to minimize protein alterations and degradations that could result in artifactual spots and to improve solubilization procedures in order to avoid loss of proteins during extraction . although 2-de is a laborious method , it is still considered as the principal tool in proteomics since it is able to separate proteins according to two independent physicochemical parameters which are isoelectric point and size , and also it provides the highest resolution in the separation of complex protein mixtures . 2-de remains however , poor resolutive in detecting proteins that are smaller than 10.000 da or are basic in charge . an alternative method for the analysis of basic proteins is found in the application of nonequilibrium ph gradient electrophoresis ( nephge ) , which can provide excellent resolution of basic proteins , offering thus a promising approach for the discovery of tumor - related alterations . in a previous study , we have shown using ief and mass spectrometry that the levels of a number of proteins including fibrinogen - e , which is a potent antiangiogenic factor , were dysregulated in idcas of the breast . in the current report , protein expression profiles issued from breast idcas and their matched surrounding histologically normal tissues were examined by nephge two - dimensional gel electrophoresis and maldi - tof mass spectrometry . using this procedure , we established a nephge-2-de map of proteins altered in idcas of the breast . by comparing 2-de protein profiles , we report herein the identification of several proteins of interest dysregulated in tumor tissues . these altered proteins belong to multiple cellular pathways and may have a pathological role in breast carcinogenesis . breast tumor samples ( n = 10 ) were selected from patients living in the middle coast of tunisia ( median age at diagnosis of 50 years ) and classified as infiltrating ductal carcinomas . breast tumors were obtained with informed consent according to protocols approved by the institutional review boards of the respective hospitals . carcinomas were pathologically staged according to the tnm ( tumors , nodes , metastasis ) classification system of uicc . all the tumors investigated were classified as t2/n0 and the grade of the tumors was g3 . breast tumors and surrounding histologically normal tissues were collected at the time of surgery and analyzed by histopathology . the first , middle , and last sections were stained with hematoxylin and eosin , and examined by a pathologist to ensure that at least 70% of the tissue sections used for the proteomic study contained tumor cells . the nontumor tissues were confirmed by cryostat section before protein extraction . for proteomic analyses , tissue sections verified histologically were suspended and homogenized in 250 l of 2-de analysis buffer : 7 m urea , 2 m thiourea , 4% chaps , 0.5% dtt , and 2% ampholytes ( 1 part ph 3/10 , 1 part ph 5/7 , 2 parts ph 6/8 ) . the clear supernatant issued after centrifugation of the homogenate at 12500 g for 15 minutes was transferred to a sterile microcentrifuge tube and immediately subjected to 2-de analysis . protein contents were determined according to the procedure described by bradford and modified by ramagli and rodriguez . equal amounts of proteins ( 100 g ) issued from tumor or nontumor breast tissues were applied to the first dimension and at least three nephge gels were run for each sample . extraction of proteins , solubilization , nephge , and sds - page were carried under similar conditions for the tumor and nontumor tissues . the alterations reported in the current study were retained after 2-de analysis of protein extracts issued respectively from different frozen sections of the 10 tumor and nontumor samples investigated . the reproducibility was about 85% or more which reflected the small variances between samples or running . the dye solution contained 17% ( w / v ) ammonium sulfate , 3% ( v / v ) phosphoric acid , 0,1% ( w / v ) coomassie g250 , and 34% ( v / v ) methanol . the staining solution was changed once after 12 hours staining and the gel slabs subjected to a 24-hour cycle for increasing dye deposition on low - abundance proteins . the detection was then increased by placing the gel into 1% v / v acetic acid for producing a better contrast between spots and gel . coomassie stained gels were scanned and spot detection and quantification were carried out using pdquest 2-d software version 7.2 ( biorad ) . proteins with significant and reproducible changes between tumor and nontumor tissues were considered as differentially expressed . the individual spot volumes were normalized by dividing their optical density ( od ) volumes by the total od values of all the spots present in the gel and expressed as % vol . the significance of expression between tumor and nontumor tissues was estimated by student 's t - test , p < .05 . picked spots were washed with 100 l of 25 mm nh4hco3 and dehydrated with 100 l of acetonitrile ( acn ) . this operation was repeated twice and the pieces of gel were dried under vacuum for 10 minutes . reduction was achieved by 1-hour treatment with 10 mm dtt in nh4hco3 buffer ( 100 l ) at 56c . after discarding the dtt solution , alkylation reaction was performed by the addition of 100 l of 25 mm iodoacetamide in 25 mm nh4hco3 buffer for 1 hour at room temperature , protected from light . finally , the excised gel pieces were again washed 3 times for 5 minutes with 25 mm nh4hco3 and acn alternately . the dried gel volume was evaluated ( about 1 to 2 l ) and 3 volumes of trypsin ( promega , v5111 ) , 12.5 ng/ l , in 25 mm nh4hco3 buffer ( freshly diluted ) , were added . afterwards , 5 l of 35% h2o/60% acn/5% hcooh were added and the mixture is sonicated for 30 minutes and centrifuged in order to extract tryptic peptides . mass measurements were carried out on a biflex iii maldi - tof ( bruker , daltonics , bremen , ge ) equipped with the scout high - resolution optics with x - y multisample probe and gridless reflector . this instrument was used at a maximum accelerating potential of 19 kv ( in positive mode ) and was operated in reflector mode . a saturated solution of -cyano-4-hydroxycinnamic acid ( sigma , saint louis , mo ) in acetone a first layer of fine matrix crystals was obtained by the spreading and fast evaporation of 0.5 l of matrix solution . on this fine layer of crystals , a droplet of 0.5 l of aqueous hcooh ( 5% ) solution was deposited . afterwards , 0.5 l tryptic digest were added and mixed to a second dried under vacuum . the sample was washed once by applying 0.7 l of aqueous hcooh ( 5% ) solution on the target and then flushed after a few seconds . in positive mode , internal calibration was performed with tryptic peptides coming from autodigestion of trypsin , with respectively monoisotopic masses at m / z = 842.510 and m / z = 2211.105 . for the mass measurements , up to 1 missed tryptic cleavage and optional methionine oxidation were considered . in most cases , the mass accuracy was less than 50 ppm , a value which is generally considered adequate for achieving statistically significant results for protein identification . these files were then fed into the search engine mascot ( matrix science , london , uk ) . ihc analysis of -b crystallin was performed on formalin - fixed , paraffin - embedded sections of idca breast tumors . briefly , five - micrometer sections were deparaffinized in toluene and subsequently hydrated with graded ethanol , and rehydrated in water . slides were microwaved in 0.01 m citrate buffer at ph 6.0 for 20 minutes at 750 w. thereafter , sections were rinsed thoroughly with water and placed in a tris buffered saline ( tbs ) solution ( 0.05 m tris - hcl ph 7.6 , 1.15 m nacl ) . endogenous peroxidase activity was blocked with hydrogen peroxide solution for 7 minutes and sections were rinsed gently with tbs . sections were then incubated at 4c overnight with the antibody against -b crystallin ( ab1546 , chemicon ) at a dilution of 1 : 1000 . after rinsing in tbs , sections were incubated with the secondary antibody , dakocytomation envision+dual link system peroxydase ( dakocytomation , glostrup , denmark ) , for 30 minutes . sections were washed 2 times with tbs , followed by application of the diaminobenzidine substrate pack according to the manufacturer 's instructions ( dakocytomation ) , yielding a brown - colored signal . standardization of the incubation and development times allowed an accurate comparison of expression levels in all cases . cases were considered positive if 10% or more of the tumor cells showed unequivocal immunoreactivity . in the current study , we have used an nephge-2-de protocol for the analyses of basic proteins , and using this procedure established an nephge-2-de map of proteins altered in idcas of the breast . since nephge is stopped before equilibrium is attained , accurate experimental estimation of the pis of the dysregulated proteins can not be made by this method . table 1 depicts the theoretical isoelectric points ( pis ) and molecular weights of altered proteins in tumor tissues , many of which are basic . in our hands , while nephge-2-de allowed the resolution of basic proteins , ief-2-de resulted as expected in the stacking of most of basic proteins at the basic end , even if the ief ph gradient has been extended to higher ph values . in our hands , a comparison of the 2-de protein patterns obtained at constant voltages of 400 v for 3 hours ( 1200 v - hr ) or 4 hours ( 1600 v - hr ) showed an optimal resolution of basic proteins at 1200 v - hr while preventing basic proteins from running off the basic end of the gels . by using the protein extraction procedure listed in section 2 , an average amount of 100 g of total proteins from each sample was used in 2-de investigations . figure 1 displays electrophoresis patterns of proteins from tumor and nontumor samples subjected to nephge-2-de electrophoresis . by using this procedure , we have found that the majority of the proteins displayed were not different in abundance between normal and cancer tissues . protein alterations occurring in the tumor and normal tissues are highlighted with arrows ( figure 1 ) . among the proteins identified , the levels of 13 proteins were increased in the tumor samples , while 7 proteins were rather observed at higher amounts in the nonneoplastic breast tissues . these alterations were regarded as serious alterations occuring between tumor and nontumor tissues . altered proteins were subsequently excised and subjected to in - gel tryptic digestion and maldi - tof analyses . figure 2 shows an example of maldi - tof peptide mass fingerprints of the tryptic digests corresponding to alpha b - crystallin . a wealth of evidence suggests that most of these dysregulated proteins are closely related to neoplastic transformation . these proteins could be classified on the basis of their functions into stress - related , chaperoning proteins , cytoskeletal - related proteins , metabolic enzymes , and other molecules related to immunological responses . some of these alterations depicted in figure 1 involve co - upregulations of glycolytic enzymes ( enolase1 , spot 8 ; phosphoglycerate kinase 1 , spot 9 ) in tumor tissues , as well as , proteins involved in antioxidation pathways including peptidylprolyl isomerase a ( spots 15 , 16 ) , which has been reported to protect cells against oxidative stress and manganese superoxyde dismutase ( mn - sod , spot 12 ) . two other newly identified proteins related to immunologic responses present at lower levels in tumor tissues include complement component c3c ( spot 1 ) and ighg1 ( spots 26 ) . more interestingly , the levels of raf kinase inhibitor protein ( spot 14 ) were found to be overexpressed in cancer tissues indicating a possible special pathological role for this protein in breast carcinogenesis . other alterations more represented with respect to the idca tissues include heat shock proteins ( hsp27 , spots 18 , 19 ) and -b - crystallin ( spot 13 ) . subsequent immunohistochemical staining against -b - crystallin has also been performed ( figure 3 ) . expression of -b - crystallin was noted in all cases investigated and positivity has been retrieved in 80 to 90% of tumor cells . the signals detected in tumor cells were strong whereas epithelial cells of the normal tissues express lower levels of this protein . proteomics - based studies are increasingly used to examine cancer - related changes in protein expression . such protein alterations may eventually contribute to increase our understanding of the disease pathogenesis as well as in the development of effective strategies for diagnosis and treatment . the present study aims to investigate protein alterations in idcas of the breast using an nephge-2-de procedure . as shown in this comparative analysis , certain specific proteins were dysregulated in idcas compared with their paired normal samples . these altered proteins were subsequently analyzed by mass spectrometry and unambiguously assigned on the basis of maldi - tof spectra obtained after trypsin digestion ( table 1 ) . the proteins identified span a wide range of functions and may have a value as diagnostic and prognostic biomarkers for breast cancer . among the proteins identified , we established that there is a decreased expression of igg1 and complement component c3c proteins in breast - tumor tissues . these alterations related to immunologic responses constitute novel identifications that were not previously reported as potential markers of idca breast tumors . as shown in figure 1 , the igg1 proteins were observed as multiple protein spots with different isoelectric points and similar molecular weights . as displayed in nephge-2-de gels , these proteins appeared intact as judged by their apparent molecular mass and were closely associated with nonmalignant tissues . consistent with our findings , several studies have shown that malignant diseases of various tissue origin are associated with a shift in the igg subclass distribution . for example , the decrease of igg1 serum levels reported in colorectal cancer has been considered as an early tissue nonspecific indicator of malignant transformation that could be useful in the primary diagnosis of colorectal tumors . such alterations were recently reported to occur in colorectal adenocarcinomas and normal colorectal tissues using suppression subtractive hybridization techniques . a highly reduction of the percent of igg1 in several gynecologic malignant diseases has also been correlated with early carcinoma stages and was found as a reliable marker that discriminates between cancer patients and controls with a specificity ranging between 90 and 100% . the reduction in igg1 plasmatic levels has also been reported as a highly sensitive tumor marker for detecting primary and metastatic squamous - cell carcinomas of the head and neck , and was significantly correlated with early stages of invasive breast cancer . up to now , such alterations in igg1 levels were found with neoplastic diseases only and may likely reflect changes in the host 's immune system linked to the presence of malignant tumors . recent data unveiled that malignant tumors of various tissues lead to irregularity of the biosynthesis of igg1 and igg2 by affecting their messenger rna levels in b - cell lymphocytes . it has been hypothesized in this context that cancer cells may affect the synthesis of such igg subclasses by acting on the t - helper 1/t - helper 2 ratio in the host , and , consequently , affect the level of specific cell - derived cytokines implicated in human igg synthesis . our observation of underexpression of igg1 in idcas with regard to nontumor tissues may therefore , indicate a similar tumor - associated mechanism by which cancer cells affect igg1 synthesis . further investigations are needed however , to elucidate how malignant breast tissues may interfere with the t - cell mediated regulation of igg suclasses . interestingly , downregualtion of igg1 levels and concomitant downregulation of complement component c3c were observed in our results . to our knowledge , no studies have yet reported the decreased expression of this protein in idcas of the breast . the differential tissue expression of this complement protein provides evidence for the involvement of anti - inflammatory processes in idca tissues and may indicate a selective adaptation by breast tumors to escape elimination by antitumor immune responses . until recently , several studies with normal and malignant tissues have disclosed the presence of several immune escape strategies by which cancer cells protect themselves from the lytic action mediated by complement . several evasion strategies to complement attack have been reported in malignant cells and include complement protective proteins ( cd46 , cd55 , cd59 ) , membrane proteases reported to confer complement resistance by removing cell - bound complement components such as c3 and tumor secreted complement inhibitors . such inhibitory mechanisms , often upregulated in tumors , are thought to increase resistance to complement in malignant tissues through inhibition of serum complement activation upon cancer cells . a previous study using a rat model of human breast cancer unveiled that inhibitors of complement activation may facilitate tumor growth and enhance tumorigenicity of breast - cancer cells by inhibiting c3 deposition . the present downregulation of complement c3 component c3c in idcas may , therefore , indicate the involvement in breast tumors of similar complement inhibitory mechanisms . the raf kinase inhibitor protein ( rkip ) known also as phosphatidylethanolamine binding protein-1 ( pebp-1 ) is another basic protein that has been described as being a component of the important feedback inhibition in g - protein coupled signal transduction . it is also involved in the regulation of the nf - kappa b pathway and is associated with resistance to tnf- by inhibiting activation of the raf 1/mek / extracellular signal regulated kinase pathway , jnk , and phosphatidylethanolamine ( pe ) externalization , which is an early symptom of apoptotic cell death resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer [ 24 , 25 ] . it has also been reported that overexpressing a new member of the pebp family ( pebp-4 ) correlates with both a decreased tnf--induced pe externalization and resistance to tnf--induced apoptosis . interestingly , recent data revealed that silencing of pebp-4 expression in mcf-7 breast - cancer cells promotes tnf--induced apoptosis , as well as tnf--induced erk and jnk activation making this pe binding protein as a promising target for the treatment of breast cancer . overall , the present findings in idcas of the breast might reflect the previously reported results for the relation between pe - binding proteins and tnf - related apoptosis pathway . the present increase in rkip may confer breast - cancer cells to an advantage for survival and proliferation . from our results , the high abundance of hemoglobin in breast tumors could not be considered as a reliable parameter for distinguishing between tumor and nontumor tissues . indeed , the relationship between tissue hemoglobin levels and breast tumors appears indirect and indicates rather an enhanced blood supply to the tumor . since tumor angiogenesis is a crucial arbiter of breast - cancer growth / metastasis , this higher level of hemoglobin is consistent with the higher vascular density of growing tumors compared with normal tissues . besides these alterations , the concomitant upregulation of two other stress - related proteins ( peptidylprolylisomerase a , mnsod ) was observed in idcas . the peptidylprolylisomerase a also called cyclophilina ( cypa ) is described as being involved in protein folding and catalyzes the cis / trans - isomerization of peptide bonds preceding a proline residue . it is secreted by cells in response to both inflammatory stimulation and oxidative stress . our observation on the expression of cypa is consistent with the published results from studies on other cancers . overexpression of cypa has been reported in colorectal carcinomas in comparison to normal mucosa and is involved in lung - tumor growth by affecting proliferation and apoptosis . a recent study has shown that both cypa and its receptor cd147 are upregulated in human pancreatic adenocarcinomas compared to normal pancreatic ductal epithelium . these authors pointed that cypa is a promoter of pancreatic tumor growth and is involved in chemotaxis and cell signalling cascades . the present finding reveals a similar correlation , implying that breast carcinomas may share a similar mechanism of cell proliferation mediated by cypa . the mnsod is a mitochondrial enzyme that has been reported to protect cells against oxidative stress by increasing the dismutation rate of superoxide anion ( o2 ) to hydrogen peroxide ( h2o2 ) which the role of this antioxidant enzyme in carcinogenesis is still however controversial and unclear . in fact although , it has been reported to suppress apoptosis and protect cells against several insults , under some circumstances , the mnsod may prevent cell proliferation . although further studies are needed , the present elevation of mnsod may indicate that the antioxidant defense system has been stimulated in invasive carcinomas of the breast , highlighting the ability of tumor cells to prevent damage due to reactive oxygen species . interestingly , the co - upregulation of two glycolytic enzymes , enolase 1 and phosphoglycerate kinase 1 ( pgk-1 ) also occurred in idca tissues . these enzymes are key components of the glycolytic pathway that have shown a prior relationship to cancer . indeed , our observation of increased expression of enolase 1 and pgk-1 is supported in various tumor tissues and may relate to the increased requirement of both energy and protein synthesis / degradation pathways during carcinogenesis . this increased glycolytic metabolism is also thought to regulate hypoxia - inducible gene expression in solid tumor tissues and participates in angiogenesis . the protein cofilin-1 also was identified as differentially expressed between tumor and normal tissues . this protein is recruited at the leading edge of cells where its activation is essential for polarized migration . cofilin-1 is directly implicated in severing actin cytoskeleton filaments and regulates the polymerization / depolymerization of actin during cell migration . it has been recently demonstrated that cofilin expression is required for tumor cell invasion since inhibition of cofilin activity in carcinoma cells inhibits cell motility whereas increased expression correlates with enhanced cell migration . have demonstrated that overexpression of cofilin-1 is necessary for the activation of the cofilin pathway resulting in cell motility , invasion and metastasis . this protein has also been associated with melanoma cell invasion and migration via its interaction with the v3 integrin . overall , the current findings support the recently identified role for cofilin activity and highlight its involvement in invasive lesions of the breast as a determinant factor required for cell migration and invasion . the molecular chaperone hsp27 and alpha b - crystallin , which is a small heat shock protein ( hsp ) are two other dysregulated proteins in tumor tissues . in a previous investigation , we have reported that three other molecular chaperones ( grp78 , calreticulin , protein disulfide isomerase ) are differentially expressed in breast tumors . the concomitant upregulation of these hsps together with alpha b - crystallin and hsp27 is not surprising since chaperones are thought to work cooperatively to fulfil their functions . due to the capacity of hsps to prevent stress - accumulated , unfolded , and nascent protein aggregation , their expression has proven to have important pathological implications such as cell proliferation and disease prognosis . the hsp27 is a molecular chaperone whose rate of synthesis increases many folds in response to environmental stress and during malignant transformation . as shown in figure 1 , this protein exists as two distinct isoforms that were confirmed by mass spectrometry as hsp27 ( table 1 ) . although no evidence of posttranslational alterations was pointed out , these isoforms as reported in renal cell carcinomas might reflect phosphorylation or other posttranslational modifications . in the last years , the role of alpha b - crystallin in cancer pathology has been widely discussed with regard to its potential oncogenic role . previous studies unveiled that this small hsp may constitute a good target for modulating cell death pathways . its expression has been shown to inhibit both the mitochondrial and the receptor death activation pathways of caspase 3 and correlates with trail resistance in a panel of cancer cell lines . this protein may also be an interesting molecular target for exploring the evolution and the origin of breast tumors since higher alpha b - crystallin levels were reported in ductal carcinoma in situ , which is an earliest form of detectable breast cancer . although further studies are needed to answer how this oncoprotein contributes to breast cancer , the data reported herein highlight the importance of this molecular chaperone in invasive carcinomas as a biomarker that may play a distinctive role in the process of carcinogenesis . in summary , our data have shown the value of using an nephge 2-de based proteomic approach in identifying potential molecular alterations and targets for breast - cancer therapeutics and diagnostics . we have identified a number of tumor - associated proteins including chaperones , energy - producing enzymes , stress - related proteins , proteins involved in apoptosis and cell motility , as well as alterations highlighting changes in the host 's immune system linked to the presence of malignant tissues . these protein variations indicate that multiple cellular pathways are involved in the process of breast carcinogenesis . such alterations may help to better understand the pathology of this disease and may have potential clinical applications as cancer biomarkers .

improvement of breast - cancer detection through the identification of potential cancer biomarkers is considered as a promising strategy for effective assessment of the disease . the current study has used nonequilibrium ph gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from tunisian women . we have identified multiple protein alterations in tumor tissues that were picked , processed , and unambiguously assigned identities by matrix - assisted laser desorption / ionization - time of flight mass spectrometry ( maldi - tof ) . the proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers . they include glycolytic enzymes , molecular chaperones , cytoskeletal - related proteins , antioxydant enzymes , and immunologic related proteins . among these proteins , enolase 1 , phosphoglycerate kinase 1 , deoxyhemoglobin , mn - superoxyde dismutase , -b - crystallin , hsp27 , raf kinase inhibitor protein , heterogeneous nuclear ribonucleoprotein a2/b1 , cofilin 1 , and peptidylprolyl isomerase a were overexpressed in tumors compared with normal tissues . in contrast , the ighg1 protein , the complement c3 component c3c , which are two newly identified protein markers , were downregulated in idca tissues .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSION

PMC4327424

over the last two decades , titanium has been applied in single - tooth and fixed partial dentures1 . the increasing dental application of titanium is due to its excellent biocompatibility , high corrosion resistance , low density , high strength / weight ratio , high ductility , low thermal conductivity and adequate mechanical properties12,28,31 . the lower density of titanium ( 4.2 g / cm ) , compared to conventional alloys , such as cobalt - chromium ( 8.9 g / cm ) and gold ( 19.3 g / cm ) , allow the fabrication of prostheses with a light weight without compromising important mechanical properties , such as tensile strength and hardness12,29 . in addition , titanium is an inexpensive metal that can be used to create custom cast restorations . although special investment and casting equipment is needed to make titanium castings , the potential benefit of inexpensive and custom - made restorations is significant22 . the low density , associated with a high melting point ( 1720c ) , and the high chemical reactivity of titanium with elements in the investment19 , however , have produced frequent defects , including porosity and castability7,19 . the high chemical reactivity of titanium at high temperatures ( above 600c ) , mainly with silica - based investments , produces a reactive surface layer on the cast ( -case layer)19 that reduces the ductility and the fatigue strength of the structure and clasps of removable partial dentures28,30 and may even compromise the crown fit30 . special investments containing magnesium oxide ( mgo ) , aluminum oxide ( al2o3 ) , zirconium oxide ( zro2 ) and calcium oxide ( cao ) have been used to produce a surface with reduced reactivity8,18 . however , these materials are expensive and their thermal expansion is not sufficient to compensate for the casting shrinkage of titianium6,17,18 . additionally , these investments do not produce a marginal fit in cast crowns of commercially pure titanium ( cp ti ) comparable with those of other alloys , such as au and pd - ag24 . the fit of cast denture restoration is essential for the evaluation of the casting technique , as it may influence the thickness of the cement margin , periodontal soundness , and the risk of secondary caries4 , which are decisive factors accounting for the clinical success of restorations . for a suitable cast crown fit , investment expansion needs to be compatible with the casting shrinkage of the metal . thus , it is necessary to establish a mold temperature that produces a suitable thermal expansion , which , associated with the setting expansion , may compensate for the cast metal shrinkage . manufacturers have suggested that the setting and thermal expansions of rematitan plus investment , specific for titanium , may be controlled by the concentration of the special liquid , and this has been confirmed by a study by soo , et al.26 ( 2001 ) , who evaluated the setting and thermal expansions of rematitan plus , rema exact and gc fujivest investments by changing the concentration of their special liquids . previous studies , considering the importance of mold temperature2526 and the concentration of the investment special liquid23 on the quality of titanium casting , found an unsatisfactory fit with the rematitan plus investment when using the conditions recommended by the manufacturer , namely , a liquid concentration of 100% and mold temperature of430c . thus , the null hypothesis of this study was that the effect caused by the concentration of the special liquid and by the temperature of the mold of rematitan plus investment on the internal fit of cp ti crowns is not significant . individual polyvinyl siloxane impressions were used to duplicate steel stylized die and create sets of epoxy resin ( epo - thin ; buehler uk ltd . , coventry , west midlands , england ) ( figure 1 ) . the steel dies represent teeth prepared for full - crown molar restoration with the following dimensions : length ( 6.0 mm ) , shoulder ( 1.0 mm ) , die diameter ( 7.0 mm ) , occlusal diameter ( 5.7 mm ) , and with a 6-degree axial surface convergence angle . labial surface was marked with a thin line , and all wax patterns received this mark to ensure proper orientation of crown casting . for waxing of each crown , a ring - shaped stainless - steel matrix , 8.0 mm internal diameter and 7.5 mm height , was adapted above the individual dies of epoxy resin ( figure 2 ) . the individual sprued pattern was mounted on crucible formers so that the length of exposed sprue ( 4 mm diameter ) was about 10 mm ( figure 3 ) . the pattern was then placed in a plastic casting ring ( number 3 ; dentaurum j. p. winkelstroeter kg , pforzheim , germany ) . individual patterns were used with the aim of preventing different specimens from showing the same internal misfit behavior . two hundred grams of rematitan plus dental casting investment ( dentaurum j. p. winkelstroeter kg , pforzheim , germany ) were weighed for each ring and two different concentrations of special liquid , 100% and 75% , diluted in water , were measured with a 25 cm pipette , and then mixed according to the manufacturer 's instructions . the molds were heated to 150c for dewaxing , and kept at that temperature for 90 minutes , then heated to 250c , and kept at that temperature for 90 minutes . heating was carried out in an electric furnace edg 7000 ( edg equipments ; so carlos , sp , brazil ) at 5c / minute . the molds were slowly cooled inside the furnace to the three different temperatures ; 430c recommended by manufacturer , 600c ( the expansion maximum , according to the expansion curve of the manufacturer ) and 515c ( an intermediate value ) . at the final temperature , the molds were inserted in the discovery plasma ar - arc vacuum - pressure casting machine ( edg equipments , so carlos , sp , brazil ) , which consists of two chambers : 1 ) an upper melting chamber , which houses a copper crucible and a tungsten electrode ; and 2 ) a lower casting chamber placed in the invested mold . all castings were carefully removed from the mold , scrubbed under running water and cleaned in water using an ultrasonic cleaner ( ultrasonic cleaner ; odontobras , ribeiro preto , sp , brazil ) . finally , each casting was ultrasonicated individually in a solution ( 1% hf + 13% hno3 ) for 10 min once again . the castings were carefully examined for any irregularities and nodules , with no internal adaptation made . a cutting disc ( dentaurum j. p. , winkelstroeter kg , pforzheim , germany ) was used to separate the crown from the sprue . subsequently , all castings were luted with zinc phosphate cement to the individual die epoxy resin under a 5 kg static load5 . the crown and die assemblies were embedded in acrylic resin and sectioned through the long axes with a slow - speed diamond saw ( isomet saw ; beuhler , ltd . , lake bluff , il , usa ) the specimens were bisected buccolingually and one of the cut surfaces of each specimen was finished with 600-grit abrasive paper ( buehler uk ltd . , coventry , west midlands , england ) , using a rotary equipment ( metaserv 2000 ; buehler uk ltd . , coventry , west midlands , england ) to remove saw - induced distortions . eight specimens per experimental condition were prepared , observed under a stereoscopy lens ( 10x ) , and the image was captured by a digital camera ( jvc tk1380u ccd ; jvc ltd . , tokyo , japan ) ( figure 4 ) . a leica qwin image analysis system ( leica microsystems imaging solutions ltd . , cambridge , england ) was used to measure the area represented by zinc phosphate cement , which corresponded to the area ( mm ) of internal misfit of the cp ti crowns . the misfit occurred in the entire infrastructure / prepared surface interface and there was uniformity between the axial surface , and between the misfit of the occlusal surface and the crown 's edge ( figure 4 ) . internal misfit means and standard deviations of the 8 specimens of each group were statistically compared using a kruskal - wallis non - parametric test ( p = 0.05 ) because the lack of homogeneous variances did not allow the use of parametric statistics . table 1 presents the results of kruskal - wallis test , comparing the concentrations of the special liquid ( 100% and 75% ) and mold temperatures ( 430c , 515c and 600c ) . tables 2 and 3 display the comparisons between both liquid concentrations at each temperature and among the three temperatures at each special liquid concentration . significant to the level of 1% ( p<0.01 ) different letters indicate statistically significant difference at 5% . different letters indicate statistically significant difference at 5% . at 430c , the concentrations of 100% ( 7.25 mm 1.59 ) and 75% ( 8.43 mm 1.77 ) presented statistically similar levels of internal misfit ( p<0.01 ) . at 515c , the concentration of 100% ( 9.94 mm 1.18 ) presented lower levels of misfit than the concentration of 75% ( 11.73 mm 1.0 ) ( p>0.01 ) . at 600c , there was no significant difference between the concentrations of 100% ( 8.8 mm 2.25 ) and 75% ( 9.33 mm 2.31 ) ( p>0.01 ) ( figures 46 ) . with regard to the different liquid concentrations , at 100% concentration , the temperature of430c ( 7.25 mm ) presented a smaller misfit than at 515c ( 9.94 mm ) and was statistically similar to the misfit obtained with 600c ( 8.8 mm ) , which did not demonstrate any significant difference at 515c . at a concentration of 75% , the temperatures of430c ( 8.43 mm ) and 600c ( 9.33 mm ) presented statistically similar levels of internal misfit , which were lower than those obtained at 515c ( 11.73 mm ) . considering the combinations of liquid concentration and mold temperatures on the casting of rematitan plus investment , the groups 430c/100% and 600c/100% were statistically similar , presenting the lowest levels of internal misfit ( 7.25 mmand 8.8 mm , respectively ) . the special liquid of rematitan plus investment consists of a colloidal silica solution in water , and it is used with the purpose of adjusting setting and thermal expansions by its dilution in water14 . the manufacturer suggests that both expansions may be increased by controlling the concentration of the special liquid , to compensate the metal and wax pattern . among the several factors that may compensate for the metal casting shrinkage occurring during the casting process , setting and thermal expansions morey20 ( 1992 ) suggested that the setting expansion of the investment should be near 0.05% , sufficient just to harmonize with wax expansion . another study3 confirmed this statement , reporting that investments with low setting expansions were less affected by restriction forces , and suggested that investments undergo the smallest setting expansion possible , to avoid distortion of the wax pattern , and high thermal expansion to compensate for the casting shrinkage of the metal20 . in addition to the concentration of the special liquid , mold temperature may be varied in an attempt to adjust the thermal expansion to the thermal shrinkage coefficient of titanium28,29 . when used as recommended by the manufacturer ( 100%/ 430c ) , rematitan plus investment , specific for titanium , did not provide fit levels that were comparable to those of conventional alloys21 . taking this into consideration , the present study investigated the effect of altering the concentration of the special liquid and the mold temperature for rematitan plus investment on both setting and thermal expansions , on the internal misfit of cp ti crowns . at temperatures of430c and 600c , the dilution of 75% special liquid did not significantly increase internal misfit levels . on the other hand , at 515c , this dilution resulted in a significant increase in internal misfit levels ( table 2 ) . these results showed that dilution of rematitan plus investment liquid did not improve the internal misfit levels of cp ti crowns ; rather , at 515c , it resulted in higher internal misfit levels . hutton and marshall9 ( 1993 ) demonstrated a large reduction in the investment setting expansion with the dilution of special liquid in distilled water . this occurs because there is higher silica content in the concentrated special liquid , which increases the silica - based investment setting expansion8 . soo , et al.26 ( 2001 ) also observed an increase in the setting and thermal expansions with the increase in the concentrations of the special liquid of rematitan plus , rema exakt and gc fujivest investments . although no significant difference was found between the 100% and 75% concentrations at 430c and 600c , a trend for increased internal misfit levels of the cp ti crowns was observed with dilution of rematitan plus investment special liquid . this may have occurred due to the decrease of total expansion with liquid dilution , confirming the findings of low and mori16(1999 ) , who reported a high correlation coefficient ( r=0.87 ) between the thermal expansion and the dimension accuracy of titanium cast crown . considering the concentrations of special liquid evaluated , 100% and 75% , the temperature of 515c resulted in a significant increase in the internal misfit levels ( table 3 ) . however , there was no statistically significant difference between these concentrations at 430c and 600c . the increase in mold temperature may cause an increase in thermal expansion and a consequently higher compensation of titanium shrinkage casting , producing crowns with lower misfit levels . on the other hand , inoue , et al.11(2001 ) evaluated the dimensional changes and setting accuracy of cp ti crowns , obtained under various mold temperatures , and observed a increase in the misfit with the increase of mold temperature . still according to inoue , et al.11(2001 ) , the misfit of the occlusal portion of the crown was greater than that of the cervical portion . this supposedly occurred due to the higher heating of the occlusal surface by the cast titanium because the sprue was located in the center of that surface11 . the results of the present study suggest that other factors may be more important than the increase in total expansion . the reaction layer of the titanium surface is a probable factor , considering that titanium has a high chemical reactivity at elevated temperatures13,24,28 . the higher internal misfit levels obtained at 515c compared to 600c , when using the 75% concentration , probably occurred because the total internal expansion at 515c was not sufficient to compensate for the effect of the reaction layer on titanium surface . on the other hand , at 600c , although the reactivity of the layer is higher due to the higher temperature , the higher expansion was probably compensated , in part , by the reaction layer . in conclusion , the changes in the concentration of the special liquid and mold temperature of rematitan plus investment did not result in any improvement in the internal misfit levels of the cp ti casting crowns , compared to the conditions recommended by manufacturers , i.e. 100% liquid concentration and a mold temperature of430c . given that conventional preheating furnaces are of low accuracy ( more than 10c / minus from the nominal temperature shown in the display ) and that it usually takes more than 2 minutes before casting is completed after removal of molds from the oven , it is difficult to determine the real temperature of the mold at the moment of casting . although the fit of titanium casting crowns has been evaluated10,14,23,30 , most available information refers to marginal fit . the lack of studies addressing internal misfit based on the evaluation of cement area ( mm ) together with differences in methodologies hinder any comparisons of the study outcomes . however , a possible relation between the values of internal and marginal misfit may be expected because the discrepancy that occurred on the occlusal surface of the die seems to correspond to that of the cervical margin . thus , the clinical benefit of the internal fit test is that it may represent an important tool to evaluate denture structure fit . al wazzan and al - nazzawi1 ( 2007 ) evaluated marginal and internal fit of cp ti and ti-6al-4v crowns and showed that ti-6al-4v had less fit discrepancy than cpti . several authors agree that a marginal gap of less than 100 m appears to be within the acceptable range for clinical longevity restorations2,14,18 . mid - occlusal internal fit has been shown to present better gap discrepancy than the axial internal fit . these results are consistent with those of the present study , as shown on figures 46 . the most plausible explanation for the difference in the internal discrepancy between occlusal and axial areas is the expansion behavior of the investment material in the casting ring . during setting expansion , the axial surfaces are substantially restricted because it is oriented towards an open end of the investment ring . moreover , thermal expansion of the investment may further allow the occlusal surface to expand more during wax elimination , consequently increasing the occlusal internal gap1 . further research is needed to clarify the favorable relationship between the cement space and the casting shrinkage value32 . dilution of the special liquid of rematitan plus investment did not result in any improvement in the levels of internal fit of the commercially pure titanium cast crowns , regardless of mold temperature . in addition , at a mold temperature of 515c , regardless of liquid concentration , a significant increase in internal fit levels was observed , thus rejecting the null hypothesis .

as the adaptation of titanium crowns obtained by rematitan plus investment , specific for titanium , is not recognized to be suitable , this study evaluated the effect of the concentration of the specific liquid and the temperature of the mold of investments on the internal misfit of crowns cast on commercially pure titanium . individual dies of epoxy resin were obtained , representing teeth prepared for full - crown restoration with a 6-degree axial surface convergence angle and shoulder ( 1.0 mm ) . for the waxing of each crown , a ring - shaped stainless steel matrix ( 8.0 mm internal diameter ; 7.5 mm height ) was adapted above the individual dies of epoxy resin . the rematian plus investment was mixed according to the manufacturer 's instructions using two different concentrations of the specific liquid : 100% , 75% . casting was performed in a discovery plasma ar - arc vacuum - pressure casting machine with molds at temperatures of 430c , 515c and 600c . the crowns were cleaned individually in a solution ( 1% hf + 13% hno3 ) for 10 min using a ultrasonic cleaner , with no internal adaptations , and luted with zinc phosphate cement under a 5 kg static load . the crown and die assemblies were embedded in resin and sectioned longitudinally . the area occupied by cement was observed using stereoscopic lens ( 10x ) and measured by the leica qwin image analysis system ( mm2 ) . the data for each experimental condition ( n=8 ) were analyzed by kruskal - wallis non - parametric test ( =0.05 ) . the results showed that liquid dilution and the increase in mold temperature did not significantly influence the levels of internal fit of the cast titanium crowns . the lowest means ( sd ) of internal misfit were obtained for the 430c/100% : ( 7.25 mm2 1.59 ) and 600c/100% ( 8.8 mm2 2.25 ) groups , which presented statistically similar levels of internal misfit .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3484937

sudden cardiac death ( scd ) is described as an event that is non - traumatic , non - violent , unexpected and resulting from sudden cardiac arrest within six hours of previously witnessed normal health . it is a rare but tragic condition usually precipitated by physical activity which occurs not only in athletes , but in all people . the growth of long distance running races has been accompanied by studies documenting that prolonged exercise can trigger unheralded ventricular arrhythmias and cardiac arrest in individuals without overt heart disease . an accurate determination of the incidence of the phenomenon is very difficult to achieve , because of the extreme differences in age , sex , race , athletes and non - athletes . literature data report that in apparently healthy long - distance runners the incidence of sudden cardiac arrest ranges from 1:15000 to 1:150000 per years , while the estimated incidence of sudden deaths in marathon runners is 0.63:100000 . scd is more frequent in male and black athletes , and increases significantly with age . the most common cardiovascular conditions predisposing to ventricular tachyarrhythmias , leading to sudden cardiac arrest in athletes , include manifest or latent primary electrical disease and latent structural causes ( table 1 ) . the most common cause of death by sudden cardiac arrest in italy is arrhytmogenic right ventricular cardiomiopathy , while in uk and in usa is hypertrophic cardiomiopathy . the diagnosis is very difficult to make as many athletes remain asymptomatic until a cardiac dysfunction takes place . moreover , even when each of the known causes of cardiac arrest without overt heart disease has been excluded , the diagnosis remains unexplained in almost half of these patients . at the finishing line of a marathon ( 42 km and 195 m ) , a 37 y runner with no history of disease collapsed due to a cardiac arrest after 236 minutes of running . the cardiac arrest was witnessed by an advanced cardiac life support ( acls ) team that immediately started advanced cardiopulmonary resuscitation ( first return of spontaneous circulation achieved in 18 ) . central venous blood gas analysis with manual ventilation was : ph 7,01 , pao2 23 mmhg , paco2 52 mmhg , na 145 meq , k 5.1 meq , ca 1.23 mmol / l , glucose 62 mg / dl , lactate 10.5 mmol / l , hco 13.1 meq / l , be -17.9 , svo2 17% . during the treatment of the refractory cardiac arrest the patient developed an impressive pulmonary edema , the patient was admitted to the nearest emergency department and then , taken into consideration the indication to veno - arterial extracorporeal membrane oxygenation ( ecmo ) , he was immediately transferred to a tertiary centre ( irccs san raffaele of milan ) to start the extracorporeal life support ( ecls ) . thanks to this procedure , it was possible to perform controlled cooling of the patient till 32c in consideration of the multi - organ damage to the heat stroke . further testing strategy included a coronary angiography that excluded coronary spasm , occlusion and anatomic anomalies , intra - aortic balloon pump positioning , transtoracic ecocardiography revealing normal kinesis . after five days on veno arterial ecmo , brain death diagnosis was established and multi - organ donation was allowed ( kidneys , bones , liver , femoral artery , cardiac valves , corneas ) . autopsy revealed a diffuse cerebral edema , acute pulmonary stasis , a focal undermucosal colic hemorrhage and no definite cause of scd . nowadays it is evident that scd is a rare but dreadful event within both athletes and non - athletes . the outcome of patients victims of sudden cardiac arrest can be largely different depending on different variables including etiology , rapidity of cardiopulmonary resuscitation and defibrillation and age . the outcome can be : survival with recovery ( 14.6% ) as well as organ donation , heart transplantation or ventricular assist device implantation . today , these results can be attained with ecls treatment , by now codified in referral tertiary care hospitals . clinicians can use different strategies to prevent scd : pre - participation screening as accurate anamnesis , medical examination , electrocardiography and echocardiogram can be life - saving and cost effective , even if contrasting results have been demonstrated ; making progress in research to allow early diagnosis of potentially lethal clinically silent cardiovascular abnormalities causing scd and detect its risk factors;guarantee an on - the - field cardiopulmonary resuscitation and early defibrillation by trained personnel ; predispose an ecmo assistance on - site in those long - distance races in which the risk of scd is increased , to provide an immediate and aggressive treatment of cardiac arrest and controlled cooling to ameliorate outcomes . pre - participation screening as accurate anamnesis , medical examination , electrocardiography and echocardiogram can be life - saving and cost effective , even if contrasting results have been demonstrated ; making progress in research to allow early diagnosis of potentially lethal clinically silent cardiovascular abnormalities causing scd and detect its risk factors ; guarantee an on - the - field cardiopulmonary resuscitation and early defibrillation by trained personnel ; predispose an ecmo assistance on - site in those long - distance races in which the risk of scd is increased , to provide an immediate and aggressive treatment of cardiac arrest and controlled cooling to ameliorate outcomes . deaths between athletes can be minimized if these conditions will be guaranteed : race organizers plan for comprehensive and advanced emergency cardiac care;the public and the participants are aware of the problem and trained in basic life support;an accurate screening of athletes is performed to detect who meets the criteria for disqualification ; ecls should be considered without delay in refractory cardiac arrest;ecls fast - track should be planned not only for large events involving thousands athletes , but also for ordinary management of refractory cardiac arrest in patient who meet criteria for enrollment to extracorporeal treatment ( actually , waiting for a consensus conference guidelines , represented by patients characterized by brain too good to die , receiving high quality cardiopulmonary resuscitation ( cpr ) and with a short road - to - pump time . race organizers plan for comprehensive and advanced emergency cardiac care ; the public and the participants are aware of the problem and trained in basic life support ; an accurate screening of athletes is performed to detect who meets the criteria for disqualification ; ecls should be considered without delay in refractory cardiac arrest ; ecls fast - track should be planned not only for large events involving thousands athletes , but also for ordinary management of refractory cardiac arrest in patient who meet criteria for enrollment to extracorporeal treatment ( actually , waiting for a consensus conference guidelines , represented by patients characterized by brain too good to die , receiving high quality cardiopulmonary resuscitation ( cpr ) and with a short road - to - pump time .

sudden cardiac death is a rare condition resulting from undetected cardiac abnormalities in athletes and non - athletes . participant screening , immediate and advanced medical management can probably reduce mortality and ameliorate outcomes . in recent years , extracorporeal membrane oxygenation has emerged as a valuable therapeutic option in patients experiencing refractory cardiac arrest as a bridge to different types of outcome , including recovery , heart transplantation or ventricular assist device and organ donations . in this report we describe a case of a sudden cardiac arrest in a marathon runner treated with extracorporeal membrane oxygenation .

Introduction Case Report Discussion Conclusions

PMC4831896

appropriate responses to exercise stressors are driven , in large part , by the autonomic nervous system ( ans ) and the resultant balance between sympathetic and parasympathetic influences on peripheral tissues ( sympathovagal balance ) . heart rate ( hr ) and the variability of heart beat - to - beat intervals , i.e. , heart rate variability ( hrv ) , are indirect metrics reflecting ans activity ( 19 ) , and reflect stress level during large muscle mass , dynamic exercise like running and cycling . however , the accuracy of these metrics in the field is complicated by the disproportionate change in the sympathovagal balance versus intensity relationship at or around the anaerobic threshold ( at ) ( 8 , 14,16,17 , 21 , 22 ) . numerous studies have identified hr threshold ( hrt ) and thresholds for various hrv metrics corresponding to at ( 6 , 2 , 10 , 11 , 13 , 18 ) . expressing stress level relative to such a hr or hrv threshold ( e.g. , power output eliciting 75% of hrt ) , as opposed to absolute cardiac metrics , would allow accurate , real - time pacing in the field . furthermore , the ability to assess at in the field using hr or hrv would be more applicable for athletes and coaches than established laboratory - based techniques such as lactate threshold ( lt ) and ventilatory threshold ( vt ) ( 6 ) . most investigations that evaluate hr or hrv versus intensity use continuous incremental exercise protocols ( reviewed in 6 ) . the exercise stages for these protocols typically last 12 min , which does not allow time for adequate sympathovagal balance . this is due to inherent fluctuations of efferent / afferent control systems , which are more dynamic during the initial response to stress . once the appropriate afferent / efferent balance is achieved , the recorded metric will not fluctuate greatly ( i.e. , steady state ) unless the stress level changes . for instance , during exercise of a given intensity , o2 consumption and hr both rise during the first minutes exercise ( 14 ) . therefore , in the first minute of a 12 min exercise stage , it is difficult to know if data collected during the first minutes of a given exercise stage reflect steady - state responses ( 5 ) . in addition , with continuous protocols it is probable that responses to one exercise stage carryover to the next stage . because of this , development of accurate linear relationships between hr / hrv and intensities below at are not possible . therefore , the ideal endurance training output at hrt derived from a continuous test protocol would be misidentified , and premature fatigue would likely occur . discontinuous incremental exercise protocols allow for longer stages at given intensities and brief recovery periods . in such a protocol , exercise performance metrics such as hr and vo2 are assessed late in the exercise stage ( e.g. , after 35 min of the same exercise intensity ) . thus , the assessment occurs after hr and vo2 responses have plateaued , reflecting appropriately balanced additionally , the recovery period in between stages ensures that the subsequent stage data is not influenced by protracted factors from a previous stage ( i.e. , lactic acid , epinephrine , etc . ) . because of these differences , discontinuous protocols allow for a true indication of steady - state physiologic stress responses , independent of the possible carryover effects from previous exercise stages ( reviewed in 5 ) compared to continuous protocols . despite the physiological benefits described above , discontinuous protocols have not been used to study simple surrogate measures of at . therefore , the purpose of the present study was to determine if changes in hr and/or hrv would reflect at using a discontinuous , incremental , cycle test . to accomplish this , exercise thresholds were determined for hr and hrv metrics and compared with the criterion at assessment , vt . using a student t - based algorithm with an alpha = 0.05 , and beta ( power level ) = 80% , a power analysis was run to determine the minimum number of subjects required for our comparison . it was determined based on available pilot data for at ( n=100 , sd=20 ) , that 15 subjects were needed to detect a 5 w difference in w at at . experienced cyclists , age 1835 y , were recruited for this study , with eight female and seven male participants subjected to the protocols described below . for this study , the term experienced cyclist was defined as an individual self - reporting moderate intensity cycling an average of three times per week . this study was approved by the institutional review board for the protection of human subjects in research of the university of texas at san antonio . written informed consent was obtained from all participants following a verbal and written briefing of all experimental procedures . participants were asked to abstain from stimulants , including caffeine , exercise , and alcohol 12 h before the cycling sessions . participants reported to the laboratory in the morning after an 8 h fasting condition on two sessions . the height and weight of the participants was recorded and their body composition was assessed using 7-site skinfolds ( 1 ) . four frame measurements were taken on each participant s bicycle were matched on a lode excalibur ( lode b.v . , groningen , netherlands ) cycle ergometer to reproduce the participants training position . participants performed all testing in their usual training attire and used their own cycle pedals and shoes . all procedures were performed in a thermoneutral environment under close supervision of laboratory assistants familiar with the testing protocol and data collection . following measurement of shoeless height and body mass , participants were outfitted with a breathing mask allowing for inhalation of room air and analysis of expired air for ventilation ( ve ; l airmin1 ) , oxygen consumption ( vo2 ; l o2min1 ) and carbon dioxide production ( vco2 ; l co2min1 ; trueone2400 metabolic measurement system , parvomedics inc . , sandy , ut ) . participants wore a chest hr transmitter ( 2-lead ecg ; polar electro , kempele , finland ) and wristwatch monitor ( polar model rs810 ) capable of measuring the time interval between successive heart beats as denoted by cardiac cycle r - waves ( i.e. , r - r interval ) . participants completed a traditional continuous cycle exercise test during which intensity was increased by 20 watts ( w ) every minute starting at 60 w and continuing until volitional fatigue ( e.g. , ~400 w for males and ~300 w for female cyclists ) . this session provided data to 1 ) determine of the cardiorespiratory fitness of each participant ( i.e. , vo2peak ) , 2 ) determine participants individual vt using ve versus w ( i.e. , vet1w ) , and 3 ) establish the intensities for the subsequent discontinuous cycle test exercise stages . individual s ve data were plotted versus w for each stage to determine vt by computer linear regression to establish vet1w ( 20 ) . the same instrumentation described above , participants performed a series of seven stages of exercise lasting 6 min each with 3 min of rest between each stage . one stage was then performed at vet1w ( i.e. , at at ) followed by three stages above vet1w ( i.e. , above at ) . for example , with vet1w considered as 100% , stage 1 w were 40% of vet1w , stage 2 w were 60% of vet1w and stage 3 w were 80% of vet1w . for the stages above at , 10% increases in intensity were used from vet1w ( i.e. , stages 5 , 6 , and 7 were 110 , 120 , and 130% of vet1w , respectively ) . exercise ve , hr , and cardiac interbeat intervals were recorded during the last 3 min of each stage . hr and interbeat interval data were downloaded to a computer for off - line analysis with kubios hrv analysis software ( biosignal analysis and medical imaging group ; department of physics , university of kuopio , finland ) . averaged for hrv metrics , the following time - domain data were assessed : mean rr interval ( mrr ) , standard deviation rr interval ( stdrr ) , and root mean square standard deviation ( rmssd ) . additionally , the following frequency - domain results were analyzed : low frequency spectral energy ( lf ; 0.0140.15 hz ) , high frequency spectral energy ( hf ; 0.150.4 hz ) , hf frequency peak ( fhf ; ms2 ) , and hffhf ( hzms2 ) . the averaged stage data for ve , hr , and each time- and frequency - domain hrv metric were plotted versus w ( microsoft office excel 2007 , microsoft corporation ) . for each parameter , two trend lines with corresponding slope - intercept ( i.e. , y = mx+b ) formulas were established for the stage data using microsoft excel ( microsoft corporation ) . one trend line was established for data points from stages known to be below and at at , as reflected by vet1w , while the other trend line was established for data points collected at stages corresponding to at and above . the intersection point of the two lines was then determined using y = ax+b and y = cx+d followed by x=(db)(ac)1 . the w corresponding to the intersection of these trend lines was considered to represent each metric s threshold . no relationships were discernible for stdrr or rmssd data versus w so these parameters were not evaluated further . once parameters thresholds were identified , they were compared to the w corresponding to the discontinuous cycle test vt ( i.e. , vet2w ) using pearson ( two - tailed ) and paired - sample t - test ( ibm spss statistic , version 19 ; ibm company ) . using a student t - based algorithm with an alpha = 0.05 , and beta ( power level ) = 80% , a power analysis was run to determine the minimum number of subjects required for our comparison . it was determined based on available pilot data for at ( n=100 , sd=20 ) , that 15 subjects were needed to detect a 5 w difference in w at at . experienced cyclists , age 1835 y , were recruited for this study , with eight female and seven male participants subjected to the protocols described below . for this study , the term experienced cyclist was defined as an individual self - reporting moderate intensity cycling an average of three times per week . this study was approved by the institutional review board for the protection of human subjects in research of the university of texas at san antonio . written informed consent was obtained from all participants following a verbal and written briefing of all experimental procedures . participants were asked to abstain from stimulants , including caffeine , exercise , and alcohol 12 h before the cycling sessions . participants reported to the laboratory in the morning after an 8 h fasting condition on two sessions . the height and weight of the participants was recorded and their body composition was assessed using 7-site skinfolds ( 1 ) . four frame measurements were taken on each participant s bicycle were matched on a lode excalibur ( lode b.v . , groningen , netherlands ) cycle ergometer to reproduce the participants training position . participants performed all testing in their usual training attire and used their own cycle pedals and shoes . all procedures were performed in a thermoneutral environment under close supervision of laboratory assistants familiar with the testing protocol and data collection . following measurement of shoeless height and body mass , participants were outfitted with a breathing mask allowing for inhalation of room air and analysis of expired air for ventilation ( ve ; l airmin1 ) , oxygen consumption ( vo2 ; l o2min1 ) and carbon dioxide production ( vco2 ; l co2min1 ; trueone2400 metabolic measurement system , parvomedics inc . , sandy , ut ) . participants wore a chest hr transmitter ( 2-lead ecg ; polar electro , kempele , finland ) and wristwatch monitor ( polar model rs810 ) capable of measuring the time interval between successive heart beats as denoted by cardiac cycle r - waves ( i.e. , r - r interval ) . participants completed a traditional continuous cycle exercise test during which intensity was increased by 20 watts ( w ) every minute starting at 60 w and continuing until volitional fatigue ( e.g. , ~400 w for males and ~300 w for female cyclists ) . this session provided data to 1 ) determine of the cardiorespiratory fitness of each participant ( i.e. , vo2peak ) , 2 ) determine participants individual vt using ve versus w ( i.e. , vet1w ) , and 3 ) establish the intensities for the subsequent discontinuous cycle test exercise stages . individual s ve data were plotted versus w for each stage to determine vt by computer linear regression to establish vet1w ( 20 ) . following the same instrumentation described above , participants performed a series of seven stages of exercise lasting 6 min each with 3 min of rest between each stage . one stage was then performed at vet1w ( i.e. , at at ) followed by three stages above vet1w ( i.e. , above at ) . for example , with vet1w considered as 100% , stage 1 w were 40% of vet1w , stage 2 w were 60% of vet1w and stage 3 w were 80% of vet1w . for the stages above at , 10% increases in intensity were used from vet1w ( i.e. , stages 5 , 6 , and 7 were 110 , 120 , and 130% of vet1w , respectively ) . exercise ve , hr , and cardiac interbeat intervals were recorded during the last 3 min of each stage . hr and interbeat interval data were downloaded to a computer for off - line analysis with kubios hrv analysis software ( biosignal analysis and medical imaging group ; department of physics , university of kuopio , finland ) . averaged for hrv metrics , the following time - domain data were assessed : mean rr interval ( mrr ) , standard deviation rr interval ( stdrr ) , and root mean square standard deviation ( rmssd ) . additionally , the following frequency - domain results were analyzed : low frequency spectral energy ( lf ; 0.0140.15 hz ) , high frequency spectral energy ( hf ; 0.150.4 hz ) , hf frequency peak ( fhf ; ms2 ) , and hffhf ( hzms2 ) . the averaged stage data for ve , hr , and each time- and frequency - domain hrv metric were plotted versus w ( microsoft office excel 2007 , microsoft corporation ) . for each parameter , two trend lines with corresponding slope - intercept ( i.e. , y = mx+b ) formulas were established for the stage data using microsoft excel ( microsoft corporation ) . one trend line was established for data points from stages known to be below and at at , as reflected by vet1w , while the other trend line was established for data points collected at stages corresponding to at and above . the intersection point of the two lines was then determined using y = ax+b and y = cx+d followed by x=(db)(ac)1 . the w corresponding to the intersection of these trend lines was considered to represent each metric s threshold . no relationships were discernible for stdrr or rmssd data versus w so these parameters were not evaluated further . once parameters thresholds were identified , they were compared to the w corresponding to the discontinuous cycle test vt ( i.e. , vet2w ) using pearson ( two - tailed ) and paired - sample t - test ( ibm spss statistic , version 19 ; ibm company ) . participants average age , height , body mass , body mass index ( bmi ) , vet1w , and vo2peak are provided in table 1 . normal while the vo2peak data is consistent with that of ~80th percentile for 2040 year olds by the american college of sports medicine ( 1 ) . the average observed w corresponding to vt during the continuous cycle test ( vet1w ) and the discontinuous cycle test ( vet2w ) were normally distributed ( shapiro - wilk=0.931 , df=15 , p>0.05 ) highly correlated ( r=0.97 ; p=0.000 ) , with individual participants vet1w and vet2w varying by less than 3 w ( p>0.05 ) . participants individual vet2w were used in the comparison of hr and hrv metrics threshold w. the w corresponding to the intersection of trend lines established for hr , mrr , lf , hf , fhf , hffhf data were considered to represent each metric s threshold ( i.e. , hrtw , mrrtw , lftw , hftw , fhftw , and hffhftw , respectively ) ( figure 1 ) . discernible linear relationships between the hrv metric data and w were not observed for all participants . namely , hrtw was not established for one female subject , mrrtw was not established for two male subjects and one female subject , hftw and fhftw were each not established for a male subject . when a hr or hrv metric threshold could not be established , the subject s data , including vet2w , were not included in the subsequent statistical analyses . comparisons of the established hr and hrv metric threshold w and the corresponding vet2w are presented in table 2 . statistically significant correlations ( p0.05 ) were observed between all hr and hrv metric threshold w and vet2w when participants data were compared . significant statistical differences ( p<0.05 ) were not observed for vet2w and hrtw ( 2.0 13.4w difference ) , mrrtw ( 10.0 26.4w difference ) , hftw ( 7.3 19.0w difference ) , and hffhftw ( 2.4 23.1w difference ) . however , the 11.3 17.8w difference for lftw and 13.9 18.7w difference for fhftw were statistically significant ( p=0.028 and p=0.015 , respectively ) . we have demonstrated that the power output at at can be determined using hrt and several hrv metric thresholds from a discontinuous , incremental cycling protocol . in the present study , thresholds for hrt and the related mrr correlated significantly with w at at as determined by vt . the inherent fluctuation of autonomic balance makes it difficult to determine if physiologic data collected early in a given exercise stage will reflect the body s definitive response . hrt and hrv threshold investigations in cycling have utilized continuous , incremental exercise protocols consisting of 12 min stages . therefore , threshold determination using blood lactate , ve , vo2 , hr , or hrv metrics may not be optimally applicable for training or competition wattage prescription in the field . for example , the hrt , or oft - termed heart rate deflection point ( hrdp ) , was identified in cyclists by bourgois and colleagues ( 7 ) using a continuous cycle test . a subsequent 30 min test at hrdp w only 6 of 11 subjects were able to complete the 30 min with 5 subjects demonstrating accumulating blood lactate values . these findings are indicative of the hrdp w , as determined by a continuous test , over - predicting the subsequent work rate leading to premature fatigue . such an at over - prediction was also observed in a large cycle study by carey et al . the agreement between time trial performance and data collected during protocols using multiple , longer stages ( e.g. , 3 min ) of exercise has also been demonstrated by bentley dj & mcnaughton lr ( 4 ) . when considering that typical endurance ( i.e. , at ) cycle training rides or races last far longer than 30 min , the potential performance impact of an over - prediction of at using continuous protocol - derived w from hrt is clear . together , the findings above suggest that at data derived from a discontinuous protocol stages that are long enough to establish steady - state may be more appropriate for exercise prescription . accurate representation of the initial , sub - at , linear relationship between intensity and ve , hr , and hrv metrics is paramount to subsequent identification of the nonlinear deflection in the data collected at intensities above at . the data from the present study were collected during the last 3 min of cycling at each 6 min exercise intensity stage . furthermore , several minutes of rest between each stage ensures that no lingering effects from an earlier stage the physiological response to a subsequent workrate . therefore , data from the sub - at intensity stages can be considered as steady - state conditions and reflective of the appropriate response level of the fast - responding physiological metrics of interest . the highest observed absolute difference in vet2w and the hrtw and hrv metrics w was > 16 w with several comparisons differing by as little as 2 w. this range is notable as bicycles used for endurance training and competition commonly will change by ~20 w per sprocket / gear shift at the same cadence . a low degree of error of the appropriate sub - at power output determined in the laboratory to the field allows cyclists to determine the optimal sprocket and cadence combination to suit their performance . we are not aware of any study reporting comparisons between hrv and ve thresholds using a discontinuous exercise protocol . several studies have focused on evaluation of hrv changes near at using continuous protocols ( 2 , 10 , 11 , 13 , 18 ) . spectral analysis of hrv has proven useful in these investigations because cardiorespiratory parameters temporal series , namely hr and ve , fluctuate dramatically during load increases . observed changes in the increment of increase in the hf domain ( > 0.15 hz ) the hf parameter reflects the amplitude of the sinus arrhythmia ( rsa ) and vagal tone . high frequency increases in vagal tone above the intrinsic sinus depolarization rate ( i.e. , ~100bpm ) is perhaps a response to venous return - mediated stretch of the right atrium and sinus node . thresholds identified in the present study using hf hrv metrics ( i.e. , hftw , fhftw , hffhftw ) all correlated with vet2w . spectral lf variations in r - r intervals reflect changes in both sns and psns activity ( 19 ) . this finding was somewhat surprising from a physiological perspective as the overall psns influence is minimal above low intensity exercise ( hr > 100 bpm ) . in such , changes in lf above at would be influenced predominantly by sns , possibly decreasing the predictive potential of lf for atw . additionally , other , non - neural factors such asepinephrine or respiratory pump on hr increase with intensity which may play a role as well , which could impact the reliability of the lf parameter to predict at . there are a few key limitations to our generalizing our findings to all adult cyclists . whether or not the findings would apply across a wider range of experience levels or age groups is not known . secondly , discernible relationships between the hrv metric data and w were not observed for all participants . namely , hrtw was not established for one female subject , mrrtw was not established for two male subjects and one female subject , hftw was not established for one male subject , and fhftw was not established for one male subject . when a hr or hrv metric threshold could not be established , the subject s data , including vet2w , were not included in the statistical analyses . furthermore , the inability of identifying a threshold in 30% of participants has important implications for the consistency of these metrics in the field . the low number of participants makes extrapolation of the findings to similar populations difficult at best . further studies are needed to ascertain if failure to identify a threshold results from the protocol , data collection techniques , or participants individual differences . at is the best predictor of performance in endurance activities such as cycling and running ( reviewed in 12 ) . knowing the w associated with at is especially relevant when cyclists want to maintain a given % of at for extended periods of time . cycling at or just under at allows for sustained performance without premature fatigue due to effects of lactic acid . using a hrv metric threshold would allow detection of changes in ans balance ( cardiac drift , dehydration , fatigue , metabolic status , etc . ) preceding premature fatigue . in such a case , appropriate adjustments to power output could be made to avoid performance detriments . future studies will need to compare the hrtws from continuous and discontinuous protocols to maximal lactate steady state ( mlss ) during a prolonged performance test . this will have further relevance to the field as mlss and mlss velocity are highly correlated with prolonged endurance performance in cyclists ( 3 , 12 ) .

the purpose was to determine if heart rate ( hr ) and heart rate variability ( hrv ) responses would reflect anaerobic threshold ( at ) using a discontinuous , incremental , cycle test . at was determined by ventilatory threshold ( vt ) . cyclists ( 30.65.9y ; 7 males , 8 females ) completed a discontinuous cycle test consisting of 7 stages ( 6 min each with 3 min of rest between ) . three stages were performed at power outputs ( w ) below those corresponding to a previously established at , one at w corresponding to at , and 3 at w above those corresponding to at . the w at the intersection of the trend lines was considered each metric s threshold . the averaged stage data for ve , hr , and time- and frequency - domain hrv metrics were plotted versus w. the w at the threshold for the metrics of interest were compared using correlation analysis and paired - sample t - test . in all , several heart rate - related parameters accurately reflected at with significant correlations ( p0.05 ) were observed between at w and hr , mean rr interval ( mrr ) , low and high frequency spectral energy ( lf and hr , respectively ) , high frequency peak ( fhf ) , and hfxfhf metrics threshold w ( i.e. , mrrtw , etc . ) . differences in hr or hrv metric threshold w and at for all subjects were less than 14 w. the steady state data from discontinuous protocols may allow for a true indication of steady - state physiologic stress responses and corresponding w at at , compared to continuous protocols using 12 min exercise stages .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4588799

the demand for restoring teeth , not only anatomically and functionally , but also esthetically , has been increased in daily clinical practice.1 resin composites have been utilized remarkably in restorative dentistry to enhance dental structure or correction of tooth color.1 a strong adhesion between tooth tissue and resin composite is essential for an ideal restoration . enamel bonding has tremendous clinical success ; however , dentin bonding can not predictably be applied for long - term interfacial integrity . since dentin is a vital part of the tooth and has complex biological structure,2 it can influence the performance of different bonding strategies . total - etch bonding systems work by removing the smear layer with phosphoric acid , applying a primer and adhesive in two different steps or even in the same step ( total etch self - priming systems ) . the self - etching approach is another technique in which increased concentrations of acidic monomers enable the primer or adhesive to etch and prime the dentin simultaneously.3 in accordance with these systems , seven main generations of dentin bonding agents have been introduced in dentistry , but the fifth generation is more common for usage in adhesive treatments . in that generation , primer and bonding agent are combined into a single solution , so separate etching step is required.4 in clinical conditions , teeth are constantly encountered stresses during mastication and parafunctional habits.5 these stresses will induce some micro cracks which subsequently jeopardize the long - term survival rate of bonding and final mechanical degradation of tooth tissue and restoration interface.6 several studies have shown that masticatory loadings could accelerate the degradation of dentin bonding interface.1,7,8 the thermal cycles in oral environment can induce deteriorating stresses between a tooth substrate and a restorative material by generating expansion / contraction stresses.1 since impossibility of constant and rapid assessment of adhesive materials in clinical trial studies , use of thermal cycling ( tc ) and mechanical cycling ( mc ) would facilitate evaluation of dental materials similar to clinical conditions.7,9,10 the porosities and other internal defects within the adhesive layer may have detrimental effects on bonding durability . the static bond strength tests can not adequately demonstrate it.11 cyclic loading could rapidly unfold the effect of these defects on long - term bonding.10 application of microtensile bond strength ( tbs ) testing allows evaluation of the in - vitro bond strength of resin / dentin under more clinically relevant conditions than those which are usually performed in static bond strength testing techniques . furthermore , this test enables researchers to measure bond strength at different regions . due to the limitations and difficulties of evaluating dentin / resin adhesion in clinical trial studies , researchers usually try to resemble oral environment in - vitro studies and attribute their results to actual conditions . the current study designed to evaluate the effects of tc and mc on tbs of single bond-2 to dentin and observing the pattern of failure in bonded samples . the null hypothesis was that there would be no differences between tbs of resin / dentin neither with nor without tc and mc . in this in - vitro study , 48 freshly extracted intact and carries - free human third molar teeth , were collected and restored in distilled water at 4c.7 the teeth with any signs of cracks or developmental defect were excluded . the teeth were cleaned and polished using slurry pumice with a brush and low - speed handpiece , and were disinfected by chloramine - t 1% ( merck , darmstadt , germany ) solution for 24 h prior to testing.12 all of the samples were mounted in self - curing acrylic resin ( flash acrylic , yates motloid , chicago , usa ) with the level of 1 mm below the cementoenamel junction ( cej ) . diamond burs ( teeskavan , tehran , iran ) and high - speed handpiece with water spray were used to remove the occlusal enamel surface , beneath the pit and fissure surfaces , and flattened surface obtained with exposed underlying superficial dentin . each bur was used for removing the enamel of five teeth . before application of bonding agent , the dentin surfaces were treated by 320 grit silicone abrasive paper ( carbimet discs , buehler , lake bluff , il , usa ) for 15 s to create standard smear layer on the each tooth surface.13 in accordance with the instructions of single bond-2 ( 3 m , espe , usa ) company , the dentin surface of each specimen was acid - etched by 37% phosphoric acid gel for 15 s and then they were rinsed for 10 s by distilled water . two layers of adhesive was applied and light cured by using light curing unit ( bluephase 20i , vivadent , lichtenestein , germany ) with a light intensity of 600 mw / cm for 10 s. 3 mm z250 resin composite ( 3 m , espe , usa ) in two layers were used to restore the bonded areas . the light curing distance was at a minimum distance for all of the samples , and each layer were light - cured for 40 s. all of the samples were mounted in self - curing acrylic resin with the level of 1 mm below the cej . specimens were divided into eight equal groups ( g1-g8 ) , by random table number ( n = 6 ) . according to the tc and mc groups were as follow : g1 ( control ) : no thermo and mc ; g2 : 50 k mc ; g3 : 100 k mc ; g4 : 500 k mc ; g5 : 1000 tc ; g6 : 50 k mc+1000 tc ; g7 : 100 k mc+1000 tc ; g8 : 500 k mc+1000 tc . for mc , teeth were subjected to load cycling ( erios , sao paulo , brazil ) . 90 n force with a frequency of 0.5 hz was applied.14 the teeth were embedded in normal saline during mc . the specimens from group g5 through g8 were thermocycled using a thermocycle machine ( erios , sao paulo , brazil ) at 5 - 55c water baths with holding and dwell time of 60 and 15 s , respectively . the mounted teeth in self - cure acrylic resin were sectioned in mesiodistal direction and parallel to the vertical plane under running water to prepare two slabs with about 1 mm thickness from each tooth . the bonding area ( 0.8 mm interface area ) of each sample were thinned to create an hour - glass shape by using a diamond cylindrical bur ( ss white , ny , usa ) . the upper and lower parts of hour - glass shaped samples were stuck to the designed arms of testing machine . then , the samples were subjected to tbs test by universal testing machine ( plus universal testing machine mtd-500 , germany ) at a crosshead speed of 0.5 mm / min in order to create fracture at the interface of composite and dentin . the kolmogorov the data were analyzed by one - way analysis of variance ( anova ) , tukey hsd and post - hoc tests . two - way analysis variance was employed to determine the reciprocal effect of tc and mc on the tbs , using spss 17 software program ( spss 17 , spss inc . , illinois , chicago , usa ) at a significant level of 0.05 . in order to determine the location and mode of fracture , all of the samples were examined by using a scanning electron microscope ( sem ) ( ht 416002 , fuji , japan ) . in this in - vitro study , 48 freshly extracted intact and carries - free human third molar teeth , were collected and restored in distilled water at 4c.7 the teeth with any signs of cracks or developmental defect were excluded . the teeth were cleaned and polished using slurry pumice with a brush and low - speed handpiece , and were disinfected by chloramine - t 1% ( merck , darmstadt , germany ) solution for 24 h prior to testing.12 all of the samples were mounted in self - curing acrylic resin ( flash acrylic , yates motloid , chicago , usa ) with the level of 1 mm below the cementoenamel junction ( cej ) . diamond burs ( teeskavan , tehran , iran ) and high - speed handpiece with water spray were used to remove the occlusal enamel surface , beneath the pit and fissure surfaces , and flattened surface obtained with exposed underlying superficial dentin . each bur was used for removing the enamel of five teeth . before application of bonding agent , the dentin surfaces were treated by 320 grit silicone abrasive paper ( carbimet discs , buehler , lake bluff , il , usa ) for 15 s to create standard smear layer on the each tooth surface.13 in accordance with the instructions of single bond-2 ( 3 m , espe , usa ) company , the dentin surface of each specimen was acid - etched by 37% phosphoric acid gel for 15 s and then they were rinsed for 10 s by distilled water . two layers of adhesive was applied and light cured by using light curing unit ( bluephase 20i , vivadent , lichtenestein , germany ) with a light intensity of 600 mw / cm for 10 s. 3 mm z250 resin composite ( 3 m , espe , usa ) in two layers were used to restore the bonded areas . the light curing distance was at a minimum distance for all of the samples , and each layer were light - cured for 40 s. all of the samples were mounted in self - curing acrylic resin with the level of 1 mm below the cej . specimens were divided into eight equal groups ( g1-g8 ) , by random table number ( n = 6 ) . according to the tc and mc groups were as follow : g1 ( control ) : no thermo and mc ; g2 : 50 k mc ; g3 : 100 k mc ; g4 : 500 k mc ; g5 : 1000 tc ; g6 : 50 k mc+1000 tc ; g7 : 100 k mc+1000 tc ; g8 : 500 k mc+1000 tc . for mc , teeth were subjected to load cycling ( erios , sao paulo , brazil ) . 90 n force with a frequency of 0.5 hz was applied.14 the teeth were embedded in normal saline during mc . the specimens from group g5 through g8 were thermocycled using a thermocycle machine ( erios , sao paulo , brazil ) at 5 - 55c water baths with holding and dwell time of 60 and 15 s , respectively . the mounted teeth in self - cure acrylic resin were sectioned in mesiodistal direction and parallel to the vertical plane under running water to prepare two slabs with about 1 mm thickness from each tooth . the bonding area ( 0.8 mm interface area ) of each sample were thinned to create an hour - glass shape by using a diamond cylindrical bur ( ss white , ny , usa ) . the upper and lower parts of hour - glass shaped samples were stuck to the designed arms of testing machine . then , the samples were subjected to tbs test by universal testing machine ( plus universal testing machine mtd-500 , germany ) at a crosshead speed of 0.5 mm / min in order to create fracture at the interface of composite and dentin . the kolmogorov smirnov test was used to evaluate the normal distribution of the groups . the data were analyzed by one - way analysis of variance ( anova ) , tukey hsd and post - hoc tests . two - way analysis variance was employed to determine the reciprocal effect of tc and mc on the tbs , using spss 17 software program ( spss 17 , spss inc . , illinois , chicago , usa ) at a significant level of 0.05 . in order to determine the location and mode of fracture , all of the samples were examined by using a scanning electron microscope ( sem ) ( ht 416002 , fuji , japan ) . kolmogorov smirnov test confirmed the normal distribution of data in all groups ( p = 0.322 ) . highest mean value in tbs was in g1 ( 29.37 2.89 ) and the lowest was in g8 ( 11.72 2.76 ) ( table 1 ) . pairwise comparison among all groups was done by tukey post - hoc test ( table 2 ) and the differences were significant ( p < 0.05 ) . pairwise comparison ( p values ) of among studied groups . with an increase in the cycles of the mechanical load , either with or without tc , furthermore , two - way anova revealed that both tc and mc decreased tbs and there was not a reciprocal difference between the effects of mc and tc ( p = 0.788 ) . so , the maximum number of fractures was in adhesive ( 70.80% ) and the minimum was in mixed types ( 3.20% ) ( table 3 ) . a reliable and durable bonding of resin materials to dentin is important in the field of adhesive dentistry . an ideal study design to evaluate the quality of new bonding systems is a long - term clinical trial.4 however , some difficulties can not be overlooked such as operator variability , substrate differences,6 matter of time and resources,6 etc . the current study was conducted to simulate clinical situations to evaluate the effects of tc and mc on the tbs of tetric n - bond to dentin . the results determined that the application of tc and mc simultaneously leads to decrease in tbs . that result was in accordance to the studies by mitsui et al.,15 abdalla et al.,14 toledano et al.,3 bedran - de - castro et al.;8 and in contrast with the result of de munck et al.9 and nikaido et al.10 some factors play a determining role in the outcomes of tbs test such as : type of tooth and adhesive resin , the elapsed time of extraction , storage condition , composite resin type , mode of curing , intensity , and magnitude of applied force , number of tc and mc.16 intraoral restorations are consistently exposed to about 1 million ( 1000 k ) mechanical strokes per year of the opposite tooth . these strokes might affect interfacial bonds of restoration and tooth surface , which may result in failure of the restoration . many studies objected tc and mc techniques to provide conditions similar to the oral environment.8 in the present study , mentioned methods used to mimic chewing condition , too . different studies have reported different mc.3,15,17 in the present study , the applied mc were 50 k , 100 k and 500 k , respectively . anderson18 stated that the chewing and swallowing force is between 70 and 150 n. in the present study , the specimens were subjected to 90 n. plastic deformation of the adhesive interface and concentration of main stresses in hybridoid layer interface could be a possible explanation for present results in total - etch adhesive dentin bonding systems . our results confirmed previous studies which indicated that fatigue could decrease resin - dentin bond strength.15,17,19 furthermore , it has been reported that demineralized dentin became weaker after cyclic loading.20 a study on a total - etch adhesive system showed that mc alone did not affect bond strength but when tc and mc were performed , bond strength decreased significantly.8 based on the iso tb 11450 standard , 500 cycles must be executed for tc . however , a review article reported that a tc of 10,000 cycles is similar to approximately 1-year fatigue in the oral environment.9 in the present study , 1000 tc were used . tc properly simulates oral condition in the laboratory.21 this process helps to induce stress to a restoration due to aging , and thermal changes.2 tc accelerates the hydrolysis of unprotected collagen fibers and removing the unpolymerized resin oligomers.1,13,22 higher amounts of thermal expansion and contraction of restorative materials in comparison to dental structures , might result in the formation of an interfacial gap . furthermore , more stresses are induced in the higher ratio of configuration factor.23 the effect of thermocycling on the bonding strength is not clear . one meta - analysis study showed that thermocycling had not a significant effect on the bond strength.24 it was reported that , higher tbs values were presented in the alcoholic base adhesives after load cycling when the dentin was etched.3 existence of alcohol in a composition of adhesive can increase the infiltration in collapsed collagen network and improve tensile bond strength.25 hence , alcoholic base adhesive was used in the current study which created higher tensile bond strength . tbs is a relatively new technique for measuring bond.26 compared to other adhesive bond strength tests , tbs test has several advantages , including the improvement of stress distribution during the test , the prevention of cohesive failures in dentin , the ability to measure regional differences in resin - dentin bond strength , and the ability to measure the higher bond strength of newly developed materials.27 the measured bond strength would be near the actual and higher than tensile bond strength test.16 therefore , surface area of about 1 mm is one of the standard criteria in these tests which was considered in this study . this test depicts more reliable values of bond strength , and it would enable comparing various types of bonding.28 on the other hand , tbs test has its own several difficulties , like required 1 mm thick slices , hour - glass or beam shaped samples , and its highly technique sensitive etc.9 nikaido et al.,10 bedran - de - castro et al.,8,17 mitsui et al.15 and abdalla et al.14 used beam shaped samples in their studies , while toledano et al.3 used hour - glass shaped samples . in the present study , dentinal surface was exposed by diamond fissure burs . in a study by ogata et al.,29 the impact of the type of bur on tbs was evaluated . in that study , samples were prepared by using various burs and final results showed no significant differences between burs , but the bond strength values were differed when different types of adhesive resin were used . in this study , the value of tbs was measured by using a cross head at the speed of 0.5 mm / min . in a study by reis et al.,30 the effect of various cross head speed on tbs were observed , and no significant differences were found among 0.5 , 1 , 2 and 4 mm / min . however , cutting speeds of 0.5 and 1 mm / min have been used in several studies . in the present study , fracture mode adhesive fracture was the most relevant fracture mode in both control and test groups which was in accordance with the bedran - de - castro et al . study15 the most common fracture mode was in a mixed pattern and after increasing the tc and mc , the rate of this failure pattern was increased . variation in classification of fractures might be the reason of different locations or types of fracture in different studies . certain studies have reported that vast numbers of cohesive fracture were detectable by low magnification stereo microscope , but adhesive and a mixed pattern of fracture would be detectable only in high magnification . the high rate of reported cohesive fractures in some studies could be due to misalignment of the samples position , formation of small cracks during slicing that could be mistakenly considered as cohesive fractures.31 variation of results in different in - vitro studies interfere with the generalized conclusion in clinical experiments . this variation might be due to : teeth type , storage environment , control infection condition , presence or absence of tc and/or mc , mechanical properties of the restorative materials , type of test ( shear , micro shear , micro tensile , and tensile ) , speed and magnitude of the cross head , design and shape of the final sample.16 with limitation of the present study , it was shown that tc and mc had an adverse effect on tbs of tetric n - bond adhesive to dentin .

background : different studies have shown the uncertain effects of thermal cycling ( tc ) and mechanical load cycling ( mc ) on the dentin microtensile bond strength ( tbs ) of composites . this study designed to investigate the effects of tc and mc on the dentin tbs of single bond-2.materials and methods : flat dentinal surface was prepared on 48 sound extracted human third molar teeth , and were bonded by single bond-2 adhesive and z250 resin composite . the teeth were randomly divided into eight equal groups , according to the thermal / mechanical protocol . tc and mc were proceeded at 5 - 55c and 90 n with 0.5 hz . then restorations were sectioned to shape the hour - glass form and subjected to tbs testing at a speed of 0.5 mm / min . to evaluate the bonding failure , the specimens were observed under the scanning electron microscope . the results were statistically analyzed with analysis of variance , t - test , tukey hsd and post - hoc by using spss software version 17 at a significant level of 0.05.results:tbs of all groups were significantly lower than the control group ( p < 0.001 ) . adhesive failure was predominant in all groups and increased with tc and mc.conclusions:tc and mc had an adverse effect on tbs of the tested adhesive resin to dentin .

Introduction Materials and Methods Specimen preparation Bonding procedure TC and MC procedure TBS Data analysis Results Discussion Conclusions

PMC3539124

within the bacillus genus , the most pathogenic species is bacillus ( b. ) anthracis , the causative agent of anthrax and a gram - positive spore - forming soil bacterium . this zoonosis and infectious disease affects a wide range of species , including humans and herbivorous mammals . the incidence of anthrax has been reduced by continuous research and disease control , but still occurs in undeveloped and developing countries . in 1905 , the first anthrax case occurred in animal was recorded in korea . during the next 5 years after the first official report in 1907 , the number of anthrax cases occurred in animal increased to 2,562 , causing serious damage to related industries that handle animal by - products like meat and animal skins . more than 1,000 cases were subsequently reported annually , and until 1924 the rate of occurrence was approximately 500 cases per year . the reduction of anthrax cases is due to development of live vaccines using non - virulent b. anthracis sterne and other methods of disease control . however , there were two cases reported in 1994 ( kyungju and hongseong , respectively ) , one in 1995 ( hongseong ) , another two cases in 2000 ( changnyung ) , and one in 2008 ( yeongcheon ) . the disease occurs annually in a seasonal manner , but the distribution of cases is most prominent in may and august . since the annual average rainfall is high in april and july to august , it can be inferred that contaminated soil is more exposed during the rainy season and pollutes pastures , thus transmitting the pathogen to cattle discriminatory markers have long been used to identify and subdivide groups of these microbial pathogens . tracking the epidemic and pandemic spread of particular strains may be possible using such markers , yielding a long - term overview of epidemiological patterns . there are regions in the b. anthracis genome that vary greatly from strain to strain . numerous studies have shown that genetic markers such as single - nucleotide polymorphisms ( snps ) , variable number tandem repeats ( vntrs ) , and single - nucleotide repeats ( snrs ) can be used to discriminate between strains in b. anthracis . canonical snps ( cansnps ) , originally described by keim et al . , are a number of snps located at key phylogenetic junctions and are sufficient to create phylogenetic trees . previously , a set of cansnps was able to distinguish three major lineages of b. anthracis isolates and 12 clonal sub - lineages . in the present study , we examined a genetic population of b. anthracis isolates from korea and compared it to a global genetic population using an eight - loci multiple - locus variable - number tandem repeat analysis ( mlva ) and 13-cansnp analysis . the b. anthracis strains used in this study included four obtained from the korean centers for disease control and prevention ( kcdc ) , five reference strains , and 17 isolates from soil of four different regions in korea ( table 1 ) . atcc14185 and atcc14578 stain obtained from american type culture collection ( atcc ) , ( usa ) , and pasteur , delta sterne , and sterne strain obtained from national veterinary research and quarantine service ( nvrqs ) , ( korea ) . the b. anthracis strains were cultured in brain heart infusion media ( becton dickinson , usa ) and grown at 37 with shaking at 200 rpm for 3 days . total genomic dna was extracted was modified by hunter 's method . to isolate total bacterial dna , bacteria cells were harvested by centrifugation at 15,000 rpm for 10 min at 4 and resuspended in 1 ml te buffer ( ph 7.4 ) containing lysozyme ( 0.1 mg / ml ) , sds and proteinase k at 55 for 3 h. after the completion of this incubation , dna was extracted with phenol - chloroform , precipitated with isopropanol and dissolved in te buffer with rnase ( 20 g / ml ) . in order to determine whether b. anthracis isolates were full virulent ( pxo1 and/or pxo2 ) , we performed a polymerase chain reaction ( pcr ) assay with a primer set specific for b. anthracis . each pcr mixture ( 20 l ) contained 10 ng of genomic dna , 10 pmole of each b. anthracis specific primer , and 2 ef - taq premix ii ( 10 l , solgent , korea ) . the following thermal cycling ( bio - rad , usa ) conditions were used : an initial denaturation step ( 94 for 5 min ) followed by 30 cycles of denaturation at 94 for 1 min , annealing at 40 for 1 min , and primer extension at 72 for 1 min . the pcr products were electrophoretically separated on 1% agarose gel and stained with ethidium bromide . mlva was performed for the eight different vntr loci described by keim et al . . after amplification , the pcr products were diluted 1 : 10 ( volume : volume ) in te buffer and 1 l of the dilution was added to a mixture containing 8 l of hi - ditm formamide ( applied biosystems , usa ) and rox - labeled mapmaker 1000 size standard [ bioventures , usa ; 20 : 1 ( volume : volume ) ] . the dna was denatured at 95 for 5 min and then immediately placed on ice . fragments were detected using an abi 3100 prism genetic analyzer ( applied biosystems , usa ) with a 36-cm capillary at a running voltage of 15 kv at 60. to determine the size of the detected fragments , peak height and peak area was measured using genescan analysis software ( ver . an mlva bank developed by the university of orsay ( university paris xi , france ) was used . for cansnp analysis , we used the primers described by van ert et al . and purchased from genotech ( korea ) in which each primer was added a t7 primer sequence as a 5 ' tail on the primers for sequencing purposes . after amplification , the pcr products were cloned using a t - a cloning vector ( rbc bioscience , taiwan ) . each pcr mixtures contained 2 ef - taq premix ii ( solgent , korea ) , forward and reverse primers ( 0.5 pmol each ) , and genomic dna ( 400 ng/l ) . the thermal cycling conditions were incubation at 95 for 5 min that was followed by 30 cycles of 95 for 1 min , 60 for 1 min , and 72 for 1 min . the plasmids were then purified with hi - yield plasmid mini kit ( rbc bioscience , taiwan ) and sequenced using the t7 primer . mlva and cansnp analysis data obtained for b. anthracis isolates from korea were used to calculate simple matching coefficients using mega ( ver . to investigate the genetic diversity of b. anthracis strains isolated in korea , we used mlva and cansnp analysis to characterize 21 strains , including four that were obtained from the kcdc ( table 1 ) . many researchers reported the pasteur strain to be pxo1 and pxo2 , but the one used in our study was pxo1 and pxo2 since pxo2 was cured during the culturing process . b. anthracis strains obtained from the kcdc were pxo1 and pxo2 while the isolates obtained in korea ( hs , bch , kj , and ch ) contained both pathogenic plasmids ( pxo1 and pxo2 ; table 1 ) . genotype analysis was performed using eight - locus mlva data acquired from an mlva bank . b. anthracis strains obtained from the kcdc were placed into the a3b cluster while the ch isolates were in a b1 cluster ( fig . 1 and table 2 ) . on the other hand , hs , bch , and kj strain were classified as a3a cluster and were not a part of the a3b or b1 clusters . b. anthracis strains obtained from the kcdc ( s0303 , s0304 , s0307 , and s0308 ) had the same mlva type with # 07 strain of ryu 's study . interestingly , all of the local isolates ( ch ) were pathogenic and contained identical sequences at eight hypervariable mlva loci . sequence repeats in eight of the vntr loci ( vrra , vrrb1 , vrrb2 , vrrc1 , vrrc2 , cg3 , pxo1 , and pxo2 ) were identical to those of the other korean b. anthracis strains from changnyung ( # 02 strain of ryu ' study ) . in our analysis , sequences of the b. anthracis strains obtained from the kcdc ( pxo1 , pxo2 ) were identical in the vntr loci ( vrra , vrrb1 , vrrb2 , vrrc1 , vrrc2 , and cg3 ) and varied at one vntr locus ( pxo2 ) with respect to the sterne strain . furthermore , the genotypes of the b. anthracis strains obtained from the kcdc and the sterne strain differed and were varied at three vntr loci ( vrra , vrrb1 , and vrrb2 ) with respect to the atcc14185 and atcc14578 strains ( table 2 ) . the hs strain had the same mlva type as # 50 strain of keim 's study except vrrc1 . the mlva type of the bch strain was identical to # 58 strain of keim 's study except pxo2 . additionally , the mlva type of the kj strain was same as # 32 strain of keim 's study except vrrc1 . cansnps were used to calculate simple matching coefficients and subdivided the b. anthracis isolates according to major sub - lineages ( a and b ) . these were further divided into six of 12 different sub - lineages ( table 3 and fig . 2 , diamond ) or sub - groups ( circle ) . in order to make comparisons with global genetic populations , data obtained for b. anthracis korean isolates from our study along with the results of an eight - loci mlva and 13-cansnp analysis for 1,033 reported b. anthracis isolates from 42 countries were used to calculate simple matching coefficients ( figs . 2 and 3 ) . b. anthracis vntr sequences found in many different chromosomal and plasmid locations exhibit a few or even dozens of different allelic states , showing that this bacterium has a degree of diversity frequently lacking among pathogenic microbes . mlva is currently utilized to identify different sub - populations and particular strains of interest . for b. anthracis , six chromosomal loci and one locus on each of pxo1 and pxo2 , a global b. anthracis ( over 400 strains ) analyzed and subdivided two major clonal lineages ( a and b ) and 86 unique mlva 8 genotype using eight - loci mlva . this eight - loci mlva has also been used to examine the genotypes of b. anthracis in france , poland , italy , and countries in southern and northern africa . in 2007 , van ert et al . , used a 15 marker - loci , mlva15 , to examine a collection of 1,033 b. anthracis isolates from 42 countries . this group described three major lineages ( a , b , and c ) that were further subdivided into 12 clonal sub - lineages or sub - groups , and finally 211 unique mlva15 genotypes . le flche et al . augmented the mlva8 assay by proposing 14 additional markers . additionally , lista et al . expanded the mlva21 assay with the addition of four markers ( 25-loci mlva ) and described two new branches , d and e. the eight - locus mlva was used in our study of vrrb1 , vrrb2 , vrrc1 , vrrc2 , and cg3 identified by sequencing b. anthracis amplified fragment length polymorphism markers . vrra was identified by andersen et al . , and the remaining two loci were identified by analysis of the pxo1 and pxo2 plasmid sequences . vrrc is located in a gene encoding a ftsk - spooiiie dna translocase family homologue . vrra , vrrb , and vrrc markers are found in protein coding regions of the b. anthracis genome . as the mlva marker expands or contracts , this genotyping method had been previously used for consistent subspecies typing worldwide for discrimination of the b. anthracis genome with the highly monomorphic nature . among the isolates analyzed in our study , eight vntr markers had different levels of variability in four loci ( vrra , vrrb1 , vrrb2 , and pxo2 ) and based on the mlva profiles , korean b. anthracis isolates were assigned to both the a and b branches . similar genetic dissimilarity has been found in b. anthracis isolates originating from several geographic regions in africa , europe , north america , and south america . in a previous analysis , b. anthracis isolates originating from asia , turkey , and china were assigned to branch a . it is interesting that our korean isolates belong to both the a and b branches although our data agree with the results of a previous study . variations in the number of repeat sequences at a given locus , or in - sequence heterogeneity among individual isolates , allows discrimination among strains . these may be due to slipped - strand mispairing and can occur in combination with inadequate dna mismatch repair during replication . however , the discriminative power of vntr markers becomes limited when analyzing strains of close geographical origins . in such situations , diversity can be demonstrated by either analyzing a greater number of vntr markers or using more sensitive markers . snps have been used in several studies as important markers that linked sequence variations to phenotypic changes , thus elucidating the molecular basis of diseases . these are also considered extremely valuable to perform phylogenetic analysis as snps have very low mutation rates and are less prone to homoplasy . comparative full - genome sequencing between eight strains of b. anthracis which led to the discovery of about 3,500 snps was reported in the study of read et al . along with 990 snps in 26 diverse isolates . these results demonstrate the usefulness of a few strategically placed snps that may replace a large number of snps for typing b. anthracis strains and generating an snp - based tree . cansnp have also been previously used to successfully differentiate b. anthracis isolates and identify worldwide patterns of distribution in which the major clonal lineage a had been shown to be widespread globally . in the study of van ert et al . , b. anthracis isolates are subdivided into two previously recognized distinct major sub - lineages ( a and b ) according to the cansnp data , and further divided into 6 of 12 different sub - lineages . in this study , data from an eight - locus mlva also clearly classified these strains into two major lineages . the more common genotype among the b. anthracis strains previously analyzed and globally distributed strains is the a genotype . strains obtained from the kcdc ( s0303 , s0304 , s0307 , and s0308 ) and pasteur strain belong to the a.br.ames sub - group , which is found in central and eastern china . later , other isolates were shown to be closely related to the ames strain , indicating the rarity of this strain in nature . despite its scarcity , the ames strain is widely used as a reference strain in many laboratories for genotyping analysis . two similar strains of b. anthracis ( 14578 ) from the kcdc and from the laboratory stock used in our study were found to belong to the a.br.vollum sub - lineage . this sub - lineage genotype is dominant in southern africa and is also present in europe . three strains ( sterne , hs , and delta sterne ) were found to belong to the a.br.001/002 sub - group , and a similar genotype has been identified in central and eastern china . cansnp analysis of one laboratory strain ( 14185 ) showed that it belongs to the a.br.003/004 sub - group in which is common in south america . one of the b. anthracis isolates ( kj ) collected recently from korea belonged to the a.br.005/006 sub - group , which is common in southern africa . the korean b. anthracis isolates from ch analyzed in our study belonged to the b.br.001/002 cansnp sub - group . the major clonal lineages found in asia are usually assigned to lineage a , which is more common than the other lineages and distributed globally . b lineage isolates have been found in southern africa ( b.br.kruger sub - lineage and b.br.001/002 cansnp sub - group genotypes ) , some parts of europe ( b.cneva-9006 sub - lineage ) , and a small region in california , usa . additionally , a major clonal lineage b genotype was identified in korean isolates from ch in this study , and these isolates were closely related to the b1 cluster of mlva reported by ryu et al . . anthrax is rare among humans but very common among grazing animals due to the natural spore transmission cycle . spores that are formed in a place where an animal died from anthrax are the main source of infection for grazing animals . it is also plausible that livestock grazing over dry , dusty , and contaminated soil inhale spores , leading to infection . humans can be infected through contact with diseased animals or animal products depending on the route of spore entry : cutaneous ( the most frequent form of natural infection ) , inhalation , and gastrointestinal . throughout history , anthrax had been widely spread around the world through the trade of infected live animals or meat , hides , hair , wool , or bones from such animals . infected animals and products are often transported in long distances for industrial , food , or handicraft purposes , and the anthrax spores may find their way to livestock in the local community , thus spreading the disease in locations distant from the original infection source . restricting the geographical distribution of b. anthracis is primarily a result of niche specialization associated with adaptive differences among the different lineages . however , genetic evidence indicates that human activities have dramatically influenced the global population structure of b. anthracis . given that natural outbreaks of anthrax are extremely rare , the introduction of new lineages in korea might be the result of agricultural development and increased international trade . results of the present study have added further insight into b. anthracis phylogeny and the distribution of korean isolates .

bacillus ( b. ) anthracis is the pathogen that causes fatal anthrax . strain - specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics . in the present study , we employed molecular approaches including multiple - locus variable - number tandem repeat analysis ( mlva ) and canonical single - nucleotide polymorphism ( cansnp ) analysis to perform molecular typing of b. anthracis strains isolated in korea . according to the mlva , 17 b. anthracis isolates were classified into a3a , a3b , and b1 clusters . the cansnp analyses subdivided the b. anthracis isolates into two of the three previously recognized major lineages ( a and b ) . b. anthracis isolates from korea were found to belong to four cansnp sub - groups ( b.br.001/2 , a.br.005/006 , a.br.001/002 , and a.br.ames ) . the a.br.001/002 and a.br.ames sub - lineages are closely related genotypes frequently found in central asia and most isolates were . on the other hand , b. anthracis ch isolates were analyzed that belonged to the b.br.001/002 sub - group which found in southern africa , europe and california ( usa ) . b.br.001/002 genotype is new lineage of b. anthracis in korea that was not found before . this discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in korea .

Introduction Materials and Methods Results Discussion

PMC4616117

one of the most important topics in the research on human muscles has been the brain s control over maximal voluntary contractions1 . this is related to whether the motoneuron pool has sufficient excitability relative to the force the muscle attempts to generate . this can be examined by analyzing the muscular response pattern upon transmission of a maximal electrical stimulus generated by a muscle undergoing a maximal voluntary contraction2 . a single transmitted electric stimulus is referred to as an interpolated twitch , and the technique is called the twitch interpolation technique . as the intensity of a muscular contraction increases , the amplitude of the twitch decreases ; the excitability of the motoneuron pool can be measured by assessing the degree of amplitude reduction . one application of this method is measurement of the level of muscular inhibition ( mi ) . muscular inhibition refers to the amount of muscle inhibited from carrying out a maximal voluntary contraction ordered by the brain . the inhibition occurs via a process in which a supramaximal electric stimulus is transmitted to the nerve connected to the muscle undergoing a maximal voluntary contraction3 . in general , resisted movement , a training method to enhance muscular functions , does not increase muscle strength by increasing the number of muscle fibers but rather increases muscle strength by expanding them . increased muscle strength during the early stages of training is merely the result of the adaptation of neural factors , not the result of muscle hypertrophy4 . such neuromuscular adaptation is known to be induced by the use of submaximal stress during the initial stages of training . similarly , increased muscle strength after long - term resistance exercise is not solely the result of muscle hypertrophy either , as neuromuscular factors may be involved5 , 6 . walking and running the two most representative aerobic exercises are becoming increasingly popular among people seeking to improve their health . resistance exercise , a type of anaerobic exercise , is attracting many health - seeking individuals as well . as is evident , many people are engaging in physical activities centered on enhancing cardiopulmonary and muscular functions . reaping the benefits from these exercises unfortunately , however , many fail to exploit these workouts to their fullest due to an insufficient number of workout sessions , short - term and intermittent workouts , and early termination . inappropriate and sudden stress , muscular fatigue , and muscular activation may increase the risk of injury by changing several properties of the muscles7 . most of the existing research has simply focused on the changes in outcomes , such as changes in muscular strength or exercise performance after resistance exercise . any form of change in outcome implies a change in the various internal environments within a muscle ; that is , it signifies that there are several neuromechanical changes within the muscle , such as neurological , functional , and mechanical changes , and that the features of these changes are dependent on the exercise load and amount of stimulation . therefore , it is essential to analyze the internal environment of muscles against a variety of external environmental factors and stimulation conditions in order to determine the causes of change . thus , this study employed a neuromechanical approach and used the interpolated twitch technique ( itt ) to conduct a comprehensive and multilateral analysis of the neurological , functional , and mechanical changes in human skeletal muscles related to resistance exercise . the subjects in this study consisted of 11 healthy adult males in their 20s , none of whom had any orthopedic history ( age , 23.4 1.4 years ; height , 178.5 5.2 cm ; weight , 75.6 8.7 kg ) at the time of the study . the subjects received a complete explanation of the objective and contents of this study , as well as the study procedures , subjects rights , and safety issues , after which they voluntarily agreed to participate and signed an informed consent in accordance with the ethical standards of the declaration of helsinki . the subjects performed a standing barbell calf raise exercise to examine the changes that occur in the ankle plantar flexors after resistance exercise . to ensure an equal amount of resistance for all of the subjects , the subjects were directed to perform sufficient warm - up activities and to choose a load equal to their weights . they placed the bar on the back of their shoulders with their legs apart at a distance equal to the shoulder length , placed the metatarsal region of their feet on top of a 5-cm block , and raised their heels at a constant speed . as they raised their heels , the subjects were directed to reach the maximum plantar flexion , utilizing the maximum range of motion ( rom ) of their ankle joint , and when lowering their heels , the heels were to be maintained at a point just above the ground . the subjects performed a total of five sets consisting of 10 repetitions per set . the present study measured two types of muscular contractions : a maximal voluntary isometric contraction and a supramaximal muscular contraction after resistance exercise , as generated by a twitch ( that the human body can not produce voluntarily ) induced by an artificial electrical stimulation . the equipment used to transmit the electrical stimulus was a grass 88 siu-5 electrical stimulator ( grass technologies , natus neurology , warwick , ri , usa ) . the pulse duration ( 0.5 s ) was equally applied to all subjects with an inter - pulse duration of 20 ms . to determine the optimal stimulation point , the ankle was fixed at 10 in the plantar flexion position , and an anode was attached to the femur above the patella , while a cathode was attached to posterior tibial nerve , which shows the maximum response in the posterior popliteal region . the maximum stimulation point was found by means of singlet stimulation , after which doublet stimulation was applied to induce a supramaximal contraction . the electrical stimulus was given two times each in consideration of the potentiated effect , and the information about the torques and angles was saved on a personal computer after computing the voltage value from a dynamometer and running it through an analogue - digital ( a / d ) converter . a bagnoli 8-channel wireless electromyography ( emg ) system ( delsys , boston , ma , usa ) with a sampling rate of 200 hz and band - pass filter of 2045 hz was used to collect electromyographic signals . the electromyography system was used to measure the level of supramaximal contraction via stimulation of the posterior tibial nerve and the levels of agonist and antagonist activation during a maximal voluntary isometric contraction ( mvic ) . the electrodes were placed parallel to the muscle fibers located in the center of the muscle belly of the tibialis anterior , soleus , lateral gastrocnemius , and medial gastrocnemius muscles . before placing the electrodes , the surfaces were shaved and sterilized with alcohol to minimize the skin resistance levels . the electromyographic signals were fed into an a / d converter and later saved on a personal computer . this study used the labview 8.0 ( national instruments , austin , tx , usa ) software to collect measurement data and save it onto a personal computer . torques and angle signals were received from the dynamometer , the signals from the four muscles were received from the electromyography system , and synchronization signals were received from the electrical stimulator . these data were fed into the a / d converter , and the resulting digital data were saved on a personal computer . the voltage value received from the dynamometer was recalibrated and converted to an nm value for analysis , while the remaining signals were extracted as real data using chart 5 for windows ( adinstruments , colorado springs , co , usa ) program . the collected data were statistically analyzed using the spss for windows , version 12.0 , software . the mean ( m ) and standard deviation ( sd ) were calculated , and a paired t - test was conducted to compare the difference in each variable before and after the resistance exercise . the mvic torque was found to have decreased from 122.06 21.05 nm ( before exercise ) to 116.80 19.71 nm ( after exercise ) , but the change was not statistically significant . the resting twitch torque also decreased , from 37.04 10.14 nm ( before exercise ) to 31.46 8.61 nm ( after exercise ) , and the change was statistically significant ( p<0.01 ) . mi appeared to have increased from 5.83 6.16% ( before exercise ) to 7.18 5.05% ( after exercise ) , but the increase was not statistically significant . moreover , muscle activation slightly decreased from 0.144 0.066 v ( before exercise ) to 0.139 0.065 v ( after exercise ) , but there was no statistical significance . the muscle contraction time decreased from 0.13 0.01 msec ( before exercise ) to 0.12 0.01 msec ( after exercise ) , and the change was statistically significant ( p<0.001 ) . similarly , the half relaxation time was also decreased , from 0.09 0.01 msec ( before exercise ) to 0.07 0.01 msec ( after exercise ) , and the difference was statistically significant ( p<0.001 ) ( table 1table 1.changes in muscle properties of the ankle plantar flexor after heel raise exercise ( meansd)variableprepostmvic ( nm)122.06 21.05116.80 19.71rtt ( nm)37.04 10.1431.46 8.61**mi ( % ) 5.83 6.167.18 5.05rms ( v)0.144 0.0660.139 0.065ct ( msec)0.13 0.010.12 0.01***hrt ( msec)0.09 0.010.07 0.01***mvic : maximal voluntary isometric contraction ; rtt : resting twitch torque ; mi : muscle inhibition ; rms : root mean square of muscle activation ; ct : contraction time ; hrt : half relaxation time . * * p<0.01 ; * * * p<0.001 ) . mvic : maximal voluntary isometric contraction ; rtt : resting twitch torque ; mi : muscle inhibition ; rms : root mean square of muscle activation ; ct : contraction time ; hrt : half relaxation time . * * p<0.01 ; * * * p<0.001 the present study sought to conduct a comprehensive and multilateral analysis of acute neurological , functional , and mechanical changes in human skeletal muscles after resistance exercise through a neuromechanical approach . the itt was developed by merton in 19548 as a means of measuring the inactivation of the adductor pollicis muscle . it has been used in several studies that have reported the level of muscular activation ( prominently in the ankle dorsiflexor and plantar flexor9 , the quadriceps femoris muscle10 , and the elbow joint flexor4 ) during a maximal voluntary contraction . furthermore , the itt has been used in analyzing the mechanisms of muscular fatigue and muscle weakness induced by a range of causes . in particular , the itt has been utilized in studies of neural adaptation during a maximal isometric contraction in relation to physical training1 . this study used a dynamometer and electrical stimulator to compare and analyze the mvic torques and twitch torques before and after resistance exercise in an attempt to ascertain the changes in muscular strength induced by resistance exercise . the mvic was interpreted as a voluntary contraction produced by neural control , and the twitch torque was interpreted as a contraction dependent on the mechanical properties of peripheral nerves without any neural control . the resting twitch torque value was calculated by averaging the two twitches induced by the electrical stimuli , and the resting mvic torque value was the value at the point of maximum contraction based on the passive torque11 . the measurements showed that the mvic torque was lower after resisted exercise , but the change was statistically insignificant . these results indicate that there is a greater reduction in muscular strength ( after resistance exercise ) due to the peripheral mechanical systems of muscles than due to neurological factors12 . they also signify that although fatigue has an effect on both factors , it has a greater impact on the mechanical factors13 . future researchers should conduct further studies on long - lasting fatigue and fatigue reduction or recovery factors . muscle activation was measured using an electromyography system and the mi value to analyze neuromuscular properties . the mi value was calculated by measuring the twitching plantar flexor torque induced by a supramaximal electrical stimulus applied to the posterior tibial nerve when the plantar flexor muscles were undergoing a maximal voluntary isometric contraction . the root mean square ( rms ) of muscular activation was computed by running the measured waveform through the emgwork 4.1 software ( delsys , boston , ma , usa ) . the results indicated no significant changes in the mi or rms in relation to resisted training . the statistical insignificance pertaining to muscle inhibition and activation should be examined in association with similar statistically insignificant changes ( caused by neurological factors ) in muscular strength during an mvic14 . neurological factors not only generate muscle strength but also play a role in the acute effects of resistance exercise . in fact , it can be said that peripheral neurological factors sustain the functional decline of mechanical factors induced by twitching stimulations and that they are less influenced by acute effects overall . the muscle contraction time and half relaxation time were analyzed to examine the functional properties of skeletal muscles . the contraction time refers to the period from rest to the onset of maximum torque , and the half relaxation time refers to the time it takes for the maximum torque value to decline by half . generally , muscle contraction times and half relaxation times are thought to be prolonged as muscular fatigue accumulates15 . likewise , the reductions in contraction time and half relaxation time found in this study appear to stem from muscular fatigue induced by the resistance exercise . similar to other factors , the measurement variables induced by twitch stimuli all have a statistically significant impact on the acute effects noted during resistance exercise . in summary , the acute effects of resistance exercise have a temporary impact on the peripheral mechanical system itself , not on the neurological factors , in terms of reducing muscle strength . future studies should more closely examine the roles of neurological factors in intensifying fatigue and several neuromechanical factors in relation to the periods and lengths of recovery exercises .

[ purpose ] the purpose of this study was to perform a quantitative assessment of neuromechanical adaptation in skeletal muscles and to propose the scientific underpinnings of the acute effects induced by resistance exercise . [ subjects ] the subjects in this study were 11 healthy adult men in their 20s who had no orthopedic history at the time of the study . to examine any signs of resistance exercise - induced changes in the ankle plantar flexor , the subjects were directed to perform a standing barbell calf raise routine . [ methods ] subjects were to carry a load equal to their weights and to perform five sets of ten repetitions . the maximal voluntary isometric contraction torque , resting twitch torque , muscle inhibition , root mean square of muscular activation , contraction time , and half relaxation time were analyzed by synchronizing a dynamometer , an electrical stimulator , and an electromyography system . [ results ] the maximal voluntary isometric contraction torque appeared to decline , but the change was not statistically significant . the decline of resting twitch torque , on the other hand , was found to be statistically significant . muscle inhibition and root mean square of muscular activation were both reduced , but both changes were not statistically significant . lastly , contraction time and half relaxation time both statistically decreased significantly after resistance exercise . [ conclusion ] these results indicate that the acute effects of resistance exercise have a greater impact on the peripheral mechanical system itself , rather than on neurological factors , in terms of the generation of muscle force .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4384850

isokinetic testing is a commonly utilized tool for the assessment of muscular strength in orthopedic and sports medicine setting abroad , but in our country or region were poor documented studies about it . in rehabilitation patients after acl reconstruction isokinetic testing isokinetic is frequently chosen because of their inherent patient safety , objectivity and reproducibility in testing measures ( 1 ) . numerous research have documented clinical outcome studies utilizing peak torque parameters , especially the bilateral comparison ration of the quadriceps and hamstring muscles ( 2 , 3 ) . a few investigations have reported outcomes utilizing muscle performance parameters such as work , power and endurance ( 4 ) . aim of this paper is to present role of isokinetic testing in evaluation thigh muscle strength in patients which underwent acl reconstruction and rehabilitation protocol . in prospective study , we evaluated 40 subjects which were divided into two groups , experimental and control . experimental group consisted of 20 recreational males which underwent acl reconstruction with hamstring tendon and rehabilitation protocol 6 month before isokinetic testing . knee muscle testing was performed on a biodex system 4 pro isokinetic dynamo - meter et velocities of 60/s and 180/s . all subjects included in this study had et least two prior testing on the biodex system to familiarize them to the machine and testing sequence . the subjects were tested for knee extension and flexion strength using the following protocol : each subject performed a 6-minute active warm - up on stationary bicycle at a submaximal work level . the subjects were tested in a seated position , hip flexion et 110 to 125 , and stabilization straps were applied to the trunk and thighs . the resistance pad was placed at a level a proximally 3 cm proximal of medial malleolus . the range of motion of the knee joint during testing was set from 0 - 90 and all limbs were gravity compensated . bilateral isokinetic ( concentric / concentric ) knee extension and flexion studies with the protocol of 60/s ( 5 repetitions ) and 180/s ( 10 repetitions ) were accomplished . between two session subjects for 30 seconds . parameters which were followed , average power and h / q ratios , automatically calculated by the device . in statistical analysis was used student s t test statistically there are significant differences in the mean value of peak torque to body weight ( pt / bw ) in patients of experimental group et velocity 60/s ( p<0.01 ) . no significant differences were found in the mean value of pt / bw knee extensor muscle between evaluated groups et velocity 60/sec ( p>0.05 ) ( figure 2 ) . the mean value of pt / bw of knee flexors et velocity 60/s the mean value of pt / bw of knee extensors et velocity 60/s in addition , the endurance of dynamic stabilizers of the knee ( extensor and flexor knee muscles ) was statistically significant better in patients of experimental group versus control ( p<0.01 ) ( figure 3 , 4 . ) the mean value of pt / bw of knee extensors et velocity 180/s the mean value of pt / bw of knee flexors et velocity 180/s observing the mean value of the classic h / q ratio , there was statistically significant differences between evaluated group of patients in favor of experimental group ( p<0.05 ) ( figure 5 ) . our primary finding was that patients after acl reconstruction and underwent rehabilitation did not have muscle disbalance in knee fexors and extensor . a major area of focus in our study was to examine effect of rehabilitation protocol in this patients which we demonstrate by measurement muscle parameters of dynamic stabilizers of knee joint on isokinetic dynamo - meter biodex 4 pro system . during the postoperative acl rehabilitation we recommend hamstring strengthening exercises to reduce anterior tibial translation . there are studies that show adverse results about return strength of hamstring muscles after acl reconstruction ( 5 , 6 ) . some of them demonstrate relatively fast hamstring strength recovery , right after 12 - 14 weeks postoperatively on the preinjury level ( 5 ) . on the other hand , seto et all reported that hamstring strength in the reconstructed limb was significantly less in patients after acl ligamentoplasty even after five years postoperatively ( 6 ) . results of our finding show complete recovery of the hamstrings muscles after 6 month postoperative and agrees to the study show by wilk et all . other than just of the recovery of hamstring muscle strength this all applies to endurance . accelerated rehabilitation protocols after acl reconstruction commonly employs immediate motion , weight bearing , neuromuscular electrostimulation and exercise to avoid inactivity and muscle hypothrophy of the quadriceps . up to 2 years may be needed to regain normal quadriceps muscle performance following acl reconstruction ( 7 ) . even though , our results demonstrate that there are no significant differences in quadriceps strength muscle between acl reconstructed patients and healthy recreational sportsmen . however , we showed that endurance of quadriceps muscle is significantly better in acl reconstructed patients versus patients in control group , so we can conclude the positive outcome of adequate and continued rehabilitation . our results do not agree with findings of hoffman et al , but we must take in account that the study takes another surgical technique ( patellar tendon graft ) ( 8) . the hamstring / quadriceps muscle strength ratio has been used as an indicator of normal balance between the knee flexors and extensors ( 9 ) . it is some of very significant parameter in estimating the time of return to sports activities . our acl reconstructed patients after 6 months of the rehabilitation have the value of h / q ratio in the recommended level which allows them to go back into sports activities . isokinetic testing of dynamic stabilizers of the knee is need in diagnostic and treatment thigh muscle imbalance after acl injury and surgical intervention . it is an objective parameter for decision of return to sports activities after acl reconstruction .

introduction : numerous rehab protocols have been used in rehabilitation after acl reconstruction . isokinetic testing is an objective way to evaluate dynamic stability of the knee joint that estimates the quality of rehabilitation outcome after acl reconstruction . our investigation goal was to show importance of isokinetic testing in evaluation thigh muscle strength in patients which underwent acl reconstruction and rehabilitation protocol.subjects and methods : in prospective study , we evaluated 40 subjects which were divided into two groups . experimental group consisted of 20 recreational males which underwent acl reconstruction with hamstring tendon and rehabilitation protocol 6 months before isokinetic testing . control group ( 20 subjects ) consisted of healthy recreational males . in all subjects knee muscle testing was performed on a biodex system 4 pro isokinetic dynamo - meter et velocities of 60/s and 180/s . we followed average peak torque to body weight ( pt / bw ) and classic h / q ratio . in statistical analysis student s t test was used.results:there were statistically significant differences between groups in all evaluated parameters except of the mean value of pt / bw of the quadriceps et velocity of 60/s ( p>0.05).conclusion : isokinetic testing of dynamic stabilizers of the knee is need in diagnostic and treatment thigh muscle imbalance . we believe that isokinetic testing is an objective parameter for return to sport activities after acl reconstruction .

1. INTRODUCTION 2. OBJECTIVE 3. PATIENTS AND METHODS 4. RESULTS 5. DISCUSSION 6. CONCLUSION

PMC3405770

the contraction of cardiomyocytes has been studied extensively because it is crucial to proper heart function . cross - bridges between actin and myosin lead to force generation and subsequently contraction . one important regulatory protein in the thin filament complex is troponin ( tn ) . tn consists of three subunits : troponin c ( tnc ) , troponin i ( tni ) , and troponin t ( tnt ) . upon binding of the signaling ion , ca , the n - terminal regulatory domain of tnc undergoes a structural reorganization that initiates myofilament contraction . calcium binding to tnc exposes a hydrophobic patch on tnc s surface that promotes its association with the switch region of troponin i ( tni ) . this in turn disturbs the interaction of the tni inhibitory region with tropomyosin and actin , hence relieving tni s inhibition of contractile activity . therefore , ca - binding to the 89 residue n - terminal regulatory domain of tnc presents a crucial step in a chain of events leading to a contractile response . elucidating its structure and dynamics is central to studies of the role of calcium in myofilament contraction . a plethora of structural studies have been carried out to elucidate the structure and function of the tnc regulatory domain of which nuclear magnetic resonance ( nmr ) has been particularly popular . three states have been identified that are considered to be important intermediates in the myofilament contraction process : the free ( apo ) state , the ca - bound state , and the ca - tni - switch - peptide - bound state . additionally , the structures of the tnc regulatory domain in complex with small molecules and mutants of tnc have been elucidated . the regulatory domain , a highly -helical molecule that constitutes the n - terminal half of the troponin c protein , consists of five -helices ( n , a the ef - hands , which are known to be metal - binding sites , are labeled sites i and ii . in contrast with the c - terminal domain of tnc , which contains two high - affinity ca - binding sites , the sites in the regulatory domain are of considerably lower affinity . interestingly , in cardiac tnc , site i is completely defunct for calcium binding that is due to several amino acid substitutions with respect to site i in skeletal tnc . site ii , the low - affinity , ca - specific ca - binding site , is generally considered to be the only site directly involved in calcium regulation of cardiac muscle contraction . ca - binding to site ii of cardiac tnc does not induce an opening transition akin to skeletal tnc but leaves the structure more or less unperturbed in the closed conformation . it is believed that the tni switch peptide has to be present to stabilize the open conformation of the ca - bound regulatory domain of cardiac tnc , suggesting that the open conformation may only be a transient state that is sampled by tnc after ca - binding . in addition to the structural properties of tnc , its calcium exchange kinetics have been the focus of much experimental investigation . the calcium association rate to site ii of isolated cardiac troponin c has been measured using stopped - flow techniques to be very high . similarly , stopped - flow techniquesand nmr spectroscopy were able to determine ca - dissociation rates from site ii much faster than the time scale of muscle relaxation . although a wide variety of experimental techniques have shed light on the structure and function of tnc , atomistic level simulations of tnc structure and dynamics in various states of calcium and tni switch peptide association are necessary for understanding the binding processes involved . as elucidation of the dynamics of the tnc regulatory domain in response to calcium binding is integral for estimating ca affinity , this work may also provide a platform for computationally calculating association and dissociation rates . additionally , relaxed complex scheme type approaches could be used to generate actionable pharmaceutical leads by docking drug - like small molecules into structurally representative snapshots from molecular dynamics trajectories . in general , therapeutic strategies aim to improve calcium affinity in compromised tissue by changing the thermodynamics and kinetics of ca binding and tni association . in this study , we use conventional and accelerated molecular dynamics ( md ) to elucidate the dynamics of tnc with and without ca bound as well as with the tni switch peptide bound . comparison of molecular dynamics trajectories with experimentally obtained order parameters is used as an assessment of how well the conformational landscape conducive to ca binding is sampled . we examine tnc for its ability of binding ca and furthermore determine the molecular contributions to ca binding kinetics . the results of these studies can provide a framework for understanding impaired ca handling in tnc mutants and knowledge gained from this study will guide the improvement of inotropic pharmaceuticals that target tnc . the apo system was modeled based on model 13 from pdb entry 1spy ( 89 residues ) . the ca - bound system was modeled based on model 14 from pdb entry 1ap4 ( 89 residues , ca - ion ) . the ca - tni - bound system was modeled based on model 18 from pdb entry 1mxl ( 106 residues , ca - ion ) . all systems were neutralized adding na counterions ( 1ap4 : 13 na , 1spy : 15 na , 1mxl : 11 na ) and solvated using a tip3p water box . the fully solvated systems contained 24 316 ( 1ap4 ) , 26 756 ( 1spy ) , and 25 994 ( 1mxl ) atoms , respectively . minimization using sander was performed in two stages : 1000 steps of minimization of solvent and ions ( the protein is restrained using a force constant of 500 kcal / mol / ) , followed by a 2500 step minimization of the entire system . a short initial 20 ps md simulation with weak restraints ( 10 kcal / mol / ) on the protein residues was used to heat up the system to a temperature of 300 k. both cmd and amd simulations were performed under the npt ensemble at 300 k for all three tnc systems using amber and the ff99sb force field . periodic boundary conditions were used , along with a nonbonded interaction cutoff of 10 . bonds involving hydrogen atoms were constrained using the shake algorithm , allowing for a time step of 2 fs . for each system , 100 ns md trajectories were generated ( for the ca - bound system md a trajectory of even 150 ns ) as well as 50 ns amd trajectories at four different acceleration levels . acceleration parameters were determined based on average potential and dihedral energies of the equilibrated md simulations . dual boost amd ( both the dihedral energy and the total potential energy are boosted ) was used . the acceleration level is defined in terms of eb and , where eb is the threshold boost energy and is a tuning parameter that determines the shape of the accelerated potential . on the basis of a comparative analysis of previous successful amd studies on a variety of systems of different sizes , the optimal boost energy for the torsional amd is usually found to be the average dihedral angle energy plus 3.5 times the number of residues in the solute , and should be 20% of eb . similarly the acceleration parameters for the acceleration of the total potential energy are dependent on the number of atoms in the entire simulation cell . for the amd simulations , a new in - house version of pmeamd the frames were aligned using all c atoms in the protein and subsequently clustered by rmsd using gromos++ conformational clustering . an rmsd cutoff of 1.6 ( 1ap4 ) , 1.7 ( 1spy ) and 1.8 ( 1mxl ) was chosen , respectively . these cutoffs resulted in 7 ( 1ap4 ) , 9 ( 1spy ) , and 8 ( 1mxl ) clusters that represented at least 90% of the respective trajectories . the central members of each of these clusters were chosen to represent the protein conformations within the cluster and thereby the conformations sampled by the trajectory . the principal component analysis ( pca ) was performed using the bio3d package in r. a blast profile of the 1ap4 sequence revealed 1056 hits . out of the 35 most similar hits , 29 hits were chosen ( excluding structures with mutations and the recently crystallized cadmium coordinating structure ) , and their experimentally determined structures were obtained from the protein data bank . the 29 structures underwent iterative rounds of structural superposition to determine the invariant core of the protein , a region that exhibits the least structural variance between the protein structures . subsequently , the experimental structures were superimposed onto this core , and a pca was employed . in this process , a covariance matrix from the coordinates of the superimposed structures is diagonalized . the eigenvectors of this matrix represent the principal components of the system ( parts of the structure within which there is the most variety among the set of superimposed experimental structures ) , whereas the eigenvalues are a measure of the variance within the distribution along the respective eigenvectors . all experimental structures have been projected into the space spanned by principal components one and two ( along which there is the most variance among the structures ) . the principal component space generated based on the similar experimental structures served as basis for projection of the molecular dynamics trajectories . for the analysis of interhelical angles , interhelical angles on the basis of the md trajectory of the ca - bound system ( 1ap4 ) , chemical shifts for the amide h and the amide n were calculated using the shiftx software . protein structures were extracted from the trajectory every 20 ps . for each of these structures chemical shifts were calculated and averaged . backbone n h order parameters were calculated from the 100 ns molecular dynamics simulation of the apo system ( 1spy ) using the isotropic reorientational eigenmode dynamics ( ired ) approach . order parameters were calculated by averaging 0.5 ns trajectory windows to ensure that the calculated s parameters do not contain any motions whose time scale exceeds the overall tumbling correlation time of the protein . the ptraj program was used to generate a list of eigenvalues and eigenvectors from all of the n h backbone vectors with the ired method . on the basis of these lists , pqr files for representative protein structures determined by a cluster analysis were generated using pdb2pqr . the calcium pqr file was generated using a charge of + 2 and an ionic radius of 1.14 . apbs was used to generate the electrostatic fields for the protein and the calcium ion in opendx format . bd_top was used to generate all necessary input files for the browndye runs . a phantom atom of zero charge and negative radius ( 1.14 ) the phantom atom was placed at the position of the calcium ion from the trajectory frame . it has no influence on the association rate constant calculation and serves solely to be able to define a reaction criterion that is spherically symmetric around the expected binding position of the calcium . the reaction criterion was chosen to be 1.2 within the calcium binding site . we performed 500 000 single trajectory simulations on 8 parallel processors using nam_simulation . the reaction rate constants were calculated using compute_rate_constant from the browndye package . a weighted average of the rate constants of each of the representative cluster centers yielded an estimate of the overall rate constant for the system . pca characterizes collective , high - amplitude structural variations based on a set of homologous protein structures . the predominant modes provide a basis for analyzing large - scale conformational changes anticipated in md simulations . in this study , nmr structures of apo - tnc , ca - bound tnc , ca / tni - bound tnc , and tnc in complex with compounds such as bepridil , w7 , and dfbp were used as inputs for pca . the first two principal components , pc1 and pc2 , are illustrated in figure 1and account for 50.9 and 13.5% , respectively , of the variance associated with known tnc structures . as the two components together account for 64.4% of the variance , it was considered to be appropriate to analyze the simulations just in terms of these components . pc3 accounts for another 10.4% of the variance but was not used for analysis because of its relatively low contribution . the quadrants along pc1 and pc2 describe apo and ca - bound tnc structures ( lower right , e.g. , 1spy , 1ap4 ) and ca - tni - bound tnc structures ( left side , e.g. , 1mxl , 2l1r ) . pca plot comparing cmd and amd trajectory sampling for the ca - bound system . the principal component space generated by known structures of tnc is shown along with molecular dynamics trajectories that are projected into the first versus second principal component space of known tnc structures . the nmr structures of 1ap4 , 1spy , and 1mxl are shown as filled green , red , and blue circles , respectively . ( b ) projection of the 1ap4 cmd trajectory ( full blue circles ) into the pc space is shown in density coloring where darker shades of blue indicate the more heavily sampled parts of the trajectory . ( c ) projection of the 1ap4 amd trajectory ( full blue circles ) into the pc space is shown in density coloring . ( d ) projections of the 1ap4 cmd trajectory ( full red circles ) and the 1ap4 amd trajectory ( full blue circles ) into the pc space are shown in density coloring , where darker shades of red and blue indicate the more heavily sampled parts of the trajectories . complete overlap of most heavily sampled regions for the amd trajectory with the cmd trajectory suggests that cmd samples most of the statistically relevant conformations for this system . pc1 corresponds to a concerted motion of helices b and c opening up the structure and changing the a / b interhelical angle . it appears that the motion connected to pc1 is primarily connected to an opening motion that presents the hydrophobic patch comprising the tnc / tni interface . the motion associated with pc2 whose amplitude is considerably smaller than that of pc1 pulls away helices b and c from helix a. together with the motion represented by pc1 this motion seems to expose the hydrophobic patch even further . motions associated with first two principal components . a principal component analysis of the conventional md trajectory of the calcium bound tnc regulatory domain ( 1ap4 ) was performed . the range of motions associated with pc1 ( a ) and pc2 ( b ) is shown as the overlay of two structures . the biggest difference between pc1 and pc2 is apparent in the movement of helix b. in the motion associated with pc1 , helix b slides with respect to helix a , whereas it is tilted toward helix a in the motion associated with pc2 . pca gives information about both the main internal protein motions during molecular dynamics simulations as well as about the overall structural space sampled by the simulations . for all three troponin c systems ( apo , ca - bound , and ca - tni - bound ) , at least 100 ns of conventional md ( cmd ) as well as 50 ns of accelerated md ( amd ) were performed . prior studies have demonstrated that predominant pca modes are related to low - frequency collective motions ; therefore , we project these data along the pc modes 1 and 2 . figure 1 shows the projection of the 1ap4 cmd and amd trajectories into pc space as well as an overlay of the two . cmd and amd share the same peak density location at about ( 25 , 5 ) . this minimum coincides with projection of the 1ap4 and 1spy crystal structures , suggesting a significant energetic barrier to sampling the tni - bound state . the amd trajectory uncovers several regions of intermediate density , most notably a possible secondary minimum around ( 25 , 0 ) , thus indicating its efficacy in traversing moderate conformational barriers . in the amd simulation , we also note a greater sampling of the pc2 conformational motion toward the ca - tni - bound state at about ( 10 , 5 ) . this suggests that the ca - bound form approaches tni - compatible states in the absence of tni , although there is a risk that the modified potential inherent to amd samples improbable reaction pathways . whereas cmd samples a lesser extent compared with amd , the vast majority of the sampled population is located within the region of pc space sampled by cmd centered at ( 25 , 5 ) . additionally the root - mean - square fluctuations ( rmsfs ) were compared between the cmd and amd simulations . the general trend for both cases is identical in that the most flexible residues are in the n - terminal and c - terminal regions of the protein ( residues 14 , 8789 ) corresponding to flexible loops at the termini . the most flexible region within the protein is the dysfunctional ca binding site i ( residues 2837 ) . in the cmd simulations , rmsf values for residues 5070 ( including the ca binding site ii ) are slightly more flexible ( rmsf 1.5 ) than the background at 1 . while leaving the general trend unchanged , the amd simulations increase the background rmsf level slightly ( to 1.5 ) and show slightly larger rmsf values for residues 5070 , which also include the ca binding site ii . later , we demonstrate that cmd - sampled states capture the dynamics observed in nmr chemical shift and order parameters . therefore , we expect that cmd is sufficient for generating thermodynamically relevant information , whereas amd may be required for assessing transient states leading to binding of tni or other small molecule ligands . pca can be used to compare the different simulated systems ( apo , ca - bound , and ca - tni - bound ) in terms of collective motions bridging the different states . to compare the dynamics of the three systems under investigation , we plotted the projection of the cmd and amd trajectories into the principal component space . in the cmd simulations , the structures and dynamics of the apo ( 1spy ) and ca - bound ( 1ap4 ) systems are very similar , as seen by their virtually overlapping projections into pc space ( figure 3 ) . the amd simulations , however , reveal subtle yet significant differences in the dynamics of the apo and ca - bound forms . the area sampled in pc space is larger for the ca - bound system . the simulations of the ca - tni - bound system ( 1mxl ) predominantly sample a different section of the principal component space generated based on known structures of tnc . these motions are indicative of the open conformation of tnc , facilitated by the binding of tni . ( see figure 3 . ) in particular , residues 3650 are nearly rigid in the tni - free simulation ( rmsf of 1 ) but become highly mobile in the presence of tni with rmsf values ranging between 1.5 and 2.5 . this suggests that the tni - bound structure exists in an energy well that is distinct from the apo and ca - bound forms of tnc . given that these dynamics were not even observed with amd sampling , we speculate that there is a high energy barrier between the apo and ca - bound states on the one side and tni - bound states on the other . pca plot comparing conformational dynamics sampling between the apo , ca - bound and ca - tni - bound system . the principal component space generated by known structures of tnc is shown along with molecular dynamics trajectories that are projected into the first versus second principal component space of known tnc structures . the nmr structures of 1ap4 , 1spy , and 1mxl are shown as filled green , red , and blue circles , respectively . ( b ) projections of the ca - bound tnc for the 1ap4 cmd trajectory ( full blue circles ) , the 1spy cmd trajectory ( full red circles ) , and the 1mxl cmd trajectory ( full green circles ) into the pc space are shown in density coloring , where darker shades of blue , red , and green indicate the more heavily sampled parts of the trajectories . ( c ) projections of the ca - bound tnc for the 1ap4 amd trajectory ( full blue circles ) , the 1spy amd trajectory ( full red circles ) , and the 1mxl amd trajectory ( full green circles ) into the pc space are shown in density coloring , where darker shades of blue , red , and green indicate the more heavily sampled parts of the trajectories . for the conventional molecular dynamics simulations , the principal component space sampled by the apo and ca - bound systems is almost identical indicating similar dynamics . in the amd simulations , however , occasional excursions of the ca - bound system into ca - tni - bound - like states are observed . the ca - tni - bound system , however , samples a different , hardly overlapping region of the pc space , suggesting a change in dynamics upon tni binding . additionally , to investigate more closely the opening transition believed to be crucial to tni binding , the interhelical angle analysis was performed . plotting the a / b angle over the course of the simulations has the potential of identifying transitions between open and closed states as well as possibly making a statement about the time scale of helical motions connecting the states . the concerted motion of helices b and c away from helix a presents the most notable structural change involved in exposing the hydrophobic spot on the tnc surface that facilitates tni binding . therefore , the interhelical a / b angle has been reported as a good indicator of the degree of opening in the tnc protein , with angles around 135 corresponding to a closed conformation and angles around 90100 characterizing the open conformation . this is corroborated by comparing the interhelical angle measurements over the course of the cmd simulations ( figure 5 ) . the interhelical a / b angles fluctuate around an average of 135 for both the apo and ca - bound system and range from 115 to 150. no opening transitions are seen . the amd simulations , however , reveal subtle yet significant differences in the dynamics of the apo and ca - bound forms . the area sampled in pc space is larger for the ca - bound system , corresponding to the apo and the ca - bound structures sampling a wider range of angles . the most open conformation seen for the apo system still has an angle of 105 , which is inconsistent with a full opening transition , yet there are several structures in the ca - bound simulation where the interhelical a / b angle is very close to the 90 of the open conformation . ( see figure 4c . ) this , in agreement with ref ( 2 ) , suggests that binding of calcium subtly changes the dynamics of the system , allowing for opening transitions that are not seen in the apo form . despite the fact that due to the acceleration we may underestimate the duration the system spends in the open state , this corroborates the notion of the open state being a nonstable transient one that has to be stabilized by the presence of the tni switch peptide . these results , to the best of our knowledge , are the first time that an opening transition for cardiac tnc has been observed in a molecular dynamics simulation . it is worth noting that the opening transition is also observed in the pca analysis . as previously noted , pc 1 corresponds to the motion associated with opening . in the projection of the amd trajectory into pc space ( figure 1c ) , there are multiple structures observed around ( 5 , 25 ) , which come very close to the open conformation ( 10 , 5 ) along pc1 , indicating a decreased a / b interhelical angle . these structures are not present in the projection of the cmd trajectory ( figure 1b ) . ( a ) nmr structure of ca - bound tnc ( 1ap4 ) , ( b ) nmr structure of ca - tni - bound tnc ( 1mxl ) , and ( c ) structure of frame 9840 of the 1ap4 amd simulation are shown . for the purpose of clarity , the tni switch peptide has been removed from panel b. for all panels , the helix assignment in the regulatory domain of cardiac tnc is shown . the most n - terminal helix is denoted by n , followed by helices a through d. the interhelical angle between helices a and b is used to measure the degree of openness of the domain . ( a ) ca - bound nmr structure is in the closed state with an a / b interhelical angle of around 135. ( b ) ca - tni - bound nmr structure is in the open state with an a / b interhelical angle of around 90. ( c ) frame 9840 corresponds to the most open conformation sampled during the 1ap4 amd simulation . the molecule adopts the open conformation with an a / b interhelical angle of 93. evolution of a / b interhelical angle over the course of the simulations . the interhelical angle between helices a and b has been calculated for every frame of the tnc simulations . results for the apo cmd simulation ( a ) , the apo amd simulation ( b ) , the ca - bound cmd simulation ( c ) , and the ca - bound amd simulation ( d ) are shown . the most open conformation ( a / b interhelical angle 93 ) observed in the simulations is labeled with a black circle in panel d. furthermore , a cross - correlation analysis was performed to elucidate intramolecular fluctuations unique to each state . the cross - correlation analysis of the trajectories ( figure 6 ) revealed positive correlated motion in the strands of the central -sheet ( c35-s37 , t71-d73 ) linking both ef - hands in the apo simulations and the cmd ca - bound simulation , as previously reported in ref ( 44 ) . this positive correlation is reversed into a negative correlation in the ca - bound amd simulation ( figure 6d ) . whereas this was not observed in ref ( 44 ) , we believe that the relatively short simulation time ( 6 ns ) would not capture longer - time scale motions that could contribute to anticorrelated motions . therefore , we speculate that the disruption of the correlated motion in the -sheet corresponds to helix b pivoting away from helix a as part of an opening event . another region of correlated motion that is consistently present in most simulations is the interface between helices n and d. this suggests the presence of strong hydrophobic contacts between the helices possibly stabilizing the fold . on the basis of the simulation results , it is hard to speculate on the time scale of the opening / closing transition , but it is very likely to be well beyond the 100 ns mark . future work will focus on running longer cmd simulations that reveal several opening / closing transitions and allow for an estimation of the [ open]/[closed ] equilibrium and a free energy difference of the transition . c-residue cross correlation plots for the ( a ) apo cmd , ( b ) apo amd , ( c ) ca - bound cmd , and ( d ) ca - bound amd simulations . green regions mark areas of positive cross correlation while red regions specify areas of negative cross correlation . chemical shifts derived from nmr data describe the local electronic environment about labeled nuclei and serve as a structure determination tool . in this experiment , we predicted chemical shifts from the md trajectories to demonstrate that states analogous to those observed in solution nmr are sampled . to this end , amide h and n chemical shifts were calculated from the cmd trajectory for the ca - bound tnc system , which are compared with chemical shifts obtained by nmr spectroscopy in figure 7 . the calculated shifts show surprisingly good agreement with experimental values , as the determined average root - mean - square deviations between the calculated and experimental chemical shifts are 0.4 ppm for the h shifts and 2.9 ppm for the n shifts , compared with the precision of shiftx of 0.49 ppm for amide h chemical shifts and 2.43 ppm for amide n chemical shifts . chemical shifts were calculated from the amd trajectory as well and are only marginally better with average root - mean - square deviations between the calculated amd and experimental chemical shifts of 0.5 ppm for the h shifts and 2.6 ppm for the n shifts . it is not surprising that no substantial differences between cmd and amd predicted chemical shifts is observed as the simulations identify the same thermodynamically dominant regions on the potential energy surface as demonstrated by pca . additionally , the majority of the slow time - scale dynamics observed in the simulations are concerned with tertiary structure and not changes in the secondary structure . therefore , they may not be picked up by shiftx , an algorithm predominantly based on local sequence and local secondary structure . residue - by - residue comparison of experimentally determined chemical shifts ( blue ) and chemical shifts calculated from the cmd trajectory of the ca - bound tnc system ( red with error bars ) . panel a shows the amide h chemical shifts alongside a ribbon representation of tnc , where the residues for which the predicted h chemical shifts deviate more than two standard deviations from the experimental value are labeled in red . panel b shows the amide n chemical shifts alongside a ribbon representation of tnc where the residues for which the predicted n chemical shifts deviate more than two standard deviations from the experimental value are labeled in red . figure 7 also shows the residues in the protein for which the chemical shifts deviate the most from experiment . chemical shifts of two of the residues whose h and n chemical shifts deviate from experimental values ( 72 and 73 ) have been previously reported to be greatly impacted by calcium binding both experimentally and using quantum mechanical calculations . in general , whereas these sites are distributed throughout the entire molecule , there is an increased occurrence of incorrectly predicted shifts in the calcium binding site ii and neighboring regions . the deviation of the predicted chemical shifts and experimental values can be attributed to a number of possible causes . it has been suggested that md - generated structures typically give inferior shifts compared with those generated from a molecular mechanics conformational search , even when using quantum mechanical estimates of the nuclear magnetic shieldings . therefore , it is possible that these simulations still undersample the conformational states contributing to the nmr observable . more specifically , the deviations close to the calcium binding site could be attributed to shiftx not being well - parametrized for the prediction of metal binding sites in general . another possible source of error is the use of nonpolarizable force fields in the simulations . for highly charged interactions like the ones between calcium and the negatively charged ef - hand residues , ignoring polarization effects might introduce additional error . use of polarizable force fields in future calculations may recover some of the error . backbone order parameters were calculated from the cmd trajectory for the apo tnc system and compared with the experimentally determined order parameters for the apo form of the regulatory domain of human cardiac troponin c. figure 8 shows the overlay of the calculated and experimental order parameters . the regions of greatest flexibility ( lowest order parameters ) are the two calcium binding loops , calcium binding sites i ( inactive ) and ii ( low affinity binding site ) , as well as the n- and c - terminal residues . the overall agreement of the calculated and experimental data is good with an average deviation of less than 0.03 . in agreement with previous experimental studies , the simulations identify positions 26 ( corresponding to residues 6670 ) in both sites i and ii as the most flexible residues of the protein . this is not surprising as these highly charged residues are very labile in the absence of ca . interestingly the most significant deviations between prediction and experiment are residues 4249 ( corresponding to helix b ) , with a general trend of overestimating order parameters ( i.e. , underestimating the dynamics ) . future work will focus on comparing order parameters calculated from longer simulations to experimental values . little overall changes can be observed when comparing the calculated order parameters for the ca - bound simulation with the apo simulation ( figure8b ) . in agreement with observations made in ref ( 48 ) , the order parameters in the calcium binding site ii decrease , consistent with decreased flexibility due to calcium binding . h order parameters were calculated for the apo tnc system and are compared with the experimentally determined nmr order parameters . calculated order parameters for all residues are shown in red , whereas the experimentally determined parameters at 25 c are shown in green . agreement of calculated and experimental order parameters is particularly striking in the n - terminal part of the protein including the inactive ( around residue 30 ) calcium binding site . ( b ) comparison of calculated order parameters for the apo ( blue ) and ca - bound ( purple ) tnc system . order parameters generally agree between the systems with the exception of the calcium binding site ii . the agreement of calculated and experimentally observed chemical shifts and order parameters indicates that the molecular dynamics simulations did cover the relevant dynamics of the isolated protein domain . therefore , these results provide an important measure of confidence that the dynamics seen in the calculations capture the predominant dynamics of the protein and that other quantities calculated based on the trajectories most likely represent thermodynamically relevant measures as well . brownian dynamics simulations have been used to estimate the association rate constant for calcium binding to troponin c. for this , the cmd trajectory for the ca - bound tnc system was clustered by rmsd , and seven representative structures ( cluster centers ) that represented more than 90% of the structures seen in the trajectory were extracted . we performed 500 000 single trajectory brownian dynamics simulations for each of these representative structures . the average calcium association rate constant was determined to be with 3.1 10 1/(ms ) . diffusion - limited association rates have been reported in the literature , for instance , ( 2 to 4 ) 10 1/(ms ) , 4 10 1/(ms ) , and 1.4 10 1/(ms ) . the calculations based on brownian dynamics and the cmd trajectories reproduce the experimentally observed rates well . to investigate whether there was any variation of the association rate over the course of the simulation , calcium association rates were calculated for a structure extracted every 1.5 ns from the trajectory . no appreciable variation in association rate between different structural states is observed , leading us to speculate that the binding site always assumes on open conformation . furthermore , in agreement with trends reported by ref ( 18 ) , we observe that ln(kon ) decays linearly with the root of ionic strength ( sqrt(i ) ) ( figure 9 ) . these results underscore that brownian dynamics simulations are an important and viable tool to investigate the calcium association in troponin c. dependence of ionic strength on calcium association rate . the square root of ionic strength is plotted versus the natural logarithm of calcium association rate . a linear dependence can be observed .

troponin c ( tnc ) is an important regulatory molecule in cardiomyocytes . calcium binding to site ii in tnc initiates a series of molecular events that result in muscle contraction . the most direct change upon ca2 + binding is an opening motion of the molecule that exposes a hydrophobic patch on the surface allowing for troponin i to bind . molecular dynamics simulations were used to elucidate the dynamics of this crucial protein in three different states : apo , ca2 + -bound , and ca2 + -tni - bound . dynamics between the states are compared , and the ca2 + -bound system is investigated for opening motions . on the basis of the simulations , nmr chemical shifts and order parameters are calculated and compared with experimental observables . agreement indicates that the simulations sample the relevant dynamics of the system . brownian dynamics simulations are used to investigate the calcium association of tnc . we find that calcium binding gives rise to correlative motions involving the ef hand and collective motions conducive of formation of the tni - binding interface . we furthermore indicate the essential role of electrostatic steering in facilitating diffusion - limited binding of ca2 + .

Introduction Material and Methods Results & Discussion

PMC3893972

at a time when there is a call for a complete and radical overhaul of the healthcare system of the country , scant attention is being paid toward the ground workers of the biggest medical colleges and hospitals . due to our crumbling healthcare system high cost of treatment in private facilities leads to an abnormally high patient load in few performing government facilities . also , for fear of mortalities and associated ill - reputation , patients in terminal stages of illnesses are often referred to the public facilities by private practitioners . however , the junior members of the medical team are not in a position to express their reservations or opinions to the senior consultants who make major patient admission- and treatment - related decisions . the authors have personally seen orders being relayed down from those in the highest ladder to their juniors . ultimately , the task is completed by the junior - most resident or even the intern . the staffing pattern is also unique ; while the senior doctors , nurses , and paramedical staff including the ward boys are permanent government employees , the medical resident is a temporary contract worker who has an annual contract subject to renewal every year . the majority of residents are academic residents who have a sword of damocles hanging on their shoulder . this sword is the fear of displeasing anyone in the old scheme of things and thus almost guaranteeing their failure in examinations . also , there is an acute shortage of postgraduate medical seats in relation to undergraduate seats in the country . in developed countries like usa , the postgraduate seats are more than the undergraduate seats . for e.g. , in 2010 , residency match 16,070 us medical school aspirants vied for 22,809 first year residency positions . seat of the course if they are not in a position to carry out their duties responsibly . often , admissions to the said courses are expensive and/or coupled with contracts and bonds with penalty clauses and fines in tens of lakhs of rupees which are impossible to pay back . this high workload leads to poor performance and tendency to make mistakes which can be and are fatal in medical environment . there are countless cases of medical negligence being regularly reported and documented which can be traced to overwork . while often there is a lack of support from senior collogues and faculty , who justify excessive work as tradition of good training process . interestingly , residents take the major burden , while senior faculty members get leisure of flourishing private practice overtly or covertly allowed in many states . medical professionals are supposed to be aware of the stresses and is expected that they take care of these issues . however , what is sad and surprising is that such a large cause of mental stress sometimes even leading to suicides is ever present and criminally ignored by the medical administrators . a typical work week of an anesthesia resident includes three duties and three regular days of work . a regular day of work includes ot duty , icu / ccu / nicu / picu duty , teaching seminars , thesis work , and running opd services . while a duty day means 24 straight hours of emergency / casualty posting and ot service and responding to cpr calls . it is hard to conceive a human being function at his optimum on that particular day and afterwards without any sleep whatsoever . and , this sleep - deprived duty roster continues to operate for a full 3 years , i.e. , the duration of residency . a patient admitted in a hospital is under great of stress and uncertainty himself . he / she is usually in great pain and is also afraid of his life ; the hospital is a strange and unwelcome place full of chaos and misery . this rude behavior toward patients is also a contributing cause of regular cases of violence toward the doctors by the patient attendants frequently reported in media . some practical implications of overwork also include unethical practices like lama ( leave against medical advice ) . to reduce work load , some residents unofficially at times instigate the patient to go somewhere else for treatment . the residents express doubt at the ability of consultants and cite examples of previous treatment failures and mortalities to scare away the patient who then leaves the hospital against official medical advice . motivation of the residents also takes a beating and they are not interested in trying out new and experimental treatments which extend the borders of medical science . thus , academic activities which include novel treatments are put on a backburner in busy government hospitals in order to continually clear the patient load . thesis work often becomes a formality which has to be completed somehow in the limited time . there has been a long - standing debate in the country for classifying the pay of medical residents as salaries instead of stipends . usually , the pay is irregular and minuscule in comparison to his engineering or management counterpart and his age . and , added to that is the long duty hours and responsibility . there are ethical and legal barriers which have been ignored in letting this practice continue . the international labor organization recommendations prohibit more than 48 hours of work each week since 1962 . similarly , the eu rules allow for only a 48-hour work week for a resident . this was followed by major changes in national health service ( nhs ) , uk . even the factories act in the country provisions for payment of overtime and holidays for work more than 48 hours a week . there needs to be a recognition and awareness of the contribution of medical residents toward the cause of healthcare in the country . in view of our economy getting bigger , healthcare also needs a bigger contribution both in terms of the size of the pie but also as a percentage . a large scale addition in the number of postgraduate residency seats coupled with clearing of the bottlenecks in the medical education system is the need of the hour . the surplus of medical graduates and shortage of postgraduates with plenty of hospitals available for training is a managerial problem . solutions like national board of examinations and faculty of family medicine in the country need to be mainstreamed . there needs to be flexibility in duty rosters being prepared with the option of post duty offs . undergraduates are losing on clinical skills due to extreme focus on postgraduate entrance examinations for which they start preparing from the first year itself . this leads to doctors who have not even conducted a single delivery in their whole career , if these doctors are unable to get a specialization ( which is true for many ) , they would be ill prepared to manage the primary health centers alone . contribution in clinical care by undergraduates will lead to them acquiring better training and easing on the residents work burden .

this paper argues that medical residents who do most of the hard work in big hospitals and medical colleges are overworked . a hierarchical organizational structure , staffing patterns , and fear of failure in examinations leads to overwork among residents going unreported . this can lead to poor academic performance and research work . gaps in communication have serious implications on patient health . undesirable practices like lama ( leave against medical advice ) also result from overwork . issues of pay and contracts including mandatory service need to be looked into carefully . national and international recommendations on work hours have consistently been ignored . the solutions suggested are simple and easy to implement .

Introduction

PMC4291515

slavery ( dulosis ) is the most spectacular form of social parasitism in ants and one of the most unusual interspecific relationships in the animal world ( e.g. wheeler , 1910 ; forel , 1928 ; buschinger , 1990 , 2009 ; hlldobler and wilson , 1990 ; dettorre and heinze , 2001 ) . slave - makers rob brood from colonies of their host species and transport them to their own nests . as a consequence , adult workers of the host species , the so - called slaves , emerge in the enslaver s nest and subsequently recognise it due to an early olfactory learning process ( le moli , 1980 ; le moli and mori , 1985 , 1987a , b ; blatrix and sermage , 2005)as their own and act for the benefit of the social parasite s colony ( e.g. wheeler , 1910 ; dobrzaski , 1965 ; wilson , 1975a ; buschinger et al . , 1980 ; hlldobler and wilson , 1990 ; dettorre and heinze , 2001 ; mori et al . , 2001 ; buschinger , 2009 ) . . colonies of facultative slave - makers , such as some species of the subgenus raptiformica , may function successfully without slaves ( e.g. dlusskij , 1967 ; marikovskij , 1967 ; savolainen and deslippe , 1996a ; deslippe , 2010 ) , but slaves further contribute to their ecological and evolutionary success by increasing their workforce ( savolainen and deslippe , 1996b ) . as demonstrated for the facultative slave - maker formica subnuda ( a nearctic blood - red ant ) and its slaves f. podzolica , additional energy contributed by slaves may be allocated to increase production of the enslaver sexual brood ( savolainen and deslippe , 1996b ) . in the case of obligate slavery , presence of slaves is an essential condition for the survival of the colony , as abilities of enslaver workers to carry out everyday tasks are usually highly restricted , among others due to morphological and functional limitations ( e.g. beck , 1961 ; kutter , 1969 ; wilson , 1975b ; dobrzaska , 1978b ; buschinger et al . , 1980 ; mori and le moli , 1988 ; buschinger , 2009 ; szczuka et al . , moreover , workers of obligate social parasites often occur in limited numbers , whereas their slaves are relatively numerous ( wheeler , 1910 ; talbot , 1967 ; wilson , 1971 ; hlldobler and wilson , 1990 ; savolainen and deslippe , 1996a ; seifert , 2007 ; buschinger , 2009 ) . slavery is not a frequent phenomenon among ants . as estimated by dettorre and heinze ( 2001 ) , there is not much more than 60 species of slave - making ants , i.e. as few as ca 0.5 % of all known extant ant species , and about one - fourth of all ant social parasites . slavery has been discovered in six genera of myrmicinae ( chalepoxenus , harpagoxenus , protomognathus , temnothorax , myrmoxenus , strongylognathus ) and three genera of formicinae ( formica , polyergus , rossomyrmex ) . whereas myrmicine slave - makers use as their hosts ant species of other myrmicine genera ( leptothorax , temnothorax , tetramorium ; e.g. buschinger et al . , 1980 ; hlldobler and wilson , 1990 ) , formicine slave - makers use some species of the genus formica ( in formica and polyergus ; e.g. buschinger et al . , 1980 ; hlldobler and wilson , 1990 ) and proformica ( in rossomyrmex ; zamora - muoz et al . , 2003 ; ruano et al . , 2013 ) . until recently , it was generally believed that individuals of a host species once enslaved serve the colony of their social parasite unconditionally , and are wholly lost for their own colony and population . it was even theoretically argued why so - called rebellions or revolts of enslaved individuals are impossible in evolutionary terms : slaves were supposed to be entirely unable to free themselves and reproduce ( gladstone , 1981 ) . this belief was additionally strengthened by frequently observed cases of direct collaboration of slaves with their enslavers . in particular , slaves were repeatedly observed to fight actively with the ants attacked by their enslavers , including their conspecifics , and possibly even their former nestmates , and to participate in capture and transport of robbed brood . such behaviour was described e.g. in temnothorax curvispinosus slaves of t. duloticus ( wesson , 1937 ; wilson , 1975b ) , formica fusca and f. cinerea slaves of formica sanguinea ( dobrzaski , 1961 ; czechowski , 1977 , 1998 ) , leptothorax acervorum slaves of harpagoxenus sublaevis ( buschinger and winter , 1977 ; winter , 1979a ) , temnothorax unifasciatus slaves of myrmoxenus ravouxi ( winter , 1979b ) , temnothorax ambiguus and t. longispinosus slaves of protomognathus americanus ( alloway , 1979 ; alloway and del rio pesado , 1983 ) , and tetramorium spp . slaves of dulotic strongylognathus spp . ( sanetra and buschinger , 1996 ) . in colonies of strongylognathus testaceus , the so - called degenerate slave - maker , with a vanishing ability to raiding tetramorium c.f . caespitum , host workers themselves and not workers of the social parasite were observed to attack their free conspecifics and rob pupae ( forel , 1874 ) . similar phenomena were observed in colonies of protomognathus americanus with temnothorax longispinosus slaves ( alloway and del rio pesado , 1983 ) . dobrzaska ( 1978a ) reported numerous observations of well - organised cooperation of slaves of polyergus rufescens ( a euro - siberian amazon ant ) with their enslavers during the raids . formica cinerea slaves were frequently seen to pick up pupae abandoned by p. rufescens on the nest . they also responded to p. rufescens returning from a raid by rushing toward them , snatching from them pupae , and retrieving the pupae to the nest . formica fusca slaves were also observed to pick up pupae dropped by p. rufescens close to the nest entrance , but less frequently than f. cinerea . they collaborated with p. rufescens mostly by uncovering and enlarging nest openings to facilitate the mass entry of amazon ants returning from the raid . picking up of booty abandoned by p. rufescens at the nest entrance mini raids , following raiding columns of the enslaver and picking up host pupae abandoned around the attacked nest , overlooked by the raiders or lost on the way ( czechowski , 2007 ) . interestingly , f. fusca workers enslaved by p. rufescens lose their characteristic mildness , and become aggressive to a degree unusual for that species . they were , for instance , observed to fight fiercely on the side of p. rufescens with f. sanguinea which attacked their mixed colony ( dobrzaska and dobrzaski , 1960 ) . workers of wood ants ( subgenus formica s. str . ) enslaved by f. sanguinea also readily participated in competitive ( territorial ) actions against neighbouring conspecific colonies , including direct fight with their free conspecifics at f. sanguinea s side ( czechowski , 1989 ) . the strength of integration of slaves with the colony of their enslaver is well illustrated also by the observations of the so - called nestmate rescue behaviour , i.e. coming to the rescue of a nestmate facing a dangerous situation . 2002 ) described that behaviour in workers of formica fusca enslaved by f. sanguinea coming at the rescue of their f. sanguinea nestmates captured by ant lion larvae . interestingly , f. fusca did not rescue their homospecific nestmates , and were themselves not rescued by f. sanguinea . workers from monospecific colonies of f. fusca also did not come to the rescue of nestmates trapped by ant lion larvae . loyalty of slaves to their enslavers was so well established that the title of a recent work reporting a finding of the contrary phenomenon starts with a phrase however , extensive and multifold evidence concerning various forms of the acts of opposition of slaves to slave - makers can be found even in very early myrmecological literature , not to mention numerous facts reported in more recent papers . in this review , all phenomena which may fall within the term slave rebellions ( or revolts ) have been classified into four main categories : ( 1 ) domestic misunderstandings : episodes of physical aggression of slaves directed to slave - maker adults , ( 2 ) slave reproduction : attempts of slaves to reproduce within a slave - maker colony , ( 3 ) sabotage : activities of slaves leading to weakening of a slave - maker colony and/or population , and ( 4 ) slave emancipation : partial or complete self - liberation of slaves from a slave - maker colony . agonistic interactions involving more or less escalated overt aggression between slave - making ants and their slaves were observed ( all under laboratory conditions ) in mixed colonies of , chronologically , temnothorax duloticus ( wilson , 1975b ) , protomognathus americanus ( alloway and del rio pesado , 1983 ) and harpagoxenus sublaevis ( heinze et al . , 1994 ) , all obligate myrmicine slave - makers , and their slaves of the genera temnothorax ( the first two of the mentioned slave - makers ) and leptothorax ( the last one ) . domestic misunderstandings are especially common in mixed colonies of the palaearctic slave - maker harpagoxenus sublaevis . in this species , intra - colony relations are characterised by frequent skirmishes among slave - maker workers themselves , between slave - makers and slaves , and among slaves of both the same and different species ( when enslaved workers of two host species , leptothorax acervorum and l. muscorum , occur in the same nest ) ( heinze et al . , 1994 ) . out of these two slave species , l. muscorum appeared to be more aggressive towards h. sublaevis in colonies in which these slaves considerably outnumbered l. acervorum . in such colonies , in other colonies , acts of aggression of slaves against slave - maker workers were sporadic and usually happened after a slave had been attacked by an enslaver . however , sometimes they were observed to catch hold of and/or pull a leg or an antenna of the opponent . there is no information on possible victims and/or body injuries of participants of these intra - colony misunderstandings between allospecific nestmates , so it seems that these agonistic interactions were relatively unescalated and did not lead to serious injuries . in contrast to harpagoxenus sublaevis , in colonies of the nearctic slave - maker protomognathusamericanus , enslavers were not observed to attack slaves , but slaves ( temnothorax ambiguus and t. longispinosus ) were frequently observed to bite p. americanus workers and to drag them out of the nest . these attacks were relatively escalated : as a result , some enslavers lost parts of their appendages . however , such hostile encounters always occurred as single events and never took on a form of a mass slave revolt . interestingly , p. americanus workers attacked by a specific slave were cared for ( fed and groomed ) by other slaves , and a slave attacking one individual treated other p. americanus workers in a friendly way ( alloway and del rio pesado , 1983 ) . intra - colony duels between slave - maker workers , between slaves and between slaves and slave - makers were also observed in mixed colonies of temnothorax duloticus , a nearctic slave - maker which , similarly as protomognathus americanus , uses ants of the genus temnothorax ( e.g. t. curvispinosus ) as its slaves . some of these incidents were violent and long - lasting , but never ended fatally . they were always initiated by slaves , and attacked slave - maker workers were never seen to fight back ( wilson , 1975b ) . workers of t. curvispinosus were even observed to attack queens of t. duloticus ( see slave reproduction ) . internal conflicts within mixed colonies , due to their frequency and a high level of aggressiveness of slaves , may escalate into such cases were observed in some nearctic polyergus species and their slaves of the formicapallidefulva species group ( king and trager , 2007 ) ( see sabotage ) . worker reproduction is common in eusocial hymenoptera ( see e.g. bourke , 1988a ; wenseleers et al . , 2004 ) including ants ( e.g. peeters , 1991 ; helanter and sundstrm , 2007 ) , and among them dulotic species ( e.g. bourke et al . , 1988 ; herbers and stuart , 1998 ; foitzik and herbers , 2001 ; blatrix and herbers , 2004 ; brunner et al . , worker egg - laying is even especially intensive in slave - makers in comparison with non - parasitic ant species , as dulotic ants are more strongly selected to reproduce ( see e.g. hung , 1973 ; wilson , 1975b ; winter and buschinger , 1983 ; heinze , 1996a , b ) . for example , in colonies of polyergus rufescens , up to 100 % of males are produced by workers ( brunner et al . , 2005 ) . similarly , in protomognathus americanus , also an obligate slave - maker , worker reproduction accounts for more than 70 % of all males ( foitzik and herbers , 2001 ) . generally , in ants , worker oviposition starts or increases when the colony is orphaned ( becomes queenless ) which suggests self - restraint or self - policing of workers in queenright conditions and/or some control from the queen(s ) ( e.g. mamsch , 1967 ; bourke , 1988b ; czechowski , 1996 ; helanter and sundstrm , 2007 ) . enslaved workers also may lay eggs in nests of their enslavers in which they are deprived from presence of homospecific queens . however , czechowski s ( unpubl . data ) intense search of formica fusca males as expected sons of the slaves in colonies of f. sanguinea living under natural conditions ended in failure . males of f. fusca were not found even in a f. sanguinea colony with an unusually high proportion ( 58 % ) of slaves . the vast majority of f. fusca workers was found in the deepest parts of the nest ( czechowski , 1996 ) which suggests that in that colony slaves consisted predominantly of young individuals , predisposed to egg - laying . ovaries of young ant workers are known to be relatively well developed and to regress as a function of individual age and behavioural specialisation ( ovaries of extranidal workers are as a rule regressed ) ( e.g. otto , 1958 ; hohorst , 1972 ; billen , 1982 ; fresneau , 1984 ; minkenberg and petit , 1985 ; tsuji , 1988 ; fnron et al . , the colony under discussion was excavated in july 1992 , transferred to laboratory and split into two parts , a queenright and a queenless one . the queenright part was initially composed of about 300 ( 37.5 % ) workers of f. sanguinea and 500 ( 62.5 % ) workers of f. fusca ; the queenless one counted about 700 ( 64 % ) workers of f. sanguinea and 400 ( 36 % ) workers of f. fusca . during the same and the next season ( 1993 ) , the queenright part was subjected to a series of adoption experiments ( see czechowski , 1996 ) , whereas the queenless one was left alone ( except an unsuccessful attempt to introduce f. fusca queens in may 1993 ; see also slave males never appeared in the colony part with a f. sanguinea queen , while in the queenless part about 100 male larvae and pupae appeared in may 1993 . then , within ca 2 months , 53 f. fusca adult males emerged there . it is puzzling that no f. sanguinea males were observed in both cultures ( czechowski , 1996 ) . data ) who transferred to the laboratory a large queenless fragment of a natural mixed colony of f. sanguinea and f. fusca . this time , too , no f. sanguinea males were produced , but after several months the mixed culture contained numerous f. fusca large males ( macraners ) . all these findings suggest that at least in f. ( raptiformica ) sanguinea , a palaearctic facultative slave - maker , the production of males by f. fusca slaves is efficiently inhibited by the presence of the dulotic queen and starts when the queen is lost . hung ( 1973 ) obtained , however , opposite results for the nearctic facultative slave - maker formica ( raptiformica ) pergandei and the obligate slave - maker polyergus breviceps , and their slave species formica canadensis ( the fusca group ) . he created mixed queenless cultures composed of slave - maker workers and their slaves out of individuals taken from natural mixed colonies collected in the field . production of slave males was fully inhibited in presence of enslaver workers even in absence of a slave - maker queen in both f. pergandei and p. breviceps . in such mixed cultures , in contrast , males of f. canadensis were produced in control colonies composed only of workers of that species . similarly , workers of f. japonica enslaved by the japanese amazon ant polyergus samurai produced their own males only in absence of the slave - maker workers ( tsuneoka , 2006 ) . an interesting situation was observed in a colony of polyergus rufescens with formica cinerea slaves found in the kampinos forest ( central poland ) . one nest of the polydomous nest system of f. cinerea was occupied by p. rufescens . nuptial flights of f. cinerea males ( and only males ) were observed to depart from all nest holes of that nest system with the exception of that single nest ( or , more precisely , a group of nest holes ) ( czechowski , 1975 ) . if in that peculiar mixed colony queen(s ) of f. cinerea were absent , these males must have been the sons of p. rufescens slaves . if so , this observation would imply that f. cinerea workers may reproduce only if they are physically separated from a p. rufescens queen . however , theoretically , according to the principle of oligogyny , a queen or queens of f. cinerea might have been present at a sufficient distance from the slave - maker queen and it can not be excluded that the observed males were her sons ( see slave emancipation ) . yet another situation was observed under laboratory conditions in colonies of the obligate slave - maker harpagoxenus sublaevis . slaves of this ant species , leptothorax acervorum and l. muscorum , reproduced even in queenright colonies of the enslaver . the majority of aggressive interactions between slave - maker workers and slaves within mixed colonies of these species ( see domestic misunderstandings ) had for their background the competition for the production of males . the enslaver workers attempted to prevent the slaves from egg - laying by attacking and dominating them . anyway , eggs laid by the slaves were usually destroyed by the enslaver or policed by other slaves ( heinze et al . , 1994 ) . slave egg - laying was also reported , under laboratory conditions as well , in colonies of temnothorax duloticus . slaves of that species , t. curvispinosus , laid eggs directly on an egg pile guarded by the slave - maker queen . before egg - laying , they frequently approached the queen and engaged in biting of her head or thorax . once , a slave was observed to lay an egg at some distance from the queen , and then to insert it peacefully into the egg pile ( wilson , 1975b ) . several laboratory studies reported that slaves may kill a large part of their enslaver s brood . this phenomenon was described for the first time by alloway and del rio pesado ( 1983 ) in an incipient colony of protomognathus americanus transferred to the laboratory from the field . the colony consisted of a slave - maker queen , her brood and some workers of temnothorax longispinosus , remnants of an originally free colony of that slave species , taken over by a p. americanus queen . due to that , despite the fact that the authors used the term slaves to denote these workers of t. longispinosis , a more general term hosts seems to be more appropriate here . these host workers of t. longispinosus cared for slave - maker brood throughout the pupal instar , but then they killed all eclosing p. americanus workers . apart from this own single observation , alloway and del rio pesado ( 1983 ) similar events in other incipient h. [ harpagoxenus = protomognathus ] americanus colonies have been observed ( but not published ) by r.j . more recently , achenbach and foitzik ( 2009 ) reported the same phenomenon ( also under laboratory conditions ) in temnothorax curvispinosus , t. longispinosus and t. ambiguus enslaved by protomognathus americanus ( see also pamminger et al . , 2013 ) . slaves of these species systematically eliminated up to two - thirds of female brood of their enslaver . worker and gyne pupae of p. americanus were either directly killed , or removed from the nest and abandoned . it should be stressed that these events took place in mature ( not incipient ) p. americanus colonies , in which temnothorax workers were real slaves , and not remnants of originally free host species colonies taken over by slave - maker queens . the authors interpreted their findings in terms of an anti - parasite strategy acting as a second line of defence of the slave species against the slave - maker which may be adopted if direct defence of the slave colony ended in failure . thanks to that strategy , slaves indirectly benefit local populations of their own species by decreasing enslaver s workforce and colony fitness . slower growth of slave - maker colonies reduces their raiding activity and , at the same time , their impact on neighbouring host colonies . the question of proximate causal factors underlying this form of sabotage ( killing of slave - maker s brood ) also needs further research . in relation to slave rebellions within laboratory mixed colonies of protomognathus americanus and their temnothorax slaves , achenbach et al . ( 2010 ) sought their basis in differential cuticular hydrocarbon profiles of the parasite and slave pupae . however , johnson et al . ( 2005 ) showed that workers of two host species of the obligate slave - maker polyergus breviceps , formica gnava and f. occulta , are able to reject eggs of p. breviceps in spite of the fact that in terms of hydrocarbon profiles parasite eggs may be locally adapted to their host species . it should also be pointed out that killing of slave - maker brood by slave workers was so far observed only under laboratory colonies and it remains still to be confirmed that it takes place also in the field ( see discussion / open questions ) . it is known that artificial laboratory conditions can disturb normal functioning of an ant colony , including slave / slave - maker relationship . james c. trager stressed the importance of that fact in his online statement : in my seemingly life - long study of polyergus , i ve observed colonies to persist for years in the field . but when brought into captive conditions , the formica host workers almost always rebel , fail to raise the parasite s brood , and begin to harass and kill off the polyergus workers this comment is most probably based on published cases of direct aggression between p. lucidus ( sensu lato ) and their formica slaves ( of the f. pallidefulva group ) observed under laboratory conditions by king and trager ( 2007 ) . slave aggressiveness escalated after several months of laboratory rearing to the point that on two occasions the queen was killed as a result of several days harassment . if to take into account the subsequent revision of the genus polyergus trager s ( 2013b ) , revision of the genus polyergus in one case it was a p. longicornis queen ( killed by f. dolosa workers ) , in the second p . oligergus queen ( killed by f. archboldi workers ) ( king and trager , 2007 ) . devouring of enslaver brood which was observed in experimental mixed colonies of formica sanguinea and wood ant workers as their untypical slaves ( czechowski , 1996 ) also fits well into the category sabotage , clearly leading to weakening of slave - maker colonies . for more information about this behaviour , recognised as one of the most important factors making possible self - liberation of slaves , see self - liberation of slaves . emancipation ( self - liberation ) of slaves is well documented in two palaearctic slave - makers : the blood - red ant formica sanguinea ( a facultative slave - maker ) , and the amazon ant polyergus rufescens ( an obligate slave - maker ) . both these enslavers use species of the subgenus serviformica as their slaves ( e.g. dlusskij , 1967 and le moli et al . , 1994a , respectively ) . formica sanguinea is additionally able , under some conditions , to enslave ant species of the subgenera formica s. str . ( i.e. wood ants ; e.g. marikovsky , 1963 ) and coptoformica ( czechowski , 1977 ) . socially parasitic relations between f. sanguinea and wood ants are discussed in detail by czechowski ( 1996 , 2001 , 2003 ) . even in the very early myrmecological literature ( starting from the nineteenth century ) , there are mentions indicating that the blood - red ant f. sanguinea colonies containing wood ant workers as their untypical slaves may split into two homospecific parts , and that slaves may also take over the colonies of the slave - maker . some of the f. sanguinea colonies experimentally mixed in the field with f. pratensis ( by supplying them with worker pupae of the latter species ) spontaneously changed with time into queenright homospecific colonies of f. pratensis ( forel , 1874 ) . forel ( 1928 ) , vaguely ( with no date ) invoking e. wasmann , explained these transformations by replacements of an accidentally lost f. sanguinea queen by a young queen of f. pratensis adopted by the slaves . presently , such taking over of f. sanguinea nests by f. pratensis as an alleged slave species seems to be relatively easy to explain as this very species of wood ants has been revealed to be an occasional temporary social parasite of f. sanguinea colonies ( at least queenless ones ) ( czechowski , 2001 , 2003 ) . it is to be expected that the probability of successful takeover of a host colony by a dependent foundatrix is higher if conspecific workers and their species - specific olfactory discriminators are already present there . however , cases of taking over f. sanguinea colonies by enslaved wood ant workers were reported in other species of formica s. str . as well in other words , not only f. pratensis as a temporary social parasite of f. sanguinea , but also other wood ants are able to take over the colonies of that species . marikovsky ( 1963 ) reported that newly established colonies of f. rufa artificially settled in western siberia were often raided by f. sanguinea and their brood was pillaged . as a consequence , mixed colonies of f. sanguinea with wood ant workers came into being . however , subsequently monospecific groups of slaves escaped out of some of these mixed colonies , built their own nests in the vicinity , and recruited conspecific queens , creating thus queenright colonies of their own species . however , members of these colonies retained a friendly attitude towards workers of f. sanguinea , their former nestmates from still queenright f. sanguinea colonies . in such a way , peculiar bi - specific polydomous nest systems came into existence . it should , however , be mentioned here that the species of wood ants investigated by marikovsky ( spelled also as marikovskij ) is uncertain : it was first labelled as f. rufa ( marikovsky , 1963 ) , and then as f. polyctena ( marikovskij , 1971 ) . czechowski ( 1996 ) provided a detailed account of his observations made in the course of numerous experiments during which field colonies of f. sanguinea were artificially supplied with large amounts of worker pupae of several species of wood ants ( f. polyctena , f. rufa , f. aquilonia and f. lugubris ) . these experiments were carried out in poland and finland in the years 19871996 . as a result , 21 mixed colonies of f. sanguinea with different proportions ( 2996 % ) of wood ant slaves were created ( czechowski , 1996 ) . mass emergence of these untypical slaves in the slave - maker nests often resulted in splitting of the mixed colonies . as a rule , the majority of the slave - maker workers stayed in the old nest ( supposedly the one containing the f. sanguinea queen ) , and slaves prevailed in newly established nests . even if a colony continued to be monodomous , high differentiation of the enslaver / slave ratio in particular sectors of the extended complex nest was observed ( czechowski , 1996 ) . similar differentiation of the slave - maker / slave ratio is also common in mixed colonies of f. sanguinea and f. cinerea inhabiting compound nest systems typical of this host species . in such nest systems , the proportion of slaves varies from a few percent in the central part of the nest system to nearly 100 % in its peripheral parts ( dlusskij , 1967 ; czechowski and rotkiewicz , 1997 ; czechowski , 1998 ) . returning to the mixed colonies of f. sanguinea and atypical wood ant slaves created by czechowski , during the following season , even though f. sanguinea workers were still present , brood of wood ants ( worker larvae and pupae ) appeared in some of the colonies providing an evident proof that the slaves had managed to adopt their conspecific ( or related ) queen(s ) ( czechowski 1990 , 1996 ) . such a situation happened in eight out of 21 mixed colonies ( 38 % ) . only f. polyctena queens were adopted ( in seven colonies by the conspecific slaves , and in one case by f. rufa slaves ; czechowski , 1996 ) . this last case was not surprising , as in wood ants heterospecific queen adoptions in orphaned colonies happen quite frequently ( e.g. hagemann and schmidt , 1985 ; czechowski , 1996 ; pisarski and czechowski , 1994 ) . takeover of an originally f. sanguinea colony by wood ant slaves may be temporary or definite . in four colonies in which brood of the enslaved wood ant species appeared during the next season , restitution of f. sanguinea ( manifesting itself by the appearance of f. sanguinea brood ) started in the following year . the remaining four colonies underwent a permanent transformation into monospecific f. polyctena colonies ( czechowski , 1996 ) . subsequent laboratory experiments revealed the existence of two mechanisms of the slave emancipation : ( a ) elimination of the slave - maker brood and ( b ) functional ( not necessarily physical ) queen replacement . these two processes were not independent : elimination of the slave - maker brood facilitated or even acted as a necessary condition for subsequent queen replacement ( czechowski , 1996 ; see below).elimination of the slave - maker brood the striking rate at which f. sanguinea workers disappeared in their own field nests after the emergence of wood ant slaves , observed in a situation in which allospecific nestmate workers of these two species did not show any signs of mutual aggressiveness , strongly suggested that this phenomenon was related to the elimination of slave - maker s brood . this hypothesis was confirmed by the results of laboratory experiments : when starvation was inflicted , mixed cultures of f. sanguinea and f. polyctena were forced to resort to brood cannibalism . in concordance with the economy of a mixed colony as the whole , younger brood was devoured first irrespectively of the species ( slave vs slave - maker ) . as a consequence of different life histories of these two species , brood of f. sanguinea is usually slightly younger than brood of f. polyctena ( czechowski , 1996 ) , and in the discussed laboratory experiment brood of f. sanguinea ( larvae and relatively young pupae ) was indeed generally younger than relatively aged pupae of wood ants experimentally delivered to artificial nests . during the field experiments reported above , elimination of slave - maker s brood might also have been related to the fact that mixed colonies of f. sanguinea and wood ants were temporarily facing hunger as a consequence of mass emergence of callow slaves , too young to act as foragers and requiring to be fed themselves ( czechowski , 1996).queen replacement severe reduction of the rising generation of slave - maker workers undoubtedly made easier the next step of the slave emancipation observed in some colonies investigated in the field experiment of czechowski ( 1996 ) : the substitution of a f. sanguinea queen by f. polyctena one(s ) . the results of subsequent laboratory experiments ( czechowski , 1996 ) shed some light on other general determinants of queen replacement taking place in the mixed colonies composed of f. sanguinea and their atypical wood ant slaves . in particular , they revealed that splitting of the mixed colony constituted the most important factor conducive to the adoption of wood ant queens . as already told , such colony splitting into two or more parts was often observed in the field , and was experimentally simulated under laboratory conditions , too . only queenless parts of the mixed colonies ( with no f. sanguinea queen ) were able to accept wood ant queens , both in the laboratory and in the field ( czechowski , 1996 ) . elimination of the slave - maker brood the striking rate at which f. sanguinea workers disappeared in their own field nests after the emergence of wood ant slaves , observed in a situation in which allospecific nestmate workers of these two species did not show any signs of mutual aggressiveness , strongly suggested that this phenomenon was related to the elimination of slave - maker s brood . this hypothesis was confirmed by the results of laboratory experiments : when starvation was inflicted , mixed cultures of f. sanguinea and f. polyctena were forced to resort to brood cannibalism . in concordance with the economy of a mixed colony as the whole , younger brood was devoured first irrespectively of the species ( slave vs slave - maker ) . as a consequence of different life histories of these two species , brood of f. sanguinea is usually slightly younger than brood of f. polyctena ( czechowski , 1996 ) , and in the discussed laboratory experiment brood of f. sanguinea ( larvae and relatively young pupae ) was indeed generally younger than relatively aged pupae of wood ants experimentally delivered to artificial nests . during the field experiments reported above , elimination of slave - maker s brood might also have been related to the fact that mixed colonies of f. sanguinea and wood ants were temporarily facing hunger as a consequence of mass emergence of callow slaves , too young to act as foragers and requiring to be fed themselves ( czechowski , 1996 ) . queen replacement severe reduction of the rising generation of slave - maker workers undoubtedly made easier the next step of the slave emancipation observed in some colonies investigated in the field experiment of czechowski ( 1996 ) : the substitution of a f. sanguinea queen by f. polyctena one(s ) . the results of subsequent laboratory experiments ( czechowski , 1996 ) shed some light on other general determinants of queen replacement taking place in the mixed colonies composed of f. sanguinea and their atypical wood ant slaves . in particular , they revealed that splitting of the mixed colony constituted the most important factor conducive to the adoption of wood ant queens . as already told , such colony splitting into two or more parts was often observed in the field , and was experimentally simulated under laboratory conditions , too . only queenless parts of the mixed colonies ( with no f. sanguinea queen ) were able to accept wood ant queens , both in the laboratory and in the field ( czechowski , 1996 ) . the experiments of czechowski ( 1996 ) also revealed that the critical moment for the newly adopted wood ant queens comes when the colony undergoes again a fusion after a temporary splitting into a queenless and a queenright part . in two cases , single f. polyctena queens that had been adopted by the queenless part of the mixed laboratory colony were subsequently killed by workers from the part containing a f. sanguinea queen . in yet another case , 30 wood ant queens , accepted by a colony part consisting predominantly of workers of f. polyctena ( 97 % ) , were attacked by the queen of f. sanguinea which managed to kill three of them and then died , too , most probably as a result of injuries sustained in the combat with wood ant queens ( czechowski , 1996 ) . the above - mentioned results strongly suggest that adoption of many conspecific queens is the most important prerequisite of successful emancipation of slaves of f. sanguinea . generally , in wood ants ( at least in polygynous species such as f. polyctena ) , queenless groups of workers very readily adopt young conspecific queens and even related heterospecific ones ( czechowski , 1994 ; pisarski and czechowski , 1994 ; yamauchi et al . , 1994 ) . during the discussed field experiments of czechowski ( 1996 ) , permanent colony takeover by f. polyctena most probably occurred only in those mixed colonies of f. sanguinea and wood ant workers which recruited numerous f. polyctena queens . it might also be supposed that the higher is the number of f. sanguinea queens present in the original colony , the more resistant is that colony against a takeover by the slave species . a single queen may not be able to survive successive , even victorious fights with several wood ant queens without serious injuries . the data on the level of gyny in f. sanguinea are very ambiguous , but monogyny seems to be prevalent in colonies of that ant species ( see czechowski , 1996 ) . however , genetic data suggest that colonies of f. sanguinea are usually oligogynous , but functionally monogynous , i.e. they contain one egg - laying queen plus a few reserve ones ( pamilo et al . , 1978 ; pamilo and varvio - aho , 1979 ) . a mixed colony of f. sanguinea and f. polyctena ( or similar atypical slaves ) represents thus a conglomerate of ant species following different social strategies : monogyny vs high degree of polygyny . the probability of successful self - liberation of slaves from a polygynous species is particularly high , as they can recruit multiple conspecific queens , whereas f. sanguinea remain monogynous , and their single queen or at best few queens are much more vulnerable than multiple queens of their highly polygynous slave species . the phenomenon of self - liberation of slaves of the obligate social parasite , the amazon ant p. rufescens , is the most spectacular and most advanced known form of a slave rebellion and represents the case of slave self - freeing from enslavement in the most literal sense of that word . the origin of that phenomenon may be sought in enormous numbers of enslaved workers which amazon ants accumulate in their nests , several times higher than the numbers of workers found in monospecific colonies of the host species . in formica fusca , one of the most common slave species of p. rufescens , colony size usually does not exceed several hundred adults , rarely reaching 1,5002,000 workers in especially large colonies ( savolainen , 1990 ) . however , colonies of p. rufescens may include more than 2,000 slave - maker workers ( czechowski et al . , 2012 ) , and the proportion of slaves in colonies of various amazon ants may amount to 7090 % of the mixed workforce ( e.g. wheeler , 1910 ; talbot , 1967 ; hlldobler and wilson , 1990 ; seifert , 2007 ; buschinger , 2009 ) . this means that p. rufescens are able to gather a dozen of thousands of slaves in their nests . such a large number of slaves , together with the slave - maker workforce and brood and amassed slave pupae , may easily lead to overcrowding in the nest . possible consequences of that phenomenon were revealed during the studies on a mixed colony of p. rufescens and f. fusca in the biaowiea forest ( n - e poland ) ( czechowski , 2005 , 2006 ) . the observed colony was very large and inhabited a nest system consisting of the main nest and several small satellite nests , arranged as a semicircular at the distances of 1.64.4 m from the main nest entrances . at least some of the satellite nests were connected with the main nest by underground tunnels . the whole system resembled nest systems typical rather of f. cinerea ( see e.g. dlusskij , 1967 ; czechowski and rotkiewicz , 1997 ) . only the entrances of the main nest were used by p. rufescens ( scouts and outbound and inbound raiding columns ) . crews of the satellite nests consisted of small groups of f. fusca slaves ( few dozens of workers with no brood ) . sometimes , a few young sexuals and , less frequently , single workers of p. rufescens were seen in these satellite nests , too . during the first observation season , a dealate f. fusca gyne was seen twice in two different satellite nests during the period of f. fusca nuptial flights . in contrast to f. sanguinea , p. rufescens never bring host sexual pupae to their nest ( basing on w. czechowski s observations of innumerable raids ) . therefore , the observed gynes must have arrived from the outside and have been adopted ( or at least temporarily accepted ) by their enslaved conspecifics . from there , there is only one step to the emancipation of individual groups of slaves inhabiting small satellite nests . p. rufescens prevented such turn of events systematically raiding its own satellite nests , and carrying adult slaves present there to the main nest . no trace of mutual aggression and/or any opposition from the slaves could be observed during such raids : f. fusca workers were carried in a manner typical for adult nestmate transport , with the carried ant curled up beneath the carrier . these peculiar raids were starting identically as regular raids ( see e.g. dobrzaska and dobrzaski , 1960 ; mori et al . , 1991a , b ; le moli et al . , 1994a , b ) , but in the course of time they were evolving into several hours long individual activity of p. rufescens workers which kept running on their own in both directions between the main nest and the target satellite nest . in this way , up to 2,0003,000 slaves were carried during a single afternoon which implies that the workers of f. fusca taken out of the satellite nests were systematically replaced by other ( or the same ) ones , coming from the main nest through underground passages . due to their supposed function , these operations of p. rufescens were called the integration raids ( czechowski , 2005 ) . further observations made during the next season dotted the i s ( czechowski , 2006 ; see below).the integration raids would be , most probably , fully effective to keep the integrity of the observed mixed colony , if a colony of f. polyctena was not present in its vicinity . a much frequented foraging route of this colony ran at a distance of 1 m from the entrances of the main p. rufescens nest . that route was entirely impassable for p. rufescens ( both scouts and raiding columns ) . as a consequence , the area on the other side of the route was never raided during the first year of the observations . formica fusca slaves were , however , able to cross freely the route of f. polyctena . this was not surprising , as f. fusca is a submissive species , well adapted to live within territories of wood ants ( e.g. savolainen and vepslinen , 1989 ; savolainen , 1990 ) . next year , the f. polyctena colony moved to another place , its former route disappeared , and the area behind it was now wide open for raiding by p. rufescens . however , still before raiding period , an increased movement of f. fusca slaves in this direction was observed . not only single f. fusca workers , but also loose columns of several ( up to 20 ) individuals were seen to leave the main nest of the mixed colony , to run outside the former wood ant route , and to disperse there . mini raids of f. fusca slaves ( see czechowski , 2007 and also introduction ) . however , they were undertaken only by f. fusca , and did not accompany the raids of the slave - maker . only when the raids of p. rufescens started , they revealed a group of at least six small cryptic f. fusca nests scattered within an area of ca 8 m , from 5.3 to 7.1 m away from the main nest of p. rufescens outside the former route of f. polyctena . some of them contained small amounts of brood . during 11 raids observed during that season , p. rufescens obtained from these nests altogether about 200 worker pupae and last instar larvae . prolonged stays of some raiders in the target nest were a peculiar feature of these raids . single p. rufescens workers remained there even on the next day , being fully tolerated by the local f. fusca . another characteristic trait of these peculiar raids consisted in gradual decline of the initial mutual aggressiveness that evolved into apparently friendly relations between the two species : allospecific workers responded to each other only by antennal palpation . at the same time , p. rufescens started to try to transport f. fusca workers to its own nest in the same way as in the course of integration raids ( see above ) . however , as a consequence of a much less strong involvement of p. rufescens in this task , and a much lower readiness of f. fusca to accept to be transported , only about 50 adult f. fusca workers were successfully carried to the p. rufescens nest ( czechowski , 2006 ) . in light of these observations , it seems very probable that small cryptic colonies of f. fusca raided by p. rufescens in this peculiar way consisted of ex - slaves which had managed to break away from the enslaver colony . these ex - slaves established their own nests and adopted conspecific queens , but they apparently partly retained olfactory connection with their former mixed colony . these colonies of the newly emancipated f. fusca slaves would most probably gain permanent autonomy , if their f. polyctena neighbour had not moved away . it may also be noted that a phenomenon belonging to the category of integration raids was signalled already by dobrzaska ( 1976 ) in her study of a colony of p. rufescens with f. cinerea slaves . these p. rufescens were observed to raid ( according to the author , by mistake ) a filial nest of the mixed colony at a distance of 1.5 m from its main nest . the target nest was inhabited by f. cinerea slaves which were carried by p. rufescens to the main nest . immediately afterwards , the majority of f. cinerea workers spontaneously returned to their own ( filial ) nest . this observation confirms the tendency of enslaved serviformica workers to leave the overcrowded ( ? ) nests of p. rufescens , and settle in homospecific groups in their vicinity , and the ability of p. rufescens to take preventive measures against that behaviour which otherwise may ultimately lead to slave emancipation . besides that exceptional case , j. dobrzaska and j. dobrzaski , who studied countless raids of p. rufescens , never reported any further cases of capture of mature ( not callow ) workers from the target host species nest . however , mori et al . ( 1991a , b ) mentioned also the cases of capture of mature adult workers of f. cunicularia during three out of 38 p. rufescens raids observed . however , it is impossible to tell whether their observations documented capture of slaves on their way to liberation , because , unfortunately , the authors did not provide detailed information about these particular raids . if the emancipation of enslaved workers is possible through their exodus from the nest of the slave - maker , a possible reverse mechanism may also be possible : slave emancipation may be achieved as a result of staying in the old nest when the mixed colony moves to a new place . such periodic colony relocations are common in slave - making ants and occur , among others , to gain access to more host colonies , as recently reported for two nearctic blood - red ant species , f. subintegra and f. pergandei ( apple et al . , 2014 ) . in the case of the nearctic amazon ants , p. lucidus , such colony relocations were accompanied by the so - called emigration raids aimed at the slaves , f. dolosa (= f. schaufussi ; see trager et al . , 2007 ) and f. archboldi which stayed in the old nest ( kwait and topoff , 1983 ; trager and johnson , 1985 ) . the biological function of these emigration raids seems to be the same as that of the integration raids observed by czechowski ( 2005 ) in p. rufescens : they prevent the process of fractionation of the mixed colony . in both cases , disturbances of the process of the mixed colony unification may result in isolation of a group of the slaves , and , subsequently , in their more or less long - lasting emancipation from the social parasite . even in the very early myrmecological literature ( starting from the nineteenth century ) , there are mentions indicating that the blood - red ant f. sanguinea colonies containing wood ant workers as their untypical slaves may split into two homospecific parts , and that slaves may also take over the colonies of the slave - maker . some of the f. sanguinea colonies experimentally mixed in the field with f. pratensis ( by supplying them with worker pupae of the latter species ) spontaneously changed with time into queenright homospecific colonies of f. pratensis ( forel , 1874 ) . forel ( 1928 ) , vaguely ( with no date ) invoking e. wasmann , explained these transformations by replacements of an accidentally lost f. sanguinea queen by a young queen of f. pratensis adopted by the slaves . presently , such taking over of f. sanguinea nests by f. pratensis as an alleged slave species seems to be relatively easy to explain as this very species of wood ants has been revealed to be an occasional temporary social parasite of f. sanguinea colonies ( at least queenless ones ) ( czechowski , 2001 , 2003 ) . it is to be expected that the probability of successful takeover of a host colony by a dependent foundatrix is higher if conspecific workers and their species - specific olfactory discriminators are already present there . however , cases of taking over f. sanguinea colonies by enslaved wood ant workers were reported in other species of formica s. str . as well in other words , not only f. pratensis as a temporary social parasite of f. sanguinea , but also other wood ants are able to take over the colonies of that species . marikovsky ( 1963 ) reported that newly established colonies of f. rufa artificially settled in western siberia were often raided by f. sanguinea and their brood was pillaged . as a consequence , mixed colonies of f. sanguinea with wood ant workers came into being . however , subsequently monospecific groups of slaves escaped out of some of these mixed colonies , built their own nests in the vicinity , and recruited conspecific queens , creating thus queenright colonies of their own species . however , members of these colonies retained a friendly attitude towards workers of f. sanguinea , their former nestmates from still queenright f. sanguinea colonies . in such a way , peculiar bi - specific polydomous nest systems came into existence . it should , however , be mentioned here that the species of wood ants investigated by marikovsky ( spelled also as marikovskij ) is uncertain : it was first labelled as f. rufa ( marikovsky , 1963 ) , and then as f. polyctena ( marikovskij , 1971 ) . czechowski ( 1996 ) provided a detailed account of his observations made in the course of numerous experiments during which field colonies of f. sanguinea were artificially supplied with large amounts of worker pupae of several species of wood ants ( f. polyctena , f. rufa , f. aquilonia and f. lugubris ) . these experiments were carried out in poland and finland in the years 19871996 . as a result , 21 mixed colonies of f. sanguinea with different proportions ( 2996 % ) of wood ant slaves were created ( czechowski , 1996 ) . mass emergence of these untypical slaves in the slave - maker nests often resulted in splitting of the mixed colonies . as a rule , the majority of the slave - maker workers stayed in the old nest ( supposedly the one containing the f. sanguinea queen ) , and slaves prevailed in newly established nests . even if a colony continued to be monodomous , high differentiation of the enslaver / slave ratio in particular sectors of the extended complex nest was observed ( czechowski , 1996 ) . similar differentiation of the slave - maker / slave ratio is also common in mixed colonies of f. sanguinea and f. cinerea inhabiting compound nest systems typical of this host species . in such nest systems , the proportion of slaves varies from a few percent in the central part of the nest system to nearly 100 % in its peripheral parts ( dlusskij , 1967 ; czechowski and rotkiewicz , 1997 ; czechowski , 1998 ) . returning to the mixed colonies of f. sanguinea and atypical wood ant slaves created by czechowski , during the following season , even though f. sanguinea workers were still present , brood of wood ants ( worker larvae and pupae ) appeared in some of the colonies providing an evident proof that the slaves had managed to adopt their conspecific ( or related ) queen(s ) ( czechowski 1990 , 1996 ) . such a situation happened in eight out of 21 mixed colonies ( 38 % ) . only f. polyctena queens were adopted ( in seven colonies by the conspecific slaves , and in one case by f. rufa slaves ; czechowski , 1996 ) . this last case was not surprising , as in wood ants heterospecific queen adoptions in orphaned colonies happen quite frequently ( e.g. hagemann and schmidt , 1985 ; czechowski , 1996 ; pisarski and czechowski , 1994 ) . takeover of an originally f. sanguinea colony by wood ant slaves may be temporary or definite . in four colonies in which brood of the enslaved wood ant species appeared during the next season , restitution of f. sanguinea ( manifesting itself by the appearance of f. sanguinea brood ) started in the following year . the remaining four colonies underwent a permanent transformation into monospecific f. polyctena colonies ( czechowski , 1996 ) . subsequent laboratory experiments revealed the existence of two mechanisms of the slave emancipation : ( a ) elimination of the slave - maker brood and ( b ) functional ( not necessarily physical ) queen replacement . these two processes were not independent : elimination of the slave - maker brood facilitated or even acted as a necessary condition for subsequent queen replacement ( czechowski , 1996 ; see below).elimination of the slave - maker brood the striking rate at which f. sanguinea workers disappeared in their own field nests after the emergence of wood ant slaves , observed in a situation in which allospecific nestmate workers of these two species did not show any signs of mutual aggressiveness , strongly suggested that this phenomenon was related to the elimination of slave - maker s brood . this hypothesis was confirmed by the results of laboratory experiments : when starvation was inflicted , mixed cultures of f. sanguinea and f. polyctena were forced to resort to brood cannibalism . in concordance with the economy of a mixed colony as the whole , younger brood was devoured first irrespectively of the species ( slave vs slave - maker ) . as a consequence of different life histories of these two species , brood of f. sanguinea is usually slightly younger than brood of f. polyctena ( czechowski , 1996 ) , and in the discussed laboratory experiment brood of f. sanguinea ( larvae and relatively young pupae ) was indeed generally younger than relatively aged pupae of wood ants experimentally delivered to artificial nests . during the field experiments reported above , elimination of slave - maker s brood might also have been related to the fact that mixed colonies of f. sanguinea and wood ants were temporarily facing hunger as a consequence of mass emergence of callow slaves , too young to act as foragers and requiring to be fed themselves ( czechowski , 1996).queen replacement severe reduction of the rising generation of slave - maker workers undoubtedly made easier the next step of the slave emancipation observed in some colonies investigated in the field experiment of czechowski ( 1996 ) : the substitution of a f. sanguinea queen by f. polyctena one(s ) . the results of subsequent laboratory experiments ( czechowski , 1996 ) shed some light on other general determinants of queen replacement taking place in the mixed colonies composed of f. sanguinea and their atypical wood ant slaves . in particular , they revealed that splitting of the mixed colony constituted the most important factor conducive to the adoption of wood ant queens . as already told , such colony splitting into two or more parts was often observed in the field , and was experimentally simulated under laboratory conditions , too . only queenless parts of the mixed colonies ( with no f. sanguinea queen ) were able to accept wood ant queens , both in the laboratory and in the field ( czechowski , 1996 ) . elimination of the slave - maker brood the striking rate at which f. sanguinea workers disappeared in their own field nests after the emergence of wood ant slaves , observed in a situation in which allospecific nestmate workers of these two species did not show any signs of mutual aggressiveness , strongly suggested that this phenomenon was related to the elimination of slave - maker s brood . this hypothesis was confirmed by the results of laboratory experiments : when starvation was inflicted , mixed cultures of f. sanguinea and f. polyctena were forced to resort to brood cannibalism . in concordance with the economy of a mixed colony as the whole , younger brood was devoured first irrespectively of the species ( slave vs slave - maker ) . as a consequence of different life histories of these two species , brood of f. sanguinea is usually slightly younger than brood of f. polyctena ( czechowski , 1996 ) , and in the discussed laboratory experiment brood of f. sanguinea ( larvae and relatively young pupae ) was indeed generally younger than relatively aged pupae of wood ants experimentally delivered to artificial nests . during the field experiments reported above , elimination of slave - maker s brood might also have been related to the fact that mixed colonies of f. sanguinea and wood ants were temporarily facing hunger as a consequence of mass emergence of callow slaves , too young to act as foragers and requiring to be fed themselves ( czechowski , 1996 ) . queen replacement severe reduction of the rising generation of slave - maker workers undoubtedly made easier the next step of the slave emancipation observed in some colonies investigated in the field experiment of czechowski ( 1996 ) : the substitution of a f. sanguinea queen by f. polyctena one(s ) . the results of subsequent laboratory experiments ( czechowski , 1996 ) shed some light on other general determinants of queen replacement taking place in the mixed colonies composed of f. sanguinea and their atypical wood ant slaves . in particular , they revealed that splitting of the mixed colony constituted the most important factor conducive to the adoption of wood ant queens . as already told , such colony splitting into two or more parts was often observed in the field , and was experimentally simulated under laboratory conditions , too . only queenless parts of the mixed colonies ( with no f. sanguinea queen ) were able to accept wood ant queens , both in the laboratory and in the field ( czechowski , 1996 ) . the experiments of czechowski ( 1996 ) also revealed that the critical moment for the newly adopted wood ant queens comes when the colony undergoes again a fusion after a temporary splitting into a queenless and a queenright part . in two cases , single f. polyctena queens that had been adopted by the queenless part of the mixed laboratory colony were subsequently killed by workers from the part containing a f. sanguinea queen . in yet another case , 30 wood ant queens , accepted by a colony part consisting predominantly of workers of f. polyctena ( 97 % ) , were attacked by the queen of f. sanguinea which managed to kill three of them and then died , too , most probably as a result of injuries sustained in the combat with wood ant queens ( czechowski , 1996 ) . the above - mentioned results strongly suggest that adoption of many conspecific queens is the most important prerequisite of successful emancipation of slaves of f. sanguinea . generally , in wood ants ( at least in polygynous species such as f. polyctena ) , queenless groups of workers very readily adopt young conspecific queens and even related heterospecific ones ( czechowski , 1994 ; pisarski and czechowski , 1994 ; yamauchi et al . , 1994 ) . during the discussed field experiments of czechowski ( 1996 ) , permanent colony takeover by f. polyctena most probably occurred only in those mixed colonies of f. sanguinea and wood ant workers which recruited numerous f. polyctena queens . it might also be supposed that the higher is the number of f. sanguinea queens present in the original colony , the more resistant is that colony against a takeover by the slave species . a single queen may not be able to survive successive , even victorious fights with several wood ant queens without serious injuries . the data on the level of gyny in f. sanguinea are very ambiguous , but monogyny seems to be prevalent in colonies of that ant species ( see czechowski , 1996 ) . however , genetic data suggest that colonies of f. sanguinea are usually oligogynous , but functionally monogynous , i.e. they contain one egg - laying queen plus a few reserve ones ( pamilo et al . , 1978 ; pamilo and varvio - aho , 1979 ) . a mixed colony of f. sanguinea and f. polyctena ( or similar atypical slaves ) represents thus a conglomerate of ant species following different social strategies : monogyny vs high degree of polygyny . the probability of successful self - liberation of slaves from a polygynous species is particularly high , as they can recruit multiple conspecific queens , whereas f. sanguinea remain monogynous , and their single queen or at best few queens are much more vulnerable than multiple queens of their highly polygynous slave species . the phenomenon of self - liberation of slaves of the obligate social parasite , the amazon ant p. rufescens , is the most spectacular and most advanced known form of a slave rebellion and represents the case of slave self - freeing from enslavement in the most literal sense of that word . the origin of that phenomenon may be sought in enormous numbers of enslaved workers which amazon ants accumulate in their nests , several times higher than the numbers of workers found in monospecific colonies of the host species . in formica fusca , one of the most common slave species of p. rufescens , colony size usually does not exceed several hundred adults , rarely reaching 1,5002,000 workers in especially large colonies ( savolainen , 1990 ) . however , colonies of p. rufescens may include more than 2,000 slave - maker workers ( czechowski et al . , 2012 ) , and the proportion of slaves in colonies of various amazon ants may amount to 7090 % of the mixed workforce ( e.g. wheeler , 1910 ; talbot , 1967 ; hlldobler and wilson , 1990 ; seifert , 2007 ; buschinger , 2009 ) . this means that p. rufescens are able to gather a dozen of thousands of slaves in their nests . such a large number of slaves , together with the slave - maker workforce and brood and amassed slave pupae , may easily lead to overcrowding in the nest . possible consequences of that phenomenon were revealed during the studies on a mixed colony of p. rufescens and f. fusca in the biaowiea forest ( n - e poland ) ( czechowski , 2005 , 2006 ) . the observed colony was very large and inhabited a nest system consisting of the main nest and several small satellite nests , arranged as a semicircular at the distances of 1.64.4 m from the main nest entrances . at least some of the satellite nests were connected with the main nest by underground tunnels . the whole system resembled nest systems typical rather of f. cinerea ( see e.g. dlusskij , 1967 ; czechowski and rotkiewicz , 1997 ) . only the entrances of the main nest were used by p. rufescens ( scouts and outbound and inbound raiding columns ) . crews of the satellite nests consisted of small groups of f. fusca slaves ( few dozens of workers with no brood ) . sometimes , a few young sexuals and , less frequently , single workers of p. rufescens were seen in these satellite nests , too . during the first observation season , a dealate f. fusca gyne was seen twice in two different satellite nests during the period of f. fusca nuptial flights . in contrast to f. sanguinea , p. rufescens never bring host sexual pupae to their nest ( basing on w. czechowski s observations of innumerable raids ) . therefore , the observed gynes must have arrived from the outside and have been adopted ( or at least temporarily accepted ) by their enslaved conspecifics . from there , there is only one step to the emancipation of individual groups of slaves inhabiting small satellite nests . p. rufescens prevented such turn of events systematically raiding its own satellite nests , and carrying adult slaves present there to the main nest . no trace of mutual aggression and/or any opposition from the slaves could be observed during such raids : f. fusca workers were carried in a manner typical for adult nestmate transport , with the carried ant curled up beneath the carrier . these peculiar raids were starting identically as regular raids ( see e.g. dobrzaska and dobrzaski , 1960 ; mori et al . , 1991a , b ; le moli et al . , 1994a , b ) , but in the course of time they were evolving into several hours long individual activity of p. rufescens workers which kept running on their own in both directions between the main nest and the target satellite nest . in this way , up to 2,0003,000 slaves were carried during a single afternoon which implies that the workers of f. fusca taken out of the satellite nests were systematically replaced by other ( or the same ) ones , coming from the main nest through underground passages . due to their supposed function , these operations of p. rufescens were called the integration raids ( czechowski , 2005 ) . further observations made during the next season dotted the i s ( czechowski , 2006 ; see below).the integration raids would be , most probably , fully effective to keep the integrity of the observed mixed colony , if a colony of f. polyctena was not present in its vicinity . a much frequented foraging route of this colony ran at a distance of 1 m from the entrances of the main p. rufescens nest . that route was entirely impassable for p. rufescens ( both scouts and raiding columns ) . as a consequence , the area on the other side of the route was never raided during the first year of the observations . formica fusca slaves were , however , able to cross freely the route of f. polyctena . this was not surprising , as f. fusca is a submissive species , well adapted to live within territories of wood ants ( e.g. savolainen and vepslinen , 1989 ; savolainen , 1990 ) . next year , the f. polyctena colony moved to another place , its former route disappeared , and the area behind it was now wide open for raiding by p. rufescens . however , still before raiding period , an increased movement of f. fusca slaves in this direction was observed . not only single f. fusca workers , but also loose columns of several ( up to 20 ) individuals were seen to leave the main nest of the mixed colony , to run outside the former wood ant route , and to disperse there . mini raids of f. fusca slaves ( see czechowski , 2007 and also introduction ) . however , they were undertaken only by f. fusca , and did not accompany the raids of the slave - maker . only when the raids of p. rufescens started , they revealed a group of at least six small cryptic f. fusca nests scattered within an area of ca 8 m , from 5.3 to 7.1 m away from the main nest of p. rufescens outside the former route of f. polyctena . some of them contained small amounts of brood . during 11 raids observed during that season , p. rufescens obtained from these nests altogether about 200 worker pupae and last instar larvae prolonged stays of some raiders in the target nest were a peculiar feature of these raids . single p. rufescens workers remained there even on the next day , being fully tolerated by the local f. fusca . another characteristic trait of these peculiar raids consisted in gradual decline of the initial mutual aggressiveness that evolved into apparently friendly relations between the two species : allospecific workers responded to each other only by antennal palpation . at the same time , p. rufescens started to try to transport f. fusca workers to its own nest in the same way as in the course of integration raids ( see above ) . however , as a consequence of a much less strong involvement of p. rufescens in this task , and a much lower readiness of f. fusca to accept to be transported , only about 50 adult f. fusca workers were successfully carried to the p. rufescens nest ( czechowski , 2006 ) . in light of these observations , it seems very probable that small cryptic colonies of f. fusca raided by p. rufescens in this peculiar way consisted of ex - slaves which had managed to break away from the enslaver colony . these ex - slaves established their own nests and adopted conspecific queens , but they apparently partly retained olfactory connection with their former mixed colony . these colonies of the newly emancipated f. fusca slaves would most probably gain permanent autonomy , if their f. polyctena neighbour had not moved away . it may also be noted that a phenomenon belonging to the category of integration raids was signalled already by dobrzaska ( 1976 ) in her study of a colony of p. rufescens with f. cinerea slaves . these p. rufescens were observed to raid ( according to the author , by mistake ) a filial nest of the mixed colony at a distance of 1.5 m from its main nest . the target nest was inhabited by f. cinerea slaves which were carried by p. rufescens to the main nest . immediately afterwards , the majority of f. cinerea workers spontaneously returned to their own ( filial ) nest . this observation confirms the tendency of enslaved serviformica workers to leave the overcrowded ( ? ) nests of p. rufescens , and settle in homospecific groups in their vicinity , and the ability of p. rufescens to take preventive measures against that behaviour which otherwise may ultimately lead to slave emancipation . besides that exceptional case , j. dobrzaska and j. dobrzaski , who studied countless raids of p. rufescens , never reported any further cases of capture of mature ( not callow ) workers from the target host species nest ( 1991a , b ) mentioned also the cases of capture of mature adult workers of f. cunicularia during three out of 38 p. rufescens raids observed . however , it is impossible to tell whether their observations documented capture of slaves on their way to liberation , because , unfortunately , the authors did not provide detailed information about these particular raids . if the emancipation of enslaved workers is possible through their exodus from the nest of the slave - maker , a possible reverse mechanism may also be possible : slave emancipation may be achieved as a result of staying in the old nest when the mixed colony moves to a new place . such periodic colony relocations are common in slave - making ants and occur , among others , to gain access to more host colonies , as recently reported for two nearctic blood - red ant species , f. subintegra and f. pergandei ( apple et al . , 2014 ) . in the case of the nearctic amazon ants , p. lucidus , such colony relocations were accompanied by the so - called emigration raids aimed at the slaves , f. dolosa (= f. schaufussi ; see trager et al . , 2007 ) and f. archboldi which stayed in the old nest ( kwait and topoff , 1983 ; trager and johnson , 1985 ) . the biological function of these emigration raids seems to be the same as that of the integration raids observed by czechowski ( 2005 ) in p. rufescens : they prevent the process of fractionation of the mixed colony . in both cases , disturbances of the process of the mixed colony unification may result in isolation of a group of the slaves , and , subsequently , in their more or less long - lasting emancipation from the social parasite . the present survey of observational and experimental data documenting various phenomena related to acts of opposition of ant slaves against their enslavers leaves unanswered some questions which remain open for future research and reflexions . first of all , it should be noticed that all known cases of direct hostility between allospecific individuals within mixed colonies of dulotic ant species were observed under laboratory conditions which unquestionably differ a lot from those encountered in natural nests . therefore , a question arises whether these in vitro observations reflect faithfully the relations between slave - makers and their slaves occurring under natural conditions . nestmate recognition is fundamental for the integrity of social insect colonies ( crozier and dix , 1979 ; crozier and pamilo , 1996 ; sturgis and gordon , 2012 ) , including mixed colonies of ant slave - makers and their slaves , in which enslaved host workers do not recognise their enslavers as strangers . only such a situation based on olfactory deception ( chemical camouflage , i.e. passive acquisition of recognition compounds , or chemical mimicry , i.e. induced biosynthesis of the proper chemical label ; e.g. lenoir et al . , 2001 ; dettorre et al . , 2002 ; akino , 2008 ; achenbach et al . , 2010 ; guillem et al for instance , dettorre et al . ( 2002 ) showed that workers of p. rufescens reared in isolation from the mother colony developed cuticular hydrocarbon profiles closely matching those of the primary host species , which indicates the occurrence of local host adaptation in the slave - maker population . on the other hand , the ability of slaves to recognise their homospecific nestmates allows them to act for the benefit of their maternal populations ( indirectly , via weakening of the slave - maker s colonies , and directly , via self - emancipation ) . therefore , in the course of the so - called co - evolutionary arms race between social parasites and their host species ( davies et al . , 2012a , b ) , the ants from the host species may develop recognition mechanisms allowing them to identify their enslavers ( for review see delattre et al . , they may display agonistic behaviour more frequently in response to parasites than in response to non - parasitic local allospecifics . such a situation was reported , among others , in temnothorax species acting as potential hosts of the dulotic species myrmoxenus ravouxi ( delattre et al . , 2012b ) . recent studies also show that host species may develop intolerance of parasite brood , which could be the first step towards slave rebellions in mixed colonies ( johnson et al . , 2005 , delattre et al . , 2012a ) so far , it is still not clear whether and to what degree nestmate recognition within mixed colonies of dulotic ants and their slaves depends of conditions encountered in natural and artificial nests . it is possible that distribution and blending of olfactory cues is achieved differently in petri dishes or test tubes than in cramped and crowded natural nest spaces with organic or mineral walls saturated with established colony odour . as already told , trager ( 2013a ) pointed out that when colonies of polyergus , which had successfully persisted for years in the field , were transferred to laboratory , the formica slaves almost always failed to raise polyergus brood and started to harass and kill off polyergus workers ( see sabotage ) . these observations suggest some disturbances of the nestmate recognition system related to some as yet unspecified differences between physical and/or social environment experienced by ants in the field and in the laboratory . let us remind that aggressive encounters observed within laboratory mixed colonies were not limited to skirmishes between allospecific nestmates , but they happened also between homospecific ants , both slave - makers and slaves ( see domestic misunderstandings ) . anyway , all results concerning ant behaviour obtained under laboratory conditions , usually very simplified , ought to be approached with a certain measure of reserve . on the other hand , it is hardly imaginable to study most of the phenomena discussed here in another way than in artificial nests . paradoxically , all known cases of true liberation of slaves ( see self - liberation of slaves)as opposed to less radical forms of the slave rebellions ( domestic misunderstandings , sabotage and partly slave reproduction)were observed in the field . all observed cases of emancipation of slaves of the obligate slave - maker polyergus rufescens belonging to a typical slave species ( formica fusca ) were fully spontaneous from the beginning to the end . in the case of the facultative slave - maker f. sanguinea , slave emancipation was observed only in untypical slave species ( wood ants ) and was initiated artificially , either accidentally ( marikovsky , 1963 ) or deliberately ( czechowski , 1996 ) . the question arises whether such latent possibility to emancipate lies dormant also in typical slaves of f. sanguinea ( i.e. serviformica species ) , to be expressed , too , in some specific circumstances . the first precondition for that ( and , at the same time , the main difficulty ) seems to be numerical predominance of slaves in the mixed colony . while in p. rufescens high ratio of slaves to slave - makers is a norm , the reverse is true for f. sanguinea ( see self - liberation of slaves ) . although slaves of f. sanguinea very rarely outnumber slave - maker workers , such exceptions happen ( e.g. up to 58 % of f. fusca and 84 % of f. japonica ; see czechowski , 1996 ) , and it can not be ruled out that typical slaves , when their proportion is high enough , might influence the f. sanguinea colony in some destructive way . czechowski ( 1996 ) observed in the field a mixed colony of f. sanguinea in which slaves of two serviformica species ( f. cunicularia and f. fusca ) altogether constituted 40 % of the workforce . although the colony was queenright , there was no f. sanguinea female brood in the nest . in september , i.e. much later than the regular period of nuptial flights of f. sanguinea , only numerous males of that species ( sons of slave - maker workers ? ) were present and their proportion reached as much as 22 % of the total number of adults of f. sanguinea . two conjectures arise : either the parasite queen was infertile or something wrong was happening to her female brood . the second possibility bears much similarity to the phenomenon studied by achenbach and foitzik ( 2009 ) ( see sabotage ) , i.e. destroying female parasite brood by temnothorax slaves enslaved by protomognathus americanus . the question of existence of such phenomena in mixed colonies of f. sanguinea as well remains , however , to be checked by future research . would serviformica slaves be able to adopt their conspecific queen in presence of formica sanguinea ? czechowski ( 1996 ) observed that in two mixed laboratory cultures of f. sanguinea and their f. fusca slaves ( a queenright one and a queenless one with 62 and 36 % share of the slaves , respectively ) all experimentally introduced f. fusca queens were killed , and interestingly they were attacked mainly by f. fusca workers . another strategy which may lead to slave emancipation , i.e. selective devouring of the slave - maker brood , observed in artificially created f. sanguinea colonies with wood ant slaves ( czechowski , 1996 ) , in typical ( i.e. serviformica ) slaves seems to be even less probable than adoption of conspecific queens . as already pointed out ( see slave emancipation ) , in the conditions of starvation , younger brood is killed first . kidnapped pupae of typical slaves are generally younger than the slave - maker s own brood . moreover , the ratios of natural slaves in f. sanguinea colonies are usually low , and during spontaneous raids the inflow of robbed pupae into the slave - maker nest is rather gradual . as a consequence , callow slaves ( still inefficient and requiring care ) do not represent an important burden for the mixed colony , differently than in the case of large numbers of artificially introduced wood ant slaves . however , so far nobody studied the functioning of field colonies of f. sanguinea artificially supplied with a surplus of typical slaves . one may also ask if adoption of a homospecific queen represents indeed the final step ( the coping stone ) of a successful slave emancipation . however , if the queen is only distantly related to emancipated ex - slaves , they may gain more in terms of inclusive fitness by remaining queenless and producing their own sons than by taking care of her offspring . in this review , we classified various phenomena documenting the resistance of slaves against their enslavers into four categories : these categories do not overlap , but they are not entirely discrete and at least some of them may be interrelated . for instance , it would be interesting to learn more about possible ultimate consequences of small injuries suffered by slave - makers during aggressive encounters with their slaves . as already pointed out , worker reproduction plays an essential role in the overall production of sexuals in slave - maker ants ( see slave reproduction ) . similarly as other ants , workers of dulotic species also stay inside the nest when they are young and switch to extranidal activities as they grow older ( formica sanguinea : dobrzaska , 1959 ; polyergus lucidus : cool - kwait and topoff , 1984 ; polyergus rufescens : le moli et al . , 1994b ) . ( 2008 , 2012 ) , even relatively small injuries may accelerate the transition of ant workers to extranidal activities and/or induce their switching to risky tasks . injured individuals may even abandon the nest ( hlldobler and wilson , 1990 ) , similarly as other moribund ant workers ( tofilski et al . as extensively documented in many ant species , transition from intranidal to extranidal activities is as a rule accompanied by degeneration of ovaries ( see slave reproduction ) . inverse relationship between fertility / ovarian size and engagement in extranidal activities / slave raids was also reported in the obligate slave - makers p. americanus ( franks and scovell , 1983 ; blatrix and herbers , 2004 ; pohl et al . , 2011 ) and harpagoxenus sublaevis ( bourke , 1988b ) . therefore , the transition to extranidal and/or risky tasks induced by injuries inflicted during seemingly harmless domestic misunderstandings with slaves may ultimately have far - reaching consequences for the slave - maker workers : it may compromise their chances to gain access to reproduction via production of male offspring . occurrence and importance of decreased fertility of slave - makers resulting from injuries inflicted by slaves remains to be checked by future studies . however , it can be reminded that workers of temnothorax ambiguus and t. longispinosus acting as slaves in protomognathus americanus colonies were frequently observed not only to bite p. americanus workers , but also to drag them out of the nest ( alloway and del rio pesado , 1983 ) . it also should be pointed out that various actions undertaken by slaves against their enslavers represent only one of the possible strategies of defence of the host species against the slave - maker . other defence strategies involve enemy recognition , adjustment of the recognition threshold , fighting , inducible aggression , escape from the attacked nest , etc . ( see pamminger et al . , 2013 and references therein ) . a recent experimental work ( pamminger et al . , 2013 ) devoted to the expression of behavioural trait slave rebellion sensu achenbach and foitzik ( 2009 ) ( i.e. killing of the enslaver brood ) , in workers of temnothorax longispinosus acting as slaves of protomognathus americanus , suggests that slaves employ that strategy only if other defence strategies proved to be unsuccessful . interestingly , the lowest survival rate of slave - maker s brood was observed in colony fragments with small numbers of slaves , and not in those with large numbers of slaves in which , theoretically , slaves could rebel most easily as a consequence of their numerical predominance . it would be interesting to check how both these matters look in other colonies of obligate social parasites . in conclusion , we would like to express our hope that observational and experimental data discussed in this review have demonstrated convincingly that ant slaves are not as helpless as they were thought to be and that they can defend themselves against the exploitation by the slave - makers in many different ways .

slavery in ants involves robbing of brood of host ant species and rearing captured individuals in the enslaver s nest . whereas slaves of facultative slave - makers increase the workforce of the colony , in obligate slave - makers presence of slaves is vital for colony survival . until recently , it was generally believed that enslaved workers act solely for the benefit of their social parasite and are wholly lost for their own colony and population . however , evidence that slaves may act also in favour of their own maternal population by engaging in various forms of the so - called slave rebellions is already quite extensive and may be found in both old and recent myrmecological literature , although , unfortunately , these data are often neglected or overlooked . they may be classified into four categories : ( 1 ) acts of physical aggression directed by slaves to slave - makers , ( 2 ) attempts of slaves to reproduce within a slave - maker colony , ( 3 ) sabotage , i.e. activities of slaves leading to weakening of the slave - maker colony and population , and ( 4 ) slave emancipation , i.e. partial or complete self - liberation of slaves from slave - maker colonies . in this review , we present and discuss all these diverse ( often interrelated ) expressions of slave opposition to their enslavers , focussing our discussion on both proximate and evolutionary causation of the discussed phenomena . we also indicate some open questions which remain to be answered by future research .

Introduction Domestic misunderstandings Slave reproduction Sabotage Self-liberation of slaves Emancipation of slaves of Emancipation of slaves of Discussion/Open questions

PMC4095915

partial atomic charges are an integral part of today s molecular mechanic force fields . although force fields that involve more rigorous potential energy functions , such as those including polarizability or multipole electrostatics , are growing in popularity and availability , fixed - charge force fields continue to receive extensive use for modeling condensed - phase systems . the increasing role of computational chemistry in drug discovery and biological modeling often requires the ability to model an arbitrary molecule which has not been explicitly parametrized . while intramolecular and lennard - jones parameters are often easily estimated or transferable for new organic functional groups or heterocycles , the assignment of partial atomic charges is not obvious . if experimental data are available , force fields can be parametrized specifically for these molecules , to reproduce liquid - state properties , but this situation is generally rare for many components of drugs such as heterocycles . alternatively , quantum mechanical ( qm ) calculations can be performed , whereby atomic charges are derived from the wave functions to reproduce gas - phase dipole moments or electrostatic potentials . for use with fixed - charge models in condensed - phase simulations , it is then necessary to scale the gas - phase charges to take into account polarization effects in an average sense . optimization of the scaling factors often focuses on minimizing errors in computed free energies of hydration . in 2004 , charge models 1 and 3 ( cm1 & cm3 ) were tested for their utility in condensed - phase simulations . charges were computed in conjunction with am1 and pm3 semiempirical quantum methods ( creating the cmxa and cmxp models ) and tested for their accuracy to reproduce experimental free energies of hydration for 25 neutral organic molecules . optimal scaling factors of 1.14 and 1.15 were shown to give the lowest mean unsigned errors ( mues ) of 1.03 and 1.13 kcal / mol for cm1a and cm3a , respectively . due to the better performance of the 1.14*cm1a charges in reproducing free energies of hydration for amides , this method was taken to be preferred for simulations of biomolecular systems in explicit water . since the introduction of the cm1a and cm1p charge models in 1995 , successive refinements have been made to produce charge models ( cmx ) 2 , 3 , 4 , and 4 m . these charge models transform mulliken , lwdin , or redistributed lwdin population analyses to derive atomic charges capable of accurately reproducing experimental gas - phase dipole moments . most recently , charge model 5 ( cm5 ) was published , which utilizes gas - phase atomic charges from a hirshfeld population analyses . in that report , hirshfeld charges were shown to be less sensitive to basis set size than lwdin or mulliken charges and more consistent across a wide range of electronic structure methods . these characteristics are maintained in cm5 , allowing the model to consist of only a single set of parameters for various combinations of electronic structure methods and basis sets . cm5 parameters were fit using a test set of 614 neutral molecules containing 26 different elements ( h ca , zn , ge br , and i ) and were extended to every element in the periodic table via recursive fitting ; the mean unsigned error is only 0.26 d. thus , a new charge model has been created for potential use for an enormous range of molecules and applications . with the noted success of cm5 for reproducing gas - phase electrostatic properties , the present investigation was initiated to determine its utility in the condensed phase , explicit solvent simulations . free energies of hydration provide an important measure for testing force field parameters , especially atomic charges , as witnessed by the large number of publications computing this property within the past few years . experimental data are available , and applications to larger systems in chemistry , biology , and medicine are clear . in this report , free energies of hydration are computed initially for an expanded set of 42 neutral , organic molecules to determine an optimal scaling factor for cm5 charges . free energies of hydration obtained using the scaled cm5 charges are compared with those derived from other charge methods including 1.14*cm1a and opls - aa . cm1a and cm5 partial atomic charges were computed via single point ( sp ) calculations on geometries optimized using the boss program with the opls - aa force field . this included the use of the nonbonded parameters for thiols and sulfides , which were updated in 2001 . hirshfeld atomic charges were obtained from a m06 - 2x density function theory ( dft ) single - point calculation using the 6 - 311+g(2df,2p ) basis set in conjunction with a hirshfeld population analysis in gaussian 09 , revision a.02 . cm5 charges were then calculated with the cm5pac software , which uses the hirshfeld charges from the gaussian output file . it should be noted that in the course of completing this study , gaussian 09 revision d.01 was released , which automatically incorporates a cm5 charge calculation with every hirshfeld population analysis . the cm1a and cm5 charges were averaged as usual for equivalent atoms , e.g. , hydrogen atoms in a methyl group . this is necessary for force fields to avoid nonphysical asymmetries , such as different energies for identical rotamers . scaled cm1a and cm5 atomic charges for the molecules considered here are recorded in table s1 of the supporting information . metropolis monte carlo statistical mechanics in conjunction with free energy perturbation ( mc / fep ) calculations were carried out using boss to compute relative and absolute free energies of hydration for the 42 molecules . the fep calculations were based upon the zwanzig equation ( eq 1 ) . the free energy difference between an initial ( a ) and final state ( b ) of a system is calculated as an ensemble average of the potential energy difference between states , sampled at the initial state . to apply fep theory to chemical equilibria , a thermodynamic cycle is employed ( scheme 1 ) in which the initial and final states could be different molecules , conformers , or different force field parameters for the same molecule . conversion of a to b in two media , g1 and g2 , provides the difference in free energy of transfer , gb ga ( scheme 1 ) . a difference in free energies of hydration can be obtained by taking medium 1 as the gas phase and medium 2 as water . to achieve good convergence , windows , categorized by a coupling parameter , i ( eq 2 ) , which scales all geometrical and force field parameters , i , as a transforms to b. such calculations were first performed in 1985.12 in boss , two sampling schemes are implemented for computing free - energy differences , double - wide sampling ( dws ) and simple overlap sampling ( sos ) . both methods deliver accurate results when more than 10 windows are used for molecular transformations involving a single non - hydrogen atom , e.g. , ch3 h. in this work , both sos and dws have been employed , as discussed below . absolute free energies of hydration can be computed in two ways . historically , relative molecular perturbations between two molecules ( a and b in scheme 1 ) have been favored due to their precision and viable computational expense . if changes between a and b are small , then sufficient convergence is achieved using ca . relative free energy values can then be added to a reference absolute free energy to derive new absolute free energies . this works well when only a few transformations are needed to connect to the reference molecule , often methane or benzene ; however , errors accumulate for longer sequences of transformations . the alternative approach is to perform complete molecular annihilations , these calculations make the entire molecule disappear from a medium by scaling all nonbonded force - field parameters to zero . while demanding long molecular dynamics or monte carlo runs and small fep steps , these calculations provide a means to calculate an absolute free energy of solvation for any molecule , independent of previous calculations . the statistical uncertainties in the results can also be systematically reduced by increasing the averaging periods . all mc / fep calculations were carried out with boss and closely followed previously reported procedures . a single solute was placed in a cubic box with 500 water molecules , and simulations were conducted at 25 c and 1 atm in the isothermal isobaric ensemble . solvent solvent and solute solvent cutoff distances , rc , for calculating nonbonded interactions were set at 10 based on all non - hydrogen atom pairs ; i.e. , if any heavy atom heavy atom distance is below rc , the entire intermolecular interaction is evaluated . as before , the intermolecular interactions are quadratically feathered to zero over the last 0.5 based on the shortest heavy atom heavy atom distance , rm ; the scaling factor is ( rc rm)/(rc rl ) where rl = rc 0.5 . all internal degrees of freedom were sampled for the solute , while tip4p water molecules underwent rigid - body translations and rotations . solute and volume moves were attempted every 100 and 3125 configurations , respectively . ranges for translations and rotations of 0.06 and 6.0 for solutes and 0.15 and 15.0 for the water molecules were chosen to produce mc acceptance rates of ca . dummy atoms were used when perturbations were performed between molecules with different numbers of atoms . dummy atoms have zero valued nonbonded parameters , equilibrium bond lengths of 0.30 , and force constants for bond stretching and angle bending as in their real counterparts . statistical uncertainties ( 1 ) were calculated from the batch means procedure using batch sizes of 1 m configurations ; additional tests of precision are described below . for all ghyd calculations , final uncertainties are below 0.25 kcal / mol , which is comparable to typical experimental uncertainties of 0.3 kcal / mol . this work began by computing ghyd for the 42 molecules using 1.14*cm1a and opls - aa charges in the a b manner . twenty - one windows of simple overlap sampling ( 21-sos ) with 5 million configurations of equilibration and averaging ( 5m/5 m ) in the gas phase and 15m/30 m configurations in water were used . previously , 10 windows of dws ( 10-dws ) were carried out with 10m/2040 m configurations in water to compute ghyd for 25 molecules . in view of the success with sos procedures , it was decided to recalculate all 1.14*cm1a and opls - aa results with this sampling method to further assess precision . in this regard , it is informative to analyze results for closed fep cycles ; the sum of the individual free energy changes for each cycle should be zero . as shown in table 1 , the mean signed errors for the cycles using sos with both charge options are near zero ; the mean unsigned errors ( mue ) are less than 0.15 kcal / mol , and the maximum individual errors are no more than 0.24 kcal / mol . absolute free energies of hydration for all 42 molecules were computed in a series of perturbations to methane . methanol and acetone were used as intermediates for many sequences , and aromatic compounds were perturbed to benzene , which was converted to methane . in addition to the relative free energy calculations , full molecular annihilations were performed for all molecules using the 1.14*cm1a charges . annihilations were accomplished by first neutralizing the atomic charges and , in a separate calculation , diminishing the lennard - jones parameters to zero . for all annihilations , 21-sos windows with 8m/8 m configurations in the gas phase and 35m/70 m configurations in water were used . as i progressed from 0 1 , each atom was transformed into a corresponding idealized sp or sp dummy atom , and equilibrium bond lengths were perturbed to 0.30 . unique to the annihilations , the sos sampling led to end - point problems when sampling occurred at i = 1 , reminiscent of earlier findings . in this case , solute and solvent molecules can occupy the same space , which introduces infinite nonbonded energies into the energy difference e in eq 1 . to correct for this , sos sampling was performed up to i = 0.99 , at which point , dws sampling was used to obtain the free - energy result for i = 0.99 1 by running from i = 0.995 to 0.99 and 1.0 . computer time for a single fep window averaged 30 min for the relative perturbations and 70 min for the annihilations in water on an intel core2 quad 3.3 ghz processor . charge perturbations were then executed to determine the ghyd results using the cm5 charges . 1.14*cm1a charges were first perturbed to unscaled cm5 charges ( 1.00*cm5 ) while keeping other force - field parameters constant . thus , 10 windows of double - wide sampling were used featuring 5 million configurations for equilibration and averaging for each window in the gas phase and 10m/15 m configurations in water . the cm5 charges were subsequently scaled in separate fep calculations from 1.00*qcm5 to 1.50*qcm5 in steps of 0.01 , where qcm5 represents an atomic charge . absolute free energies of hydration were computed for each scaling , and the optimal scaling factor was chosen to minimize the mean unsigned error for all 42 molecules . absolute free energies of hydration were computed using two cmx charge models and the opls - aa force field for the initial set of 42 molecules . the absolute free energy of hydration of methane was computed to be 2.51 0.07 kcal / mol via annihilation with the 1.14*cm1a charges . this value is in good agreement with previous results of 2.32.6 kcal / mol for opls - based models and experiment ( 2.01 kcal / mol ) . all other absolute free energies of hydration were anchored by this value via relative fep transformations . in summary , a mean unsigned error of 1.17 kcal / mol is obtained for the 42 absolute free energies of hydration with the 1.14*cm1a charges . an optimal scaling factor of 1.27 was determined for the cm5 charges , which yields a mue of 1.10 kcal / mol for ghyd . table 2 compares the previous and recomputed 1.14*cm1a results ; table 3 records the full results for the 1.14*cm1a and 1.27*cm5 free energies of hydration . a more thorough analysis is made in the discussion section and focuses on comparing the 1.14*cm1a and 1.27*cm5 results . in addition , results for 44 relative free - energy calculations , ghyd , which yielded mues of 1.08 and 0.66 kcal / mol using the 1.14*cm1a and opls - aa charges , are listed in table s2 . from ref ( 13 ) : 8m/8 m gas - phase and 10m/2040 m aqueous - phase configurations . this work : 5m/5 m gas - phase and 15m/30 m aqueous - phase configurations . results obtained from a series of fep / sos calculations ; computed uncertainties are less than 0.25 kcal / mol . in 2004 , the optimal scaling factor of 1.14 for cm1a charges was determined from fitting to free energies of hydration for 25 molecules using series of fep calculations with 10 windows of dws . the cm1a results for these molecules were recalculated in this work with 21 windows of sos , and then the coverage was expanded to 42 molecules . overall , the dws and sos results are mostly the same within the statistical limits of about 0.3 kcal / mol . improvements of around 0.50 kcal / mol or greater , nearer experimental values , are noted for methanethiol and methyl acetate , while reductions in accuracy of 0.50 kcal / mol or greater are found for acetonitrile , acetone , and ( e)-n - methylacetamide . the variations in the results are in - line with previous findings ; they are attributed to the differences in numbers of windows and run lengths , which favor the 21-sos procedure . the net result is an insignificant change in the overall mue from 1.03 to 1.06 kcal / mol . additional problem cases are methanethiol , aniline , and pyridine with errors around 2 kcal / mol . expanding the calculations from 25 to 42 molecules increases the mue from 1.06 to 1.17 kcal / mol using the 1.14*cm1a charges ( table 3 ) . the mue was not helped by the addition of nitrobenzene and the methylanilines , which continue the pattern of being computed to be too well hydrated with an error now of nearly 4 kcal / mol for nitrobenzene . for the added sulfur - containing molecules , the ghyd for dimethyl sulfide is too positive by 2.79 kcal / mol ; however , the errors for thiophenol and thioanisole are less than 0.6 kcal / mol . the errors for most of the other new additions are under 1.5 kcal / mol ( propene , methyl formate , fluorobenzene , bromobenzene , benzonitrile , acetophenone , -methylstyrene , and methyl benzoate ) . the error for acetaldehyde is 1.57 kcal / mol , and it is 2.77 kcal / mol for dimethyl sulfoxide . though the error for acetamide is only 0.43 kcal / mol , the newly added benzamide is computed to be too well hydrated by 2.25 kcal / mol . the results are plotted in figure 1 ; a linear fit gives an r of 0.87 for the correlation of the computed and experimental data . correlation between experimental and computed free energies of hydration ( kcal / mol ) : 1.14*cm1a results ( top ) , 1.27*cm5 results ( bottom ) . the solid line shows the ideal y = x line ; the dashed line represents the best fit line for the computed data . the optimal scaling factor for the cm5 charges turned out to be 1.27 to minimize the mue for the computed free energies of hydration . the 1.27*cm5 results in table 3 lead to mean signed and unsigned errors of 0.49 and 1.10 kcal / mol , respectively . the mue is a little lower than for the 1.14*cm1a results ; however , the r for the linear fit to the experimental data in figure 1 improves to 0.95 from 0.87 . it may be noted that the choice of the cm5 scaling factor benefited from use of the results for the full set of 42 molecules , while the cm1a scaling factor was derived from the results for the original 25 molecules . for the 25 molecules , the mue is 0.96 kcal / mol using the 1.27*cm5 charges , which is again a little lower than the mue of 1.06 kcal / mol with the 1.14*cm1a charges . in comparing several electrostatic - potential and am1-based charge models , mobley and co - workers suggested that a root - mean - square error of ca . 1.0 kcal / mol may be the current limit for fixed charge models in predicting free energies of hydration . it is apparent that some functionality is not well represented by the single charged site per atom model . as discussed more below , the principal problems with the 1.27*cm5 charges occur for sulfur and halogen containing molecules . the cmx results are also compared with published data from molecular dynamics calculations using am1-bcc / gaff charges in tip3p water and opls_2005 charges in spc water in table 3 . although results from other methods exist for comparison , the chosen ones are viewed as relatively accurate . the mean unsigned errors for the cmx models fall between those for the opls_2005 and am1-bcc / gaff methods . both cmx methods have signed errors less positive than the opls_2005 and gaff results . furthermore , the r values for the opls_2005 and gaff results are 0.96 and 0.92 versus the 0.87 and 0.95 for 1.14*cm1a and 1.27*cm5 . the results can be easily biased by selective addition and removal of compounds , e.g. , removal of nitro compounds for 1.14*cm1a and dmso for 1.27*cm5 . completion of the missing numbers in table 3 , e.g. , for dmso , the methyanilines , and thiophenol with opls_2005 , can also be expected to affect the results , adversely in this case . overall , the results in table 3 for the different methods can be viewed as similar and reflecting the 1 kcal / mol limit for general charge methods . dipole moments were calculated for all molecules with the 1.14*cm1a , 1.27*cm5 , and 1.00*cm5 charges ( table 4 ) . the charge scaling for neutral molecules is needed to incorporate solute polarization in an average sense for condensed phase modeling with fixed - charge force fields . as shown in table 4 , the unscaled cm5 charges yield excellent values for gas - phase dipole moments with a mue of only 0.16 d. it is noted that some differences occur between 1.00*cm5 dipole moments computed here and those in the original cm5 report . the differences of 0.30 d or more for dmso , nitroethane , nitrobenzene , and n , n - dimethylacetamide can be attributed to the variations in optimized geometries and electronic structure methods used for the cm5 charge computations ( see tables s4 and s5 ) . it is also apparent that in comparing the 1.14*cm1a and 1.27*cm5 dipole moments , the larger scaling factor for the cm5 charges does not always lead to higher dipole moments . the most striking exceptions are for amines and nitro compounds . the lower dipole moments for methylamine and dimethylamine with the 1.27*cm5 charges do correlate with the overly positive computed free energies of hydration ( table 3 ) , while the dipole moments for the nitro compounds are clearly too large with the cm1a charges and contribute to the far too favorable computed ghyd values . the 1.14*cm1a charges for nitrogen and oxygen are 0.68 and 0.46 , while they are 0.54 and 0.37 with opls - aa and 0.11 and 0.23 with 1.27*cm5 . however o bonds in dimethyl sulfoxide , too large dipole moments , and too negative free energies of hydration . mean unsigned errors in ghyd for compound classes are listed in table 5 to help identify systematic strengths and weaknesses for the charge models . the 1.27*cm5 model yields lower average errors than the 1.14*cm1a alternative for most classes except hydrocarbons and halogen and sulfur containing molecules . ghyd for less polar benzene derivatives is uniformly too positive by 12 kcal / mol with 1.27*cm5 ( table 3 ) , which contributes significantly to the errors for hydrocarbons and halides . the clear failing of the 1.14*cm1a charges is nitro compounds , while both cmx approaches show significant deviations for sulfur - containing molecules . dimethyl sulfoxide ( dmso ) is particularly problematic with errors of 2.8 and 3.6 kcal / mol . unfortunately , additional sulfoxides and sulfones could not be tested due to a lack of experimental data . the 1.27*cm5 method shows higher unsigned errors for the sulfur - containing molecules due to its larger deviations for thiophenol and thioanisole . there is no problem with the computed dipole moments ( table 4 ) , so it is in the details of the charge distributions . for thiophenol , the thiol sulfur and hydrogen have charges of 0.14 and 0.16 with 1.14*cm1a and 0.21 and 0.17 with 1.27*cm5 . these differences are not striking , though the larger charge on sulfur would diminish the hydrogen - bond donating ability of the thiol group . however , there is a general pattern that the charges on the carbons and hydrogens in phenyl rings are smaller in magnitude with 1.27*cm5 than with 1.14*cm1a by ca . remarkably , this translates to less favorable free energies of hydration for benzene , toluene , and -methylstyrene by 1.21.5 kcal / mol with 1.27*cm5 ( table 3 ) and presumably a similar offset for the other substituted benzenes . additional insights can be obtained by considering computed interaction energies for some hydrogen - bonded complexes in the gas phase . results are reported in table 6 for nine representative complexes using opls - aa and the opls / cmx models . opls - aa interaction energies correlate well with high - end ab initio results and are given for comparison . the variation of the results for each of the nine complexes is notably small , within 1 kcal / mol for most cases . for both methanol and methanethiol , water is preferred as the hydrogen - bond donor by about 1 kcal / mol , and the interactions are more favorable with the 1.27*cm5 charges than 1.14*cm1a by a few tenths of a kilocalorie per mole . however , in both cases , the free energies of hydration are too positive , though the results are improved with 1.27*cm5 ( table 3 ) . cbs - q results for the four complexes of methanol and methanethiol in order are 5.69 , 5.27 , 3.90 , and 2.50 kcal / mol , and mp2/6 - 31++g(2d(x+),p ) gives very similar values . thus , the raw interaction energies are not overly weak , and the conclusion is that the effects of polarization in water are not sufficiently compensated for by the charge scaling in these cases . for dimethyl ether and dimethyl sulfide , the best ab initio and dft results indicate interaction energies with water of about 5 and 4 kcal / mol , while the scaled cmx results in table 6 are stronger and weaker , respectively . the consequences are reasonably computed ghyd values for dimethyl ether , but overly positive results for dimethyl sulfide by about 2.7 kcal / mol . for dimethyl sulfide , the sulfur atom is not a sufficiently good hydrogen - bond acceptor with the cmx charge models . the larger size and increased polarizability of sulfur relative to oxygen likely make its condensed phase electrostatics more difficult to model with fixed charge approximations . the free energies of hydration of aliphatic amides , a functional group particularly important for biologically related studies , are well reproduced by the 1.14*cm1a model ( table 3 ) . with the 1.27*cm5 charges , the ghyd values for acetamide and z - nma are too favorable by 2.4 and 1.1 kcal / mol . for the water acetamide complexes in table 6 , the hydrogen bonds to the carbonyl oxygen are significantly stronger with 1.27*cm5 than with opls - aa or 1.14*cm1a . if the scaling factor for the cm5 charges were reduced to 1.20 , the ghyd errors would decrease to 0.58 kcal / mol for both acetamide and z - nma . benzamide turns out to be too well hydrated for both charge models with errors of 2.3 and 1.8 kcal / mol ( table 3 ) . in general , the 1.14*cm1a charges for amide nitrogens seem large in magnitude at ca . e with 1.27*cm5 and 0.76 and 0.50 e for primary and secondary amides with opls - aa . it is also noted that both charge models predict poorer hydration of the e conformer of nma by about 1.2 kcal / mol , while the available experimental data indicate there is no e / z differential . this discrepancy is a recurrent one going back to the earliest fep calculations for amide e / z interconversions . the experimental measurements are challenging owing to the low population of the e conformer . as an additional check of the reported free energies of hydration in table 3 , full molecular annihilations were performed for all molecules . for molecules with up to 10 heavy atoms , sufficient convergence required sampling roughly three - times the number of configurations for a typical single heavy atom perturbation . the free energy results from the sequential and annihilation procedures with the 1.14*cm1a charges are compared in figure 2 ; a similar plot for the 1.27*cm5 charges is shown in figure s2 . excellent correlations are observed for both cmx methods ; the linear fits have slopes of 1.0 , intercepts near 0.0 , and r values of 0.995 . the consistency confirms the precision of the results in table 3 , and it also provides validation of the annihilation methodology itself . this permits more automated calculations of free energies of hydration without the need to devise a mutation sequence . comparison of computed free energy of hydration ( kcal / mol ) using a series of fep calculations or annihilation with 1.14*cm1a charges . an additional 20 neutral , organic molecules were studied by molecular annihilation to ascertain the validity and transferability of the 1.27 scale factor . molecules were chosen to exhibit molecular diversity and for which independent free energies of hydration have been previously computed . both e and z isomers have been included for n - methylformamide , although no am1-bcc / gaff data are available for this molecule . the free energies of hydration are reported in table 7 ; scaled cmx charges are reported in table s7 . the mue with the 1.27*cm5 charges is 1.31 kcal / mol , while it is again higher , 1.47 kcal / mol , for the 1.14*cm1a charges . am1-bcc / gaff has the lowest mue for this set , though based on the error of 1.7 kcal / mol for n - methylacetamide in table 3 , inclusion of results for nmf could increase the mue by ca . the results indicate that average errors of 1.01.5 kcal / mol can be expected for application of these charge models to other small molecules . for the 1.27*cm5 charges , aliphatic amines are under - hydrated with all methods , while the performance for amides in tables 3 and 7 is notably improved with the cmx charges . the cumulative errors for polypeptides can be expected to be large with many charge models , which has implications for modeling protein stability and folding . results obtained from a fep annihilations ; computed uncertainties are less than 0.25 kcal / mol . a promising attribute of charge model 5 is the ability to provide atomic charges for any electronic structure method in conjunction with a hirshfeld population analysis . for the present context , tests were performed to analyze the transferability of the 1.27 scale factor for alternative density functional methods . specifically , m06 , m06-l , b3lyp , and mpw1pw91 were utilized to derive cm5 charges for a subset of 10 molecules . the same procedure for obtaining the cm5 charges was followed as before , only the m06 - 2x method was replaced by the alternatives . absolute free energies of hydration were determined by charge perturbations from 1.14*cm1a to the new 1.27*cm5 charges for each molecule in the gas phase and in water . the computed ghyd was added to the ghyd(1.14*cm1a ) for each molecule to determine the new ghyd(1.27*cm5 ) . the final results are summarized in table 8 including the standard deviation for the five computed values for each molecule . the effects of changing the basis set on the ghyd results were also examined with the m06 - 2x functional . three additional basis sets , 6 - 311g(d , p ) , 6 - 31+g(d , p ) , and 6 - 31g(d ) , were used to compute new 1.27*cm5 charges . the absolute free energies of hydration for the 10 molecules were again computed by charge perturbations . overall , little effect is found on the computed free energies of hydration from changes in the density functional method or basis set . the standard deviations average below 0.20 kcal / mol , which is near the level of the statistical uncertainties in the results . the conclusion is that the scale factor of 1.27 for cm5 charges is valid for a wide range of dft methods . the ease of access to cm5 charges from the gaussian 09 program coupled with its implementation of continuum solvation models raised the possibility of combining these methods to compute solvent - polarized charges . such charges might be appropriate for direct use in molecular mechanics based fluid simulations . single - point calculations were carried out for the 42 molecules using m06 - 2x/6 - 311+g(2df,2p ) and the conductor - like polarizable continuum model ( cpcm ) with the dielectric value for water , 78.3 . the hirshfeld charges were computed and processed with cm5pac to derive new cm5 charges ( cm5/cpcm ) . perturbations from the 1.14*cm1a to the cm5/cpcm charges were then performed to obtain the ghyd values for the cm5/cpcm model . the resultant ghyd mue for the cm5/cpcm approach is 2.11 kcal / mol . this represents improvement from the mue of 3.08 kcal / mol with unscaled cm5 charges ; however , it is not competitive with the mue of 1.10 kcal / mol using the 1.27*cm5 charges ( table 3 ) . table s8 reports the cm5/cpcm enhanced dipole moments and resultant free energies of hydration . cm5/cpcm dipole moments are significantly different from the hirshfeld / cpcm dipole moments due to empirical adjustments by cm5 . although the cm5/cpcm dipole moments are enhanced by about 20% over cm5 and gas - phase experimental values , this is insufficient for optimal modeling of chemistry in aqueous solution with fixed - charge force fields . since the average errors for free energies of hydration with the 1.14*cm1a and 1.27*cm5 methods are similar and near the 1 kcal / mol level , both approaches are reasonable for use in simulations of aqueous systems . a simple welch two - sample t test comparing cm1a and cm5 signed errors of the training set produces a p - statistic of 0.328 and a t - statistic of 0.985 . while no statistical significance can be made for the training set based on this t test , correlation with experimental data and precision are improved with the 1.27*cm5 charges ( figure 1 , table s3 , figure s3 ) . a smaller standard deviation of unsigned errors for 1.27*cm5 versus 1.14*cm1a , 0.78 vs 0.89 kcal / mol , respectively , and a repeated reduced mean unsigned error in the validation set , 1.31 vs 1.47 kcal / mol , respectively , support this conclusion and highlight cm5 improvements as important . first , the cm1a method is we d to semiempirical am1 wave functions , while the cm5 charges can be derived from hirshfeld charges obtained from a wide variety of dft methods and basis sets . the hirshfeld charges , cm5 charges , and , as shown here , the resultant ghyd values are insensitive to reasonable choices for the dft method and basis set . second , the variety of elements is limited with am1 , while the cm5 model has been parametrized for all atoms of the periodic table . third , the cm5 method yields more reasonable charges for buried atoms , e.g. , amide nitrogen atoms as noted above , and less dependence on conformation than alternative charge models . finally , incorporation of cm5 charges into molecular mechanics software is straightforward given access to gaussian 09 . for boss , the subroutine that requests quantum mechanics calculations was modified to invoke an external call to a linker script , which interfaces with gaussian . the script ( i ) creates a gaussian input file using solute coordinates and variables specifying the calculation type from boss , ( ii ) initializes and executes a single - point calculation in gaussian , and ( iii ) reads the output and loads the total energy and cm5 charges back into boss . though the present testing has only considered a few elements , optimism can be expressed for broader utility given the excellent performance of cm5 for dipole moments for a wide range of molecules . the recently reported cm5 charge model was tested for performance in computing free energies of hydration in conjunction with the opls - aa force field for the remaining force - field parameters . an optimal scaling factor of 1.27 for the cm5 charges was determined to minimize the error in the computed free energies of hydration for 42 neutral organic molecules . the mean unsigned error of 1.10 kcal / mol from the 1.27*cm5 calculations compares favorably with the error of 1.17 kcal / mol from the well - established 1.14*cm1a alternative . both approaches give larger errors for sulfur - containing molecules , and the 1.14*cm1a method makes nitro groups much too polar . additional attractive features of the cm5 approach include the transferability of the 1.27 scale factor to a wide range of density functional methods and basis sets , coverage of all elements of the periodic table , and the availability of cm5 charges from the gaussian program . use of the scaled cm5 charges in conjunction with fixed - charge force fields should provide a viable approach to modeling an enormous range of molecular systems in solution .

the recently developed charge model 5 ( cm5 ) is tested for its utility in condensed - phase simulations . the cm5 approach , which derives partial atomic charges from hirshfeld population analyses , provides excellent results for gas - phase dipole moments and is applicable to all elements of the periodic table . herein , the adequacy of scaled cm5 charges for use in modeling aqueous solutions has been evaluated by computing free energies of hydration ( ghyd ) for 42 neutral organic molecules via monte carlo statistical mechanics . an optimal scaling factor for the cm5 charges was determined to be 1.27 , resulting in a mean unsigned error ( mue ) of 1.1 kcal / mol for the free energies of hydration . testing for an additional 20 molecules gave an mue of 1.3 kcal / mol . the high precision of the results is confirmed by free energy calculations using both sequential perturbations and complete molecular annihilation . performance for specific functional groups is discussed ; sulfur - containing molecules yield the largest errors . in addition , the scaling factor of 1.27 is shown to be appropriate for cm5 charges derived from a variety of density functional methods and basis sets . though the average errors from the 1.27*cm5 results are only slightly lower than those using 1.14*cm1a charges , the broader applicability and easier access to cm5 charges via the gaussian program are additional attractive features . the 1.27*cm5 charge model can be used for an enormous variety of applications in conjunction with many fixed - charge force fields and molecular modeling programs .

Introduction Computational Details Results Discussion Conclusion

PMC3610307

although it has constant mean and median the menopause in the recent century approves its occurrence as a phenomenon affected by ethnic and genetic factors , and environmental factors ( 25 ) . in some studies the role of some associated factors in defining menopausal time have been investigated in various nations which showed changes in life style and social status may affect menopause age as an inevitable occurrence in women 's life ( 68 ) . according to the previous studies , menopause age in iran is relatively within earlier ages compared to other countries ( 913 ) . these differences imply that menopause age may be affected by environmental factors ( 4 ) . in spite of recent improvements in diagnosis of associated hormonal changes in menopause and accessible treatments for its symptoms , based on the previous studies , factors such as smoking , nutrition , socioeconomic status , fertility rate and anthropometric factors can affect menopause age ( 416 ) ; however there is not a general consensus about all of them , but most of the studies confirmed role of smoking . some chronic diseases such as osteoporosis , cardiovascular diseases and some cancers are related to continuation of ovarian function ; in the other hand early menopause can increase risk of cardiovascular diseases and osteoporosis , but endometrial and breast cancers have increased incidence rates in higher menopause ages ( 12 ) . thus the age of menopause as a result of ovarian dysfunction is important in epidemiology of these diseases . in fact , the importance of menopause in family and society health is due to its complications in women and its health - threatening problems ; however these complications are preventable . considering to physical , emotional and psychiatric complications of early and delayed menopause , it is necessary to recognize associated factors of menopause to provide appropriate awareness and treatment before and after menopause . during 2009 in this cross - sectional population - based study , 804 menopausal women in gorgan , north - east of iran were selected via two - stage sampling method . in gorgan there are 8 health - treatment centers and 12 health bases . first using cluster sampling method , health centers and bases were selected and then required samples were recruited from each region via random sampling method . ethical approval was obtained by research ethics committee of golestan university of medical sciences ( goums ) . data was gathered using a structured questionnaire which its content validity was approved by expert opinion ( gynecologists opinion and ) . in a pilot study on 30 subjects , a test - retest analysis with an interval of 10 - 14 days approved an excellent reliability with a kappa coefficient of 0.82 and a correlation coefficient of 0.78 . the questionnaire consisted of information about demographic data ( age , sex , ethnicity , educational level , etc ) , socioeconomic characteristics , menstrual and fertility characteristics and climacteric complaints . the study included only women who had undergone natural menopause and with their last menstrual bleeding at least one year in past and they settled in gorgan at least for one year and experienced menopause in this city at age less than 60 years and were local resident . whenever a woman was not willing to participate in the interview she replaced with a woman in the same place . if person reported lack of activities such as running , jogging , swimming , bicycling or walking at least 30 minutes at a time per day , we classified them as inactive person . the body mass index was calculated ( bmi = weight ( kg ) / height ( m ) ) . smoking status ( current smoker vs. non smoker ) and physical activity were considered as binary variables . early menopause was defined as menopause age with less than 40 years and menopause age more than 50 years considered as late menopause . in order to construct socioeconomic status variable using educational level , woman and her husband 's occupation and family income principal component analysis ( pca ) persons were categorized into low , intermediate and high socioeconomic status using 25% and 75% percentiles . results were reported as mean standard deviation ( sd ) or median for quantitative variables and percentages for categorical variables . data were analyzed by student 's t- test , anova ( using tukey as post hoc ) , chi square and pearson correlation test . a p - value less than 0.05 was considered as statistically significant . all the statistical analyses were performed by spss version 16 ( spss inc , chicago , il , usa ) for windows . results were reported as mean standard deviation ( sd ) or median for quantitative variables and percentages for categorical variables . data were analyzed by student 's t- test , anova ( using tukey as post hoc ) , chi square and pearson correlation test . a p - value less than 0.05 was considered as statistically significant . all the statistical analyses were performed by spss version 16 ( spss inc , chicago , il , usa ) for windows . in this study 804 women at menopausal age were investigated including 127 sistanian women ( 15.8% ) , 56 turkaman women ( 7.0% ) , 606 fars women ( 75.3% ) and 15 cases from other ethnicities ( 1.9% ) . the mean age of menopause was 47.64.45 years and the median age 48 years ( range 30 - 60 years ) . early and delayed menopauses were detected among 10.1% and 19.25% of women , respectively . regarding the occupation , 91.9% of the women were housekeepers ( n=734 ) , 3.0% ( n=24 ) , had a job and 5.1% were retired ( n=41 ) . in this study , the association between body mass index ( bmi ) and age of menopause was investigated . results showed no significant relation between the age of menopause and bmi ( p > 0.05 ) . the association between menopausal age anddemo - socio characteristics among women . in order to study the association between socio - economic status and age of menopause , socio - economic status was determined through principal component analysis ( pca ) . using this method , five dependent variables including female educational level ( fe ) , male educational level ( me ) , female occupation ( fo ) , male occupation ( mo ) , and family income ( fi ) were summarizedin one main component presented below : principal component= 0.86 fe+ 0.85 me + 0.67 fo + 0.54 mo + 0.75 fi this component explained 72.0% of the variance . pearson correlation test showed an increase in this component which was associated with the decrease in age of menopause , but the correlation coefficient was not significant ( p>0.05 ) . using the 25and 75 percentiles of the principal component of socio - economic status , women in our study were categorized into three groups of low , intermediate , and high socioeconomic status . there was no significant difference in the age of menopause between these 3 groups ( p>0.05 ) . the association between family income and the age of menopause was separately examined , which revealed no significant association ( p>0.05 ) . although the correlation in women with a history of smoking had lower menopause age , the association was not significant , neither with cigarette nor with tobacco smoking . no significant relation was found between menopause age and physical activity ( p>0.05 ) ( table 1 ) . in this study a significant association was not found between menopause age , age of first delivery and menopause age ( p > 0.05 ) . an increase in number of pregnancies and deliveries associated with higher menopause age . nonetheless , last pregnancy in older ages was significantly associated with higher menopause ages ( p < 0.01 ) . in this study the mean age of menopause age was non - significantly lower in those who consumed oral contraceptive pills ( ocp ) compared to those who did not ( p>0.05 ) . our results showed that menopause age was not significantly different between women with sudden changes in menstrual cycles and those with gradual changes in menstrual cycles ( p>0.05 ) . no statistical association was found between mean menopause age and menstrual duration , history of miscarriage , history of dilation and curettage , marriage age and duration of breast feeding , regular menstrual cycle and twin pregnancy ( p>0.05 ) , ( table 2 ) . in this study the mean age of menopause was 47.64.45 years and the median age 48 years , which was relatively lower compared to other provinces studies in iran ( 4 , 6 , 7 , 11 , 12 , 14 ) and some other countries ( 8 , 1722 ) . the mean menopause age in this study was quite similar to the results reported in a study in birjand , iran ( 47.19 years ) ( 13 ) . these two provinces are geographically neighbors , which implies the impact of cultural and climate determinants on the age of menopause . the means menopause age reported from south korea ( 21 ) and pakistan ( 23 ) were lower than our findings . thus , the impact of climate and cultural determinants should be considered in this context . our results revealed no statistical association between bmi and menopause age ; however women with lower weight had earlier menopause . meanwhile , there was no significant relation between height and weight with menopause age , which was consistent with a study by reynolds et al in lebanon ( 24 ) . however , other studies showed opposite results ( 11 , 22 , 25 , 26 ) . the discrepancy is probably due to differences in socioeconomic status , since most obese women have higher socio - economic status and hence the menopause age is higher among them . an association might be expected between married and unmarried groups ( 27 ) , but the limited number of the unmarried group ( n=3 ) made the evaluation impossible . these results congruent with some previous study in iran ( 4 , 7 ) and some other countries ( 15 , 16 ) . based on finding , it is seen that the women who had low household income had earlier age at natural menopause than the others , and these differences were close to be statistically significant ( p=0.07 ) . in some studies , it was shown that the low socioeconomic level may be effective on earlier age at menopause ( 28 , 29 ) . the variable that we created as an indicator of socio - economic status in this study did not show any significant association with menopause age ( p>0.05 ) . the increase in the values of this variable was not associated with an increase in menopause age . this result was not in accordance with previous studies ( 6 , 8 , 19 , 21 , 26 ) . the components of socio - economic status in our study were not the same as those used in previous studies and this might explain the observed differences to some extent . although women who were current or past smokers had lower menopause age , but no significant association was found between menopause age and smoking ( cigarette or tobacco ) . pokoradi and et al found that current smokers reached menopause at a significantly younger age than nonsmokers ( 30 ) . only four women in our study had a history of smoking , which made it hard to compare our results with other studies . our results in accord with previous findings illustrated no relation between menopause age and physical activity ( 6 , 12 , 22 , 27 ) . in contrast , one study in turkey has reported significant correlation concerning this relation ( 31 ) but studies concerning this relation are controversial ( 32 , 33 ) . our study confirmed a significant association between menopause age and age of last pregnancy ; however , higher age of last pregnancy was associated with delayed menopause . this finding was similar to those previously found in shiraz ( 9 ) and birjand ( 13 ) , but different from a number of studies in italy ( 22 ) and poland ( 20 ) . moreover , there was a significant association between number of pregnancies and deliveries with menopause age ; higher number of pregnancies and deliveries was associated with higher age of menopause . this finding was similar to studies in yazd ( 7 ) , and poland ( 20 ) . excess activity of ovaries and uterine and breastfeeding which are related to progesterone and estrogen levels can affect menopause . the mean menopause age was not significantly different between participants who consumed ocp and those who did not . in contrast , previous studies in birjand ( 13 ) , ahvaz ( 11 ) , shiraz ( 19 ) and poland ( 20 ) had reported that ocp consumption can be associated with delayed menopause age . menstrual cycle duration , history of miscarriage , history of curettage , age of marriage , and duration of breastfeeding were found to have no significant association with menopause age , which is in accord with previous studies in shiraz ( 9 ) , yazd ( 7 ) , meshhad ( 12 ) and birjand ( 13 ) . a previous study in poland ( 20 ) showed that longer menstrual cycle was associated with earlier menopause . in our study , women with longer menstrual cycles had earlier menopause , but this association was not statistically significant . it should be mentioned that some studies have showed that women with shorter menstrual cycle have earlier menopause by at least 1 - 2 years . in summary , in this study socio - economic status was not found to have any relation with age of menopause , which was in contrast with some previous studies ( 4 , 6 , 15 , 20 ) . in comparison to other provinces and cities in the country , age of menopause thus , genetic and ethnic heterogeneity should be considered as determinants of menopause age which requires further investigation . one of main important limitation of our study was being cross - sectional and recall - bias about the associated factors due to the duration . the mean age menopause of the study participant was lower than other provinces of iran and some other countries . this study illustrated that menstrual and fertility factors have influence on menopausal age while socioeconomic factors were not . therefore , according to the consequences of menopause and lower menopause age , the women in this study environment require special attention .

backgroundconsidering the physical , emotional and psychological complications of early or delayed menopause on womens life , it is necessary to determine associated factors of menopause age . this study designed to determine menopausal age and associated factors in women of gorgan , i.e. the capital of golestan province in the north - east of iran.methodsin this cross - sectional study , 804 menopausal women in gorgan were selected via two - stage sampling method in 2009 . the study included only women who had undergone natural menopause and had their last menstrual bleeding at least one year before . data were gathered through structured questionnaire that included individual characteristics , socioeconomic characteristics , menstrual and fertility characteristics and climacteric complaints . socioeconomic status was defined using principal component analysis . data were analyzed with t - student 's and anova tests using spss version 16 ( spss inc , chicago , il , usa ) for windows.resultsthe mean menopause age was 47.64.45 years with the median age of 48 years . the mean menopause age in women with first pregnancy before 30 years ( 47.584.47years ) , without pregnancy ( 46.264.90years ) and without delivery ( 46.304.47 years ) was significantly lower than others ( p<0.01 ) . the mean menopause age was lower in single women ( 46.62.80 years ) , with low income level ( 47.74.39 years ) and smokers ( 47.64.45 years ) compared to others ; but these differences were not statistically significant ( p>0.05 ) . socioeconomic status was not associated significantly with menopause age ( p>0.05).conclusionthis study illustrated that menstrual and fertility factors have influence on menopausal age while socioeconomic factors were not effective .

Introduction Methods Statistical analysis Results Discussion Conclusion

PMC3237138

for this study , a 31-item self - rated questionnaire was designed . for the same , all the available internet addiction questionnaires were reviewed . in addition , items were generated keeping the icd-10 criteria of substance dependence in mind . this questionnaire was reviewed by a panel of six qualified psychiatrists , including those with a specialist experience of dealing with addictive disorders . in addition , the questionnaire was reviewed by a clinical psychologist and a psychiatric social worker working in this field . all the panelists were requested to critically review the questionnaire for completeness of addiction criteria as per substance dependence syndrome criteria of icd-10 , for completeness of excessive behaviors that could be associated with internet use , and for overlapping questions , if any . after receiving the feedback , a 42-item questionnaire was generated , which was again reviewed by all the panelists for its completeness . no. the questionnaire covered all the icd-10 criteria ( which were covered by 1 - 14 questions of the questionnaire ) and young 's criteria for internet addiction . later , basic demographics ( age , gender , level of education , and occupation ) , basic internet use pattern ( duration of internet use in years , for how long are you using internet daily , number of internet use hours per day , and various purposes for which you use internet ) , and one question tapping family history of substance dependence were added . the questionnaire was sent to 284 persons through e - mail and was given to 38 internet users in person over a period of 2 months by one of the authors ( k.c . ) . of the 284 persons approached through e - mail , 11 e - mails met with failure notice generated by the system ( due to nonexistent / wrong e - mail addresses ) . the subjects were informed about the purpose of the study and were given freedom of choice to respond or not to respond . they were also assured about maintenance of confidentiality and anonymity so that no individual responder could be identified from the collective data . the questionnaire was sent to the subjects one to four times ( those who responded back were not sent reminder mails ) at a weekly interval . it was presumed that the persons who would respond would provide implied consent to participate in the study . the responses on the questionnaire were evaluated on the icd-10 substance dependence criteria and young 's iadq . the data were analyzed by using spss version 14.0 for windows ( spss , chicago , il ) . mean and standard deviation were calculated for the continuous variables and frequencies , and percentages were computed for the discontinuous variables . comparisons were done by using unpaired student 's t test and chi - square test . spearman 's rank order correlation analysis was used to study the relationship between parametric variables ( age , total duration of internet use , duration of daily internet use , time spent in internet use everyday ) and internet addiction . the data were analyzed by using spss version 14.0 for windows ( spss , chicago , il ) . mean and standard deviation were calculated for the continuous variables and frequencies , and percentages were computed for the discontinuous variables . comparisons were done by using unpaired student 's t test and chi - square test . spearman 's rank order correlation analysis was used to study the relationship between parametric variables ( age , total duration of internet use , duration of daily internet use , time spent in internet use everyday ) and internet addiction . of the total 312 persons contacted , 104 ( 33.4% ) responded to the survey . males ( 58.7% ) outnumbered females ( 41.3% ) , and most of them had completed their graduation ( n=101 ; 97.1% ) . half of the sample comprised of doctors , about a fifth ( 20.2% ) of sample comprised other professionals ( nurses , engineers , and scientists ) , and the rest were students ( 29.8% ) . the mean age of the sample was 27.73 years ( sd 5.14 ; range 20 - 49 ) , with females ( mean 24.79 ; sd 4.64 ) being significantly younger ( t - test value 5.56 , p<0.001 ) than males ( mean 29.80 ; sd 4.44 ) . the mean duration of internet use was 73.43 months ( sd 44.51 ) , and this was significantly longer ( t - test value 3.00 ; p<0.001 ) for males ( mean 84.03 , sd 43.75 ) compared to females ( mean 58.39 , sd 41.58 ) . two - thirds ( 65.38% ) of them were using internet on a regular basis for a period of more than a year , and the mean duration of daily internet use was 39.13 months ( sd 35.97 ) and this was significantly longer ( t - test value 3.43 ; p=0.001 ) for males ( mean 48.80 , sd 36.48 ) than for females ( mean 25.41,sd 30.73 ) . the average time spent in internet use was 2.13 h everyday ( sd 1.98 ) , with no significant difference between males ( mean 2.44 , sd 2.17 ) and females ( mean 1.69 , sd 1.60 ) . more than half ( 56.73% ) of the sample was using internet at least for 2 h / day , and the daily internet use of more than half of the sample ( 58.6% ) was in the range of 1 - 3 h. nine ( 8.65% ) subjects had a first - degree relative who was currently using or had used alcohol or any other drug of abuse , leading to marked dysfunction in social and occupational functioning . when the respondents were asked to rank the purposes of their internet use on a scale of 1 ( i.e. , most common purpose for use ) to 5 ( i.e. , least common purpose for use ) , the most common purpose of use was educational for two - thirds ( 62.5% ) of the sample . other common purposes of use were chatting ( 50.9% ) , recreational ( 37.5% ) , and e - mailing ( 33.6% ) . table 1 shows the items of internet use questionnaire grouped under suitable criteria of icd-10 substance dependence . the five most commonly endorsed items were as follows : the need to use the internet everyday ( 53.8% ) , internet use helping to overcome bad moods ( 50% ) , staying online longer than one originally intends to ( 43.3% ) , eating while surfing ( 24% ) , and physical activity going down since one has started using the internet ( 22.1% ) . items of internet use questionnaire for assessing the icd-10 criteria , any subject who answered positively on any 1 item covering a particular criterion was considered to be fulfilling that criterion . accordingly , the icd-10 dependence criteria that were fulfilled by about half of the sample were persistence despite harm ( 54.8% ) , craving ( 53.8% ) , preoccupation with use ( 51% ) , and dyscontrol ( 49% ) . the icd-10 criteria of withdrawal ( 20.2% ) and tolerance ( 14.4% ) were fulfilled by only a few subjects . overall , 51.9% of the sample met any 3 of the total 6 icd-10 criteria together , and hence could be considered as cases of internet addiction according to the definitional equivalent of the icd-10 diagnostic guidelines for substance dependence . from the 31 items of the internet use questionnaire , items similar to young 's iadq [ table 2 ] were chosen ; the prevalence of internet addiction as per iadq ( i.e. , had at least five criteria out of eight ) was only 3.8% . young 's criteria for internet addiction significant association was seen between dependence as per icd-10 criteria and male gender ( spearman 's rank order correlation 0.325 ; p<0.01 ) , duration of daily internet use ( spearman 's rank order correlation 0.228 ; p<0.05 ) , and average time spent in internet use everyday ( spearman 's rank order correlation 0.273 ; p<0.01 ) . significant association was seen between dependence as per icd-10 criteria and male gender ( spearman 's rank order correlation 0.325 ; p<0.01 ) , duration of daily internet use ( spearman 's rank order correlation 0.228 ; p<0.05 ) , and average time spent in internet use everyday ( spearman 's rank order correlation 0.273 ; p<0.01 ) . this was a preliminary survey to evaluate the internet addiction and impact of internet use in a purposive indian sample . in the present study , available data from the community and online surveys as well as clinical samples[3338 ] suggest that internet addiction appears to have a male preponderance . in a finnish study , men had significantly higher mean score on the internet addiction test ( iat ) than did women . a study that included adolescents revealed 50% increased odds for males to be addicted to the internet ( or=1.5 , 95% ci=1.12.2 ) when compared with females . it is suggested that the gender distribution may be explained by the fact that men are more likely to express interest in games , pornography , and gambling activities that have all been associated with problematic internet use . studies have found that the internet addiction usually manifests itself in the late 20s or early 30s . the mean age ( 27.735.14 years ) of the subjects in the index study also support the same . black et al . reported a lag time of 11 years from the initial use to the problematic use . other studies have also reported a 3-year history of problematic use at the time of interview . subjects in the present study had a lag period of approximately 6 years ( 73.4344.51 months ) from the initial computer use to the index assessment for internet addiction , which is in the range of that reported in the other studies . subjects in the present study used internet for 2.13 h / day , which translates into 14.91 h / week , which is lower compared with that reported by other studies . in the index study the most commonly endorsed items on internet use questionnaire were the need to use the internet everyday ( 53.8% ) , internet use helping to overcome bad moods ( 50% ) , staying online longer than one originally intends to ( 43.3% ) , eating while surfing ( 24% ) , physical activity going down since one has started using the internet ( 22.1% ) , using internet to escape from problems ( 18.3% ) , becoming upset on attempting to cut down internet use ( 17.3% ) , surfing being responsible for spending less time with family members ( 16.3% ) , surfing causing a change in sleep pattern ( 16.3% ) , and so on . previous studies from other countries have also reported similar positive and negative impact of internet use as found by us . a study from india too reported that those who were dependent on internet would delay their work to spend time online , lose sleep due to logging in till late night , feel lonelier , and feel life would be boring without the internet as compared with nondependent subjects . the studies that have estimated the prevalence of internet addiction have come up with varying results ( 0.9 - 38% ) depending on the criteria used and the sample studied . in a methodologically rigorous study that involved a random telephone survey of 2513 adults aged 18 years and older and employed four criteria sets , prevalence rates varied from 0.3 to 0.7% . the only published study from india , which evaluated internet addiction by using davis online cognition scale in school - going children aged 16 - 18 years , reported a prevalence of 18% . the reasons for huge variation in the prevalence rates could be as follows : difficulty in conceptualizing internet addiction , heterogeneity of population studied , lack of availability of standard diagnostic criteria , studies failing to differentiate between essential and nonessential internet use , and nonconsideration of psychiatric comorbidity in some of the studies.[13163336384548 ] more surprisingly , the studies that have used young 's iat have also come up with varied prevalence rates . this suggests that a single instrument can not reliably pick up the cases of internet addiction . another fact that contributes to a wide variation in the prevalence rates is the fact that internet addiction has been viewed from different theoretical perspectives such as an impulse control disorder , obsessive compulsive disorder , and substance use disorder . however , there is very little agreement between these on the crucial components and dimensions of internet addiction . in the present study , the prevalence rate of 51.9% as per icd-10 substance dependence criteria seems to be overinflated whereas the prevalence of 3.8% as per iadq is probably an underestimate , although both the figures are very near to range reported in the literature . this wide variation suggests that some conceptual issues have to be addressed before we even plan to define internet addiction and estimate its prevalence . for example , are we going to diagnose internet addiction in people who are compelled to use internet for prolonged periods of time because of the nature of their occupation ? what about people who are logged on to internet all the time just checking their mails intermittently ? should it be appropriate to diagnose internet addiction in a job seeker or a researcher who try to keep abreast of the recent developments by logging on to the internet ? the internet , like mobile phones and other electronic gadgets , has become a part and parcel of modern life . an in - depth interview of the subject , with corroboration of history from the family members , might be a more reasonable approach to diagnose internet addiction . a diagnostic questionnaire at best can serve as a screening instrument to detect the population at risk of internet addiction rather than identifying the persons having full blown disorder . no face - to - face interview was conducted to explore the phenomenology of internet addiction ; data were exclusively gathered by the self - reporting internet use questionnaire . items were generated from various questionnaires developed to identify internet addiction and some items were constructed keeping the icd-10 criteria of substance dependence in mind . it can be argued that icd-10 dependence criteria are developed for psychoactive substance dependence and hence can not be applied to those substance - neutral behaviors commonly performed in daily life . thus , the item for assessing the criterion craving ( item 1 : do you feel the need to use the internet everyday ? ) would easily capture the work - related internet users for whom daily internet use is essential . the questionnaire is self - administered and thus the authors can not clarify the meaning of such items . yes. however , when the hardcore physical dependence features , i.e. , tolerance and withdrawal , are concerned , only about 15% of subjects fulfill such criteria . as mentioned earlier , this study did not differentiate between essential and nonessential internet use . the issue of the presence of psychiatric comorbidity in the subjects was also not considered . nevertheless , in view of the current ongoing debate on internet addiction after its inclusion in the proposal draft of dsm - v , this study from india could provide some insights into the impact of internet use and , more importantly , the issues facing the delineation of a syndrome of internet addiction. the sharp difference in the prevalence estimates of internet addiction depending on the type of criteria used shows the fragility of the construct of internet addiction . this study should not be cited as a usual prevalence study of a particular disorder , because of both sampling issues and methodological issues as mentioned . moreover , the issue of whom to treat and whom not to still remains open . before embarking on any kind of major manipulation to the diagnostic system , a through understanding of the concept of internet addiction is required . this study brings into focus some of these conceptual and methodological issues that must be debated before any decisive action in this regard is taken .

background : to study the pattern of internet use across people of various professions who have access to it ; the impact of internet use on their personal , social , and occupational life ; and to evaluate their internet use on the international classification of diseases tenth revision ( icd-10 ) dependence criteria and young 's internet addiction diagnostic questionnaire ( iadq).materials and methods : hundred four respondents were assessed on a 31-items self - rated questionnaire covering all the icd-10 criteria and young 's criteria for internet addiction.results:the typical profile of an internet user was as follows : the mean duration of internet use was 73.43 months ( sd 44.51 ) , two - thirds ( 65.38% ) of them were using internet on a regular basis for a period of more than a year , the mean duration of daily internet use was 39.13 months ( sd 35.97 ) , the average time spent in internet use was 2.13 h ( sd 1.98 ) everyday , more than half ( 56.73% ) of the sample was using internet at least for 2 h / day , and the most common purpose of internet use was educational for two - thirds ( 62.5% ) of the sample . the five most commonly endorsed items were as follows : the need to use the internet everyday ( 53.8% ) , internet use helping to overcome bad moods ( 50% ) , staying online longer than one originally intends to ( 43.3% ) , eating while surfing ( 24% ) , and physical activity going down since one has started using the internet ( 22.1% ) . when evaluated on icd-10 substance dependence criteria and young 's iadq separately , the prevalence of the cases of internet addiction came out to be 51.9 and 3.8% , respectively.conclusions:the internet affects the users life in multiple ways . the sharp difference in the prevalence estimates of internet addiction depending on the type of criteria used shows the fragility of the construct of internet addiction . a cautious approach should be adopted while revising the nosological system to differentiate users from those who are dependent .

MATERIALS AND METHODS Data analysis RESULTS Correlation analysis DISCUSSION

PMC4021678

the most common chronic and currently regarded as potentially irreversible disease that affects the joints is osteoarthritis ( oa ) [ 1 , 2 ] . the constantly growing number of causes for the development of the disease includes , for example , genetic predisposition , aging , obesity , trauma , and other systemic diseases ( figure 1 ) . although we are dealing with a diverse aetiology , which is most often the result of a number of overlapping factors , processes occur during the development of the pathomechanisms of disease at the tissue , cellular and ultrastructural level that gradually take a similar nature , resulting in the phenotypic image of oa . according to the latest medical knowledge , the participation of the immune system in the development and progression of oa is one of the key elements in the pathogenesis of the disease . currently , many independent authors are focused on identifying and describing the factors responsible for the development of inflammatory processes involved in oa . an analysis of the ever - increasing number of reports directs attention to the special role of the cytokine network in the pathogenesis of oa . during the progression of oa , the production and operation of various cytokines can vary depending on the duration and severity of the disease . the most important effect that cytokines have involves disturbing the catabolism and anabolism processes , particularly important in tissues that are often subject to high mechanical load , such as human joints . as a result of disrupting the said balance , there is a progressive degeneration of articular cartilage performing a key role in the biomechanics of each joint and other components of the joint , which results in the development of a difficult - to - interrupt disease process that involves both inflammatory , degradation , and production processes , which together lead to a gradual loss of joint function and pain . due to the effect of cytokines in the context of an inflammatory disease such as oa , they can be divided into inflammatory and anti - inflammatory . it should be noted that the pathophysiological processes occurring in the joint affected by oa are largely mediated by inflammatory cytokines and other mediators intensifying catabolic effects . at the same time , one should not underestimate the role of anti - inflammatory cytokines that may modulate an inflammatory response and act protectively on joint tissue . although we are not able to prevent some of the causes of oa ( genetic factors , aging , and trauma ) , we can try to alter the course of disease at the level of cellular communication through a better understanding of the sensitive dependences in the network of cytokines . this opens the possibility of using suitable targeted treatment with the use of antibodies and other recombinant biological factors that interrupt the self - perpetuating processes of the closed illness process . this review aims to provide accurate and current knowledge on the role of inflammatory and anti - inflammatory cytokines in the pathogenesis of oa . it is worth noting that the role of anti - inflammatory cytokines in the context of oa has not been studied comprehensively so far . the group of inflammatory cytokines is the most important group of compounds participating in the pathogenesis of oa . they are responsible to the greatest extent for the loss of metabolic homeostasis of tissues forming joints by promoting catabolic and destructive processes . the key role they play in the pathogenesis of oa is a result of the effect these compounds have on the majority of cells that are in the joint and the impact via intracellular pathways of signal transduction on the production of cytokines as well as other inflammatory compounds and enzymes . among the many representatives of this group , the greatest importance is attributed to il-1 , tnf , il-6 , il-15 , il-17 , and il-18 . interleukin-1 beta ( il-1 ) is considered one of the key cytokines involved in the pathogenesis of oa ; it induces inflammatory reactions and catabolic effect independently as well as being combined with other mediators with respect to the articular cartilage and other elements of joints . it is one of the 11 representatives of the il-1 family ( il-1f ) ( figure 2 ) . it is originally formed as a cytosolic precursor protein ( pro - il-1 ) consisting of 269 amino acid residues [ 9 , 10 ] . the preparation of the active form of il-1 with 153 amino acid residues is the result of intracellular proteolysis carried out by the enzyme caspase 1 ( il-1 converting enzyme , ice ) , followed by the release into the extracellular space . its synthesis in the joint is governed by chondrocytes , osteoblasts , cells forming the synovial membrane , and mononuclear cells that were previously present in the joint or infiltrated its structure during the inflammatory response [ 4 , 1216 ] . patients with oa have an elevated level of il-1 in both the synovial fluid , synovial membrane , cartilage , and the subchondral bone layer [ 12 , 13 , 15 , 17 ] . the biological activation of cells by il-1 is mediated by interaction with the membrane receptor , namely , the il-1r1 ( il-1ri , cd121a ) , which can also bind il-1 and il-1ra . another receptor capable of binding il-1 is il-1r2 ( il-1rii , cd121b ) , which after binding a ligand , such as the il-1 , il-1 , or il-1ra , forms an inactive ligand - receptor complex , showing no ability to communicate and activate the intracellular signal . at this point it is worth mentioning the presence of the receptor antagonist il-1ra produced by certain cells of the joint , which ( as noted above ) can bond with il-1r1 and il-1r2 , thereby blocking their connection with il-1 , which may to some extent affect the decrease in the activity of il-1 [ 20 , 21 ] . the expression of the il-1r1 receptor is increased in patients with oa on the surface of chondrocytes and fibroblast - like synoviocytes ( fls ) compared to treatment groups [ 22 , 23 ] . binding the il-1 to a receptor of the tlr family , such as il-1r1 , is followed by recruitment of additional il-1r3 chain ( il-1racp ) , thereby forming a complex which , through its intracellular domain toll - il-1r ( tir ) , recruits the adapter protein myd88 . the entire previously described complex binds serine - threonine kinases of the irak group , which affect the traf6 protein , which induces further binding of tak1 , tab1 , and tab2 . tak1 affects the phosphorylation of the ib kinase complex ( ikk ) , thereby activating the transcription factor nf-b . moreover , p38mapk and c - jun n - terminal kinase ( jnk ) are also activated [ 24 , 25 ] . activation of these transcription factors very quickly results in the expression of hundreds of genes whose products include other cytokines , chemokines , adhesion molecules , inflammatory mediators , and enzymes . the effect of il-1 is manifested by its significant effect on the metabolism of cells and the extracellular matrix ( ecm ) . in the course of the disease , many studies confirm that the effect of il-1 blocks chondrocytes in the context of the synthesis of ecm components , interfering with the synthesis of the key structural proteins such as type - ii collagen and aggrecan [ 28 , 29 ] . in addition to the decrease in the synthesis of the building blocks , the il-1 affects the operation of chondrocytes in the synthesis of enzymes from the group of metalloproteinases ( mmps ) , mainly interstitial collagenase ( mmp-1 ) , stromelysin-1 ( mmp-3 ) , and collagenase 3 ( mmp-13 ) , which have a destructive effect on cartilage components [ 3032 ] . besides the induction of enzymes of the mmps family , il-1 affects the chondrocytes ' production of adamts metalloproteinases , which are responsible for the proteolysis of aggrecan molecules . a major role is attributed to adamts-4 , whose production is stimulated by both il-1 and tnf , while adamts-5 has no correlation with the influence of cytokines and is produced constitutively [ 33 , 34 ] . chondrocytes subjected to the effect of il-1 and tnf also tend to age more rapidly and to induce apoptosis [ 3537 ] . when analyzing the above information , one can observe the manifold effect of il-1 on cartilage by inhibiting its restoration possibility , intensifying its deterioration by enzymes and a direct adverse effect on chondrocytes . in the cells of the joint , il-1 is able to induce its own secretion in an autocrine manner to stimulate the synthesis of other cytokines such as , for example , tnf , il-6 , il-8 , and ccl5 chemokine [ 3842 ] . il-1 has been shown to inhibit the signal pathway of the receptor - regulated smads ( r - smad ) , crucial to the activation of transcription factors associated with tgf- . this is done by increasing the expression of the inhibitor protein smad7 and inhibition of synthesis of the tgf- type ii receptor in chondrocytes . in addition , effects are observed on the secretion of a number of other enzymes and mediators involved in the pathophysiology of oa . these compounds may include the inos generating no , phospholipase a2 ( pla2 ) , cyclooxygenase-2 ( cox-2 ) , prostaglandin e synthase 2 ( pge2 synthase ) producing prostaglandin e2 ( pge2 ) [ 4446 ] . during the course of the disease , il-1 stimulates the production of reactive oxygen species ( ros ) , which generate the formation of , for example , peroxides and hydroxylated radicals , which directly damage the articular cartilage ; the intensification of this process is also associated with decreased expression of oxidative enzymes , which is observed in the joint affected by the disease . tumor necrosis factor alpha ( tnf ) , together with il-1 , is considered a key inflammatory cytokine involved in the pathophysiological processes occurring in the course of oa ( figure 3 ) . it is one of the 19 ligands within the tumour necrosis factors superfamily ( tnf superfamily ) . it is originally formed as a homotrimeric transmembrane protein type ii ( mtnf ) , which can be secreted into the environment in the next stage involving the tace / adam17 metalloproteinase , creating a free form of tnf ( stnf ) [ 4951 ] . tnf is secreted by the same cells in the joint that synthesize il-1 , and its increased concentration is also observed in the same elements , such as synovial fluid , synovial membrane , cartilage , and subchondral bone layer , where increased levels of il-1 are also detected [ 12 , 13 , 15 , 17 ] . the cytokine has the ability to bind to the two isotypes of membrane receptors located on the surface of almost every nucleated cell tnf - r1 ( p55 , cd120a , and tnfrsf1a ) and tnf - r2 ( p75 , cd120b , and tnfrsf1b ) [ 52 , 53 ] . the tnf - r1 receptor can be efficiently activated by the soluble and membrane forms , while tnf - r2 mainly binds the membrane form . so far the participation of tnf - r1 seems to have a greater impact on the local loss of articular cartilage than tnf - r2 ; this , however , does not change the fact that both receptors are involved in signal transduction after being activated by tnf in the processes occurring in oa [ 54 , 55 ] . the expression of tnf - r1 is also increased within the fls cells [ 56 , 57 ] . as mentioned above , both receptors differ in their affinity to the tnf and also in their amino acid composition , degree of glycosylation , and structure , primarily in their intracellular part , which within the tnf - r1 includes the so - called death domain ( dd ) , which does not occur in the tnf - r2 receptor [ 51 , 58 ] . in view of the significant lack of a homology in the intracellular portion tnf - r1 is able to recruit two different tnf - r1 signalling complexes ( tnf - rsc ) [ 59 , 60 ] . complex i is mainly involved in the activation of pathways whose end products stimulate an inflammatory response , especially the production and secretion of cytokines and the production of proteins that prevent apoptosis , whereas the main function of complex ii involves signal transduction leading to cell disintegration [ 60 , 61 ] . the association of tnf with tnf - r1 causes interaction between the tradd adapter protein with the dd domain and gradual binding of other adapter proteins , such as traf2 , c - iap1 , c - iap2 , and rip1 [ 6264 ] . the creation of the complex is followed by ubiquitination of the rip1 protein , which also binds tak1 , tab1 , and tab2 , resulting in the phosphorylation of the ikk complex and ultimately , the activation of one of the most important transcription pathways in the course of oa nf-b [ 65 , 66 ] . furthermore , during the formation and effect of complex i , the other important signalling pathway is also activated by jnk kinase , as well as others such as extracellular - regulated kinase ( erk ) pathway and p38mapk [ 60 , 67 , 68 ] . the formation of complex ii is accompanied by endocytosis of the activated receptor , changing its conformation and recruitment of fadd and pro - caspase 8 , which ends with cell death [ 59 , 69 ] . in turn , binding mtnf to the tnf - r2 receptor will result in the binding and mutual interaction of proteins , which includes traf2 , which is crucial in the signal transduction and others such as traf3 , c - iap1 , and c - iap2 . the effect of interaction of the complex is the activation of the jnk kinase and the transcription factor nf-b [ 7073 ] . it has been proven that polymorphism in the gene ( m196r ) encoding the receptor protein tnf - r2 may predetermine the development of oa by increasing the number of receptor proteins on the surface of chondrocytes , which leads to disturbance of their functions due to excessive activation by mtnf . moreover , not only polymorphism in the receptor but also in the tnf ligand may favour the occurrence of oa in relation to the specific populations studied [ 75 , 76 ] . it is worth to mention the presence of an additional ligand capable of incorporating both tnf - r1 and tnf - r2 with still increasing importance in the pathogenesis of oa , which is progranulin ( pgrn ) also known as granulin epithelin precursor ( gep ) , pc - cell derived growth factor ( pcdgf ) , proepithelin ( pepi ) , or acrogranin [ 7780 ] . it was shown that pgrn level is significantly elevated in patients suffering from cartilage arthropathies including oa . the pgrn binding ability to the dedicated tnf receptors , as well as its elevated levels during the course of the disease , makes it a natural antagonist of tnf that interfere with the signaling pathway tnf/tnf - r1 and tnf/tnf - r2 . this implies that an imbalance of tnf/pgrn can both accelerate and inhibit the development of oa . in addition , the presence of pgrn autoantibodies was observed in some joint diseases with an autoimmune component [ 8486 ] . the effect of tnf in most cases coincides with the action of il-1 , and in the case of many phenomena occurring in the course of oa there is a marked synergism between the two cytokines . this effect is the result of activation of the same group of intracellular signalling pathways , which in turn triggers similar effects that increase the inflammation and catabolism in joint tissues [ 26 , 27 ] . tnf affects blocking the chondrocytes ' synthesis of proteoglycan components , proteins binding proteoglycans , and type ii collagen [ 88 , 89 ] . activated chondrocytes also produce mmp-1 , mmp-3 , mmp-13 , and adamts-4 [ 33 , 90 , 91 ] . as described earlier , there is an induction of chondrocyte death and a disorder in the migration of chondrogenic progenitor cells ( cpcs ) , which strips the cartilage of any possibility of regeneration [ 3537 , 92 ] . a clear impact of tnf and il-1 on reducing the efficiency of the respiratory chain was also observed , and hence the decrease in atp produced within the mitochondria located in chondrocytes ; additionally , there is a decrease in the potential of the mitochondrial membrane . tnf is responsible for increased synthesis of , for example , il-6 , il-8 , rantes , and vegf [ 3941 , 94 ] . moreover , as described above , il-1 and tnf induce the production of inos , cox-2 , and pge2 synthase , thereby increasing the amounts of their products [ 44 , 46 , 95 , 96 ] . interleukin-6 ( il-6 ) is a compound characterized by omnidirectional interactions in the processes occurring in the human body . it is considered a cytokine that strongly activates the immune system and enhances inflammatory response , although considering some of its effects , it may be classified as anti - inflammatory interaction . il-6 is a glycoprotein consisting of 184 amino acid residues , which in the process of posttranslational processing ultimately becomes an interconnected structure of four -helices . the production of il-6 in the tissues of the affected joint is usually in response to il-1 and tnf and is mainly implemented by chondrocytes , osteoblasts , fls , macrophages , and adipocytes [ 39 , 98102 ] . the increased concentration of il-6 is present in both the synovial fluid and serum and is positively correlated with the intensity of lesions in x - ray imaging [ 103107 ] . there are two subtypes of the il-6r receptor ( gp80 , cd126 ) , namely , the membrane form of mil-6r and the soluble sil-6r , which may be formed by cutting or by the adam17/tace by metalloproteinase or by means of alternative splicing . transferring the signal into the cell requires the association of additional gp130 protein ( cd130 ) [ 109 , 110 ] . the gp130 protein is also present in the membrane form mgp130 and in soluble form sgp130 [ 111 , 112 ] . the association of the il-6 and il-6r with sgp130 is associated with inhibiting the signalling pathway of il-6 [ 111 , 112 ] . recruitment of the ligand - receptor complex to mgp130 causes homodimerization and consequently the formation of hexamer that allows effective signal transduction to the cell . jak kinase mediates the phosphorylation of tyrosine residues that are an integral part of the gp130 , which in further stages results in the activation of stat3 , phosphorylation of mapk , and activation of the pi3 k / akt pathway [ 114 , 115 ] . while analyzing the role of il-6 in oa , one should first note that the polymorphism of gene ( 174g / c ) encoding il-6 may predetermine the development of oa [ 116 , 117 ] . the effect of il-6 on joint cartilage is not different from other cytokines and , in synergy with them , causes a decrease in the production of type ii collagen and increases the production of enzymes from the mmps group [ 118120 ] . it was also found that these effects can be enhanced under the influence of injury . il-6 is considered to be the key cytokine , which causes changes in the subchondral bone layer [ 122 , 123 ] . its effect is largely based on promoting the formation of osteoclasts and thus bone resorption while showing synergism with il-1 and tnf [ 122 , 124 ] . osteoblasts stimulated by il-1 , tnf , and il-6 become a source thereof and may also produce mmps by adversely affecting the cartilage located near it . in addition to the demonstrated role of other cytokines in inducing the production of il-6 , its secretion by osteoblasts , chondrocytes is also affected by prostaglandin e2 [ 125 , 126 ] . in turn , while analyzing tests on animal models , it can be observed that il-6 may have a different effect in some situations . an experiment performed on mice lacking the gene for il-6 showed that they exhibited a tendency to develop much more advanced degenerative changes than healthy mice . during another experiment , it was also demonstrated in mice lacking the gene for il-6 that intra - articular injection of il-6 reduces the loss of proteoglycans in the acute phase of chronic joint inflammation and induces the formation of osteophytes . interleukin-15 ( il-15 ) is a glycoprotein that takes the form of four interconnected -helices with a mass of 14 - 15 kda [ 129 , 130 ] . its action is mainly based on the stimulation of differentiation and proliferation of t cells and nk cells . it is one of the better documented cytokines involved in the pathogenesis of rheumatoid arthritis ( ra ) [ 132 , 133 ] . while analyzing the involvement of il-15 in the pathogenesis of oa , its increased concentration was found in the synovial fluid in the early stages of the disease . it has also been shown that the increased il-15 level in the serum correlates with both the sensation of pain and the severity of lesions in the x - ray image . it has also been noted that its presence can stimulate the secretion of certain types of metalloproteinases from the mmps group . the family of interleukins-17 ( il-17 ) is a group of cytokines with inflammatory effect , which draws more and more attention of researchers for its participation in the pathogenesis of oa . it consists of six members ( il-17a - f ) that can interact through five types of receptors ( il-17ra - e ) [ 136 , 137 ] . the source of il-17 is mainly stimulated cd4 t cells and mast cells that infiltrate the synovial membrane and the entire joint through blood vessels [ 138141 ] . the main cells in the joint that are affected by il-17 are chondrocytes and fls exhibiting the expression of il-17r on their surface . in addition , studies have demonstrated direct cellular immune response of those t cells against membrane antigens of chondrocytes and fibroblasts in the course of oa . the level of il-17 measured in the serum and the synovial fluid of patients is elevated and shows a positive correlation with the radiographic image of lesions in oa . il-17 has been shown to inhibit the synthesis of proteoglycans by chondrocytes and promotes the production of enzymes of the mmps group [ 145147 ] . furthermore , il-17 influences the secretion of other cytokines and compounds negatively affecting the cartilage , such as il-1 , tnf , il-6 , no , and pge2 [ 148150 ] . the effect of il-17 on the secretion of vegf by both chondrocytes and fls is also characteristic ; it favours the excessive development of blood vessel network within the synovial membrane , leading to its hypertrophy [ 94 , 142 ] . it was also confirmed recently that the polymorphism the gene il-17a g-197a in certain populations may correlate with the susceptibility to the development of oa . it is generated as a precursor form of the pro - il-18 , consisting of 192 amino acid residues , which is transformed into a biologically active form after the activation of caspase 1 or proteinase 3 , comprising 157 amino acid residues [ 152 , 153 ] . it has been noted that the level of caspase 1 is elevated both in the articular cartilage and synovium in oa patients which greatly promotes the formation of il-1 and il-18 . the production of il-18 in the joint is determined by chondrocytes , osteoblasts , fls , and macrophages [ 155157 ] . its increased concentration is evident in the synovial fluid , synovium , cartilage , and blood serum and shows a positive correlation with the degree of severity of the disease seen in radiographic images [ 158160 ] . the effect of il-18 is mediated by the il-18r receptor , which bears structural and functional similarity to il-1r . as in the case of il-1 , efficient signal transmission by il-18 requires not only the presence of il-18r but also the additional recruitment of the il-18r ( il-18racp ) chain [ 161 , 162 ] . intraventricular signal transmission through a heterodimeric receptor complex il-18 does not show significant differences from the mechanism of signal transduction by the il-1 receptor complex . studies have shown that polymorphism of genes encoding both il-18r and the il-18 interleukin in some variants may predetermine both the development of oa and lumbar disc degeneration ( ldd ) [ 164 , 165 ] . il-18 affects chondrocytes by inducing the upregulation of il-18r on their surface and stimulation excess synthesis of metalloproteinases mmp-1 , mmp-3 , and mmp-13 . in addition to increasing the concentration of cartilage degrading enzymes , there is an inhibition of production of proteoglycans , aggrecan , and type ii collagen ; moreover , chondrocytes exhibit morphological changes typical of cells entering apoptosis [ 167169 ] . il-18 affects chondrocytes and synovial cells , increasing the production of a range of compounds and enzymes , such as the il-18 in an autocrine manner , il-6 , inos , pge2 , cox-2 , and vegf [ 155 , 170173 ] . the main representatives of the group of anti - inflammatory cytokines involved in the pathogenesis of oa are il-4 , il-10 , and il-13 . in this chapter , the authors focus on presenting the effect of only those of the above cytokines , whose biological activity is the most important in the light of the relevant problems ( figure 4 ) . however , it should be noted that il-4 , il-10 , and il-13 belong to a broader group of anti - inflammatory cytokines and are not the only representatives . interleukin-4 ( il-4 ) is a protein composed of 129 amino acids , which takes the form of four interconnected -helices additionally stabilized by three disulfide bonds [ 174176 ] . il-4 is a ligand whose biological activity is mediated through a receptor system dedicated to both il-4 and il-13 . there are two types of receptors forming heterodimers which are structurally a combination of two or three receptor chains , such as il-4r , il-13r1 , and il-2rc . the dimerization of il-4r and il-2rc ( type 1 complex ) enables the attachment of il-4 , while the interaction between il-4r and il-13r1 ( type 2 complex ) enables efficient attachment of both il-4 and il-13 [ 177179 ] . after the formation of the receptor type 1 complex , an intracellular signal transduction takes place by gradual phosphorylation of the il-4r/jak1/stat3/stat6 cascade , leading to the expression of several proinflammatory genes [ 180 , 181 ] . the observed polymorphism of the genes encoding both il-4 and il-4r may predetermine the development of oa in the joints of the hand , knee and hip joint gives reason for claiming that the interaction between il-4 and its dedicated receptor complex is one of the elements of the pathogenesis of the disease [ 182184 ] . the production of il-4 is primarily determined by t cells ( th2 ) infiltrating the synovium of the joint by blood vessels [ 185 , 186 ] . it was further found that the level of soluble il-4r ( sil-4r ) is elevated in the serum of patients suffering from oa as compared to the healthy control group . the increased concentration of il-4 is also observed in the synovial fluid and synovial cells [ 185 , 188 , 189 ] . it was found that il-4 has an inhibiting effect on the degradation of proteoglycans in the articular cartilage , by inhibiting the secretion of mmps metalloproteinases , as well as reducing the variation in the production of proteoglycans that are visible in the course of oa [ 190193 ] . interestingly , in the course of oa , chondrocytes showed decreased susceptibility to the effects of il-4 which may be responsible for the rapid degeneration of the articular cartilage [ 194196 ] . in addition , il-4 alone or in combination with il-10 exhibits properties inhibiting the apoptosis of both the chondrocytes and fls [ 191 , 197 , 198 ] . considering the effect of il-4 on cell cultures of chondrocytes and fls treated with it , there is a decrease of synthesis of inflammatory cytokines such as il-1 , tnf , and il-6 [ 191 , 197 , 199 ] . simultaneously , il-4 can induce upregulation of the expression of tnf receptors , such as tnf - r1 , and tnf - r2 . in addition to a direct decrease in the secretion of inflammatory cytokines , there is also a decrease in the secretion of other inflammatory mediators such as pge2 , cox-2 , pla2 , and inos [ 191 , 197 , 200 , 201 ] . it seems reasonable to cite a study , which analyzed the effect of rmil-4 ( recombinant mouse il-4 ) in combination with rmgm - csf for the dendritic cells cd11bf4/80idc in a mouse model , where inflammatory joint disease was induced artificially . the observed results seem to confirm the positive effect of il-4 and gm - csf on restoring the balance between the secretion of proinflammatory and anti - inflammatory factors in the immune cells in mice with cia ( collagen - induced arthritis ) . cd11bf4/80idc cells exposure to said effect showed an increased ability to synthesize the anti - inflammatory il-10 and tgf- as well as an inhibitory or excitatory effect relative to other cells of the immune system . it has been noted that proliferation of th17 lymphocytes has been reduced and that they showed the ability to synthesize and secrete proinflammatory il-17 . macrophages showed a reduced ability to produce and secrete il-1 and tnf. in turn , the regulatory t cells were stimulated to increase the production of il-10 and tgf-. the test mice showed reduced morphological markers of inflammation of the synovial membrane and articular cartilage and decreased immunological activity of the thymus , relative to the control group ( unexposed to rmil-4 rmgm - csf ) . although the mouse model with cia corresponds mainly to ra in the world literature , the dependencies between the effect of il-4 and gm - csf observed for cells of the immune system may be a reference to some of the processes occurring in oa . in the context of other studies , this gives us a rough idea about the anti - inflammatory effect of il-4 in the course of oa , although it certainly requires further study . interleukin-10 ( il-10 ) is a cytokine structurally related to interferons , which is in the form of a homodimer wherein every monomer is a polypeptide chain consisting of 160 amino acid residues , taking the structure of 6 interconnected -helices [ 203205 ] . initiation of il-10 is made possible by binding it to the receptor il-10r , which is a heterodimer composed of subunits il-10r1 and il-10r2 [ 206 , 207 ] . the first step of activation of the receptor involves binding il-10 to the il-10r1 subunit , which then changes its conformation in such a way as to allow binding the il-10 and il-10r1 complex to the il-10r2 subunit [ 206 , 207 ] . at this point a cascade of intracellular signal transmission is started . this leads to the activation of the tyrosine kinase jak1 ( associated with the il-10r1 subunit ) and tyk2 ( associated with il-10r2 ) . this in turn implies sending the cellular signal further by the transcriptional activators stat3 , which upon binding to sbe ( stat - binding elements ) of the cell nucleus stimulate the synthesis of the products of genes dependent on il-10 . it has been proven that il-10 is involved in stimulating the synthesis of type ii collagen and aggrecan . following the administration of il-10 in vitro conditions , both healthy articular cartilage and one in the course of oa demonstrated an increase in proteoglycan synthesis and its percentage share in the extracellular matrix . furthermore , the synovium of patients with osteoarthritis in the context of hemophilia , which showed a tendency to excessive production of tnf and il-1 following the in vitro administration of il-10 significantly reduced the average secretion of the mentioned proinflammatory cytokines by 60% and 83% , respectively . it has been shown that il-10 is responsible for inhibiting the production of mmps family of metalloproteinases . it was confirmed that il-10 ( similar to il-4 ) inhibits the apoptosis of chondrocytes . these properties of il-10 are likely the result of stimulation of the synthesis of il-1 antagonist , which is il-1ra and the tissue inhibitor of metalloproteinases-1 ( timp-1 ) as well as growth factors [ 214 , 215 ] . the study of the effect of il-10 on the stimulation of chondrocyte proliferation in a mouse model shows further signaling pathways of il-10 . it has been proved that il-10 activates the kinase pathway smad1/smad5/smad8 and erk1/2 map and induces the expression of bone morphogenetic proteins 2 and 6 ( bmp-2 , bmp-6 ) . they are part of the signaling pathway that affects many genes and proteins responsible for the regulation of the mesenchymal cell transformation in chondrocytes , such as nkx-3.2/sox9 , sox5 , and sox6 [ 218 , 219 ] . in addition to determining the transformation of multipotent cells towards chondrocytes , bmp proteins stimulate their proliferation both directly and through the ihh / pthrp - dependent pathway [ 220 , 221 ] . the involvement of il-10 in the induction of differentiation and proliferation of chondrocytes through the bmp signaling pathway was thus confirmed . the study authors deduce that the observed mechanisms may be partly responsible for the chondoprotective effects of il-10 in oa . in another study , the aim was to figure out how lps stimulation affects the synthesis and level increase of il-10 in blood samples from patients with oa and healthy subjects . based on the results of il-10 levels and information on the health status of the subjects , a specific prediction model of oa was developed . persons whose blood samples showed no significant increase in the level of il-10 relative to the starting point ( the lowest quartile of results ) were running even a 3-fold risk of developing oa compared to those whose levels of il-10 after stimulation were in the highest quartile . this is manifest in a significant decrease in their secretion of pge2 , cox-2 , and pla2 . interestingly , il-10 does not have those properties with respect to the fibroblasts not treated with tnf. it was confirmed that il-10 reduces the expression of tnf receptors and thus their ability to bind to the surface of fibroblasts . the anti - inflammatory and chondroprotective nature of il-10 is composed of results of interesting studies of the influence of exercise on the secretion of cytokines in joint fluid and periarticular tissues in patients with oa of the knee joints [ 223 , 224 ] . in these studies , patients were divided into a study group , which has exercised knee joints affected by oa for 3 h and a control group , which had to refrain from exercise during the same time . it is worth noting that in the control group there were patients with the same degree of severity of oa of the knee joints . knee joints affected by oa were connected to catheters in both the study and control groups . catheters , placed into the joints and periarticularly , were used to examine the levels of il-10 and other cytokines before exercise , during exercise , and after exercise relative to the control group . a significant increase in the level of il-10 in the synovial fluid and periarticular tissues was observed in patients in the study group as compared to the control group , in which the levels of il-10 observed before and after exercise showed no significant difference . on the basis of these observations , it seems reasonable to advocate the medicinal effect of physical exercise on joints affected by oa . in truth , it is not clear what mechanism leads to an increased secretion of il-10 . some authors argue that this is due to the inductive effect of increased intra - articular pressure on cellular secretion [ 225 , 226 ] . interleukin-13 ( il-13 ) is a compound that takes the structure of four interconnected -helices , which is very similar in its effect to il-4 [ 227 , 228 ] . similar to il-4 , the action of il-13 as a ligand is mediated through a receptor system that combines both cytokines . in this case it requires the recruitment of a ligand , namely , il-13 , and in addition the chains il-4r and il-13r1 to create a type 2 complex [ 177 , 178 ] . with this combination , il-13 has the capacity to transfer the intracellular signal both by the cascade il-4r/jak2/stat3 and il-13r1/tyk2/stat1/stat6 . the anti - inflammatory and chondoprotective effects of il-13 on the cells of the immune response , articular cartilage , and synovium in oa have been fairly well documented [ 229 , 230 ] . the anti - inflammatory effect of il-13 in the context of oa seems most important with respect to fibroblasts included in the synovium . note , however , that the anti - inflammatory properties based on the inhibition of the secretion of inflammatory cytokines relate to a whole range of cells such as macrophages , monocytes , b cells , nk cells , and endothelial cells . in one of the most important studies into the effects of il-13 on oa , synovium samples were taken from 16 patients with oa during the knee endoprosthesis implantation procedure ( tkr ) . patients were diagnosed based on the criteria of the acr diagnostic subcommittee for oa , revealing stage iii or iv of the disease according to radiological imaging . the synovial samples taken were administered proinflammatory lps ( control samples ) , followed by il-13 ( test samples ) . after 72 h incubation , laboratory tests were performed including binding tests , northern blotting , and elisa . it has been shown that , compared to the control samples , il-13 showed inhibitory effects on the synthesis of proinflammatory il-1 , tnf , and mmp-3 with a simultaneous increase in the level of il-1ra . in the studies synovium cells , the amount of mrna for il-1 was reduced , while the mrna level for il-1ra was increased relative to control samples . moreover , synovial fibroblasts showed a reduction in binding between il-1 and its receptor , which was caused by the increased production and action of il-1ra . it has been demonstrated that il-13 has the ability to inhibit the proinflammatory effect of tnf relative to fibroblasts from patients with oa . a significant decrease in pge2 synthesis by blocking the synthesis of cox-2 was observed . interestingly , compared to il-4 and il-10 , il-13 does not affect the production level of pla2 . this may indicate the selective effect of il-13 that only involves cox-2 and , more specifically , the expression of its gene . the study proved that il-13 has the ability to decrease the nuclear concentration of transcription factors c / ebp , which may affect the inhibition of the synthesis of cox-2 . in this context , fibroblasts may exhibit properties similar to osteoblasts . the analysis of these results indicates the potential utility of il-13 in the treatment of oa , as a compound that inhibits the inflammatory processes , protects chondrocytes , reduces the secretion of inflammatory cytokines and metalloproteinases , while stimulating the synthesis of il-1ra . oa is a set of complex and difficult - to - identify processes leading to progressive degeneration of joints . but , contrary to what it may seem to be , it is not only a set of catabolic effects . evident imbalance in metabolism observed in oa is the final manifestation of the dysregulation within the so - called cytokine network between anti - inflammatory and inflammatory cytokines . in this paper , current knowledge on the impact of the most important cytokines in the pathogenesis of oa has been presented , based on world literature survey . cytokines were categorized with respect to their biological effect as inflammatory and anti - inflammatory . however , this criterion is not so obvious in particular conditions . respective cytokines should be analysed together with the nature and structure of specific receptors and the intracellular signalling pathways triggered by their activation in the specific tissue . thus , particular attention was paid to the potential impact of cytokines on articular cartilage , synovium and the cells of the immune system . in the context of developing optimal treatment tactics for oa , the authors of this paper have tried to summarize and comment on the most important information resulting from the collected above ( published ) data . due to their effects within the joint it is a multilevel impact that involves not only the induction of aging and apoptosis of chondrocytes , but also a decrease in the synthesis of the key components of ecm , such as proteoglycans , aggrecan , and type ii collagen . in addition , inflammatory cytokines contribute to the increased synthesis and release of many proteolytic enzymes that decompose articular cartilage , which include the metalloproteinases family mmps and adamts . apart from their effect on chondrocytes , synovial cells and other articular , and periarticular tissues , inflammatory cytokines affect the cells of the immune system migrating to the site of inflammation . due to the action of inflammatory cytokines , all of these cells tend to produce excessive amounts of inflammatory pge2 , cox-2 , phospholipase a2 , no , and free radicals . it is worth noting that inflammatory cytokines stimulate cells to synthesize other inflammatory cytokines , namely , il-1 , tnf , il-6 , il-8 , and chemokines ccl5 ( rantes ) . they can thereby also promote their own production , thus demonstrating an autocrine , paracrine , and self - propelling effect on the inflammation process . the action of anti - inflammatory cytokines mainly involves inhibiting the synthesis of inflammatory cytokines , particularly il-1 and tnf. observed effects of anti - inflammatory cytokines include increased proteoglycan synthesis , inhibited apoptosis of chondrocytes , decreased synthesis and secretion of metalloproteinases , and decreased level of pge2 . however , these properties should be treated as an inhibitory effect on inflammatory cytokines rather than direct chondoprotective capabilities . it should be added that it has been proven that the action of anti - inflammatory cytokines is mainly performed relating to cells stimulated by inflammatory cytokines , whereas no significant differences are noted in the metabolism of cells not subject to such stimulation . such a relationship has been observed , for example , regarding synovial fibroblasts obtained from patients with oa . however , certain properties of anti - inflammatory cytokines go beyond the simplest idea of antagonism with respect to inflammatory cytokines . for example , il-10 stimulates the synthesis of growth factors of the bmp family ( bmp-2 , bmp-6 ) and affects the regulatory proteins such as nkx-3.2/sox9 , sox5 , and sox6 which induces and regulates the process of chondrogenesis . in turn , rmil-4 in a mouse model stimulates regulatory t cells to synthesize il-10 and the growth factor tgf- , which at least corresponds to the properties of inflammatory cytokines in the context of stimulating their own synthesis . the dominant observation concerning the effects of inflammatory and anti - inflammatory cytokines in the course of oa is that the inflammatory response within the affected joints is never controlled completely . many authors have confirmed that the effect of anti - inflammatory cytokines and the treatment based on them can not positively stop the progression of the disease by blocking the promotion of catabolic pathways . so far , there have been no comprehensive answers to this issue . according to the standpoint of the authors of this study , the insufficient effect of anti - inflammatory cytokines in the joint may be determined by the characteristics of their action . relative to healthy joint tissue , anti - inflammatory cytokines show no significant anabolic effect likely to create a sufficient biochemical advantage against the effects of inflammatory cytokines . it is only in a joint affected by the disease in the presence of inflammatory mediators that the role of anti - inflammatory cytokines becomes noticeable . the effect of anti - inflammatory cytokines therefore shows no primary prophylactic capability ; however , it has the properties of a secondary response to combat the symptoms of the disease . such blocking effect of the activity of inflammatory cytokines on the action - reaction principle , already in the early stages of the disease places the anticatabolic processes as secondary in relation to the catabolic effect and is therefore late or delayed . in search of a better understanding of the pathomechanism of oa , one should pay attention to the quantitative changes that occur in the so - called cytokine network . the abundance or deficiency of any element can cause disturbances of the whole structure and ultimately its collapse . in oa , there is a lot of missing building blocks that contribute to preservation of the metabolic balance in the joint . the main figure in this group is primarily tgf- , a cytokine and growth factor responsible for stimulating the production of proteoglycan , type ii collagen , and chondrogenesis [ 235 , 236 ] . its presence in the joints is observed in healthy subjects , as opposed to patients with oa , where the amounts of tgf- are low or even undetectable . some authors believe that reducing the level of this cytokine is responsible for sensitizing the cartilage to the effect of inflammation mediators and for the progress of the disease . the explanation for the reduced amount of tgf- in the course of oa and thus also its biological activity may involve intercepting the cell signalling pathways by inflammatory cytokines , although this certainly requires further investigation [ 238 , 239 ] . another factor , which may contribute to the unrestrained progression of oa , is the cell type responsible for the production of specific cytokines . namely , in the oa model , the production of anti - inflammatory cytokines is determined primarily by cells of the immune system , which are largely infiltrating cells . in turn , inflammatory cytokines such as il-1 or tnf are synthesized and secreted by the cells of the immune system , as well as locally by articular cartilage cells . this dual model of release seems to create favourable conditions for the development of oa . add a slightly visible influence of anti - inflammatory cytokines on stimulating their own secretion and thus the lack of a self - supplying mechanism ( as opposed to inflammatory cytokines ) , and we will see the image of the disease , in which it is not possible to bring about the advantages of inflammatory processes . such a scheme of the progress disease quite accurately corresponds to the image of oa progression . currently we are trying to seek appropriate treatment likely to stop the progress of oa or at least slow it down to a satisfactory degree . the tactics used by researchers are to influence cell transduction pathways that are crucial to the induction of inflammatory process . therefore , research is underway , aimed at obtaining more accurate knowledge of the effect of inflammatory and anti - inflammatory cytokines . the emerging picture shows the multiplicity , complexity , and the multilevel nature of processes taking place with the participation of cytokines in the course of oa . their discovery and understanding is a big challenge for today 's medicine ; however , it seems absolutely crucial to finally develop effective methods of treatment .

osteoarthritis ( oa ) is the most common chronic disease of human joints . the basis of pathologic changes involves all the tissues forming the joint ; already , at an early stage , it has the nature of inflammation with varying degrees of severity . an analysis of the complex relationships indicates that the processes taking place inside the joint are not merely a set that ( seemingly ) only includes catabolic effects . apart from them , anti - inflammatory anabolic processes also occur continually . these phenomena are driven by various mediators , of which the key role is attributed to the interactions within the cytokine network . the most important group controlling the disease seems to be inflammatory cytokines , including il-1 , tnf , il-6 , il-15 , il-17 , and il-18 . the second group with antagonistic effect is formed by cytokines known as anti - inflammatory cytokines such as il-4 , il-10 , and il-13 . the role of inflammatory and anti - inflammatory cytokines in the pathogenesis of oa with respect to inter- and intracellular signaling pathways is still under investigation . this paper summarizes the current state of knowledge . the cytokine network in oa is put in the context of cells involved in this degenerative joint disease . the possibilities for further implementation of new therapeutic strategies in oa are also pointed .

1. Introduction 2. Inflammatory Cytokines 3. Anti-Inflammatory Cytokines 4. Summary

PMC4479904

yoga originated around 3000 years b.c . , under mystical and philosophical concepts in the hindu tradition . its principles rest in metaphysics , which are hard to understand in western countries and for those who do not practice it . some authors , who have gone deep into its study and practice , have tried to explain it . etymologically , yoga means to add , join , unite or attach ( sanskrit , ioga ) where the body ( anga ) , mind ( chitta ) , emotions and the soul ( atma or atman ) . a complete explanation of this ancient discipline was given by eliade in his treatise yoga , immortality and freedom , and defined as a collection of specific techniques to seek a truth hidden in the silence and in the inner calm of people , a fundamental truth which enables one to free the soul from false reality , a state of liberation of the waves of thought or ecstasy ( samadhi ; sanskrit , sam o samialk [ complete ] and dhi [ mentally absorbed ] ) . the one practiced in western societies is an integral yoga described by patajali ( ii century b.c . ) . he condenses in his yoga stras , a collection of aphorisms in a buddhist / hindi text or manual , the traditions and practices of ancient and contemporary practitioners ( yogis ) . this type of yoga was brought to the american continent by swami vivekananda at the end of the 19 century ( 18941896 ) and was scientifically and philosophically enriched by eliade . however , through the years yoga has undergone many transformations and adaptations , thereby changing its original principles and fundamentals . as opposed to the traditional practice , physical focus on yoga became very popular in the west beginning in the second half of the 20 century and is often referred simply as to hatha yoga ( hy ) . hatha yoga refers to a set of physical ( asanas ) and mental exercises , designed to align the body and mind , in such way the vital energy ( pra ) can flow freely . it consists of respiratory exercises ( prnymas or shatkarma ) , physical stretching postures , isometric force , balance , relaxation ( yoganidra ) and concentration ( dharana ) , whose purpose is to ensure that anga is fit for meditation ( dhyana ) . these elements are conducive to a unique level of consciousness and self - realization , leading to liberation ( kaivalya ) of the self ( atman ) . hatha yoga reduces stress , improves overall physical fitness and reduces some risk factors for cardiovascular diseases . other health effects include prevention of cardiac arrhythmias , hypertension , insomnia , cardiopulmonary disorders , depression and anxiety , epilepsy , cancer , menopause symptoms and chronic back pain . that is why it is adopted as part of a healthy lifestyle or as a therapeutic resource in alternative medicine . to give just an example , ross et al . , postulate that the frequency of yoga practice at home favorably predicted ( p < 0.001 ) : mindfulness , subjective well - being , healthy body mass index ( bmi ) , fruit and vegetable consumption , vegetarian status and vigor . moreover , specifics components of yoga practice ( e.g. , physical poses , breath work , meditation , and study of yoga philosophy ) improve health behaviors or lifestyle - related health conditions . however , its benefits in other physical and mental disturbances remain not conclusive , and even harmful effects have even been reported when practiced incorrectly , by unskilled people or disabled . anyhow , hy should be considered as a preventive strategy for improving several metabolic conditions although its utility in complementary medicine , as compared with conventional medical therapies , is under - recognized by the health care community . lastly , yoga is also a lifestyle , so the physiological events are complemented with other environmental factors such as the change in eating patterns . here 's a conceptual review of this subject , with particular emphasis on the changes in eating behaviors ( ebs ) and bioenergy ( be ) management in yogis and the practice of hy as a mean to improve lifestyle and eating patterns in nonyoga practitioners . the issues to be addressed in the following sections of this critical review , derived from a systematic search for information on 5 databases ( medline [ pubmed ] , lilacs [ scielo ] , latindex , science direct , google scholar ) are recognized in the field of yoga and its health impacts . the following medical subject headings ( mesh , tree number ) were used in combination with yoga and hy with the purpose of gathering and evaluating judiciously : energy metabolism ( em ) ( mesh : g03.495.335 ) , energy expenditure ( ee ) ( mesh : g03.495.335 ) , food ( mesh : j02.500 ) , diet ( mesh : g07.610.240 ) , eating disorders ( ed ) ( mesh : f03.375 ) , ebs ( mesh : f01.145.113.547 , f01.145.407 ) , eating ( mesh : g07.610.593.260 , g07.700.620.260 , g10.261.326.240 ) , anorexia nervosa ( mesh : f03.375.100 ) , bulimia nervosa ( mesh : f03.375.250 ) and binge eating . anticipating the specificity and unexplored nature of certain topics , the information gathered from these databases within the philosophy of yoga , the body 's energy is studied from a more subtle and difficult way to measure , which plays a part on the control of total energy intake ( tei ) and total energy expenditure ( tee ) . so , a more holistic view of energy balance has to be addressed , here referred to as be or kundalini energy . with the purpose of achieving the desired freedom ( kaivalya ) of the inner self ( atman ) , the yogi tries to control the body 's energy centers ( kundalini - chakras ) and senses ( jnanendriya ) . the kundalini energy comes in three states : the common dormant , the aroused and the awakened states . when dormant , one 's spiritual understanding is restricted , and everything is perceived and interpreted according to a mundane and selfish perspective . when aroused , it gives a sudden temporary state of spiritual insight and spiritual energy , but it is not stable . only the awakened kundalini energy gives stable transformations of consciousness and progressive realization . the yogi is not interested in developing physical strength or athletic abilities , at least not in the way they are perceived in the west . the yogi is only interested in the control of its body for the development of atman . to achieve this bioenergetic level , the yogi integrates abstinence ( yamas ) , purity , moderation , and modesty ( niyamas ) into his / her daily life , and even some dietary and physical activity aspects rely on these principles . therefore , it is somewhat meaningless for the yogi to seek athletic ability using kundalini energy . nevertheless , some studies using subjective methods to study the effect of hy in be demonstrate that systematic practice improves the yogi`s vitality and perception of its own physical condition , social functioning and quality of life . also , because of the nature of physical exercises ( asanas ) performed in hy , it is common to find exceptional physical abilities in trained yogis especially in muscular flexibility , strength and stress control . given the mystical - philosophical roots of hy , the contemporary yogi continues to strive for something more than merely physical and mental health . however , due to the benefits of overall health , it is important to continue to study in detail the subject of be of hy as compared to other exercise protocols . in the following paragraphs , only the measurable energetic aspects are evaluated from the tee point of view and in terms of changes in ebs which in turn modify tei . on the metaphysical aspect of hy , psychology and anthropology can provide better arguments and theories , an aspect that escapes the purpose of this review scientific studies on em are focused on measuring tei or tee . the latter is generally measured at rest ( resting energy expenditure [ ree ] ) or at sleep ( basal , basal energy expenditure ) or as a result of different pathological , pharmacological , physiological or nutritional modifications . at cellular and molecular levels , many ionic , enzymatic , biosynthetic and genetic mechanisms are involved with either tei or tee . consequently , several metabolic indicators , forms of measurement and equations to estimate the study of the body`s energy balance and body weight control have been generated . recently goshvarpour et al . reported chaotic heart rate signals as a result of kundalini meditation , which are quite different from those observed in chinese chi meditation . from a physiological perspective , meditation is a physiological state of demonstrated reduced metabolic activity different from sleep that elicits physical and mental relaxation . therefore , the ee involved in kundalini meditation is ree . also , while performing hy ( asanas ) the physical intensity measured as consumption of oxygen ( vo2 ) or metabolic equivalents ( mets ) , is low ; in fact , it is lesser than that expended in other physical activities such as walking , jogging , running , cycling and swimming . , while studying young adults , found that the mets while performing hy ( asanas + pranayamas + dhyana ) is 53% less than jogging at 3.5 mill / h ( 2.2 vs. 3.3 mets ) ; hagins et al . , found that it is similar to walking at 2 mill / h ( ~2.5 mets ) , and that asanas performed in sitting or lying position expend lesser energy ( 1.5 mets ) than those performed in standing positions ( 2.3 mets ) . on the other hand , and wallace and benson , found relevant reductions in vo2 during meditation and relaxation ( yoganidra ) as compared to resting conditions ( ~2.6 vs. 4.0 ml of o2/kg of body mass / min ) . all of the above indicates that common protocols of hy are characterized as being of very low intensity , with little possibility of cardiovascular benefits . however , there is a possibility to improve the physical performance , hemodynamic function and increased cardio - respiratory reserve in hy , in spite of the low exercise stimulus ; this as a consequence of concerted physical and mental events , such as local muscular adaptation during some physically intense asanas , breathing exercises ( pryma ) and psycho - physiological control ( concentration ) . it is noteworthy that asanas performed at different intensities may increase tee up to 3.0 kcal / min while that expended on breathing exercises or during meditation is 2.0 and 1.4 kcal / min , respectively . in view of these arguments , from a cardiopulmonary conditioning standpoint , it is necessary to include complementary aerobic exercises into the hy routine or to perform it with greater intensity . sun salutation ( surya namaskar ) is one of the oldest yoga exercises known to man and is one of the most popular and well - acclaimed yoga postures . it has been practiced for centuries and consists of 1012 different postures which are preferably performed at dawn . each posture counteracts the preceding one producing a balance between flexion and extension with synchronized breathing and aerobic activity . the posture cycle can be repeated several times and at different velocities in the same workout , thereby placing more emphasis on increase of tee and cardiovascular conditioning . however , surya namaskar requires , for its proper performance , an adequate amount of flexibility and muscular strength which is why the studies of this practice have only been done on people who are young or physically fit . further , the execution must be rhythmic in nature , with each posture and its transition being executed in smooth cadence , and the postures must be performed with minimal jerks or ungainly movements . mody , reported vo2 of 26 ml / kg / min during each round , resulting in an tee of 234 kcal during a 30 min session for a 60 kg individual . that ee is enough to maintain body weight or to improve aerobic conditioning . in view of this and in accordance with its intensity and tee , surya namaskar is classified as a moderate - to - high intensity exercise that can be used , when performed at high rhythms , as a form of cardiopulmonary conditioning for people who are young or physically fit . furthermore , it could be included in contemporary hy sessions . in order to demonstrate that surya namaskar is a safe exercise , omkar et al . , studied the force and moment effects on specific joints ( wrist , elbow , shoulder , hip , knee and ankle ) during practice of surya namaskar . using a mathematical model , they found that none of the joints were overstressed during surya namaskar practice , and concluded that the joints involved are subjected to submaximal loadings as compared to more high impact exercises for which the ee is comparable . this is of particular importance for older people and for those who have functional limitations in performing aerobic training . other alternatives for increasing tee and cardiovascular conditioning while performing hy , is increasing its intensity and duration of the sessions or adding complementary aerobic and muscular resistance exercises . ray et al . , also demonstrated improved aerobic capacity and decreased perceived exertion after the maximal exercise of hy . ramos - jimnez et al . , found that 11 weeks of an intensive hy program , under a more intense protocol than usual performed by trained practitioners of yoga , produced an increase in vo2 max ( ~3 ml / kg / min ) , a decrease in body fat ( ~1.5 kg ) , systolic blood pressure ( ~5.5 mmhg ) and diastolic blood pressure ( ~3 mmhg ) , as well as a weekly tee of ~1000 kcal . so , intense hy would fulfill the minimum guidelines of the american college of sports medicine for maintaining body weight . in conclusion , given the growing popularity of hy , it can be considered as an alternative to increase the level of physical activity . however , it is recommended to increase the intensity and duration and to include alternative exercises like surya namaskar to ensure a maximal tee and cardiovascular fitness . for instance , the practice of asanas could be an optimal method for preserving the physical function in older people if exercise series are adapted to muscle and join performance as demonstrated in the yoga empowers senior study . lastly , the surya namaskar could be a better alternative for cardiovascular health , but this should be practiced with caution , especially in people with low fitness levels . the dietary pattern of a person is one of the most important predictors of health risk . there is substantial evidence that a diet rich in fruits , vegetables , whole grain cereals , lean meat , and fish are inversely associated with the risk of chronic illnesses like cardiovascular disease , cancer , or diabetes . however , the food selection is a complex behavioral process since individuals and groups make dietary choices based on food familiarity , availability , cost , cultural norms , taste preferences , health , and convenience , among other factors . the current environment of modern food , with the wide variety of food options , can be so large that it can become difficult to identify a consistent food pattern among people . healthy eating can also be considered a practice to seek for and attain harmonic body / mind balance . according to yoga philosophy , there are intimate connections of diet with mind , and foods have an unknown subtle essence difficult to prove through modern scientific methods . according to yoga , there are three types of foods : sattvic , rajasic and tamasic . the sattvic diet ( pure and balanced ) is believed to increase energy , produces happiness , calmness , and mental clarity . it could enhance longevity , health , and spirituality . according to maha narayana upanishad ( ~5000 b.c . ) it promotes a life expectancy of 100150 years and it is recommended for saints . all foods included in this diet are fresh , juicy , nutritious , and tasty , thus including the consumption of fresh fruits and vegetables , sprouted grains , roots , tubers , nuts , cow milk , curd , and honey . the sattvic dietary pattern appears to be similar to a modern but prudent dietary pattern.the rajasic diet ( over stimulating ) is believed to produce jealousy , anger , unfaithfulness , fantasies , and selfishness . it is recommended to leaders and fighters since it may cause excitement , confidence and increase in intelligence . the foods in this diet are bitter , tart , salty , spicy , hot , and dry ; they also include white sugar , radishes , and fried foods.the tamasic diet ( weakens and makes sleepy ) is believed to increase pessimism , weakness , laziness , and doubt . the yoga practitioners mention that this dietary pattern makes one dull , enhances anger and criminal tendency and impedes spiritual progress . the foods in this diet include meats from big tamed animals , onions , mushrooms , stale , undercooked - and highly fried foods , high fat fried foods , salt , sugar , spices , chilies pepper , butter and liquor ; medicines and stimulants are also included.agte and chiplonkar compiled a database of nutrient contents of 110-food items in two nonconsecutive 24 h - dietary recall of 109 apparently healthy adults . they classified the foods according to their gunas ; the sattvic food had the highest micronutrient density , followed by rajasic and tamasic . although fiber content was quite similar ( ~14 mg / kcal ) , the fat content was 18% , 42% , and 72% , respectively . dietary intake of sattvic , rajasic and tamasic were ~802 , ~61 and ~213 g / d . sattvic food intake had the highest correlation with food micronutrients ( ~r = 0.5 , p < 0.01 ) , rajasic only with thiamin intake ( r = 0.47 , p < 0.01 ) and tamasic with zinc ( r = 0.23 , p < 0.01 ) , iron ( r = 0.30 , p < 0.01 ) and the presence of anxiety ( r = 0.37 , p < 0.01 ) . from this evidence , the authors argued that sattvic food have the better health benefits . the authors included in their study a diet plan that would allow reduction of tamasic and rajasic foods and an increase in those sattvic . the sattvic diet ( pure and balanced ) is believed to increase energy , produces happiness , calmness , and mental clarity . it could enhance longevity , health , and spirituality . according to maha narayana upanishad ( ~5000 b.c . ) it promotes a life expectancy of 100150 years and it is recommended for saints . all foods included in this diet are fresh , juicy , nutritious , and tasty , thus including the consumption of fresh fruits and vegetables , sprouted grains , roots , tubers , nuts , cow milk , curd , and honey . the sattvic dietary pattern appears to be similar to a modern but prudent dietary pattern . the rajasic diet ( over stimulating ) is believed to produce jealousy , anger , unfaithfulness , fantasies , and selfishness . it is recommended to leaders and fighters since it may cause excitement , confidence and increase in intelligence . the foods in this diet are bitter , tart , salty , spicy , hot , and dry ; they also include white sugar , radishes , and fried foods . the tamasic diet ( weakens and makes sleepy ) is believed to increase pessimism , weakness , laziness , and doubt . the yoga practitioners mention that this dietary pattern makes one dull , enhances anger and criminal tendency and impedes spiritual progress . the life expectancy is low , and it is bad for health . the foods in this diet include meats from big tamed animals , onions , mushrooms , stale , undercooked - and highly fried foods , high fat fried foods , salt , sugar , spices , chilies pepper , butter and liquor ; medicines and stimulants are also included . it is noteworthy that sattvic food included a significant amount of functional foods such as soy milk ( flavonoids ) , tomatoes ( lycopene ) , herbal teas ( polyphenols ) and red amaranth ( bioactive peptides ) . there is also some evidence that practicing other disciplines in which the mind - body axis is used ( e.g. tai chi ) , changes the pattern of food consumption . in this respect , practitioners ( mainly from asian countries ) of these disciplines have diets based on a wide variety of vegetables , fruits , vegetables , and spices , thereby obtaining a high intake of micronutrients and functional ingredients , in addition to a reduced amount of dietary fat . cross - sectional studies show that yoga practitioners have better dietary patterns than their sedentary counterparts . palasuwan et al . , when evaluating dietary intake and cardiovascular risk factors in pre and postmenopausal thai women who practice yoga versus . tai chi practitioners or sedentary women , found that yoga practitioners have lower intakes of fats and ( bmi , kg / m ) ; the enzymatic antioxidant activity were similar among groups . when multidisciplinary interventions in which dietary habits , physical activity , stress management and hy are included , significant improvements in overall health is shown as a result of the intervention . postulate that home practice of yoga predicts healthy lifestyle changes including an increased intake of fresh fruits and vegetables . also , preliminary data obtained from a dietary evaluation in mexican hy practitioners indicate important changes toward a healthier and adequate diet [ table 1 ] and the gradual inclusion of functional ingredients such as fiber foods , lignans and flavonoids [ figure 1 ] . daily nutrient adequacy ( % ) of mexican yoga practitioners * from northern mexico lignans ( g / d ) and flavonoid intake ( mg / d ) of mexican yoga practitioners * from northern mexico . source : author 's unpublished data ; * 52 16 years old , 65% female however , there are still important questions about the relationship between diet and yoga , particularly on how a practice of yoga changes other specific aspects of the diet ( e.g. , antioxidant intake ) or how it modifies other biomarkers of dietary change ( e.g. , homocysteine ) . studies on these issues should not only consider the qualitative and quantitative aspects involved but also the holistic nature of the phenomenon . nevertheless and despite very few studies involving dietary assessment in yoga practitioners ( beginners and/or advanced ) , it can be safely concluded that the improvement in the spiritual well - being results in a healthier eb in the long - term . in this sense , ayurvedic ( sanskrit : ayus [ meaning life ] and veda [ knowledge ] ) treatments which consists of use herbal preparations , diet , yoga , meditation , and other practices , is gaining recognition in western societies as a holistic alternative intended to treat many metabolic and neuropsychiatric disorders from a predictive , preventive and personalized medicine standpoint . the diagnostic and statistical manual of mental disorders - fourth edition , text revision , lists three major types of ed : anorexia nervosa , bulimia nervosa , and unidentified disorders . anxiety and depression are common neuropsychiatric conditions in individuals with ed , being seen in ~60% of patients with anorexia and bulimia nervosa . on the other hand , binge eating is a disorder that is characterized by several criteria which include consuming large amounts of food accompanied by feeling of a lack of control . clinical , community , and population studies have reported that this disorder is associated with being overweight and severe adiposity . the treatment for the different forms of ed is based mainly on cognitive - behavioral and interpersonal therapy with the purpose of inducing positive behavioral changes concerning the people 's food intake . however , the lack of progress in treatment development , at least in part , reflects the fact that little is known about the pathophysiologic mechanisms that account for the development and persistence of ed . yoga , while seeking for the harmony of the mind and body , benefits people at risk or with established ed . dittmann and freedman , when studying body self - perception , attitudes toward food , and the spiritual beliefs of 158 female yoga practitioners , observed improvements in body satisfaction and self - acceptance along with reduced disordered eating associated to their yoga practice . similarly , intervention programs in which yoga is included as an alternative to the treatment of ed in persons with chronic obesity have shown that 12 weeks of hy practice reduces compulsive eating ( binge eating ) , lengthens meal times and improves food quality . other interventions , in which problems of anorexia and bulimia nervosa are dealt with yoga practice , also show similar results . carei et al . , when studying 54 girls ( 1121 years ) with ed , found that 1 h/2 times / week sessions of yoga for 8 weeks reduces symptoms of depression , anxiety , and worries about food as compared to girls with ed treated with conventional clinical methods . however , other studies have shown the opposite , especially when yoga is compared to other psychological strategies . , when studying ed in school age women through yoga and cognitive dissonance techniques , found that yoga fails to change these disorders , but cognitive dissonance reduced anxiety and the inability to express emotions ( alexithymia ) , improving self - perception of the body as well . it is important to note at this point that the success of clinical interventions for patients with ed , depends on many factors , but some of them have to do with age , the type of disorder and the severity of symptoms that often accompany them . in conclusion , these and other studies show that the common practice of hy can impact positively on several eds . however , more studies are needed that compare hy versus alternative clinical treatments for ed . contemporary hy ( asanas + pranayamas + dhyana ) , seen holistically , is effective for certain health problems such as hypertension , ed , stress , among others . however , due to their low intensity and low ee , they are not recommended for weight loss or improving cardiovascular conditioning . there are alternative exercises like surya namaskar , which can be included in its everyday practice , thereby improving health benefits . also , the practice of yoga is associated to healthy ebs such a higher consumption of fresh vegetables , dairy products , whole - grains and functional foods ( e.g. soy - based products ) , which could help in ed , but more case control studies are needed to recommend it as a clinical approach in eating disorders .

yoga is an ancient oriental discipline that emerged from mystical and philosophical concepts . today it is practiced in the west , partly due to the promotion of its benefits to improve the lifestyle and overall health . as compared to non - hatha yoga ( hy ) practitioners , healthier and better - eating patterns have been observed in those who practice it . agreement with the brought benefits , hy can be used as a therapeutic method to correct abnormal eating behaviors ( aeb ) , obesity , and some metabolic diseases . however , the energy expenditure during traditional protocols of hy is not high ; hence , it is not very effective for reducing or maintaining body weight or to improve cardiovascular conditioning . even so , several observational studies suggest significant changes in eating behaviors , like a reduction in dietary fat intake and increments in that of fresh vegetables , whole grains and soy - based products , which in turn may reduce the risk for cardiovascular diseases . given the inconsistency of the results derived from cross - sectional studies , more case control studies are needed to demonstrate the efficacy of hy as an alternative method in the clinical treatment of disordered eating and metabolic diseases .

INTRODUCTION METHODOLOGY YOGA BIOENERGETICS YOGA AND ENERGY EXPENDITURE YOGA AND EATING BEHAVIOR YOGA AND EATING DISORDERS CONCLUSION

PMC5031375

when think about individiually , it is easily understood that athletes injury prevention and sports performance development is very crucial performance in sport is closely related with lactate metabolism and level in the blood ( karlsson et al . , 1975 ; elimination of the lactate in the blood is the most effective way to increase the muscle performance and strenght ( stamford et al . , 1978 ) . in our study ; our aim was to explain the effects of compression garment and electrostimulation on athletes recovery period by evaluating blood lactate and isokinetic peak torque parameters . we investigated the effects of compression stocking and electrostimulation on blood lactate value and isokinetic strength value during the first 30 min of recovery after 30 min of aerobic running the treadmill at anaerobic threshold heart rate of 85% . nine female volleyball players ( age , 15.890.33 yr ; height , 171.789.52 cm ; weight , 60.444.97 kg ) and 11 male basketball players ( age , 15.270.46 yr ; height , 182.555.75 cm ; weight , 70.7312.81 kg ) totally 20 athletes ( age , 15.550.51 years ; height , 177.79.26 cm ; , weight , 66.111.15 kg ) were included the study . none of the participants had a history of heart or circulation disorder . before the study , each participant was informed about the purpose and risk of the study and signed an informed consent . methodologically , athletes were conducted two different laboratory test in the first week of laboratory visit . it was performed for the determination of maximal oxygen consumption ( vo2 max , ml / kg.min ) and anaerobic threshold pulse . it is performed for determination of the triceps surae and tibialis anterior physiologic power and endurance parameters . then , the athletes were conducted specific mask ( metalyzer 3b , cortex , leipzig , germany ) for determination of anaerobic threshold pulse and vo2 max by evaluating co2 and o2 levels ( metasoft 3 , cortex ) via v slope method . during cardiopulmonar exercise testing , all participants were monitored and electrocardiography changes and blood pressure were recorded every 3 min . the participants were run until personal exhaustion happened unless identification of any symptom or sign required to stop bruce test protocol . in our study , during cardiopulmonar exercise test , the last 10-sec maximal average value on the peak exercise was determined vo2 max . eight - five percent of anaerobic threshold pulse was identified personally in order to make athletes exercise on the aerobic metabolism for the next experimental test protocols . the isokinetic dynamometer test was planned as a second test for determining triceps surae and tibialis anterior physiologic power and endurance parameters . before isokinetic testing , participiants cycled 10 min on 5060 rpm for warming on the horizantal exercise cycle ( bike 5150r , sportsart , tainan , taiwan ) . after warming period , the participants performed low extremity stretching exercises for 5 min . immediately after stretching period , the isokinetic test ( cybex extremity system ) was performed for right and left ankle . the right and left triceps suare and tibialis anterior peak torque was evaluated on the 30/sec , endurance was evaluated on the 120/sec and physiological datas were recorded . sigvaris compression stocking ( poliamid % 64 , polyester % 17 , elastane % 19 ; ganzoni & cie ag , galen , switzerland ) was used ( 2030 mmhg ) . veinoplus neuromuscular stimulator was used for electrostimulation ( low - frequency electrostimulation ) ( ad rem technology , rue de faubourg saint honore , paris , france ) . experiment protocol was performed with the usage of electrostimulation device ( low - frequency electrostimulation ) . isokinetic test results for both ankle were planned as performance indicators during 15th min . and at the end of recovery period . during three sessions , the first isokinetic test was planned on the 15th min of the recovery period because the half time of the most team sport and individual sport lasts 15 min . the second isokinetic test was planned on the 30th min of the recovery period ( fig . athletes were exercised on treadmill on the pulse of 85% anaerobic threshold threshold value during 30 min in order to provide aerobic exhaustion . during exercise , athletes pulse value was monitored . after exercise period , athletes were sat on a chair for 30th min recovery period . during recovery period , blood samples were taken on the 0th , 3rd , 5th , 15th , 30th min from athletes finger and this blood samples were evaluated and results were recorded . first isokinetic test was performed to the right ankle of the athletes during the 15th min of recovery period and numeric datas were recorded . second isokinetic test was performed to the left ankle of the athletes during the 30th min of recovery period and numeric datas were recorded . after exercise period , compression stockings were worn before athletes were sat on a chair for 30th min recovery period , and same protocol applied after wearing compression stockings . electrostimulation was applied on to the bilateral calf muscles , same protocol applied after applying electrostimulation . the blood lactate values and isokinetic parameters during sessions , two tests were used to statistical analysis . methodologically , athletes were conducted two different laboratory test in the first week of laboratory visit . it was performed for the determination of maximal oxygen consumption ( vo2 max , ml / kg.min ) and anaerobic threshold pulse . it is performed for determination of the triceps surae and tibialis anterior physiologic power and endurance parameters . then , the athletes were conducted specific mask ( metalyzer 3b , cortex , leipzig , germany ) for determination of anaerobic threshold pulse and vo2 max by evaluating co2 and o2 levels ( metasoft 3 , cortex ) via v slope method . during cardiopulmonar exercise testing , all participants were monitored and electrocardiography changes and blood pressure were recorded every 3 min . the participants were run until personal exhaustion happened unless identification of any symptom or sign required to stop bruce test protocol . in our study , during cardiopulmonar exercise test , the last 10-sec maximal average value on the peak exercise was determined vo2 max . eight - five percent of anaerobic threshold pulse was identified personally in order to make athletes exercise on the aerobic metabolism for the next experimental test protocols . the isokinetic dynamometer test was planned as a second test for determining triceps surae and tibialis anterior physiologic power and endurance parameters . before isokinetic testing , participiants cycled 10 min on 5060 rpm for warming on the horizantal exercise cycle ( bike 5150r , sportsart , tainan , taiwan ) . after warming period , the participants performed low extremity stretching exercises for 5 min . immediately after stretching period , the isokinetic test ( cybex extremity system ) was performed for right and left ankle . the right and left triceps suare and tibialis anterior peak torque was evaluated on the 30/sec , endurance was evaluated on the 120/sec and physiological datas were recorded . sigvaris compression stocking ( poliamid % 64 , polyester % 17 , elastane % 19 ; ganzoni & cie ag , galen , switzerland ) was used ( 2030 mmhg ) . veinoplus neuromuscular stimulator was used for electrostimulation ( low - frequency electrostimulation ) ( ad rem technology , rue de faubourg saint honore , paris , france ) . experiment protocol was performed with the usage of electrostimulation device ( low - frequency electrostimulation ) . isokinetic test results for both ankle were planned as performance indicators during 15th min . and at the end of recovery period . during three sessions , the first isokinetic test was planned on the 15th min of the recovery period because the half time of the most team sport and individual sport lasts 15 min . the second isokinetic test was planned on the 30th min of the recovery period ( fig . athletes were exercised on treadmill on the pulse of 85% anaerobic threshold threshold value during 30 min in order to provide aerobic exhaustion . during exercise , athletes pulse value was monitored . after exercise period , athletes were sat on a chair for 30th min recovery period . during recovery period , blood samples were taken on the 0th , 3rd , 5th , 15th , 30th min from athletes finger and this blood samples were evaluated and results were recorded . first isokinetic test was performed to the right ankle of the athletes during the 15th min of recovery period and numeric datas were recorded . second isokinetic test was performed to the left ankle of the athletes during the 30th min of recovery period and numeric datas were recorded . after exercise period , compression stockings were worn before athletes were sat on a chair for 30th min recovery period , and same protocol applied after wearing compression stockings . electrostimulation was applied on to the bilateral calf muscles , same protocol applied after applying electrostimulation . the blood lactate values and isokinetic parameters during sessions , two tests were used to statistical analysis . there was no significant difference on blood lactate levels within measurements ( table i ) . at women ; there was no significant difference on isokinetic peak torques within compression garment measurements and control measurements . at electrostimulation measurements there was a significant increases on right plantar flexion ( rpf ) ( p<0.01 ) , right dorsal flexion ( rdf ) ( p<0.1 ) , and left plantar flexion ( lpf ) ( p<0.1 ) values compared to control measurements . at men , with compression garment measurements , there was a significant increase on lpf values compared to control measurements ( p<0.1 ) . at electrostimulation measurements , there was a significant increases on rpf ( p<0.1 ) and lpf ( p<0.1 ) values compared to control measurements . compression stocking and electrostimulation intervention effects on force generating capacity on isokinetic device during recovery period had statistically significant compared to passive recovery . in terms of the force generating capacity , electrostimulation intervention at 15th min of recovery and wearing compression stocking at the 30th min of recovery were more statistically significant ( tables 2 , 3 ) . it was shown that there is a close relationship between lactate metabolism and exercise performance ; work capacity and performance are affected negatively by increased blood lactate level ( bangsbo et al . , 1996 ; klausen et al . , 1972 ; stamford et al . , 1978 ) . the impact of the various recovery protocols on blood lactate elimination following maximal exercise was investigated . active recovery process was showed to be the most effective way in ensuring the elimination of lactate . to be sure , assuming that the active recovery has contributed to the increase of working muscles metabolism and blood flow in aerobic environment , the active recovery is believed to result in lactate distribution and transformation as a substrate for oxidation ( bangsbo et al . , 1994 ; brooks , 1986 ; hatta , 1990 ; martin et al . , 1998 ; monedero and donne 2000 ) . in our study ; it was thought that the lactate value differences between 0th min control measurement belonging to same female athletes and measurements of compression stocking and electrostimulation application could depend on sleep and rest durations of the previous night , eating habits , rest status for the last 24 hr and nourishment situation during that day of the athlete . in the application of control and electrostimulation during the process between the 0th and the 5th min of the recovery , blood lactate level , while tending to fall , were detected to increase a little amount when compression stocking is worn . this increase might be due to the difficulty and stress during wearing of compression stocking . in the period from the 5th to 15th min of the recovery , it was observed that blood lactate values obtained from compression stockings and electrostimulation applications are in a downward tendency while blood lactate values in control application are in an upward tendency . this change is not statistically significant . in the 15th min of the recovery , while the lowest blood lactate values are observed in the measurements to which the electrostimulation applied , the highest values are seen in control measurements . according to the control measurements ; this decrease observed on the people who are wearing compression stockings and to whom electrostimulation is applied is not statistically significant . blood lactate values in the people wearing compression stockings were found to be higher than those to whom electrostimulation is applied . between the 15th and the 30th min of the recovery period , while blood lactate values of the subjects wearing compression stocking and undergoing electrostimulation application tend to increase , blood lactate values of the subjects in control group tend to decrease . when blood lactate levels of male and female athletes are evaluated together , the highest blood lactate values are observed in control measurements in the 15th min of the recovery while the lower lactate values are observed in the athletes wearing compression stockings . these differences are not statistically significant . after 15 min , while the blood lactate values tend to decrease in control measurements ; they tend to increase in those wearing compression stockings and undergoing electrostimulation application . to evaluate the effects of compression stockings on the changes of athletic performance indication , lower lactate and haematocrit levels were detected after exercise in those wearing compression stockings ( berry and mcmurray , 1987 ; chatard et al . , 2004 ) . in our study , in the 15th min of the recovery after 30 min of aerobic exercise , blood lactate values are observed to be lower in females and total compression stockings implementation than those observed in control group , but the difference was not statistically significant . among many possible effective recovery modalities there are limited studies and scientific evidence related to the effect of many physiological variables on the increase of recovery kinetic , continuance of athletic performance and the decrease of rating of perceived exertion . in the studies comparing the different postexercise recovery periods , no significant difference in lactate levels were determined ( cortis et al . , 2010 ; tessitore et al . , 2008 ) . active recovery and electrostimulation application were observed to have longer climbing periods and more positive effects on blood lactate concentrations than passive recovery ( heyman et al . , 2009 ; , we found that the highest lactate values in the 15th min of recovery period after 30th min of aerobic exercise is in control measurements at rest sitting . even if blood lactate levels were determined to be lower in those wearing compression stockings and higher in those experiencing electrostimulation application , these differences were statistically significant . we used isokinetic strength values as a second parameter in researching the effects of compression stockings electrostimulation application on the recovery . in the 15th min of the recovery in electrostimulation application of females likewise , in the 15th min of recovery , we determined the statistically significant increase in the peak rdf . we did not determine significant changes in the peak rpf and peak rdf values arising from compression stockings implementation for both parameters as well . in the peak lpf values evaluated in the 30th min of recovery , we determined significant increase compared to the control measurements . in the peak lpf values of compression stockings application , we did not find a significant change compared to control measurements . according to the peak ldf values evaluated in the 30th min of recovery ; we did not identify significant changes in compression stockings and electrostimulation applications . in the peak rpf values evaluated in the 15th min of recovery in males , we identified a significant increase in electrostimulation applications compared to control measurements . we did not identify a significant change in compression stockings application compared to control measurements . in the peak lpf values evaluated in the 30th min of recovery , we identified significant increases both in compression stockings and in electrostimulation applications compared to control measurements . in the peak ldf values , another parameter evaluated in the 30th min of recovery compression stockings used before exercise , during exercise and after exercise were showed not to have an effect on blood lactate levels and not to have developed the recovery strength in the first 30 min ( duffield et al . 2006 ) . in the studies evaluating the effects of the use of compression stockings on muscle fatigue and muscle concentric strength , compression stockings were found to alleviate the decrease in concentric strength and muscle fatigue compared to control group ( ali et al . , 2007 ; duffield and portus , 2007 ; jakeman et al . , 2010 ; perrey et al . , 2008 ) . in our study , we used compression stockings with a pressure capacity of 30 mmhg at the level of ankle , and compression stockings with a pressure capacity of 20 mmhg whose intensity gradually decreases up to the knee , even if we did not observed a significant strength increase in females compared to control group , we found a significant increase in lpf strength parameters of males . in the peak strength values of rpf , rdf , and ldf which are other strength parameters , although increases were found in the group wearing compression stockings in comparison with control groups , there was not a statistically significant difference . in recovery process ; passive recovery , active recovery ( running on 50% of aerobic capacity ) and the effect of low - frequency electrostimulation were compared with each other . when knee extensor contractions with maximal voluntary were compared , it was stated that electrostimulation approach leads to a better contraction intensity , not showing a significant difference though , on the other hand , there is a need for further studies on this issue ( cortis et al . , 2010 ; grunovas et al . , 2007 ; lattier et al . , 2004 ; mcloughlin et al . , 2004 ; tessitore et al . , 2007 ; vanderthommen et al . , 2007 ) . in our study , with low - frequency electrostimulation during 30th min of recovery process after 30th min of aerobic exercise , we compared the control measurements in terms of isokinetic values for right ankle in the 15th min . we identified significant increases in rpf , rdf , and lpf isokinetic strength values in females undergoing electrostimulation compared to control measurements . as for males undergoing electrostimulation , we identified significant increases in rpl and lpl isokinetic strength values compared to control measurements . when we evaluate the results of the study that we conducted and compare this with the other studies ; we consider that electrostimulation application can be an alternative method in recovery period . during recovery , when compared to control measurement , there is not any beneficial effect seen on blood lactate level within compression garment measurements and electrostimulation measurements . when compared to passive rest , compression garments and electrostimulation interventions effects on force generation capacity at recovery are statically significant . if we consider the low level of lactate at the 15th min of recovery period especially on electrostimulation group , applying electrostimulation during halftime of activities like football , basketball seems to be beneficial . there has to be more comprehensive studies needed among bigger populations to evaluate the effectiveness of compression garments and electrostimulation on athletes recovery period .

in this study , we explained the effects of compression garment and electrostimulation on athletes recovery period by evaluating blood lactate and isokinetic peak torque parameters . twenty volunteers ( 15.55 0.51 yr ) were included to study . at recovery period , blood samples was taken for lactate values at 0th , 3rd , 5th , 15th , 30th min . the isokinetic strength test was performed on right ankle at 15th min and on the left ankle at 30th min . the same protocol was performed for compression garment on 2 weeks and for electrostimulation on third weeks and results were compared . there was nt any significant difference on blood lactate levels within groups . at women ; there was not any significant difference on isokinetic peak torques within two groups . but at electro - stimulation usage we found significant increases on right plantar flexion ( p<0.1 ) , right dorsal flexion ( rdf ) ( p<0.1 ) and left plantar flexion ( lpf ) ( p<0.1 ) values compared to control measurements . at men ; with compression garment usage , there was significant increase on lpf values compared to control measurements . at electrostimulation usage , we found significant increases on rdf ( p<0.1 ) and left dorsal flexion ( p<0.1 ) values compared to control measurements . during recovery , there is not any beneficial effect seen on blood lactate level within two groups . when compared to passive rest , compression garments and electrostimulation interventions effects on force generation capacity at recovery are statically significant . also in terms of force generation capacity ; usage of electrostimulation during 15 min and compression garments during 30 min were statically more significant .

INTRODUCTION MATERIALS AND METHODS Preliminary testing Compression stockings Electrostimulation device Expermental desgn RESULTS DISCUSSION