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PMC4780664

since 2010 , the us national health interview survey ( nhis ) has included questions assessing pain persistence and pain bothersomeness . these questions were developed and piloted as part of an international initiative under the purview of the washington group on disability statistics,1 constituted by the united nations statistical commission . the goal of the washington group is to promote and coordinate international cooperation in the area of health statistics focusing on disability measures suitable for censuses and national surveys and to develop tools to collect the basic data necessary to provide information on disability that is comparable throughout the world.2 based on qualitative and mixed - method studies performed in the united states and other countries affiliated with the washington group , miller and loeb3 have proposed a coding system that combines data from the nhis questions on pain persistence and bothersomeness to create discrete categories of increasing pain severity . in a previous study,4 we not only demonstrated the concurrent validity of the coding system , with the pain categories discriminating between different levels of health status , disability , and health care use , but also identified steps that needed to be taken before the coding scheme could be fully incorporated into the armamentarium of the pain clinician or scientist . in particular , we noted that the category cut points for the coding system needed to be confirmed in quantitative analyses and in different anatomical sites of pain . such data would offer additional support as to whether the coding system proposed by miller and loeb3 provides a valid measure of an individual s current pain experience . should the miller and loeb pain categories eventually fulfill all the criteria of a well validated and clinically useful pain scale , then the brevity of the miller and loeb approach will make it an excellent choice in situations where clinician time and patient burden are limiting factors . in the current analyses , using data from the 2012 nhis , we quantitatively assessed the pain category definitions proposed by miller and loeb3 by adapting a statistical approach widely used in the pain field for grading pain intensity with functional interference.5 three goals that guided this study are 1 ) to assess whether the cut point analysis method proposed by serlin et al5 can be applied to pain severity categories defined by self - reported pain persistence and bothersomeness , using measures of health status and health care use as cut point criteria ; 2 ) to determine whether the optimal pain category definitions identified in the entire 2012 nhis adult samples would be evidenced in randomly derived subsamples of the full sample ; and 3 ) to determine if the same optimal pain category definitions would be evidenced in back pain and joint pain subpopulations . the data used in this study are from the 2012 nhis sample adult core and the nhis adult functioning and disability ( afd ) supplement.6 the nhis is an annual survey of the health of the us civilian , noninstitutionalized population conducted by the national center for health statistics , centers for disease control and prevention . this in - person survey contains four main modules : household , family , sample child , and sample adult . the first two modules collect health and sociodemographic information on each member of all families residing within a sampled household . within each family , additional information is collected from one randomly selected adult ( the sample adult ) aged 18 years or older . bilingual interviewers or interpreters were recruited to interview all respondents who preferred the use of a language other than english . the survey uses a multistage clustered sample design and oversamples black , asian , and hispanic populations . when combined with centers for disease control and prevention - derived sampling weights , this design allows accurate extrapolation of findings to the civilian , noninstitutionalized us adult population . for the 2012 interview sample , there were 42,366 households consisting of 108,131 persons in 43,345 families . the total household response rate was 77.6% . from the households interviewed , 34,525 adults completed interviews , resulting in an overall sample adult response rate of 79.7% . approximately one - quarter of sampled adults were randomly chosen to participate in the afd supplement . almost all chosen adults ( 8,781 ) completed the supplement resulting in a 98% supplement response . the 2012 nhis was approved by the national center for health statistics research ethics review board . the afd collected information on the persistence and bothersomeness of self - reported pain in the previous 3 months . respondents were first asked how often they had pain in the previous 3 months : never , some days , most days , or every day . data from this question provide estimates of the 3-month persistence ( period prevalence ) of pain . for those who had pain on at least some days , a follow - up question assessing bothersomeness was asked : thinking about the last time you had pain , how much pain did you have a little , between a little and a lot , or a lot . ninety - six percent of afd participants completed these pain questions . given this high response rate , no attempt was made to impute missing data . miller and loeb3 have suggested a coding scheme that combines persistence and bothersomeness of pain to create four discrete categories of increasingly severe pain ( figure 1 ) . this coding scheme was tested and validated using a variety of qualitative assessments such as cognitive testing and mixed methods analyses2,3,7 and has been shown to have concurrent validity.4 in order to be consistent with the original pain category definitions of miller and loeb,3 the alternative definitions studied were restricted to four discrete categories that varied in only in their distributions of pain persistence and bothersomeness . to aid in identifying a set of alternative pain category definitions , we visually explored the relationship between nine pain persistence / bothersomeness combinations and measures of health status and health care use as defined later . chronic pain often predicts the onset of psychological distress.8,9 in turn , psychological distress has been identified as one of the factors mediating pain s relationship to disability.10 the nhis measures nonspecific psychological distress over a 30-day recall period with the kessler 6 ( k6 ) scale.11 the k6 scale asks respondents about six manifestations of psychological distress : during the past 30 days , how often did you feel ( a ) so sad that nothing could cheer you up ? possible responses are all of the time , most of the time , some of the time scoring of the individual questions is based on a scale of between 0 and 4 points , according to increased frequency of the problem , yielding a total score on the scale from 0 ( no psychological distress ) to 24 ( extreme psychological distress).11 this was examined as a continuous variable . pain often results in disability days.1214 health - related bed - disability days were assessed with the survey question : during the past 12 months , about how many days did illness or injury keep you in bed more than half of the day ( include days while an overnight patient in a hospital ) ? pain is associated with increased health care use,13,15,16 including visits to emergency departments.17,18 health care use was examined with two nhis survey questions : 1 ) how many times did you visit a doctor or other health care professional during the last 2 weeks ? and 2 ) during the past 12 months , how many times have you gone to a hospital emergency room about your own health ( this includes emergency room visits that resulted in a hospital admission ) ? the 2012 nhis adult core included questions on the presence of low back pain within the previous 3 months ( yes and no ) , and on the presence of joint , aching or stiffness in the last 30 days ( yes and no ) . we studied the alternative definitions of the four pain categories using a variation of the statistical method described by serlin et al.5 each alternative definition was related simultaneously to four dependent measures the k6 score , the number of health related bed - disability days , the number of visits to a health professional , and the number of emergency room visits using multivariate analysis of variance ( manova ) . the manova yields f values , based on wilk s lambda , for the between - category effect on the dependent variables . following serlin et al,5 the pain category definition yielding the best model fit , as measured by the f value , is considered the optimal definition . as suggested by hirschfeld and zernikow,19 we quantified the variability in the f value by running manova in 100 random samples ( with replacement ) of 2,000 participants chosen from the entire sample population of 8,781 . for each alternate definition , we calculated f score means and 95% confidence intervals ( cis ) around the means . also as suggested by hirschfeld and zernikow,19 we counted the number of times that each alternate definition was identified as the optimal definition based on manova f scores generated from the random samples . in the 12 cases where f scores were tied , each definition was counted as optimal . finally , using the approach described above , the alternative pain category definitions were further explored in two disease - specific populations : 1 ) those with back pain and 2 ) those with joint pain . the data used in this study are from the 2012 nhis sample adult core and the nhis adult functioning and disability ( afd ) supplement.6 the nhis is an annual survey of the health of the us civilian , noninstitutionalized population conducted by the national center for health statistics , centers for disease control and prevention . this in - person survey contains four main modules : household , family , sample child , and sample adult . the first two modules collect health and sociodemographic information on each member of all families residing within a sampled household . within each family , additional information is collected from one randomly selected adult ( the sample adult ) aged 18 years or older . bilingual interviewers or interpreters were recruited to interview all respondents who preferred the use of a language other than english . the survey uses a multistage clustered sample design and oversamples black , asian , and hispanic populations . when combined with centers for disease control and prevention - derived sampling weights , this design allows accurate extrapolation of findings to the civilian , noninstitutionalized us adult population . for the 2012 interview sample , there were 42,366 households consisting of 108,131 persons in 43,345 families . the total household response rate was 77.6% . from the households interviewed , 34,525 adults completed interviews , resulting in an overall sample adult response rate of 79.7% . approximately one - quarter of sampled adults were randomly chosen to participate in the afd supplement . almost all chosen adults ( 8,781 ) completed the supplement resulting in a 98% supplement response . the 2012 nhis was approved by the national center for health statistics research ethics review board . the afd collected information on the persistence and bothersomeness of self - reported pain in the previous 3 months . respondents were first asked how often they had pain in the previous 3 months : never , some days , most days , or every day . data from this question provide estimates of the 3-month persistence ( period prevalence ) of pain . for those who had pain on at least some days , a follow - up question assessing bothersomeness was asked : thinking about the last time you had pain , how much pain did you have a little , between a little and a lot , or a lot . miller and loeb3 have suggested a coding scheme that combines persistence and bothersomeness of pain to create four discrete categories of increasingly severe pain ( figure 1 ) . this coding scheme was tested and validated using a variety of qualitative assessments such as cognitive testing and in order to be consistent with the original pain category definitions of miller and loeb,3 the alternative definitions studied were restricted to four discrete categories that varied in only in their distributions of pain persistence and bothersomeness . to aid in identifying a set of alternative pain category definitions , we visually explored the relationship between nine pain persistence / bothersomeness combinations and measures of health status and health care use as defined later . chronic pain often predicts the onset of psychological distress.8,9 in turn , psychological distress has been identified as one of the factors mediating pain s relationship to disability.10 the nhis measures nonspecific psychological distress over a 30-day recall period with the kessler 6 ( k6 ) scale.11 the k6 scale asks respondents about six manifestations of psychological distress : during the past 30 days , how often did you feel ( a ) so sad that nothing could cheer you up ? possible responses are all of the time , most of the time , some of the time , a little of the time , and none of the time . scoring of the individual questions is based on a scale of between 0 and 4 points , according to increased frequency of the problem , yielding a total score on the scale from 0 ( no psychological distress ) to 24 ( extreme psychological distress).11 this was examined as a continuous variable . pain often results in disability days.1214 health - related bed - disability days were assessed with the survey question : during the past 12 months , about how many days did illness or injury keep you in bed more than half of the day ( include days while an overnight patient in a hospital ) ? pain is associated with increased health care use,13,15,16 including visits to emergency departments.17,18 health care use was examined with two nhis survey questions : 1 ) how many times did you visit a doctor or other health care professional during the last 2 weeks ? and 2 ) during the past 12 months , how many times have you gone to a hospital emergency room about your own health ( this includes emergency room visits that resulted in a hospital admission ) ? the 2012 nhis adult core included questions on the presence of low back pain within the previous 3 months ( yes and no ) , and on the presence of joint , aching or stiffness in the last 30 days ( yes and no ) . we studied the alternative definitions of the four pain categories using a variation of the statistical method described by serlin et al.5 each alternative definition was related simultaneously to four dependent measures the k6 score , the number of health related bed - disability days , the number of visits to a health professional , and the number of emergency room visits using multivariate analysis of variance ( manova ) . the manova yields f values , based on wilk s lambda , for the between - category effect on the dependent variables . following serlin et al,5 the pain category definition yielding the best model fit , as measured by the f value , is considered the optimal definition . as suggested by hirschfeld and zernikow,19 we quantified the variability in the f value by running manova in 100 random samples ( with replacement ) of 2,000 participants chosen from the entire sample population of 8,781 . for each alternate definition , we calculated f score means and 95% confidence intervals ( cis ) around the means . also as suggested by hirschfeld and zernikow,19 we counted the number of times that each alternate definition was identified as the optimal definition based on manova f scores generated from the random samples . in the 12 cases where f scores were tied , using the approach described above , the alternative pain category definitions were further explored in two disease - specific populations : 1 ) those with back pain and 2 ) those with joint pain . figure 2a d shows the relationships between the nine combinations of pain persistence and bothersomeness and measures of health status and health care use . those with a lot of pain either most days or every day had the highest mean k6 scores , 6.28 and 6.23 , respectively ( figure 2a ) , indicating they were in more psychological distress then individuals with other combinations of pain persistence and pain severity . conversely , those individuals with a little pain some days had the lowest mean k6 score ( 2.07 ) and least amount of psychological distress . similar patterns were seen when examining the mean number of health - related bed days ( figure 2b ) , the mean number of office visits ( figure 2c ) , and the mean number of emergency room visits ( figure 2d ) , with those with a lot of pain either most days or every day having the highest values ( mean number of bed days , 9.73 and 17.31 , respectively ; mean number of office visits , 0.69 and 0.77 , respectively ; and for mean number of er visits , 1.35 and 1.38 , respectively ) and those with a little pain some days having the lowest values ( mean number of bed days , 2.41 ; mean number of office visits , 0.24 ; and for the mean number of er visits , 0.27 ) . two other consistent patterns were seen 1 ) individuals with between a little and a lot of pain either most days or every day had the third and fourth highest mean k6 score ( 3.74 and 4.07 , respectively ) and had the third and fourth highest mean number of bed days ( 8.74 and 9.27 , respectively ) and office visits ( 0.56 and 0.69 , respectively ) and 2 ) those with a lot of pain some days had the fourth lowest value for the k6 score ( 3.29 ) and the fourth lowest mean number of bed days ( 5.5 ) and office visits ( 0.46 ) . the ranking of the other categories defined by pain persistence and pain severity varied considerably depending on the specific dependent measure . given the variability in the data , visual inspection of the graphs in figure 2a d was used to suggest alternative definitions of miller and loebs3 original pain categories ( figure 1 ; alternative definitions 110 ) . for instance , the original definition and alternative definitions # 1 and # 2 defined pain categories 1 and 2 identically but varied in how they defined pain categories 3 and 4 . conversely , alternative definitions # 8 and # 10 had pain categories 3 and 4 in common with the original definition but varied in how they defined pain categories 1 and 2 . alternative definition # 5 coded pain category 1 and pain category 4 identical with the original definition but varied in the other pain categories . alternative definitions # 3 , # 4 , # 6 , # 7 , and # 9 shared only one pain category in common with the original definition . along with the original definition table 2 shows the wilk s lambda f values calculated from the entire sample of 8,781 for each of the alternative definitions . the original definition produced the largest f value ( 185.87 ) followed consecutively by alternative definitions # 5 ( 184.17 ) , # 10 ( 180.95 ) , and # 9 ( 179.5 ) . nearly identical ordering was found when looking at the mean f value means generated from 100 random samples ( table 2 ) . however , considerable overlap was seen between the 95% confidence interval ( ci ) for the original definition and alternative definition # 5 , # 9 , and # 10 . figure 3 presents the frequencies with which each alternative definition achieved the optimal f value over the 100 random samples . interestingly , while there are only small differences seen in either the overall f values or mean f values ( table 2 ) , large differences were seen in the observed frequencies only two definitions had achieved the optimal f value > 5% of the time : the original definition ( 46% ) and alternative definition # 5 ( 41% ) . table 2 also shows the overall f values and the mean f values in those with back pain or joint pain , while figures 4 and 5 show the frequency of the optimal f values in those with back pain and joint pain , respectively . for both those with back pain ( table 2 ; figure 4 ) and those with joint pain ( table 2 ; figure 5 ) , the original definition had the highest overall f value ( back pain , 72.42 ; joint pain , 97.18 ) and the highest mean f value ( back pain , 16.44 ; joint pain , 22.71 ) . as in the overall adult sample , small differences in f values translated into large differences in the percent of time a given definition had the optimal f value . in the random samples of those with back pain ( figure 4 ) , 38% of the time the original definition was identified as having the optimal f value across the 100 samples , followed by alternative definition # 5 at 36% . in those with joint pain ( figure 5 ) , the frequencies were more divergent : 49% for the original definition and 28% for alternative definition # 5 . using a series of manova , we sought to identify the optimal definition of discrete pain categories using existing nhis questions that assessed pain persistence and severity . the results of manova on a large nationally representative sample of adults indicated that the original pain category definitions suggested by miller and loeb3 provided the best model fit versus ten alternative definitions . we confirmed this finding in 100 random samples of the total sample , as well as in two disease - specific subpopulations , those with back pain and those with joint pain . therefore , the present data provide additional support that the nhis coding system proposed by miller and loeb3 is a valid measure of an individual s current pain experience . given the large number of statistical tests employed in this analysis ( > 300 ) , it is possible that some of our observations occurred purely by chance . this multiple - test issue has been suggested as a possible weakness of the serlin approach.19 to overcome the multiple - test issue , hirschfeld and zernikow19 suggest examining the variability of the observed optimal definitions , which we have done in the present report . data based on f values for the entire sample , mean f values with 95% cis derived from the random samples , and the frequency with which optimal definitions are observed produced the same conclusion , that the coding scheme proposed by miller and loeb3 is the optimal definition . however , the data additionally show that one alternative definition ( # 5 ) also does very well across comparisons . the original definition and alternative definition # 5 code pain category 1 ( least severe pain ) and pain category 4 ( most severe pain ) identically and vary only slightly in how pain persistence and bothersomeness are combined to create pain categories 2 and 3 ; specifically , the original definition places slightly more emphasis on grouping by pain bothersomeness , while alternative definition # 5 places slightly more emphasis on grouping by pain persistence . future analyses , such as that using receiver operating characteristic curves , might help clarify the test characteristics of these two definitions . first , the categories we used were based on only pain persistence and bothersomeness , and therefore do not reflect the multidimensional nature of pain . second , potential confounders or effect modifiers , such as age , education , race , and sex were not included in this study as we were first concerned with exploring alternative cut points in the general population versus optimizing the manova for specific demographic groups . whether the miller and loeb definition remains the optimal definition in specific demographic populations will need to be assessed in future . third , the nhis did not include a well - validated scale for measuring pain - related disability . instead , we were limited to only a handful of variables measuring health status and health care use and for which continuous data were collected ( required for manova ) . as shown by zelman et al20 and fejer et al,21 the conclusions from cut point analyses vary depending on the dependent variables used . other results may have been found using the brief pain inventory , other pain - related disability measures , or more widely used generic measures of function such the sf-36 . therefore , the present results are suggestive rather than definitive and await corroboration in different samples . finally , in order to be consistent with the original pain category definitions suggested by miller and loeb,3 we have limited our analyses to alternative definitions derived to create four discrete pain categories . it is not known if fewer or greater numbers of categories would better explain the variance in manova . this study provides additional support for the coding of nhis - derived pain as proposed by miller and loeb.3 we demonstrated that the miller and loeb3 coding scheme provided optimal manova cut points for pain severity relative to health status and health care use , both in the general population and in disease - specific subpopulations . still to be explored is how well the nhis questions correlate with established validated measures of pain and whether they are sensitive enough to assess changes in pain severity over time . with further validation , the miller and loeb approach may prove to be a brief , viable option for assessing pain severity in clinical and research settings .

backgroundbased on qualitative and mixed - method approaches , miller and loeb have proposed a coding system that combines questions on pain persistence and bothersomeness to create discrete categories of increasing pain severity for use in large population - based surveys . in the current analyses , using data from the 2012 national health interview survey , we quantitatively assess the pain category definitions proposed by miller and loeb and compare this original definition to ten alternative definitions.methodsusing multivariate analysis of variance , each definition was related simultaneously to four dependent measures the kessler 6 score for measuring psychological distress , the number of health - related bed - disability days , the number of visits to a health professional , and the number of emergency room visits . following the protocol of serlin et al , the definition yielding the largest f score was considered the optimal definition.resultsthe miller and loeb definition produced the largest f value ( 185.87 ) , followed consecutively by several alternative definitions # 5 ( 184.17 ) , # 10 ( 180.95 ) , and # 9 ( 179.5 ) . a nearly identical ordering was found when looking at the mean f value generated from 100 random samples . we also examined the frequencies with which each alternative definition achieved the optimal f value over the 100 random samples . only two definitions had achieved the optimal f value > 5% of the time : the miller and loeb definition was optimal 46% of the time , while alternative definition # 5 was optimal 41% of the time . similar results were seen in subpopulations with back pain and joint pain.conclusionadditional support was provided for the miller and loeb coding of pain persistence and bothersomeness to produce discrete categories of increasing pain severity . this two - question coding scheme may prove to be a viable option for assessing pain severity in clinical settings where clinician time and patient burden are limiting factors .

Introduction Methods Population Dependent variable: assessment of pain Alternative definitions of the four pain categories Psychological distress Health-related disability Health care use Pain-related health conditions Statistical analyses Results Discussion Conclusion

PMC4834157

leptospirosis is one of the most important zoonoses infecting both developing and developed countries in the world . , leptospirosis was also associated with recreational activities through exposure to the contaminated soil or water [ 35 ] . an increasing number of people involved in outdoor activities have increased the chances of infection . high density of rats in the markets poses potential threats to visitors and town service workers . the expansion of housing areas also increases the opportunistic contact between humans and the infected wildlife . environments are frequently associated with nonpathogenic leptospires . however , a novel pathogenic species , l. kmetyi , has been isolated from environmental samples in malaysia . malaysia is a tropical country with high seasonal rainfall , warm temperatures , and wet and humid climate . it is common that floods occur following heavy rainfall during monsoon season in the east coast . the presence of leptospires in the environment during flooding may potentially cause an outbreak of leptospirosis . kelantan which is located in the east coast of malaysia is among the affected states . the number of cases increased during flooding from on average 20 cases to 31 cases . many inhabitants in this state are also involved in agricultural activities which pose the highest risk as 50 cases were reported from the workers in this sector . it was also reported that , in 2011 , one death was recorded out of 276 cases . previously , one death was reported from a waterfall in kelantan which is jeram pasu . in order to identify the potential threats of leptospirosis in the environments , the present study aims to detect and characterize leptospira spp . samplings were conducted from december 2012 to november 2013 at the selected recreational areas ( waterfalls with spots of running and still water ) and markets in the north - eastern state of malaysia , kelantan ( figure 1 ) . those sampling sites , which cover urban and rural areas of three districts , were selected based on previous reports of leptospirosis cases , rat infestations , and improper waste management . water samples in recreational areas were collected from shaded , suspected areas for the presence of animals and in - between rock areas . water samples were filtered through 0.2 m nalgene filter unit and 40 ml of the samples were transferred into centrifuge tubes and then centrifuged at 4000 g , 27c for 20 mins . two ml of the samples were inoculated into 5 ml of liquid emjh media with the addition of 5-fluorouracil ( 100 g / ml ) . for soil samples , sampling locations were selected from wet and shaded areas , from garbage sites , and in the places where spoiled food was spotted . in sterile 50 ml falcon tubes , the soil samples were mixed with sterile water and shaken vigorously . the suspension was allowed to settle for 510 minutes before being filtered using 0.2 m nalgene filter unit . several drops of the filtrates were inoculated into emjh media supplemented with antimicrobial agents ( 5-fluorouracil , 100 g / ml ) and incubated at 30c in a shaker incubator at 25 rpm to accelerate the growth of leptospires . the presence of leptospires was examined under dark - field microscopy using 20x and 40x magnification daily for 28 days . leptospires can be distinguished by other spirochetes based on their characteristic thin helical structures with prominent hooked ends and motility . in an event of contamination , 1000 l of contaminated cultures were transferred into fresh liquid emjh media supplemented with sulfamethoxazole and trimethoprim ( 40/8 g / ml ) , amphotericin b ( 5 g / ml ) , and 5-fluorouracil ( 100 g / ml ) according to chakraborty et al . the concentration of trimethoprim was changed from 20 g / ml to 8 g / ml and fosfomycin was not added to the media . the cultures were examined under dark - field microscopy using 20x and 40x magnification daily for 28 days . if the contaminants were still present , the cultures were diluted in sterile distilled water using a serial dilution technique starting from dilutions 10 , 10 , 10 , and 10 and then transferred into solid emjh media . the diluted culture was incubated for 3 weeks or until the leptospires colonies were observed on plates . single , isolated colonies were transferred from the plates into liquid media using sterile pasteur pipettes . the microscopic agglutination test ( mat ) was performed using a set of hyperimmune rabbit antisera purchased from the queensland health clinical and statewide services , australia , including autumnalis , tarassovi , pyrogenes , javanica , grippotyphosa , copenhageni , canicola , ballum , hardjoparjitno , celledoni , patoc , djasiman , icterohaemorrhagiae , pomona , australis , and hebdomadis using the available method the plates were gently shaken to mix contents , covered to exclude debris and prevent evaporation , and incubated at 30c for 2 hours . all of the isolates were screened for agglutination at titre 1 : 100 under dark - field microscope . agglutination of at least 50% of the leptospires was considered as positive . dna extraction was performed on isolates by using qiagen dneasy blood & tissue kit ( qiagen , usa ) according to the manufacturer 's protocols for gram negative bacteria and stored at 20c until use . a 20 l - pcr reaction mixture containing 1x pcr buffer , 2.5 mm mgcl2 , 0.16 mm dntp 's premixed , 0.04 m of each primer , and 0.75 units of taq polymerase was used in all pcr amplifications . amplification was performed in mj research thermocycler with an initial denaturation at 94c for 5 minutes , followed by 30 cycles of 94c for 30 seconds , annealing for 30 seconds at ta of the primers ( table 1 ) , and extension at 72c for 30 seconds . the primer sets sapro 1 and sapro 2 were used to detect the nonpathogenic leptospires . amplified products were characterized by electrophoresis of 5 l of each reaction on 1.5% agarose gel for 50 min at 90 v. the pcr product amplified by bak2 primer pair was purified using qiaquick pcr purification kit ( qiagen , usa ) and subsequently sequenced using sanger sequencing kits and instruments supplied by applied biosystems , us , performed by first base laboratories ( selangor , malaysia ) . the dna sequences were edited in bioedit and compared against the genbank database using blast . the 16s rrna gene partial sequences of all isolates were aligned with the 16s rrna sequences of the type strains obtained from genbank by using multiple sequence comparison by log - expectation ( muscle ) in mega 6 software . a total of 144 samples comprised of 72 water ( markets , n = 36 ; recreational areas , n = 36 ) and 72 soil ( markets , n = 36 ; recreational areas , n = 36 ) samples were collected . out of those samples , 33 were positive for leptospires based on their characteristic morphology and motility . among positive samples , 7 ( 5% ) were water samples and 26 ( 18% ) were soil samples . a total of 29 positive samples ( water , n = 7 ; soil , n = 22 ) were collected from markets and only four positive samples ( water , n = 0 ; soil , n = 4 ) were collected from recreational areas . all of the leptospiral isolates in this study were not agglutinated with any of the reference hyperimmune sera used in mat ( table 2 ) . out of 33 isolates , 18 isolates were detected as leptospira spp . using g1/g2 and b64-i / b64-ii primers ( table 2 ) . none of the isolates were found to contain the virulence gene . molecular identification by 16s rrna verified that , from 33 positive samples in this study , 31 isolates were identified as leptospira spp . and two isolates were identified as leptospiral closest relatives , leptonema illini . a pathogenic leptospire , l. alstonii ( 3% ) , was isolated from a soil sample in a market . eight isolates ( 24.2% ) were identified as intermediate pathogenic species comprised of seven l. wolffii and one l. licerasiae . a total of 22 isolates ( 66.7% ) were identified as nonpathogenic leptospires , l. meyeri . the phylogenetic tree constructed using the neighbour - joining method was illustrated in figure 2 . ls12 , ls18 , ss5 , ss6 , ws5 , ws6 , ws9 , and ws15 were grouped into intermediate pathogenic clade . ls1 , ls7 , ss4 , ws1 , ws2 , ws3 , ws4 , ws10 , ws11 , ws12 , ws13 , ws14 , ws16 , ws17 , ws18 , ww2 , ww3 , ww4 , ww5 , ww6 , ww7 , and ww8 were grouped into nonpathogenic clade . even though this is not the first study reporting the detection and characterization of leptospiral isolates from environmental samples , its isolation from recreational and market areas would give an impact to the community . reported that the prevalence of leptospires in water samples from markets was 67.9% . in the present study , the water was found to contain detergent and appeared oily , most probably due to its close proximity to the nearby food stalls . it has been reported that 30 ppm of detergent ( ceepryn , fixanol , and sapamine ) were lethal to leptospires in water in 5 minutes . the prevalence of leptospires in water samples from recreational areas in malaysia was 11.67% . in the current study , a lower prevalence of 5.56% was noted , which could be due to the effect of inappropriate choice of sampling points . all isolates recovered in this study showed negative reactions to 16 reference hyperimmune sera tested in mat . similarly , negative results of mat for environmental isolates were also reported from another study in which eight isolates from the selected urban sites in malaysia were negative for a total of 25 hyperimmune sera tested . the availability of the reference hyperimmune sera was very limited because none of local reference laboratories supplies the hyperimmune sera . since the sera used in this study were procured overseas , the available panel of sera may not cover the circulating local serovar . a total of 37 serovars of leptospira from 13 serogroups have been identified in malaysia . this highlights the importance of having locally produced hyperimmune sera available for local use . in our study , g1/g2 and b64-i / b64-ii primers detected 18 out of 33 isolates as leptospira spp . however , 16s rrna gene sequencing identified 31 out of 33 isolates as leptospira spp . g1/g2 and b64-i / b64-ii primers were not able to detect all strains of leptospira spp . the occurrence of new strains of leptospires in humans , animals , and environment has increased the need for new primers that are more specific than g1/g2 and b64-i / b64-ii . leptospiral putative virulence gene , lipl32 , rises as a new target for the detection of pathogenic leptospires though its role in virulence mechanisms remains unknown . this is further complicated by the absence of this gene in pathogenic and intermediate leptospires isolated in this study . this gene was either absence or undetectable by pcr because in some cases the lipl32 gene was not detectable in l. licerasiae by pcr but its product was detectable by both southern blot hybridization and western immunoblot . in the previous study by murgia et al . , primer sets sapro 1 and sapro 2 were used to detect the saprophytic leptospires . however , these primers were found to be unspecific as pathogenic leptospire , l. alstonii , was amplified by this primer in this study . in this study , a pathogenic species , l. alstonii , was isolated from a soil sample in market area . our finding was in line with previous studies that also isolated l. alstonii from the environment . exposure to the soil in market areas which harboured pathogenic strains may explain the high seropositivity of leptospiral antibodies among garbage collectors and town cleaners in kelantan . the isolation of leptospires from frogs specifically from kidney was reported as early as 1964 . several pathogenic serovars or serogroups such as bim , australis , and ballum have been isolated from frogs [ 27 , 28 ] . however , inoculation of frogs with pathogenic serovars to establish experimental leptospirosis led to unsatisfactory results as leptospires were not recovered in their organs . the intermediate species , l. wolffii and l. licerasiae , were isolated from markets and recreational areas . they are able to cause diseases in humans although less frequent . the presence of pathogenic and intermediate species warrants adherence to precautions and preventive measures among the visitors to those areas . it was reported that malaysians who were involved in recreational activities especially water related are 2.4 times more likely to acquire leptospirosis compared to those who were not involved in similar activities . leptospirosis was detected in wildlife in malaysia including wild mammals - monkeys , bats , squirrels , and mongoose . those animals could be the reservoirs for leptospires in recreational areas located in remote areas . a previous study reported high numbers of l. meyeri ( 88.1% ) from environmental samples . l. meyeri serovar ranarum icf which was previously considered as nonpathogenic was shown to be related to pathogenic strain suggesting that this species consisted of both pathogenic and nonpathogenic strains . this strain was also amplified by sets of primers specific for pathogenic leptospira and not the sets of primers specific for saprophytic strain . this disagreement demands a novel typing method of leptospira spp . which covers not only pathogenic species but also intermediate and nonpathogenic strains to resolve the uncertainties in species ' designation in the genus of leptospira . the accurate species designation and classification are crucial because the presence of clinically imperative leptospires in the environment would not be overlooked . phylogenetic analysis of the studied isolates using 16s rrna gene sequences concurred with the findings of previous report by morey et al . in 2006 isolates in the nonpathogenic group were closely related to both l. meyeri and l. yanagawa . it was reported that species within the nonpathogenic groups were separated by no more than 10 bp . using the 16s rrna gene , l. meyeri recovered in this study were not separated . in order to demonstrate intraspecies genetic variations , it was also suggested that a cut - off point of 1,000 base pairs ( bp ) was applied for leptospiral species identification . the phylogenetic analysis of environmental isolates using 16s rrna was also found to be better than using pfge profiles . the data presented in this study demonstrates the presence of a clinically significant pathogenic l. alstonii and the predominance of l. meyeri in the environment . the pathogenic strain was found to not contain one of the highly conserve putative leptospiral virulence genes . hence , continuous control and surveillance are essential in lowering the burden of the disease .

the presence of pathogenic leptospira spp . in the environment poses threats to human health . the aim of this study was to detect and characterize leptospira spp . from environmental samples . a total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north - eastern state in malaysia . samples were cultured on ellinghausen and mccullough modified by johnson and harris media . leptospires were positive in 22.9% ( n = 33 ) of the isolates . based on partial sequences of 16s rrna , a pathogenic leptospire , leptospira alstonii ( n = 1/33 ) , was identified in 3% of the isolates followed by intermediate leptospire ( l. wolffii , n = 1/33 , and l. licerasiae , n = 7/33 ) and nonpathogenic leptospire , l. meyeri ( n = 22/33 ) in 24.2% and 66.7% , respectively . this study demonstrates the presence of a clinically significant pathogenic l. alstonii in the environments which could pose health risks to the occupants and visitors .

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

PMC4848104

obesity is an epidemic and a significant health threat both in the united states and all around the world . poor dietary habits and lack of physical activity are the two main contributors to this growing health crisis . new smartphone applications and research projects aim at helping people track their daily food intake and activities . a growing number of smartphone apps moderate and vigorous physical activity and lifestyle changes can lead to health promotion and disease prevention , while aerobic exercise alone has not shown to be effective . additionally , increased portion sizes and high caloric intake are important contributors for developing obesity . provision of tools to accurately measure ee would allow people to actively track expenditure of calories relative to the amount of calories ingested , creating awareness of personal habits that can be modified to promote personal health . however , it is generally very difficult to know exactly how many calories people expend during daily physical activity as it depends on the age , gender , weight , height , type and intensity of activity . indirect calorimetry is a commonly used reference method to estimate energy expenditure . a cosmed k4b2 calorimeter uses pulmonary gas exchange to measure caloric expenditure with a very high correlation of 98.2% . such techniques are not exact and measure pulmonary gas exchange ( they measure oxygen exchange vo2 , not actual energy expenditure ) . the factors such as ratio of macro - nutrients burnt and any lactate accumulation during more intense exercises affect ee values but are not accounted in ee estimation using pulmonary gas exchange . accounting for this deficiency , indirect calorimetry is the closest we can get to actual ee values in an ambulatory setting . however , such a calorimeter is still impractical for use in daily life settings because of the high cost , complexity and difficulty of use . many wearable devices , such as fitbit , jawbone up and nike + fuelband provide a practical solution to monitor the dynamic energy expenditure by unobtrusively collecting data from wearable sensors to estimate ee . additionally , individuals will need to purchase and carry these devices with them all the time to get a comprehensive assessment of energy expenditure value . however , the main shortcoming of pedometers or any step - counting algorithms is their poor accuracy in detecting steps at slow speed and insensitivity to gait differences such as the length of the stride . another approach uses the accelerometer values directly , and attempt to find an empirical relation between accelerometer data and energy expenditure data measured by a calorimeter , e.g. cosmed k4b2 , . with smartphones becoming ubiquitous devices , they are conveniently suited personal devices to measure ee , rather than using dedicated wristbands , heart rate monitors or other tracking devices . users have to remember to carry them , not lose them and they are prone to damage . although the cost of commercially - available sensor products are coming down , many of these sensors cost $ 100 or more at this time and can be prohibitive for some of the population . on the other hand , people already have smartphones and the habit of carrying them along . however , further work needs to be done to improve ee accuracy using smartphone sensors . in this study , our focus is more on ambulatory activities a person engages in during the course of an individual s daily life such as walking , climbing upstairs or downstairs etc . currently existing smartphone apps utilize only the accelerometer data to estimate steps and converts them into mets ( metabolic equivalent values ) and calories estimates . however , this approach is quite inaccurate . new smartphones such as galaxy s3 , galaxy nexus , iphone 5 and later models have an integrated barometer sensor in the phone which passively measures atmospheric pressure . slight variations in atmospheric pressures can be used by these apps / algorithms to detect the work done against gravity , hence improving the results . however , caution should be exercised to not rely on absolute barometer values since these can vary depending on environmental factors as well as differences among devices . despite these limitations , variations of sensor readings within a time period and extracted features can be useful . barometer has been used for aiding gps , the main reason behind its introduction into smartphones . due to its excellent relative accuracy , differential in barometer has been used for floor - change detection , and activity classification . existing accelerometry equations require heavy computations or require high sampling frequency , both of which will drain the battery of smartphones quickly . the main contributions of this paper are as follows : 1)we propose and advance the use of machine learning techniques for ee . we have developed an initial linear regression , ann - based and bagged - regression tree based regression model to obtain a 96% correlation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } ) accuracy . we demonstrate high accuracy and low error ( root mean square error , rmse = 0.73).2)trials were conducted on 12 individuals and validated against reference ee data ( provided by cosmed k4b2 calorimeter ) . we can demonstrate high correlation using basic features and low sampling frequency , which will lead to battery efficiency.3)we demonstrate the benefit of incorporating barometer smartphone sensor in addition to accelerometer to improve ee estimation accuracy . unlike many of the currently available calorimetry equations , or usage of complex feature sets ( computationally unfeasible on smartphones ) , our approach uses simple features extracted from barometer and accelerometer sensors , fed to machine learning algorithms to obtain high accuracy and low rmse values . we demonstrate that using barometer sensor , in addition to accelerometer , improves correlation without computational overhead . we propose and advance the use of machine learning techniques for ee . we have developed an initial linear regression , ann - based and bagged - regression tree based regression model to obtain a 96% correlation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } ) accuracy . we demonstrate high accuracy and low error ( root mean square error , rmse = 0.73 ) . trials were conducted on 12 individuals and validated against reference ee data ( provided by cosmed k4b2 calorimeter ) . we can demonstrate high correlation using basic features and low sampling frequency , which will lead to battery efficiency . we demonstrate the benefit of incorporating barometer smartphone sensor in addition to accelerometer to improve ee estimation accuracy . unlike many of the currently available calorimetry equations , or usage of complex feature sets ( computationally unfeasible on smartphones ) , our approach uses simple features extracted from barometer and accelerometer sensors , fed to machine learning algorithms to obtain high accuracy and low rmse values . we demonstrate that using barometer sensor , in addition to accelerometer , improves correlation without computational overhead . we would also like to point out the scopes and limitations of our described model . first and foremost , our analysis have been built and analyzed on the basis of the most basic activities of otherwise normal and healthy human beings . the results can be extended to other physical activities like running , biking , etc , however , these will need further validation testing . secondly , our proposed model requires an individual to carry a smartphone at all times . this can be problematic as a smartphone may not always be carried by individuals and the sensor location will not always be known . recognizing the activity type with a non - fixed location of sensor on the body is complex task that will require further work . the rest of the paper is organized as follows : section 2 provide an overview of related works in this area . section 4 gives a brief summary of the prediction models used in the paper followed by experimental results in section 5 . numerous methods have been proposed to measure short- and long - term exercise energy expenditure . most are compared to either doubly - labeled water ( dlw ) or indirect calorimetry ( using a device such as cosmed k4b2 calorimeter ) as reference to actual ee values . pedometer estimates of ee are weak and have a weak correlation to actual values ( 0.530.61% , ) . similarly , the activity monitor estimates of ee are also low ( correlation 0.480.60 ) . it is possible to improve ee accuracy using multiple body sensors ( correlation of 0.90.95 ) , but it becomes inconvenient to use multiple body sensors in daily living conditions . multiple calorimetry equations from accelerometry data have been developed in research literature , but all tend to over- or under - estimate ee depending on type and intensity of the activity . a group of investigators evaluated machine - learning based approaches to estimate ee but considered only treadmill walking and leaves out activities of daily living . however , in prior work , the authors determine that height , weight and bmi ( body mass index , defined later ) are the best indicators for personalized ee for each individuals . fitbit is a highly popular commercial device which uses accelerometer and altimeter sensors to capture personal activity , a significant improvement over traditional pedometers . however , some experiments have demonstrated that fitbit is not very accurate as it lacks activity - classification algorithms . existing body - sensor based ee estimation employs a body - worn accelerometer and performs signal analysis to estimate calories expended in real - time using regression formulas . however , using a single sensor on the body is not enough to provide accurate measurement for body movement . instead , we found many of these devices were accurate in step counts but inaccurate in ee estimation . step - count based algorithms show high degrees of correlation with ee in scenarios such as walking , running and standing . however , active lifestyle often involves climbing up or down stairs . in these scenarios , for example - in a sample trial we asked some study subjects to climb up 4 flight of stairs and then to climb down the same number of stairs . the ee estimate obtained using commercial products such as fitbit and nike + fuelband ( which use pedometer based approach ) are shown in table 1 . it is counter - intuitive that one will spend more calories climbing down than upwards . existing algorithms used in these devices appear to count steps and speed of the movement and attribute higher expenditure based on these variables . given that our volunteers moved faster when climbing down stairs versus up stairs these devices measured higher caloric expenditure for the less vigorous activity of climbing down versus up . moreover , the two devices have huge variations in their measurements , leaving user to wonder about their accuracy . table 1.average measurement of ee ( cal ) and step counts using commercial devices ( nike + fuelband and fitbit one ) over volunteers performing climbing task . the devices report more work done in climbing down than up , and also have huge disparity in measurements.activity in stairs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rightarrow $ \end{document}updownsteps ( nike+)54.46 22.5859.83 11.45ee ( nike+)3.32 2.154.38 0.67steps ( fitbit)61.96 20.4471.38 13.18ee ( fitbit)7.82 2.859.14 2.92 heart rate monitors have also been used as stand - alone devices or along with accelerometer sensors to collect data and predict energy expenditure . some devices such as wahoo heart rate monitor , acquire heart rate data by measuring pulse rate and use a linear relation between heart rate and oxygen uptake to predict energy expenditure . however , heart rate monitors have low accuracy during sedentary behavior and require individual calibration , . calfit is a widely used android application that tracks time , location and physical activity patterns of users for health and wellness studies . it uses smartphones gps receiver to get the location information and the accelerometer for obtaining motion data . it uses a prior algorithm to estimate energy expenditure strictly based on accelerometer data . another previous work shows how smartphones , along with gps data , can be used to effectively estimate ee of individuals during biking . it is also used as pressure sensor which measures relative and absolute altitude through the analysis of changing atmospheric pressure . the barometer sensor can be used for motion detection , but it is mostly used by location - based applications to evaluate elevation . have first introduced the concept of combining barometer with accelerometer for detecting ambulatory movements , where authors embed a barometer sensor into a portable device to evaluate daily physical activity and classify the activity type . uses barometer in addition to accelerometer , but consider only linear regression models , thus limiting its accuracy . our primary aim was to build an application capable of accurately estimating ee without leveraging significant computational resources on the smartphones . low computational and power requirements will make such an algorithm more usable and attractive to consumers . there are three components of a system developed to estimte ee : ( a ) sampling and sensing of signal ( b ) feature calculation and ( c ) the machine learning algorithm itself . although the machine learning algorithm is computationally expensive in training , its complexity is very low for testing and practical use . therefore , we have focused on the other two aspects in this paper : ( a ) we have used a very low sampling rate of 2hz and ( b ) we choose a subset of features which give high correlation to ee but have minimal computational requirements . however , studies have shown that 0.120 hz is appropriate for most human activities . in this study , however , we restrict our measurements to the default smartphone sampling rate of 2hz which corresponds to low battery consumption and processing overhead . both accelerometer and barometer sensors are sampled at 2hz ( corresponding to 2 samples per second ) . we use a window of time equivalent to 4 seconds ( 8 samples ) to obtain different feature vectors required for our analysis . the basic features involve direct calculations of mean values from the tri - axial accelerometer and barometer sensors and these computations are simple . the derived features are obtained from basic accelerometer data and selected from existing studies in this domain , which we believe will improve the accuracy of our algorithm . we also collect anthropomorphic inputs about the users and use them as feature vector in our machine learning algorithm . the study involved 12 otherwise healthy volunteer subjects each performing a specified protocol of human activities . the participants are students in computer science department and were recruited based on open announcement in research group meetings . participants were made fully aware of the nature of the study , the purpose and any risks / concerns involved . the range ( minimum and maximum ) are mentioned in parenthesis.attribute\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ~\pm ~\sigma $ \end{document } ( min , max)age25.98 3.3 ( 22,34)height ( m)1.7621 0.06 ( 1.60,1.84)weight ( kg)73.01 17.58 ( 52,109)bmi ( kg/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m^{2}$ \end{document}23.44 5.32 ( 18.08,35.59 ) one critical task is to measure reference ee values which are used for training and testing the machine learning algorithm . direct calorimeter requires observations in a confined metabolic chamber and is therefore impractical in our scenario . doubly labeled water techniques are also inappropriate in our seting because they calculate ee over a long duration instead of for a single activity . to calibrate energy expenditure values over small time intervals , we used cosmed k4b2 indirect calorimeter , which is portable and can be used with our setup . we used samsung galaxy nexus smartphones to record observations of barometer and smartphone sensors . during each person s exercise protocol in the laboratory , smartphone - based tri - axial accelerometer and barometer were measured and recorded simultaneously along with the cosmed k4b2 system . data were downloaded from the devices after the prescribed physical activity protocol and merged based on time stamps for comparison . before each test , the oxygen and carbon dioxide analyzers and the flow turbine in the cosmed k4b2 were calibrated according to the manufacturer s instructions . subjects were asked to perform a series of activities in our exercise laboratory while being simultaneously monitored by the cosmed k4b2 portable metabolic system and a smartphone with built - in accelerometer and barometer sensor ( a galaxy nexus android - based phone running a customized software to record and extract accelerometer readings for ee estimation ) . , subjects were asked to be well hydrated and to rest or perform only light exercise the day before the test . subjects were advised to not perform any vigorous exercise and take at a normal meal 23 hours prior to testing . for each subject , we recorded age , gender , body height and body mass . body mass index ( bmi ) was calculated according to the formula : body mass ( kg ) divided by square of height ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m^{2}$ \end{document } ) . during each person s exercise protocol in the laboratory , smartphone - based tri - axial accelerometry and barometer data were downloaded from the devices after the prescribed physical activity protocol and merged based on time stamps for comparison . heart rate , oxygen consumption , carbon dioxide production , respiratory exchange ratio ( rer ) and ventilation rate were continuously monitored . , the oxygen and carbon dioxide analyzers and the flow turbine in the cosmed k4b2 were calibrated according to the manufacturers instructions . energy expenditure was calculated using the following equation : cosmed k4b2 ee ( kcalmin1 ) = ( 3.815 kcallo\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 2- 1+$ \end{document } ( 1.232 kcallo\documentclass[12pt]{minimal } 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a_{x}$ \end{document } : mean of x component of accelerometer signal.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{y}$ \end{document } : mean of y component of accelerometer signal . x and y refer to horizontal components of accelerometer signal , which are fixed to the local coordinates of smartphone.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } : mean of z component of accelerometer signal . this refers to the up - down movement of the human body.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } mean of barometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{x}$ \end{document } : mean of x component of accelerometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{y}$ \end{document } : mean of y component of accelerometer signal . x and y refer to horizontal components of accelerometer signal , which are fixed to the local coordinates of smartphone . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } : mean of z component of accelerometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } mean of barometer signal . the fv above are calculated easily from sensor data and are referred to as basic fvs . next , we define the additional fvs we derived from tri - axial accelerometer data . these features have been useful in human activity recognition and possibly also improve accuracy in our scenario . these features are calculated from the data collected from accelerometer and barometer , which are treated as time series signal . here , the word energy refers to energy of time - series signal and not the physical ee values . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{z}$ \end{document } : absolute mean of energy deviation from average of 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} \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{x}$ \end{document } = mean of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ |a_{x}-\mu a_{x}|$ \end{document})\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm$ \end{document } : signal vector magnitude is the root mean square value of ac component along all three axis.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } 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\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{z}$ \end{document } signals in given window . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek 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\usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{z}$ \end{document } : entropy of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{y}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{z}$ \end{document } signals in given window . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { p}^{2}$ \end{document } : variance of barometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ran_{p}$ \end{document } : range of barometer signal ( in given window ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ mgh$ \end{document } : work done against gravity . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ mgh = ran_{p}\times wei$ \end{document}. the activity counts or acceleration values collected using accelerometer can be combined with demographic information and regression techniques , or physical models of the human body to produce energy expenditure estimates . this model is also deployed in calfit used by researchers in california to assess associations between the built environment and physical activity in many case studies . ee estimates given by this method uses the following heuristic relation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \overbrace { ee}= a a_{h}^{k } + b a_{z}^{m } , \end{equation*}\end{document } where , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{h}= ( a_{x}^{2 } + a_{\vphantom { r_{j_{j}}}y}^{2})^{0.5}$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { a}=.01281^{*}\textit { wei } + 0.84322 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { b}=.0389^{*}\textit { wei } - 0.68244^{*}\textit { gen } + 0.69250 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { k}=.0266^{*}\textit { wei } + 0.14672 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { m}=-0.00285^{*}\textit { wei } + 0.96828 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{h}= ( a_{x}^{2 } + a_{\vphantom { r_{j_{j}}}y}^{2})^{0.5}$ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { a}=.01281^{*}\textit { wei } + 0.84322 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { b}=.0389^{*}\textit { wei } - 0.68244^{*}\textit { gen } + 0.69250 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { k}=.0266^{*}\textit { wei } + 0.14672 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { m}=-0.00285^{*}\textit { wei } + 0.96828 $ \end{document } researchers have reported 6095% correlation using equation 1 for ambulatory activities such as walking or running . artificial neural networks are a family of statistical learning models , which have excellent prediction ability . we also use an ensemble technique bagging in conjunction with regression trees with reduced - error - pruning ( rep ) . pruning is used to avoid over - fitting . bagging technique ensembles or merges the output of multiple such models to obtain the final prediction . the relative advantage of bagged rep regression trees is in low computational complexity and ability to identify high information attributes . the smartphone sensors logged their data using a native android app into a csv file while cosmed k4b2 calorimeter was used to validate the readings and measure actual ee . the smartphone was kept in waist pouch by the participants , as shown in figure 1 . unlike , activity specific classification and ee estimation algorithms , our focus here is on designing a single robust ee algorithm , that can be applied to a combination of all regular ambulatory activities in a combined manner . table 3 gives the performance results using artificial neural networks and simple linear regression models . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } indicates correlation between predicted output and actual ee values . raw accelerometer means that only the mean accelerometer values are provided as inputs to machine learning algorithm . all fv refer to the case when all 35 fvs mentioned earlier are included in ann.table 3.regression results indicate superior performance of machine learning algorithms over linear regression in both cases ( a ) using only accelerometer mean values and ( b ) all features.model used\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document}rmsemaebagged regression treeraw accelerometer ( only)0.90041.03020.5201all feature vectors ( fv)0.94680.79100.4012artificial neural networkraw accelerometer ( only)0.71891.62351.2244all feature vectors ( fv)0.87941.12660.7347linear regressionraw accelerometer ( only)0.60281.82511.4611all fvs0.58071.86431.4797demographic features ( only)0.60401.82311.4425 it can be clearly seen that linear regression gives very poor performance with only accelerometer as well as all fvs . in both cases there is no improvement in linear regression performance with increase in feature vectors . on the other hand , brt ( bagged rep - regression trees ) and ann ( artificial neural networks ) thus , the utility of using non - linear models for regression is clear . using ann model , we are able to achieve 72% correlation with actual ee values with a rmse of 1.62 using only accelerometer equations . when all fvs are used , correlation increases to 88% and rmse reduces to 1.13 . the rmse reduces to 0.79 when building brt with all features , while it is 1.03 when using only raw accelerometer values . regression trees are built using information - theoretic criterion and pick high entropy features on the top . reduced - error - pruning algorithm prunes the redundant leaves of the tree to guard against over - fitting . bagging is a meta - ensemble algorithm and the results of multiple rep - regression trees are pooled to obtain a higher accuracy than a single rep - regression tree . thus , we observe that ensemble technique gives higher correlation than anns . here , we observe that using all fvs does not improve the accuracy of linear regression model for ee over raw accelerometry values , because their impact on ee is non - linear . machine learning algorithms are able to capture the non - linearity and thus the correlation is higher ( and consequently rmse is low ) when we see the case of neural networks and bagged regression trees . figure 2 shows the results when we compare the rmse for leave one out sampling . in leave - one - out sampling , the model is trained with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n-1 $ \end{document } users and tested on 1 user , and this procedure is repeated for each user . leave - one - out technique is therefore good to find out the generality of our technique on new users , for whom the model has not been trained . the figure shows that bagging ( brt ) gives lesser rmse than ann and linear regression techniques . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r^{2}$ \end{document } statistics gives the coefficient of determination ( range usually 0 to 1 ) . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r^{2}$ \end{document } coefficient for ann was 0.87 , but was 0.96 ( close to 1 ) for brt . figure 2.plot of rmse ( root mean square error ) comparison of three techniques using leave - one - out sampling . the bars show the mean rmse value while error bars show the variance in values . plot of rmse ( root mean square error ) comparison of three techniques using leave - one - out sampling . the bars show the mean rmse value while error bars show the variance in values . the models generated using linear regression technique and single accelerometer can be used as a baseline ee algorithm . it obtains 60.28% correlation with output ee values and the root mean square error ( rmse ) is around 1.8251 . to serve as another useful baseline , we also tried to study the results when using only a model with demographic information of the participants . it evaluates the worth of a fv by measuring the gain ratio with respect to the class . the information gain is equal to the total entropy for an attribute if for each of the attribute values a unique classification can be made for the result attribute . in this case the relative entropy subtracted from the total entropy are 0.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \textrm { gainratio}(c , fv ) = ( h(c ) - h(c|fv ) ) / h(fv ) . \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c$ \end{document } is set of all training examples , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h()$ \end{document } denotes entropy . figure 3.relative information gain ratio of input feature vectors ( fvs ) obtained using gainratio criterion . relative information gain ratio of input feature vectors ( fvs ) obtained using gainratio criterion . we can observe that many input features have high information gain ratio in the range of 0.700.80 . while collecting data , we had annotated different set of activities using manual markers . here we find that the gainratio for annotations is pretty low ( 0.08 ) indicating that knowledge of activity - type has little correlation to ee values . however , since most of the features such as mean , variance , range , entropy and correlation have high gainratio , we argue that many of them are correlated and hence redundant . next , we use a twofold approach to reduce the attributes to be used in the model . first , we used feature selection algorithms to find the independent features which can give high predictive power . feature selection aims at reducing the number of attributes to be used in the model , while trying to retain the predictive power of the original set of attributes in the pre - processed data . we use the correlation feature selection ( cfs ) strategy to identify a subset of attributes which were highly correlated with the outcome variable while having low inter - correlation amongst themselves . the cfs technique was used in conjunction with a greedy stepwise search to find the subset \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document } with the best average merit , which is given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } merit_{s}=\frac { n\overline { r_{fo}}}{\sqrt { n+n(n-1)\overline { r_{ff } } } } \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$ \end{document } is the number of features in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { r_{fo}}$ \end{document } is the average value of feature - outcome correlations , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { r_{ff}}$ \end{document } is the average value of all feature - feature correlations . we used the entire dataset for feature selection , which can potentially bias the results and should be avoided in general ( cross - validation should be used for feature selection as well ) . the reason to use the entire dataset for feature selection process is our multi - step strategy , which included a manual screening to eliminate computationally - inefficient coefficients . using cross - validation for cfs would give slightly different subsets for each fold , which would complicate the manual screening step , and each resulting subset would again give different subsets after the second round of cfs . to simplify the process , we used the entire data at each step and got a single subset of features for the final model . extracting each feature vector from raw sensor inputs particularly , on an embedded device like a smartphone , such operations may drain the battery . since the exact speed of computation is device dependent , we report relative speed ( time of execution relative to time of execution of raw acc . values ( to compute the mean ) . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{acc}$ \end{document } denote the time required to process accelerometer values along one dimension . for a feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } the computation ratio is calculated as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } r_{f_{i}}=\log _ { 10}\left({\frac { t_{f_{i}}}{t_{acc}}}\right ) \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{f_{i}}$ \end{document } refers to time taken by computation of feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } on same device . these computations are performed with a desktop processor running at 2.6 ghz and averaged over 200k computations . the relative computation time is same for mobile processors , but desktop values are reported because we could average them over large sample size . next , our goal is to prune the fvs with higher computational cost without sacrificing the accuracy of ee estimation . as shown in figure 4 , correlation , pitch and range have an - order of magnitude higher computational cost than mean , energy and entropy . different features have different cost of computation , making low - cost features such as energy , svm , entropy and mean preferred to other features . the y - axis shows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } metric which is zero for feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } if the computational cost is same as that of mean of accelerometer values ( simplest operation ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } is in logarithmic scale , indicating that increase by 1 indicates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 10\times $ \end{document } increase in computational requirement on mobile devices . different features have different cost of computation , making low - cost features such as energy , svm , entropy and mean preferred to other features . the y - axis shows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } metric which is zero for feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } if the computational cost is same as that of mean of accelerometer values ( simplest operation ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } is in logarithmic scale , indicating that increase by 1 indicates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 10\times $ \end{document } increase in computational requirement on mobile devices . we would desire to use the features with least computation cost and high predictive power to form the ee estimation model . using cfs strategy in conjunction with our knowledge of computational cost , we obtain the following reduced 6 fvs in our final model : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ age$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ wt$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ bmi$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu p$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma p$ \end{document}. we note that all derived fvs for acceleromter data are pruned by the cfs algorithm . both mean and variance of barometer reading is used for ee estimation . generating a bagged rep - regression tree ( brt ) from these fvs the tree is pruned and only the top attributes , used in tree - branching are shown . it can be seen that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } is most significant predictor followed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ age$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ wt$ \end{document } and barometer readings ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu p$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma p$ \end{document } ) . we can clearly see mean barometric values ( meanp ) in the third level of hierarchy , implying clear contribution of barometer sensor in precision of regression tree ( brt ) . we can clearly see mean barometric values ( meanp ) in the third level of hierarchy , implying clear contribution of barometer sensor in precision of regression tree ( brt ) . figure 6 shows the actual ee values and the values estimated using brt ( final model ) . there is a close agreement between the values . figure 6.sample plot of ee estimates ( using bagged regression tree ( brt ) , a machine learning scheme ( final model ) . ) with actual ee values ( cosmed ) . sample plot of ee estimates ( using bagged regression tree ( brt ) , a machine learning scheme ( final model ) . ) with actual ee values ( cosmed ) . calorimetry equations ( ce ) proposed in literature , such as the one used in and have very high computational complexity as they involve fractional arithmetic and are not efficient on smartphone processors . we want to quantify the impact of these calculations on the accuracy of ann and brt models . although ce estimates roughly followed the trend for most activities , the average correlation was low . estimation error ( along the range of activities ) was 89.9788% and correlation was found to be 0.5027 . the correlation was particularly low for climbing activities while it was close to 0.65 for other activities , . next , we ran the models with and without this equation along with accelerometer ( basic ) features . it can be seen that including ce has no ( or negative ) impact on the accuracy of ann/ brt model with raw acc . table 4.impact of barometer values on ee prediction using machine learning approaches.model used\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document}rmsemaeartificial neural networks ( ann)raw accelerometer ( only)0.77791.65281.2321raw acc . + ce0.91010.97010.4920final model ( brt reduced)0.95600.73000.4015 the experimental results validated our assertion that barometric sensor ( bar . ) has high correlation with ee accuracy . appending the mean of barometer values ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } ) improve the correlation of ee to actual energy expenditure for both ann and brt as shown in table 4 . it is not possible to obtain second by second ee from commercial devices such as fitbit or nike + fuel band . however , we did calibrate these values before and after each trial and normalized for each subject s weight . the errors in individual measurements sum up and ce algorithm ( calorimetry equation used in calfit ) presents an estimate which is within 25% of the cosmed values . ann and brt values have same mean as the cosmed values . however , variance of error is smaller in brt . we can see that nike + fuelband tends to underestimate the ee for each individual while fitbit tends to overestimate the value . the error bars in the figure show the standard deviation for each device / algorithm . our algorithm has a smaller deviation over the population considered , which is comparable to actual cosmed values . figure 7.overall absolute ee comparison of cosmed , brt and ann with nike + , fitbit and calorimetry equation for two tasks across subjects . all three have same mean estimates but the variances are larger with ann as against brt . the variance in cosmed readings reflect range across subjects . the calorimetry equations and nike + product underestimates while fitbit overestimates the results . overall absolute ee comparison of cosmed , brt and ann with nike + , fitbit and calorimetry equation for two tasks across subjects . all three have same mean estimates but the variances are larger with ann as against brt . the variance in cosmed readings reflect range across subjects . the calorimetry equations and nike + product underestimates while fitbit overestimates the results . one of the most power consuming operation in ee estimation is sampling of smartphone sensors . unlike recent works that use gyroscope sensor in addition to accelerometer , we directly used the accelerometer and barometer sensor , and use machine learning algorithms to estimate ee . in next set of experiments , we tried to quantify the power savings in making this choice . we used monsoon power monitor to measure the energy consumption of sensors on the smartphone , turned off network interfaces and display and keep all system settings same during the experiments . the sampling rate was kept to be low ( at 2 hz ) and the results are averaged over few minutes of observations . figure 8 shows the relative power expenditure of these sensors ( when the smartphone os is on ) . these readings clearly demonstrate how accelerometer and barometer sensor has very low power consumption than gyroscope . figure 8.power consumption of smartphone ( with display , network and other apps off ) reading individual sensor signal at 2 hz . power consumption of smartphone ( with display , network and other apps off ) reading individual sensor signal at 2 hz . next , we justify our use of low sampling rates to obtain power - efficiency . the impact of different sampling rates on power consumption can be done using the emulation method described in and . figure 9.power consumption of smartphone ( with display , network and other apps off ) reading individual accelerometer sensor signal at different sampling rates . power consumption of smartphone ( with display , network and other apps off ) reading individual accelerometer sensor signal at different sampling rates . the training of machine learning algorithm is a power consuming task , but it can be done offline in the cloud . overall , our framework can be easily implemented in smartphones and has a power consumption less than 30 mw ( which will vary a bit with smartphone model and operating system ) . in this work , we proposed a technique for accurate ee estimation in ambulatory settings utilizing machine learning techniques and combining the sensor readings from both the accelerometer and barometer sensors of smart phones . to emulate a practical low - power setting , we used a smartphone and sampled the accelerometer and barometer sensorss at 2hz only . we then used these values to extract feature vectors ( fvs ) and fit a bagged regression tree which can yield up to 96% correlation and rmse of 0.70 with very small computational overhead . we observed significant benefits in fusing the input of barometer sensor to an accelerometer sensor as it allows , with use of simpler fvs , achievement of higher correlation and accuracy to reference ee values . improved ee estimation will provide important information regarding energy balance for individuals during various physical activities in both clinical and community living settings . in general , they rely on step counts and are expensive . building accurate ee models is helpful for the masses to measure their caloric expenditure using their already purchased smartphones and make healthy decisions about lifestyles . it is also of great interest to researchers , who can use smartphone - sensed data of individuals to gain insight into ee and gait features as well as trends . commercial devices such as fitbit have shared apis but do not allow access to sensor data , limiting clinical research explorations . motivated by the present study s encouraging results , we plan to collect a more extensive dataset , using a larger sample size of study subjects , along with other physical activities like biking , running , walking / running on inclines , and everyday chores such as gardening / cleaning . in the future , using this larger data set with wider range of activities , we wish to build a more representative model for ee ( using bagged regression trees ) . we then envision building a smartphone application which can be used to obtain accurate ee without significantly impacting the battery life of smartphones .

energy expenditure ( ee ) estimation is an important factor in tracking personal activity and preventing chronic diseases , such as obesity and diabetes . accurate and real - time ee estimation utilizing small wearable sensors is a difficult task , primarily because the most existing schemes work offline or use heuristics . in this paper , we focus on accurate ee estimation for tracking ambulatory activities ( walking , standing , climbing upstairs , or downstairs ) of a typical smartphone user . we used built - in smartphone sensors ( accelerometer and barometer sensor ) , sampled at low frequency , to accurately estimate ee . using a barometer sensor , in addition to an accelerometer sensor , greatly increases the accuracy of ee estimation . using bagged regression trees , a machine learning technique , we developed a generic regression model for ee estimation that yields upto 96% correlation with actual ee . we compare our results against the state - of - the - art calorimetry equations and consumer electronics devices ( fitbit and nike+ fuelband ) . the newly developed ee estimation algorithm demonstrated superior accuracy compared with currently available methods . the results were calibrated against cosmed k4b2 calorimeter readings .

Introduction Related Work Methodology Prediction Models Experiments and Results Discussions and Future Work

PMC55329

the statistical model proposed in for one probe set in multiple oligonucleotide arrays has the form it states that the perfect match ( pm)/mismatch ( mm ) difference in array i , probe j of this probe set is the product of model - based expression index ( mbei ) in array i ( i ) and probe - sensitivity index of probe j ( j ) plus random error . fitting the model , we can identify cross - hybridizing probes ( j with large standard error ( se ) , which are excluded during iterative fitting ) and arrays with image contamination at this probe set ( i with large se ) , as well as single outliers ( image spikes ) which are replaced by the fitted values . in effect the estimated expression index i is a weighted average of pm / mm differences : with larger weights given to probes with larger . the image of outliers ( array and single outliers ) identified through model - fitting can be used to assess the quality of an experiment and to identify unexpected problems such as a misaligned corner of a dat file . we have investigated several important properties of the model , including the reliability and stability of the fitted parameters mbei ( ) and probe sensitivity indexes ( ) , the performance of mbei compared to the commonly used average difference ( ad ) , and how the availability of se facilitates downstream comparative and clustering analysis . in practice , in an array experiment , a researcher hybridizes tissue or cell line samples , corresponding to different treatments or conditions , to a batch of arrays . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . this condition , however , may not hold for those probes that have cross - hybridization affinity to non - target genes . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . we apply the model ( equation 1 ) independently to six sets of hu6800 arrays ( 21 leukemia , lymphoma and mantle cell samples , 20 prostate cancer cell lines , 17 brain tumor samples , 55 cancer cell lines , 58 brain samples , and 55 lung tumor samples ) . figure 1a shows the values fitted for probe set 6457 ( used in figure 1 and 2 of ) in the six array sets . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . the model identifies this probe as a ' probe - outlier ' only for array set 5 and excludes it when calculating mbei ( 0 ) for array set 5 . values estimated for probe sets ( a ) 6457 , ( b ) 1248 , and ( c ) 6571 in six array sets ( shown in panels 16 from left to right for each probe set ) . values ( constrained to have sum square equal to number of probes used in each array set ) are on the y - axis , and probe pairs are labeled 1 to 20 on the x - axis . the title of each panel ( for example , p = 0 ) indicates the proportion of arrays ' present ' for the target gene in the array set . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . the average is taken over c(6 , 2 ) = 15 pairwise comparison of two array sets for each probe set , and the correlation is calculated using probes that are not identified as an outlier in both array sets . the range of the average lower presence proportion for the six boxplots are : ( 0 , 0.17 ) , ( 0.17 , 0.34 ) , ( 0.34 , 0.51 ) , ( 0.51 , 0.68 ) , ( 0.68 , 0.85 ) , ( 0.85 , 1 ) . the title of each boxplot is the number of probe sets classified into this boxplot . eleven probe sets with too few non - outlier probes to calculate correlations for all 15 comparisons are not included in the boxplots . the average lower presence proportion and average pairwise correlation for probe sets in figure 1 are ( a ) 1 , 0.95 ; ( b ) , 0.93 , 0.94 ; and ( c ) 0 , 0.86 . in figure 1a , b the target gene is present in most samples of all array sets . for a probe set whose target gene is mostly absent throughout samples ( figure 1c ) , many probes are identified as probe - outliers because of their negative indexes . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . nevertheless , the pm - mm values for these probes are random fluctuations around zero , leading to a correct expression index close to zero . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . occasionally , a responsive probe set may give rise to very different estimates in two array sets . in figure 1b , probes 8 and 13 have different relative responses in array set 1 and 4 , leading to different probe - response patterns . this might be due to the possibility that the probes in this probe set are differentially cross - hybridized in different array sets , or that the same probe in different batches of arrays may systematically behave differently . identification and flagging such probe sets is desirable and essential if we want to compare arrays hybridized to different tissue samples . figure 2 shows the boxplots of average pairwise correlations of values between two array sets , stratified by average lower presence proportion in the two sets . in general , when a gene is present in many samples of two array sets , the patterns estimated from the two sets are very similar . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . this phenomenon raised questions on the efficiency of using mm probes , and led some investigators to design custom arrays that use pm probes exclusively ( r. abagyan and yingyao zhou , personal communication ; b.r . conklin , personal communication ) , and others to calculate fold changes using only pm probes ( f. naef , personal communication ) . this design greatly increases the number of genes that can be studied on one array . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . the mm probe responses have a similar form as equation 2 except for different probe - sensitivity indexes . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . we assumed the 20-probe difference model provides the most accurate expression estimates . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . boxplot of correlations between values estimated using the 20-probe difference model and s estimated using different models , stratified by presence proportion . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . the number of presence calls for a probe set in the 21 arrays and the subpopulation size for the six boxplots are : 03 , 4,385 ; 47 , 693 ; 811 , 413 ; 1215 , 488 ; 1619 , 497 ; and 2021 , 323 . this comparison corroborates the basic notion of the technology : the pm probes hybridize more strongly to the target signals than mm probes and contain most of the information . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . each pair consists of two arrays hybridizing to samples replicated at total mrna level ( the total mrna sample is split and then amplified and labeled separately , and hybridized to two different arrays ) . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . for comparison , we also used the method in to calculate ads for all probe sets and plot them in figure 5b ( ad is based on normalized probe values , see methods and materials section for the normalization method . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . this might be explained by the relatively larger amplification variation for weakly expressed genes , given a constant success rate of amplifying a sequence by a certain fold . log ( base 10 ) expression indexes of a pair of replicate arrays ( array 1 and 2 of array set 5 ) for different statistical methods . only 6,695 ( a ) and 4,696 ( b ) probe sets with positive values in both arrays are used . the center line is y = x , and the flanking lines indicate the difference of a factor of two . researchers often use ' log ratio ' between expression values of a gene in two arrays as the criterion for identifying differentially expressed genes . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . thus for every probe set we calculated its average absolute log ( base 10 ) ratio of 29 pairs of duplicates as a statistic to compare the variation in expression levels between duplicates using the ad or the mbei method . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . the same boxplots ( figure 7 ) for another set of 60 human u95a arrays consisting of 30 replicate pairs conveys similar information . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . boxplots of average absolute log ( base 10 ) ratios between replicate arrays stratified by presence proportion for different statistical methods . ( a ) mbei method ; ( b ) ad method . the number of presence calls for a probe set in the 58 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5058 . the title of each boxplot is the number of probe sets used for the boxplot . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . 744 probe sets are not included as their average absolute log ratios can not be calculated for all the 29 pairs using either method . similar plots as in figure 6 for another set of 30 pairs of duplicated human u95a arrays . ( a ) mbei method ; ( b ) ad method.the number of presence calls for a probe set in the 60 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5060 . the title of each boxplot is the number of probe sets used for the boxplot . after obtaining expression indexes using ad or mbei , fold changes can be calculated between two arrays for every gene and used to identify differentially expressed genes . usually , low or negative expressions are truncated to a small number before calculating fold changes , and genechip also cautions against using fold changes when the baseline expression is absent . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . for example , gene 1 has fold change 2.47 and a tight confidence interval ( 2.06 , 3.02 ) . in contrast , gene 11 has a similar fold change of 2.48 but a much wider confidence interval ( 0.96 , 18.18 ) . thus the fold change around 2.5 for gene 11 is not as trustworthy as that for gene 1 . further examination reveals that this is due to the large ses relative to the expression indexes for gene 11 . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in addition , when image contamination results in unreliable expression values with large ses , the fold changes calculated using these expression value are attached with wide cis . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . in such a case , it is more helpful to filter genes by presence calls . cluster analysis is a popular method for analyzing the data of a series of microarrays [ 6 , 7 ] . if two genes are co - regulated at the transcription level , their expression values across samples are likely to be correlated . clustering algorithms use these correlations ( or monotone transformation of correlations ) to cluster co - regulated genes together the correlation based on the estimated expression levels may , however , be different from that based on the real but unobserved expression levels . also , the commonly used hierarchical clustering algorithm is an irreversible process : once two genes or nodes are merged , they will stay together , even if later on there is good reason to adjust previous clustering . a global way of using se in hierarchical clustering is to resample or bootstrap the whole ' gene by sample ' data matrix and redo the clustering , then investigate the overall properties emerging from this repertoire of clustering trees . in bittner et al . , the data matrix coming from cdna microarray experiments is resampled using the estimated variation derived from the median sd of log ratios for a gene across samples . as we now have ses for all data points , we can resample each expression value from a normal distribution with mean equal to the estimated expression value and sd equal to the attached se . figure 8a shows a hierarchical clustering tree of 225 selected genes with presence proportion > 0.5 and coefficient of variation ( sd / mean ) > 0.7 across the 20 samples in array set 2 . in trying to interpret this tree , we may be interested in the gene cluster colored in blue and the reliability of the gene members belonging to this cluster . the whole data matrix is resampled , and the clustering is performed again ( figure 8b ) . we notice that some blue genes ( genes in the original cluster are colored blue ) are clustered with other non - blue genes , and some non - blue genes are mixed into the main body of the blue genes . after each resampling , we identify a cluster that contains more than 80% of all the blue genes , but as few non - blue genes as possible ( measured as a percentage of all genes in this cluster ) . this cluster is considered to be the cluster that corresponds to the original one in figure 8a . in figure 8b the root node of the ' corresponding cluster ' is marked with small horizontal line intersecting the vertical line ( representing the range of the cluster ) on the right of the clustering picture . then , for each of all the 225 genes , if it belongs to this ' corresponding cluster ' , we increase its ' in - cluster ' count by 1 . after resampling 30 times , the in - cluster counts are indicated in gray - scale on the left side of the original clustering ( figure 8c ) , with black representing 30 and white representing zero . a high ' in - cluster ' count indicates a gene ' remains ' in the original cluster in most of the resampled clustering trees . gene clustering ( a ) 225 filtered genes are clustered based on their expression profiles across 20 samples . each gene 's expression values are standardized to have mean 0 and sd 1 across 20 samples . although the original ' blue ' genes are scattered to various places , we can still determine where the original cluster is , using the criteria described in the text . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . we can see from figure 8c that most genes in the original cluster are reliable members , whereas a few genes at the bottom of the cluster are not ( in fact they are merged into the original cluster last ) . interestingly , some genes originally not in the original cluster group with the ' corresponding clusters ' during resampling many times and have gray ' in - cluster ' marks . these genes may be related to the original cluster in some way . in summary , this method can help us to distinguish reliable and unreliable gene members of a cluster , as well as draw our attention to related genes originally clustered somewhere else because of the accidental nature of hierarchical clustering . in practice , in an array experiment , a researcher hybridizes tissue or cell line samples , corresponding to different treatments or conditions , to a batch of arrays . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . this condition , however , may not hold for those probes that have cross - hybridization affinity to non - target genes . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . we apply the model ( equation 1 ) independently to six sets of hu6800 arrays ( 21 leukemia , lymphoma and mantle cell samples , 20 prostate cancer cell lines , 17 brain tumor samples , 55 cancer cell lines , 58 brain samples , and 55 lung tumor samples ) . figure 1a shows the values fitted for probe set 6457 ( used in figure 1 and 2 of ) in the six array sets . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . the model identifies this probe as a ' probe - outlier ' only for array set 5 and excludes it when calculating mbei ( 0 ) for array set 5 . values estimated for probe sets ( a ) 6457 , ( b ) 1248 , and ( c ) 6571 in six array sets ( shown in panels 16 from left to right for each probe set ) . values ( constrained to have sum square equal to number of probes used in each array set ) are on the y - axis , and probe pairs are labeled 1 to 20 on the x - axis . the title of each panel ( for example , p = 0 ) indicates the proportion of arrays ' present ' for the target gene in the array set . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . the average is taken over c(6 , 2 ) = 15 pairwise comparison of two array sets for each probe set , and the correlation is calculated using probes that are not identified as an outlier in both array sets . the range of the average lower presence proportion for the six boxplots are : ( 0 , 0.17 ) , ( 0.17 , 0.34 ) , ( 0.34 , 0.51 ) , ( 0.51 , 0.68 ) , ( 0.68 , 0.85 ) , ( 0.85 , 1 ) . the title of each boxplot is the number of probe sets classified into this boxplot . eleven probe sets with too few non - outlier probes to calculate correlations for all 15 comparisons are not included in the boxplots . the average lower presence proportion and average pairwise correlation for probe sets in figure 1 are ( a ) 1 , 0.95 ; ( b ) , 0.93 , 0.94 ; and ( c ) 0 , 0.86 . in figure 1a , b the target gene is present in most samples of all array sets . for a probe set whose target gene is mostly absent throughout samples ( figure 1c ) , many probes are identified as probe - outliers because of their negative indexes . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . nevertheless , the pm - mm values for these probes are random fluctuations around zero , leading to a correct expression index close to zero . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . occasionally , a responsive probe set may give rise to very different estimates in two array sets . in figure 1b , probes 8 and 13 have different relative responses in array set 1 and 4 , leading to different probe - response patterns . this might be due to the possibility that the probes in this probe set are differentially cross - hybridized in different array sets , or that the same probe in different batches of arrays may systematically behave differently . identification and flagging such probe sets is desirable and essential if we want to compare arrays hybridized to different tissue samples . figure 2 shows the boxplots of average pairwise correlations of values between two array sets , stratified by average lower presence proportion in the two sets . in general , when a gene is present in many samples of two array sets , the patterns estimated from the two sets are very similar . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . this phenomenon raised questions on the efficiency of using mm probes , and led some investigators to design custom arrays that use pm probes exclusively ( r. abagyan and yingyao zhou , personal communication ; b.r . conklin , personal communication ) , and others to calculate fold changes using only pm probes ( f. naef , personal communication ) . this design greatly increases the number of genes that can be studied on one array . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . the same outlier exclusion procedure in is applied . the mm probe responses have a similar form as equation 2 except for different probe - sensitivity indexes . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . the mm - only model yields noticeably lower correlations , however . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . boxplot of correlations between values estimated using the 20-probe difference model and s estimated using different models , stratified by presence proportion . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . the number of presence calls for a probe set in the 21 arrays and the subpopulation size for the six boxplots are : 03 , 4,385 ; 47 , 693 ; 811 , 413 ; 1215 , 488 ; 1619 , 497 ; and 2021 , 323 . this comparison corroborates the basic notion of the technology : the pm probes hybridize more strongly to the target signals than mm probes and contain most of the information . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . each pair consists of two arrays hybridizing to samples replicated at total mrna level ( the total mrna sample is split and then amplified and labeled separately , and hybridized to two different arrays ) . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . for comparison , we also used the method in to calculate ads for all probe sets and plot them in figure 5b ( ad is based on normalized probe values , see methods and materials section for the normalization method . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . this might be explained by the relatively larger amplification variation for weakly expressed genes , given a constant success rate of amplifying a sequence by a certain fold . log ( base 10 ) expression indexes of a pair of replicate arrays ( array 1 and 2 of array set 5 ) for different statistical methods . ( a ) mbei method ; ( b ) ad method . only 6,695 ( a ) and 4,696 ( b ) probe sets with positive values in both arrays are used . the center line is y = x , and the flanking lines indicate the difference of a factor of two . researchers often use ' log ratio ' between expression values of a gene in two arrays as the criterion for identifying differentially expressed genes . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . thus for every probe set we calculated its average absolute log ( base 10 ) ratio of 29 pairs of duplicates as a statistic to compare the variation in expression levels between duplicates using the ad or the mbei method . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . the same boxplots ( figure 7 ) for another set of 60 human u95a arrays consisting of 30 replicate pairs conveys similar information . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . boxplots of average absolute log ( base 10 ) ratios between replicate arrays stratified by presence proportion for different statistical methods . ( a ) mbei method ; ( b ) ad method . the number of presence calls for a probe set in the 58 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5058 . the title of each boxplot is the number of probe sets used for the boxplot . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . 744 probe sets are not included as their average absolute log ratios can not be calculated for all the 29 pairs using either method . similar plots as in figure 6 for another set of 30 pairs of duplicated human u95a arrays . ( a ) mbei method ; ( b ) ad method.the number of presence calls for a probe set in the 60 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5060 . the title of each boxplot is the number of probe sets used for the boxplot . after obtaining expression indexes using ad or mbei , fold changes can be calculated between two arrays for every gene and used to identify differentially expressed genes . usually , low or negative expressions are truncated to a small number before calculating fold changes , and genechip also cautions against using fold changes when the baseline expression is absent . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . for example , gene 1 has fold change 2.47 and a tight confidence interval ( 2.06 , 3.02 ) . in contrast , gene 11 has a similar fold change of 2.48 but a much wider confidence interval ( 0.96 , 18.18 ) . thus the fold change around 2.5 for gene 11 is not as trustworthy as that for gene 1 . further examination reveals that this is due to the large ses relative to the expression indexes for gene 11 . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in addition , when image contamination results in unreliable expression values with large ses , the fold changes calculated using these expression value are attached with wide cis . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . in such a case , it is more helpful to filter genes by presence calls . cluster analysis is a popular method for analyzing the data of a series of microarrays [ 6 , 7 ] . if two genes are co - regulated at the transcription level , their expression values across samples are likely to be correlated . clustering algorithms use these correlations ( or monotone transformation of correlations ) to cluster co - regulated genes together . the correlation based on the estimated expression levels may , however , be different from that based on the real but unobserved expression levels . also , the commonly used hierarchical clustering algorithm is an irreversible process : once two genes or nodes are merged , they will stay together , even if later on there is good reason to adjust previous clustering . thus there is a need to assess the reliability of clusters . a global way of using se in hierarchical clustering is to resample or bootstrap the whole ' gene by sample ' data matrix and redo the clustering in bittner et al . , the data matrix coming from cdna microarray experiments is resampled using the estimated variation derived from the median sd of log ratios for a gene across samples . as we now have ses for all data points , we can resample each expression value from a normal distribution with mean equal to the estimated expression value and sd equal to the attached se . figure 8a shows a hierarchical clustering tree of 225 selected genes with presence proportion > 0.5 and coefficient of variation ( sd / mean ) > 0.7 across the 20 samples in array set 2 . in trying to interpret this tree , we may be interested in the gene cluster colored in blue and the reliability of the gene members belonging to this cluster . the whole data matrix is resampled , and the clustering is performed again ( figure 8b ) . we notice that some blue genes ( genes in the original cluster are colored blue ) are clustered with other non - blue genes , and some non - blue genes are mixed into the main body of the blue genes . after each resampling , we identify a cluster that contains more than 80% of all the blue genes , but as few non - blue genes as possible ( measured as a percentage of all genes in this cluster ) . this cluster is considered to be the cluster that corresponds to the original one in figure 8a . in figure 8b the root node of the ' corresponding cluster ' is marked with small horizontal line intersecting the vertical line ( representing the range of the cluster ) on the right of the clustering picture . then , for each of all the 225 genes , if it belongs to this ' corresponding cluster ' , we increase its ' in - cluster ' count by 1 . after resampling 30 times , the in - cluster counts are indicated in gray - scale on the left side of the original clustering ( figure 8c ) , with black representing 30 and white representing zero . a high ' in - cluster ' count indicates a gene ' remains ' in the original cluster in most of the resampled clustering trees . gene clustering ( a ) 225 filtered genes are clustered based on their expression profiles across 20 samples . each gene 's expression values are standardized to have mean 0 and sd 1 across 20 samples . although the original ' blue ' genes are scattered to various places , we can still determine where the original cluster is , using the criteria described in the text . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . we can see from figure 8c that most genes in the original cluster are reliable members , whereas a few genes at the bottom of the cluster are not ( in fact they are merged into the original cluster last ) . interestingly , some genes originally not in the original cluster group with the ' corresponding clusters ' during resampling many times and have gray ' in - cluster ' marks . these genes may be related to the original cluster in some way . in summary , this method can help us to distinguish reliable and unreliable gene members of a cluster , as well as draw our attention to related genes originally clustered somewhere else because of the accidental nature of hierarchical clustering . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . our experience is that more than 10 arrays are appropriate for model training , outlier detection and mbei calculation . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . as array images usually have different overall image brightness ( figure 9a ) , especially when they are generated at different times and places , proper normalization is required before comparing the expression levels of genes between arrays . model - based expression computation requires normalized probe - level data ( from affymetrix 's dat or cel files ) . for a group of arrays , we normalize all arrays ( except the baseline array ) to a common baseline array having the median overall brightness ( as measured by the median cel intensity in an array ) . ( a ) the cel intensities ( see text ) of a pair of replicate arrays ( array 11 and 12 in array set 5 ) are plotted against each other . the baseline array 11 ( shown on the y - axis ) is not as bright as array 12 ( shown on the x - axis ) . the smoothing spline ( green curve ) deviates from the diagonal line y = x ( blue curve ) , indicating the need for normalization . ( b ) the same plot as ( a ) with superimposed circles representing the invariant set , on the basis of which a piecewise linear normalization relationship is determined ( black dotted line , whose y - coordinate is the normalized value of array 12 ) . the normalization curve is close to the smoothing spline curve in ( a ) as the two arrays are replicated arrays and all probes should be invariant . ( c ) after normalization ( y - axis is the baseline array 11 , and x - axis the normalized value of array 12 ) , the scatterplot centers around the diagonal line and the array 12 is adjusted to have the similar overall brightness as array 11 . ( d ) the q - q plot of probe intensities of array 11 and normalized array 12 shows the probes in the two sets have almost the same distribution . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis . a straight line running through the origin is a multiplicative normalization method ( genechip 's scaling method ) , and a smoothing spline through the scatterplot can also be used ( figure 9a , also see ) . we should base the normalization only on probe values that belong to non - differentially expressed genes , but generally we do not know which genes are non - differentially expressed ( control or housekeeping genes may also be variable across arrays ) . nevertheless , we expect that a probe of a non - differentially expressed gene in two arrays to have similar intensity ranks ( ranks are calculated in two arrays separately ) . we use an iterative procedure to identify a set of probes ( called the invariant set ) , which presumably consists of points from non - differentially expressed genes ( figure 9b ) . specifically , we start with points of all pm probes ( about 140,000 for hu6800 array ) . if a point 's proportion rank difference ( prd , absolute rank difference in two arrays divided by n = 140,000 ) is small enough , it is kept for the new set . here the threshold of being small is prd < 0.003 when a points 's average intensity ranks in the two arrays is small and prd < 0.007 when it is large , accounting for fewer points at high - intensity range ; and the threshold is interpolated in between . we chose these parameters empirically to make the selected points in the invariant set thin enough to naturally determine a normalization relation . in this way we may obtain a new set of 10,000 points , and the same procedure is applied to the new set iteratively , until the number of points in the new set does not decrease anymore . a piecewise linear running median line figure 10 shows another pair of arrays where the normalization relationship is non - linear . similar plots as in figure 9 for arrays hybridized to two different samples ( array 24 and 36 of array set 5 ) . ( a ) cel intensities ; ( b ) same plot as in ( a ) with superimposed circles representing the invariant set ; ( c ) after renormalization ; ( d ) q - q plot of normalized probe intensities . note that the smoothing spline in ( a ) is affected by several points at the lower - right corner , which might belong to differentially expressed genes . the invariant set , on the other hand , does not include these points when determining the normalization curve , leading to a different normalization relationship at the high end . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . our experience is that more than 10 arrays are appropriate for model training , outlier detection and mbei calculation . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . as array images usually have different overall image brightness ( figure 9a ) , especially when they are generated at different times and places , proper normalization is required before comparing the expression levels of genes between arrays . model - based expression computation requires normalized probe - level data ( from affymetrix 's dat or cel files ) . for a group of arrays , we normalize all arrays ( except the baseline array ) to a common baseline array having the median overall brightness ( as measured by the median cel intensity in an array ) . ( a ) the cel intensities ( see text ) of a pair of replicate arrays ( array 11 and 12 in array set 5 ) are plotted against each other . the baseline array 11 ( shown on the y - axis ) is not as bright as array 12 ( shown on the x - axis ) . the smoothing spline ( green curve ) deviates from the diagonal line y = x ( blue curve ) , indicating the need for normalization . ( b ) the same plot as ( a ) with superimposed circles representing the invariant set , on the basis of which a piecewise linear normalization relationship is determined ( black dotted line , whose y - coordinate is the normalized value of array 12 ) . the normalization curve is close to the smoothing spline curve in ( a ) as the two arrays are replicated arrays and all probes should be invariant . ( c ) after normalization ( y - axis is the baseline array 11 , and x - axis the normalized value of array 12 ) , the scatterplot centers around the diagonal line and the array 12 is adjusted to have the similar overall brightness as array 11 . ( d ) the q - q plot of probe intensities of array 11 and normalized array 12 shows the probes in the two sets have almost the same distribution . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis . a straight line running through the origin is a multiplicative normalization method ( genechip 's scaling method ) , and a smoothing spline through the scatterplot can also be used ( figure 9a , also see ) . we should base the normalization only on probe values that belong to non - differentially expressed genes , but generally we do not know which genes are non - differentially expressed ( control or housekeeping genes may also be variable across arrays ) . nevertheless , we expect that a probe of a non - differentially expressed gene in two arrays to have similar intensity ranks ( ranks are calculated in two arrays separately ) . we use an iterative procedure to identify a set of probes ( called the invariant set ) , which presumably consists of points from non - differentially expressed genes ( figure 9b ) . specifically , we start with points of all pm probes ( about 140,000 for hu6800 array ) . if a point 's proportion rank difference ( prd , absolute rank difference in two arrays divided by n = 140,000 ) is small enough , it is kept for the new set . here the threshold of being small is prd < 0.003 when a points 's average intensity ranks in the two arrays is small and prd < 0.007 when it is large , accounting for fewer points at high - intensity range ; and the threshold is interpolated in between . we chose these parameters empirically to make the selected points in the invariant set thin enough to naturally determine a normalization relation . in this way we may obtain a new set of 10,000 points , and the same procedure is applied to the new set iteratively , until the number of points in the new set does not decrease anymore . a piecewise linear running median line figure 10 shows another pair of arrays where the normalization relationship is non - linear . similar plots as in figure 9 for arrays hybridized to two different samples ( array 24 and 36 of array set 5 ) . ( a ) cel intensities ; ( b ) same plot as in ( a ) with superimposed circles representing the invariant set ; ( c ) after renormalization ; ( d ) q - q plot of normalized probe intensities . note that the smoothing spline in ( a ) is affected by several points at the lower - right corner , which might belong to differentially expressed genes . the invariant set , on the other hand , does not include these points when determining the normalization curve , leading to a different normalization relationship at the high end . we thank sven de vos , dan tang , nik brown , stan nelson , jae k. lee , yaron hakak , john walker and arindam bhattacharjee for providing data , and the editor and referees who provided valuable suggestions . this work is supported in part by nih grant 1 ro1 hg02341 - 01 and nsf grant dbi-9904701 .

backgrounda model - based analysis of oligonucleotide expression arrays we developed previously uses a probe - sensitivity index to capture the response characteristic of a specific probe pair and calculates model - based expression indexes ( mbei ) . mbei has standard error attached to it as a measure of accuracy . here we investigate the stability of the probe - sensitivity index across different tissue types , the reproducibility of results in replicate experiments , and the use of mbei in perfect match ( pm)-only arrays.resultsprobe-sensitivity indexes are stable across tissue types . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . we extended the model to obtain expression values for pm - only arrays , and found that the 20-probe pm - only model is comparable to the 10-probe pm / mm difference model , in terms of the expression correlations with the original 20-probe pm / mm difference model . mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . the standard errors of mbei can be used to construct confidence intervals of fold changes , and the lower confidence bound of fold change is a better ranking statistic for filtering genes . we can assign reliability indexes for genes in a specific cluster of interest in hierarchical clustering by resampling clustering trees . a software dchip implementing many of these analysis methods is made available.conclusionsthe model - based approach reduces the variability of low expression estimates , and provides a natural method of calculating expression values for pm - only arrays . the standard errors attached to expression values can be used to assess the reliability of downstream analysis .

Background Results and discussion Probe-sensitivity indexes are stable across tissue types Model-based analysis for PM-only arrays MBEI reduces variability for low expression estimates Confidence interval for fold change Standard errors help to assess clustering results Methods and materials Software Normalization of arrays based on an 'invariant set' Acknowledgements

PMC3877460

primary dysmenorrhoea , in absence of a diagnosable pelvic disease and secondary dysmenorrhoea , results from pathologic pelvic problems like endometriosis and inflammatory diseases . pain starts either before or concurrently with onset of menstruation and lasts for 12 - 72 hours . although primary dysmenorrhoea is not life threatening and does not lead to individuals defect , it highly affects the daily life among the young women . it is reported that 1% of the women at reproductive age do not go to work for 1 - 3 days in a month as a result of acute dysmenorrhoea , school girl absenteeism is estimated to be 14% due to painful contractions , and those who attend their work face a severe reduction in their efficiency . this fact is of great importance from socioeconomic view and is supposed to be the main reason for waste of time at work and school . for instance , in usa , where women are accounted for 42% of total manpower , the burden is estimated to be 600 million work hours . in contrast , dysmenorrhoea can cause psychological and mental problems in some women and leads to their social isolation and lack of their active participation in the society . existence of pain , as a serious health problem , is considered important and should be relived due to ethical reasons and the reduction effect on psychological and physiological status promotion . evidences show that prostaglandin f2 , which is secreted from endometrium , is responsible for dysmenorrhoea and uterine contractions stimulation . uterine is sensitive to prostaglandin f2 in all women either with dysmenorrhoea or without , but the difference in its amount of production in dysmenorrhoea group is significant . due to lack of a unique treatment method for dysmenorrhoea and individuals various responses to the treatment different treatment methods including medication , acupuncture , skin electrical stimulation , surgery , and prescription of various vitamins and mineral have been suggested . these medications have some side effects such as nausea , digestive system dysfunction , diarrhea , and sometimes fatigue . in addition , consumption of these medications is forbidden among those with peptic ulcer , or aspirin - sensitive asthmatic patients . another suggested method is physical activity , which has been noticed as a nonmedicational method . physical activity can help venous return through muscular contraction that leads to an increase in production of prostaglandins and other substances and finally prevents their collection in the pelvis . reduction of dysmenorrhoea in women who play sport may be resulted from the effect of hormonal changes on endometrium or increase of endorphins . bouyancy reduces the imposed pressure to cardiovascular system so that floating in water up to neck , imposes the pressure to the lower limbs , facilitates venous return , and diminishes the load to cardiovascular system through hydrostatic pressure . heart rate drops by 5 - 8 pulses in resting position so that the exercises , done in water accompanied at a specific maximum heart rate , have less pulse increase ( 10 - 12 pulses ) compared with the same exercises done out of water . hydrostatic resistance also improves muscular endurance and power if aquatic exercises are designed based on scientific principles and personal characteristics and differences . aquatic exercises have been vastly recommended to various age groups due their lower weight bearing . barabosa studied the effect of aquatic exercises in a pool with temperature of 28 - 30c in healthy individuals and concluded that water temperature as well as doing exercises in deep part of the pool and using assistive tools ( for more buoyancy and doing power exercises with weights in water ) are of great importance . the above study indicates aquatic exercises as an important therapeutic program for some diseases based on various researches . numerous studies have emphasized on the association between dysmenorrhoea and regular sport activities in recent years . showed that the needed rest time for pregnancy low back pain was reduced in water . a comparison on 390 healthy pregnant women who exercised once a week in two separate groups of aquatic and land exercise showed that aquatic exercise during pregnancy reduced pelvic pain and low back pain leading to fewer sick leaves in this group compared with the land exercise group . in another study titled the effect of a set of isometric exercise on dysmenorrhoea , it was concluded that isometric exercises reduced length and severity of dysmenorrhoea as well as consumption of medication among girls . investigated the effect of a set of pilates exercises on primary dysmenorrhoea and observed a significant reduction in intensity and length of pain in pilates group after intervention . aquatic exercise can be effective on individuals pain reduction and improvement of quality of life through empowerment of muscles and decreased pressure on the joints . with regard to the specifications of the water , and as the effect of aquatic exercises among various exercises on primary dysmenorrhoea has not been already investigated , this study aimed to define the effect of a 12 week aquatic exercise on primary dysmenorrhoea among nonathlete girls in direction of women 's health and ability promotion . this is a pretest posttest quasi experimental action clinical trial with control group conducted during 2010 - 2011 . study population comprised all female nonathlete students aged 18 - 25 years in sama school of najafabad azad university and private shahid ashrafi esfahani university . inclusion criteria were existence of primary dysmenorrhoea in most of menstruation cycles , personal desire to use a method to relieve pain , regular menstruation cycles in each 24 - 35 days , onset of pain prior to or concurrent with bleeding lasting for 12 - 72 hours , 26 > body mass index ( bmi)>19 , and being single and nonathletic . exclusion criteria were pelvic pain during reproductive age ( having no pelvic infection or being involved in pain not related to dysmenorrhoea ) , history of a diagnosed endometriosis in immediate relatives , history of chronic diseases such as diabetes , hypertension , cardiovascular and pulmonary diseases , history of professional sport ( playing sports for 1 hour or more a day or 7 days or more a week ) and being employed . diagnosis of primary dysmenorrhoea and the entrance of the subjects to the study was conducted based on primary dysmenorrhoea evaluation form , checking the positive answers ( yes ) to the first five questions and responding negative ( no ) to four questions out of five in the second five questions . then , based on the above evaluation form , 40 subjects were randomly selected and assigned to a group of aquatic exercise ( n = 20 ) and control ( n = 20 ) . the questionnaires had been already numbered and the subjects were classified based on even or odd numbers . since in the present study , it was tried to measure dysmenorrhoea , which is a subjective issue , objectively as much as possible , severity of subjects dysmenorrhoea was investigated by present pain intensity ( ppi ) and visual analogue scale ( vas ) indexes of mcgill standard pain questionnaire , which is a precise and valid tool to measure phenomenon of pain objectively . reliability and validity of this questionnaire have been calculated by valiani et al . in 2009 mcgill standard questionnaire was given to the subjects in both groups ( as a pretest ) after menstruation to determine dysmenorrhoea signs . it included a vas with a 5 cm ruler scored zero ( no pain ) at one end and 5 ( intolerable pain ) at the other end . subjects ppi was measured by selecting an option from 0 to 4 ( no pain , mild pain , suffering pain , and killing pain ) . then , the researcher obtained subjects informed written consent and give them nutritional instructions including tips on salt intake , cereals , vegetables , etc . ( some nutrient are effective on decrease or increase of pain ) to make the conditions identical for both groups . in the next step , physical exercises for the subjects were precisely and regularly conducted between two menstruations with the attendance of the researcher in the study group for three straight cycles . at the beginning of the exercises then , the aquatic exercises were conducted for one hour , three sessions a week in the pool . in the first week , aquatic exercises were conducted in shallow part of the pool and in other weeks in the 3-meter deep part with temperature of 28 - 30c . meanwhile , control group had no aquatic exercise at this time interval . at the end of menstruation cycle , the questionnaires were collected and next posttest questionnaires ( for the following months ) were given to both groups . these exercises were educated to strengthen pelvic area muscles in order to modify blood stasis in pelvic veins and facilitate quicker blood circulation . the aquatic exercises included 10 minutes of warming up in form of walking and running in water , 40 - 45 minutes of aerobic , endurance , flexibility , power , coordination , speed and agility in addition to other specific exercises for abdominal and pelvic muscles and thighs . heart rate was measured by a polar pulse watch among the subjects . at the end of exercises descriptive and analytic statistical tests were used to analyze the data through spss version 16 . mean and standard deviation ( sd ) were adopted in descriptive statistics , one way analysis of variance ( anova ) to compare the variable in two independent groups and anova repeated observations was adopted to compare each group before and after intervention . subjects mean age and sd in aquatic exercises and control groups were 20.25 ( 2.02 ) and 20.50 ( 1.79 ) years , subjects mean age and sd of the first menstruation were 12.58 ( 1.72 ) and 12.30 ( 1.92 ) , and mean bmi and sd were 20.72 ( 1.80 ) and 20.33 ( 2.44 ) , respectively . one way anova showed no significant difference in mean age ( p = 0.85 , f = 0.16 ) , mean age of the first menstruation ( p = 0.61 , f = 0.49 ) , and bmi ( p = 0.83 , f = 0.18 ) in both groups before intervention . mean pain intensity , measured with scale of ppi , showed no significant difference before intervention in two groups of aquatic exercises and control ( p = 0.21 ) , and after intervention in the first menstruation than before intervention ( p = 0.48 ) , but showed a significant difference in the second and the third periods of menstruation , respectively ( p = 0.05 ) and ( p < 0.001 ) [ table 1 ] . table 2 presents mean pain intensity of dysmenorrhoea measured by vas showed no significant difference before intervention in two groups ( p = 0.057 ) , but after intervention , it showed a significant difference in all three periods of menstruation than before intervention ( p = 0.03 ) ( p 0.001 ) . as seen in table 3 , mean length of pain showed no significant difference before intervention ( p = 0.907 ) and after intervention in the first and second periods of menstruation than before intervention ( p = 0.52 ) ( p = 0.58 ) , but it showed a significant difference in the third period of menstruation ( p = 0.002 ) . the results of the present study showed that pain intensity and length were reduced after 2 months of aquatic exercises in the experimental group ( through 8 weeks of aquatic exercises ) so that if physical exercise continued , dysmenorrhoea would decrease constantly . comparison of subjects mean dysmenorrhoea pain intensity measured by ppi scale in two groups before and after intervention in each period of menstruation comparison of subjects mean dysmenorrhoea pain intensity measured by vas scale in two groups before and after intervention in each period of menstruation comparison of subjects dysmenorrhoea pain length in two groups before and after intervention in each period of menstruation long - term effect of these exercises should be investigated by further studies . in the present study , positive effect of 12 week aquatic exercises on nonathletic girls dysmenorrhoea was observed , which is consistent with the findings of some previous studies investigating the positive effect of exercises on primary dysmenorrhoea . soltani in her study titled effect of a period of isometric exercises on primary dysmenorrhoea showed that these exercises could reduce low back pain and abdominal discomfort . shahrjerdi investigated the effect of 2-month stretching exercises on primary dysmenorrhoea and concluded that these exercises reduced pain intensity and length and consumption of sedatives among school girls . rakhshaee in her investigation on the effect of three yoga postures ( cobra cat and fish ) on primary dysmenorrhoea concluded that these exercises reduced pain intensity and length in study group . dysmenorrhoea is possibly made due to an increase in uterine muscles contractions under influence of sympathetic system . stress has potentiality to increase sympathetic system activity and can enhance dysmenorrhoea through increase of sympathetic system stimulation . in a study titled stress and dysmenorrhoea , wang et al . concluded that there was a significant association between stress and dysmenorrhoea . they also reported that dysmenorrhoea increases among the women at higher ages and argued that sport can diminish sympathetic system stimulation and help improvement of dysmenorrhoea signs . with regard to the obtained results by vas , aquatic exercises reduce dysmenorrhoea in the first 4 weeks of the exercise . the reason for this difference is that vas is a quantitative scale while ppi is qualitative . the obtained results are in line with those of researches investigating the positive effect of stretching exercises on dysmenorrhoea . rasoolzade et al . investigated the effect of relaxation on primary dysmenorrhoea and concluded that muscular relaxation can reduce dysmenorrhoea . in another study on the effect of 6-week flexibility exercises on dysmenorrhoea , the researchers concluded that dysmenorrhoea signs reduced after 6 weeks of flexibility exercises . some of the pilates exercises including active stretching and isometric exercises , pelvic muscles empowerment , and mental and physical relaxation can be helpful in reduction of stress . in the present study , it was shown that pain intensity and length in aquatic exercises group was decreased after 2 months of exercises and if the exercises continued , dysmenorrhoea would be reduced constantly . kardavani investigated the effect of aquatic exercises on lumbar herniated disc pain and concluded that aquatic exercises improved pain in these patients . in another study , the effect of 8-month aquatic exercises in warm water in the patients with fibromualgia was investigated and their physical function ( aerobic capacity , balance , muscular tone ) was improved . reported the effect of 8-week swimming , as an aerobic sport , on reduction of physical and psychological premenstruation signs . kanda in her study on comparison of lower limb muscles activity during walking in water and walking on land showed that aquatic exercises can stimulate flexor and extensor muscles of hip further . she indicated the difference between walking in water and on land is due to the fact that walking in water is one of the major aquatic exercises . it is clear that in water with lower gravity power and hydrostatic resistance as well as lower load on joints and muscle , muscles , tendons , and ligaments can be easily strengthened . physiologic responses of the body to floating in warm water are quite similar to those to local heat , but with less concentration . physical properties of combination of water and heat , is the reason for most of general physiologic responses affecting various systems in the body . therefore , aquatic exercise with regard to their potential environment for activities , compared with land exercises , can be helpful for stress and pain reduction . in the present study , pain intensity and length in the study group was reduced through 8-week aquatic exercises and if the exercises continued , dysmenorrhoea would be diminished constantly . overall , the findings suggest that aquatic exercise in patients with primary dysmenorrhea can reduce the symptoms . since the primary dysmenorrhea can have a negative impact on women 's employment centers , can be an affordable way to make a positive impact on other aspects of women 's health is also proposed .

background : primary dysmenorrhoea without any specific pelvic disease is one of the common complaints in women`s medicine . the general purpose of this research is to define the effects of 12-week aquatic exercises on nonathletic girls primary dysmenorrhoea.materials and methods : this quasi - experimental was conducted on 40 nonathletic girls aged 18 - 25 years . data gathering tools were : evaluation form of primary dysmenorrhoea and the pain evaluation tool based on the mcgill standard pain questionnaire completed before and after the intervention in 3 months ( first , second , and third run ) . then , 20 subjects were assigned to aquatic exercise group and the other 20 to control group . the subjects in experimental group did aquatic exercise for three sessions a week for 60 minutes for 12 weeks between two menstruations . kruskal wallis and one way analysis of variance ( anova ) tests were used to analyze the data.results:the results of this research indicated that severity and duration of pain decreased after 12 weeks of aquatic exercises . comparison of the two groups showed a significant difference in pain intensity based on visual analogue scale ( vas ) scale after these exercises ( first , second , and third runs ) . present pain intensity ( ppi ) scale after these exercises ( second and third runs ) showed a significant difference . comparison of the two groups showed a significant difference in length of pain after these exercises ( third run).conclusions : totally , the findings of the present study showed that 12-week regular aquatic exercises are effective on decrease of the severity of the symptoms of primary dysmenorrhoea .

I M R D C

PMC3580342

the limiting factor in these flaps is the unpredictable blood supply , which , if insufficient , may produce irreversible damages to the microcirculation . this damage results in partial or complete flap necrosis and renders the wound more susceptible to infection , thereby causing further healing impairment ; thus , improving distal blood supply in the random skin flap is an important goal . the cellular damage that occurs during tissue reperfusion after ischemia is the result of a cascade of events involving free - radical oxygen and inflammatory mediators . molecular oxygen plays a central role in the reparative healing process and is one of the critical nutrients of the wound . hyperbaric oxygen ( hbo ) increases the tolerance of tissue to ischemia and enhances free - radical formation ; however , hyperoxia can increase the biochemical defence mechanisms against free radicals and improve the survival probability of ischemic tissue . the hbo has a protective effect on microcirculation possibly by interfering with the deleterious action of activated neutrophils on microvascular endothelium . one of the most important components of the intracellular antioxidant system is glutathione , a powerful active radical scavenger that is depleted in ischemia - reperfusion ( ir ) injury . n - acetylcysteine ( nac ) is a prodrug that supplies bioavailable cysteine for glutathione replenishment . in the presence of overwhelming active oxygen species ( ros ) , the antioxidant nac prevents some of these deleterious effects , indicating an involvement of oxidative stress during hbo exposure . the effects of hbo in improving the survival of a random skin flap are already known . thus , it is important to study its combination with other substances , such as enzymes , nac , and so on , to potentiate their effect to be applied to patients . thus , in this study , our aim is to investigate the role of hbo , nac , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design . the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) . all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) . thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum . the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo , n = 8) , and g - hn ( hbo plus nac , n = 8) . after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m . subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m . a rectangular area ( 2 8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] . a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] . an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd . , so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization , 100% medical oxygen was flushed through the chamber for 5 min to displace the room air . the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere . all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days . the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days . g - nh : flap proceedings and eight daily injection of nac , followed by hbo every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded . once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format . under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest . on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment . the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups . the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) . significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) . all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) . thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum . the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo , n = 8) , and g - hn ( hbo plus nac , n = 8) . after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m . subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m . under general anaesthesia , the dorsal regions were shaved and the animals were fixed in the prone position . a rectangular area ( 2 8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] . a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] . an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd . , so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization , 100% medical oxygen was flushed through the chamber for 5 min to displace the room air . the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere . all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days . the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days . g - nh : flap proceedings and eight daily injection of nac , followed by hbo every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded . once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format . under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest . on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment . the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups . the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) . significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p in figure 4 , the mean of necrosis ( % ) is shown by groups . the average flap necrosis area was 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn group . means and standard deviation of the percentage of necrosis on the random skin flaps in g - s , g - nac , g - hbo , and g - hn groups . the means of necrosis in all the experimental groups were lesser than those of the g - s group . there was a significant difference between g - hbo < g - nac ( p < 0.01 ) and g - hn < g - nac ( p < 0.001 ) ( anova test ) in table 1 , the results of macroscopic evaluation ( area in mm ) for each group are presented . means , standard deviation , and range of necrosis area ( mm ) in the skin flaps of g - s , g - nac , g - hbo , and g - hn on day 8 g - hbo group showed a smaller area of flap necrosis , when compared with g - s , but was not significant ( p = 0.12 ) ; however , when compared with the g - nac group , a significant difference was noted ( p < 0.01 ) . the flap survival in g - hbo was the same as that noted in g - hn , and when compared with the g - nac , the g - hn group showed significant difference ( p < 0.01 ) . in this study , however , the combination of nac and hbo , in contrast to the expectations , had no better survival , when compared with administration of hbo alone . there has been much controversy over the angiogenic properties of hbo . in skin wounds , the angiogenic properties induced by hbo are derived from the increase in oxygen tension that may persist for several hours after hbo . comparison of g - s and g - nac groups revealed that repeated on - off exposures produce a favourable environment in random flaps . in this study , hbo administered after surgery improved the survival of random skin flaps . this finding is in contrast to the original hypothesis that suggests that extra oxygen increases the production of free radicals that would increase flap necrosis . hong jp showed that when hypoxic tissue is exposed to hbo , there is an increase in po2 in the plasma and a reduction in po2 , and the rate of diffusion of oxygen increases . nac is a precursor of glutathione , a potent antioxidant that inhibits the induction of pro - inflammatory cytokines . glutathione also induces the production of nitric oxide synthase ( inos ) as well as adhesion molecule 1 . this antioxidant also induces the production of a vascular cell adhesion molecule 1[101517 ] and stimulates the production of nitric oxide ( no ) . in this study , the flaps treated with hbo alone led to improvement in the average survival than those treated with a combination therapy with both hbo and nac , suggesting that these agents do not potentiate one another . the groups treated with distilled water and nac alone exhibited the worst results in this experiment . the doses of nac ( 300 mg / kg / day ) utilized in this study were chosen owing to the reported low toxicity of this drug and favourable results in the protection of random skin flaps in rat model . the plastic barrier interposed between the flap and donor bed hbo in association with nac may be responsible for a protective effect on the deleterious outcome of nac . when nac is used alone , the measures of necrotic areas were found to be higher in the g - nac ( 24% ) . on the other hand , with the combination of hbo and nac , the measures of necrotic areas probably , in this study , the correct concentration of nac could have inhibited angiogenesis and wound - healing response . this inhibition is considered to occur through an imbalance in the cellular redox state or an undetermined mechanism . it has been demonstrated that hbo can increase the tolerance of tissue to ischemia , diminish metabolic disturbances , improve tissue microcirculation , and reduce platelet aggregation . these characteristics , combined with the ability of plasma to carry dissolved oxygen to areas where red blood cells can not reach , have been shown to have a beneficial effect on oxygenation of many hypoxic tissues . in the present study , the combination of hbo and antioxidants therapy did not improve survival above and beyond the effect of hbo administered alone , suggesting that the potential toxic effects of hyperoxia from ros were not minimized by antioxidant therapy with nac . oxidizing species , such as free radicals and hydrogen peroxide , may serve as cellular messengers mediating complex redox - sensitive processes , such as extracellular matrix formation , cytokine action , angiogenesis , and cell motility . knight et al . demonstrated in rabbits that high dose of nac has no significant difference in the survival of flaps . furthermore , ramon et al . showed an increase in the survival of the transverse rectus abdominis myocutaneous flap treated with hbo alone . demonstrated in rats that hbo associated with vitamin e and c could improve the survival of epigastric island skin flaps , while rocha et al . reported that hbo alone showed a protective effect in the ischemic skin flap , which was associated with reduced expression of apoptosis however , despite these results , multicenter prospective clinical studies are clearly needed to compare hbo treatment with other mechanical or pharmacologic interventions , to improve wound healing for grafting or to support flap survival . with the experimental model , it can be concluded that hbo produced an improvement in the distal blood supply of the random skin flap ; however , this improvement was not significant . furthermore , the effect of combination with nac was not significantly different on the survival of flap , when compared with the hbo therapy alone .

objective : our aim is to investigate the role of hbo ( hyperbaric oxygen ) , nac ( n - acetylcysteine ) , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design.materials and methods : thirty - two male wistar rats were randomly divided into four groups : g - s ( sham : n = 8) , g - nac ( nac : n = 8) , g - hbo ( hbo : n = 8) , and g - hn ( hbo plus nac : n = 8) . a rectangular skin flap ( 2 8 cm2 ) was dissected from the muscular dorsal layer , preserving the cranial pedicle . polyethylene film was placed over the muscular layer and an interrupted 3.0 nylon suture was employed to fix the flap into the original place . on the eighth day , full - thickness biopsy samples ( 2 1 cm2 ) were collected from the proximal , middle , and cranial areas of the skin flap , and in a site away from the flap labelled as the control area.results:the measurements of necrotic areas in the groups were 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn . significant difference was observed between the groups g - hbo and g - hn as well as g-nac.conclusion:hbo is associated with reduced area of necrosis of skin flap . the g - nac group was associated with poor results when examined in isolation . the association between hbo and nac did not produce favourable results with respect to the use of hbo alone . these findings suggest that the diffusion of oxygen through the interstitial space was the determining factor of more favourable results of hbo .

INTRODUCTION MATERIALS AND METHODS Ethical committee Animal housing and groups Anaesthesia Surgical procedure NAC and distilled water administration procedures HBO procedure Sequential daily procedures Seven days follow-up Euthanasia Determination of flap necrosis area Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC4414945

all - ceramic restorations have gained more popularity due to their high esthetic , high improvement in their fracture strength and good biocompatible properties . recently , yttria - stabilized tetragonal zirconia polycrystals ( y - tzp ) have been introduced to the dental professionals . these materials have to be fabricated in cad / cam ( computer - aided design / computer - aided manufacturing ) procedures that have been investigated under in vivo conditions.1 the partially stabilized zirconia shows high fracture strength and structural reliability compared to glass - ceramics when fabricated into prostheses framework . however , due to their low translucency of the light , all zirconia frameworks have to be veneered with glass - ceramics or porcelain for esthetic reasons . these veneering materials have to directly face with chewing force and moisture , resulting in cracks or chipping.2 chipping of a veneering material is a typical failure of all types of ceramic covered dental prostheses . these chipping problems have been reported with porcelain fused to metal ( pfm ) restorations . various factors that might have an effect on chipping have been proposed , such as adhesion between framework and veneering , veneering thickness , and supporting morphology of the finish line.2 nowadays , frameworks for all - ceramic crown design by cad / cam have been based upon empirical machine guidelines rather than clinical scientific data . most of all cad / cam systems , the frameworks of the crowns are design to arbitrary thicknesses of 0.4 to 0.6 mm . like porcelain fused to metal restorations , zirconia frameworks should be designed to provide the appropriate veneering porcelain thickness and support to minimize internal stress , reduce mechanical failures , and optimize esthetics of the veneering porcelains.4 the objective of this study is to compare the failure load and failure characteristics of two different zirconia framework designs of premolar crowns when subjected to static loading . cobalt - chromium casting master die was replicated from prepared preformed plastic maxillary right second premolar for all ceramic crowns . the preparation was prepared leaving a 0.8 mm deep chamfer finishing line ; 1.5 mm of occlusal reduction ; 6 occlusal convergence angle . the master die was scanned using the ineos blue scanner ( sirona , long island city , ny , usa ) . scanned data were computed and then designed for all - ceramic crown framework using the cerec 3d software ( sirona , long island city , ny , usa ) . first , the 0.5 mm thick framework ( ev ) including 0.5 mm thick crown margin was prepared ( fig . second , cutback design was prepared as same as that of metalceramic crowns to obtain uniform , adequate thickness and support of 0.8 mm thick of the veneering porcelain ( cb ) with the same crown margin 0.5 mm . subsequently , zirconia frameworks ( 10 frameworks / each group ) were fabricated from pre - sintered zirconia ingot ( incoris zi , sirona , long island city , ny , usa ) by a cerec inlab mc xl machine ( sirona , long island city , ny , usa ) . after milling , the framework was removed from the machine and final sintering in an infire htc speed furnace ( sirona , long island city , ny , usa ) . the framework was veneered with vita vm9 porcelain ( vident , brea , ca , usa ) using brushing technique to derive the anatomical crown shape . the veneering porcelain was constructed under vacuum - former sheet to obtain the same total thickness of the crown . all crowns were calibrated by measuring at references point on buccal and lingual incline of the buccal cusp . several pilot specimens ( 4 specimens ) were cut at the reference points to measure the real thickness of both zirconia framework and veneering ceramic . the thickness of veneering ceramic of the ev design crowns was 1.4 0.06 mm whereas those of cb design crowns was 0.8 0.05 mm , and those of zirconia frameworks were 0.5 0.04 mm and 1.1 0.07 mm respectively . before cementation , the internal walls of the crowns and the metal dies were cleaned with water steam . each crown was cemented using resin cement ( relyx u-100 , 3 m espe , st . paul , mn , usa ) onto the metal die , using finger pressure then placed under a 2 kg load for 15 minutes . all specimens were subjected to the universal testing machine ( instron model 5566 , instron corp , norwood , ma , usa ) . the cylinder rod with rounding head , radius 5 mm , was placed on the lingual incline plane of the buccal cusp of the crown . therefore , the load was applied at 45 degree to the long axis of the crown at a crosshead speed of 0.5 mm / min until crown fracture occurred . all specimens were examined under an optical light microscope ( nikon mm-11 , nikon corp , tokyo , japan ) and a scanning electron microscope ( jsm-5410lv , jeol ltd . , cohesive failure : fracture occurred within veneering materials more than 80%adhesive failure : fracture occurred along the zirconia and veneering materials interface more than 80%failure through the zirconia framework cohesive failure : fracture occurred within veneering materials more than 80% adhesive failure : fracture occurred along the zirconia and veneering materials interface more than 80% failure through the zirconia framework the load to failure data was recorded in newtons ( n ) . the difference between the even thickness framework design and cutback framework design of all ceramic crowns were examined using independent sample t - test at confidence interval of 95% ( =.05 ) . the mean and standard deviations of the failure load and failure characteristics of the crowns are shown in table 1 . n while those of cb design showed a bit higher at 1450.4 175.7 n. the t - test showed the significant differences in the failure load among the tested groups ( p<.05 ) . under visual examination , 2 ) , however cohesive or adhesive failure could not be classified by visual inspection . the results in table 1 showed that all ev design crown found cohesive failure while the cb design crown found majority failure through the zirconia framework . all of the crown fracture was found in splitting into a buccal and a lingual half pattern . the fracture lines ran from the position of the loading into the mesio - distal orientation towards the crown margin ( fig . the cutback framework design is based on what has been proposed previously for porcelain fused to metal restorations to overcome the porcelain chipping experienced at that times.5,6 clinically , delamination and minor chip - off fractures of the veneering ceramic were found as the major reason for failures of zirconia fixed restoration.1,7,8 one of the reasons for porcelain fracture is improper framework design . the improper framework design causes the improper support for the porcelain veneer layer and also the nonappropriate thickness of the veneering layer . the modification of the framework design by creating an appropriate support and allowing the proper veneering thickness has been proved to reduce the porcelain chipping rates.4 although there is no control group to compare the result , a clinical study on zirconia modified framework design on fixed partial dentures showed high survival rates in 3 years that can be speculated on the potential of the framework design for providing better porcelain support during function9 . in this study , a significant higher failure load was found for crowns with cutback design with optimal cusp support than the crowns with even thickness design . this result conformed to the previous studies that a framework with anatomical shape crown ( cut back framework ) withstood significantly higher loads before fracture than did crowns with even thickness framework.10 they explained the differences in fracture load by the thicker layer of veneering ceramic on crowns with even thickness framework . the larger the volume of ceramic , the larger the size of the flaw population and the higher the risk of prevalence of critical flaw.11,12 however , the failure characteristic in this study seems contradictory to the bonfante et al . 's study.13 they predominantly found smaller veneer layer fracture on the crowns with cutback design framework than the crowns with even thickness framework . while in this study the cutback design framework crowns predominantly found failure through the zirconia framework whereas all the even thickness framework crowns found only veneer fracture . this may be due to the thick veneering ceramic ( 1.4 mm ) with poor support is acting as the force defender of the crown , leading to veneering ceramic fracture before the loads are high enough to affect the framework . for cutback design crowns , the design creates an appropriate support and appropriate thickness of veneering ceramic ( 0.8 mm ) . the difference of both studies is the position of the load applied onto the crown . in this study , the force was applied on the buccal cusp with 45 degree to the long axis of the crown instead of the central fossa of the occlusal surface . in this study , the force was applied 45 degrees to the long axis of the tooth because clinically , the shear force is the frequent force that occurred on this tooth . the alignment of this tooth is generally inclined buccally at an average 9.5 degrees to the occlusal plane.14 however , the chewing force is not occurred vertically but sliding contact motion . theoretically , the maximum angle that can cause the highest force is 45 degree.15 as a consequence , it was assumed that this angle should cause the maximum load in the clinical application . the force was applied on the lingual incline plane of buccal cusp of premolar crown due to the clinical situation , the shear force occurs on this cusp . maxillary premolar has been found that non - functional cusps of restored teeth have a fracture more frequently than functional cusps.16 according to the studies of farah and craig,17 craig et al.,18 and nally et al.,19 the stress distribution on the porcelain shows isochromatic shearing - stress trajectories radiating from the edges of the punch when loaded in compression . therefore , fracture in porcelain would start at these trajectories running mesio - distal orientation , leading to crown fracture found in splitting into half pattern in this study . the employment of the single load to failure tests to understand the performance of dental materials has its limitations . in clinic , ceramic failure occurs from cumulative damage and slow crack growth20 when it is subjected to cyclic chewing force , approximately 380 n in the premolar area.21 this amount of force is much lesser than the failure force in this study . thus , the results in this study need to be interpreted with caution . in order to obtain more clinically relevant results , further research is necessary , including fatigue loading prior to static load tests for zirconia crowns . within the limitation of this study , the conclusions are as follows different framework designs have influence on the failure load and failure characteristics of all ceramic zirconia crowns . the cutback zirconia design for all ceramic crowns is a promising way to reduce veneer chipping failures .

purposethis in vitro study aimed to compare the failure load and failure characteristics of two different zirconia framework designs of premolar crowns when subjected to static loading.materials and methodstwo types of zirconia frameworks , conventional 0.5 mm even thickness framework design ( ev ) and 0.8 mm cutback of full contour crown anatomy design ( cb ) , were made for 10 samples each . the veneer porcelain was added on under polycarbonate shell crown made by vacuum of full contour crown to obtain the same total thickness of the experiment crowns . the crowns were cemented onto the cobalt - chromium die . the dies were tilted 45 degrees from the vertical plane to obtain the shear force to the cusp when loading . all crowns were loaded at the lingual incline of the buccal cusp until fracture using a universal testing machine with cross - head speed 0.5 mm / min . the load to fracture values ( n ) was recorded and statistically analyzed by independent sample t-test.resultsthe mean and standard deviations of the failure load were 1,170.1 90.9 n for ev design and 1,450.4 175.7 n for cb design . a significant difference in the compressive failure load was found ( p<.05 ) . for the failure characteristic , the ev design was found only cohesive failures within veneering porcelain , while the cb design found more failures through the zirconia framework ( 8 from 10 samples).conclusionthere was a significant difference in the failure load between two designs , and the design of the framework influences failure characteristic of zirconia crown .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4526216

myocardial infarction ( mi ) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and a poor quality of life . features of the mi include an increase of cardiac sympathetic nerve activity to elicit inotropic positive response for the maintenance of cardiac output ; this compensatory mechanism plays a major role in the progression of heart failure . recent data showed the role of the hypothalamic paraventricular nucleus ( pvn ) , a key neurohumoral integrative nucleus in the brain , responsible for the connections to sympathetic preganglionic motor neurons [ 3 , 4 ] . moreover , high percentage of mi patients undergo late depressive state . brain inflammation , driven by nonneuronal cells such as astrocytes and microglia , has been demonstrated to be associated with several neurological diseases including depression and anxiety , with neuropsychiatric pathologies such as mood disorders and psychosis , and with neurodegenerative diseases such as parkinson , alzheimer , and huntington diseases [ 79 ] . indeed , all these pathologies are also associated with plastic changes which involve the activation of glia and microglia in the early sensitization and the subsequent neuronal suffering . moreover , glial and microglial cells have been shown to be associated also with the chronicity of pain that could be consequence of heart failures such as myocardial infarction [ 1012 ] . recent reports have analyzed the possible changes in microglial phenotypes after myocardial infarction at different time stages , demonstrating a microglial cytoskeletal rearrangement in a late phase after mi induction in the pvn whereas no significant differences were observed in the cortex [ 13 , 14 ] . this microglia activation seems to be mediated by the p2x7 receptor and also an attenuation of microglial and neuronal activation in the brain by icv minocycline following myocardial infarction has been reported . previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after mi [ 17 , 18 ] . in this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after mi in pvn , thalamus , prefrontal cortex , and hippocampus through immunofluorescence approach . all the experimental procedures were approved by the animal ethics committee of the second university of naples . all efforts were made to minimize animal suffering and to reduce the numbers of animals used . sixty rats ( 225250 g ; n = 60 ; harlan italy ) were randomly assigned to two main groups : sedentary ( sed ; n = 30 ) and exercise trained ( tr ; n = 30 ) . briefly , trained rats were acclimated to training by walking at a speed of 10 min / day on a treadmill for 2 weeks ( panlab / harvard apparatus treadmills , holliston , ma , usa ) . from week 3 , speed and time of running were gradually increased ( 30 m / min , 45 min / day , 5 days / week , for 6 weeks ) . the sedentary rats remained in the cages for all the duration of the training protocol . twenty - four hours from the last session of exercise training 20 rats of both experimental groups were undergoing myocardial infarction by surgical occlusion of the left anterior descended ( lad ) coronary artery , according to previous described procedures . briefly , after induction with intraperitoneal injection of ketamine hydrochloride ( 100 mg / kg ) and xylazine ( 2.5 mg / kg ) supplemented as needed , the rats were intubated and ventilated with room air by a small animal ventilator ( harvard apparatus , model 623 ) . after performing the thoracotomy in the third and fourth intercostals spaces , the pericardium was incised and a 6 - 0 silk suture ( johnson & johnson ) was placed around the proximal portion of the left coronary artery . formalin - fixed , paraffin - embedded myocardial samples were cut in 5 m thick sections and stained with hematoxylin and eosin . for fluorescence imaging , tissue sections were deparaffinized and labelled with apoalert dna fragmentation assay kit ( clontech ) . subsequently , samples were incubated with an anti--sarcomeric actin ( sigma ) antibody followed by tritc - conjugated secondary antibody ( jackson immuno , west baltimore pike , west grove , pa , usa ) . samples were analyzed with a leica dm 5000b microscope and a zeiss lsm 700 confocal microscope . under pentobarbital anesthesia ( 50 mg / kg , i.p . ) , animals were transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1 m phosphate buffer . the brains were excised , postfixed for 3 h in the perfusion fixative , cryoprotected for 72 h in 30% sucrose in 0.1 m phosphate buffer , and frozen in optimal cutting temperature ( o.c.t . ) embedding compound . transverse sections ( 20 m ) were cut using a cryostat and thaw - mounted onto glass slides . slides were incubated overnight with primary antibody solutions for the microglial cell marker iba-1 ( rabbit anti - ionized calcium binding adapter molecule-1 ; 1 : 1000 ; wako chemicals , germany ) and gfap ( rabbit polyclonal antiglial fibrillary acidic protein ; 1 : 1000 ; dako cytomation , denmark ) , according to previously reported protocols [ 21 , 22 ] . possible nonspecific labeling of mouse secondary antibody was detected by using secondary antibody alone . following incubation , sections were washed and incubated for 2 h with secondary antibody solution ( donkey anti - rabbit or igg - conjugated alexa fluor 488 ; 1 : 1000 ; molecular probes , usa ) . slides were washed , coverslipped with vectashield mounting medium ( vector laboratories , usa ) , and visualized under a leica fluorescence microscope . the number of cells positive for iba-1 or gfap was determined within a box measuring 2 10 m that was placed in the lateral , central , and medial areas of cortex , hippocampus , thalamus , and hypothalamic paraventricular nucleus . eight sections were assessed from one animal and three animals were used for each group . to avoid cell overcounting , gfap - positive cells were identified as resting ( with small somata bearing long , thin , and ramified processes ) , activated microglia and astrocytes ( with hypertrophy together with retraction of processes to a length shorter than the diameter of the somata ) , or dystrophic microglia ( no dystrophic astrocytes were detected ) . dystrophic microglia was recognized by debris consisting of several cells displaying fragmented processes and an irregularly shaped cell body as previously demonstrated in humans . forty - eight hours after coronary artery ligation , the presence of myocardial infarction was confirmed by histological analysis . this was confirmed by the detection of apoptotic cardiomyocytes with a terminal deoxynucleotide transferase- ( tdt- ) mediated dutp nick - end labeling ( tunel ) assay and confocal microscopy ( figure 1 ) . mi procedure did not change the number of both total iba-1 positive cells in the cortex . however , mi animals showed a significant increase of the number of activated as well as dystrophic microglia cells in the same area . these effects were significantly prevented by applying the exercise training protocol ( figures 2(a ) and 2(b ) ) . interestingly , the exercise alone exhibited significantly effects on cells morphological changes , as compared with sedentary animals . in particular , it reduced the number of activated microglia cells and modulated the number of astrocytes , by increasing the total number of gfap - positive cells , and reduced the number of hypertrophic cells ( figures 2(c ) and 2(d ) ) . we observed a significant increase of total , activated , and dystrophic microglia cells in the hippocampus of mi rats . the exercise counteracted the microglia activation and reduced the number of dystrophic cells , without affecting the total cells number in the same area . moreover , the exercise reduced per se the number of activated microglia as compared with sedentary animals ( figures 3(a ) and 3(b ) ) . no changes in astrocytes the mi induction did not change the number of the total or activated microglia cells in the thalamus as compared with control . however , the number of dystrophic microglia dramatically increased in mi animals 2 days after surgery . exercise trained animals showed a significantly attenuated number of dystrophic microglia induced by mi by more than 50% . interestingly , the exercise erased per se the number of activated microglia in sedentary rats ( figures 4(a ) and 4(b ) ) . the total numbers of astrocytes counted in the thalamus of the different treatment groups were similar . however , mi induced an increase in the number of hypertrophic gfap - positive cells that were significantly reduced in the trained mice . moreover , the exercise training abolished the number of hypertrophic astrocytes in sedentary rats ( figures 4(c ) and 4(d ) ) . mi and sedentary rats did not differ in the number of total microglia as well as astrocytes in the pvn . furthermore , mi surgical procedure did not change the number of activated microglia cells found in the control animals ( ~10% ) . however , mi rats showed an increased number of dystrophic microglia cells as well as hypertrophic astrocytes which were not affected by exercise training ( figures 5(a)5(d ) ) . this study provides the first evidence that the possible changes in glia and microglia brain areas after myocardial infarction could be , at least in part , prevented by the prolonged moderate exercise . in this study we analyzed the morphological changes of the microglia and astrocytes in four brain areas involved in the integration of external emotional stimuli , learning and memory , sensorial perception , and metabolism , before and 48 hours after myocardial infarction and with or without 8-week training protocol . in particular , we observed that in the prefrontal cortex the 48 h mi induced an increased number of hypertrophic and dystrophic - like microglia in sedentary mice . both microglia phenotypes were significantly reduced in the prefrontal cortex in the 48 h mi trained group . no significant changes in the hypertrophic / resting astrocytes ratio were observed in the prefrontal cortex in all experimental groups except for the trained mice without mi in which we found an increase and decrease in the total and hypertrophic astrocytes , respectively . in the thalamus and hippocampus , which represent key brain areas in the sensorial and pain - associated synaptic plasticity , we observed an increased number of dystrophic microglia . furthermore , in the thalamus , microglial changes were also accompanied by an augmented number of hypertrophic astrocytes , in the mi group as compared to the sedentary animals . the prolonged exercise significantly reduced both the dystrophic microglia and reactive astrocytes induced by mi . finally , in the pvn we observed increased number of dystrophic microglia and hypertrophic astrocytes in the sedentary animals that were not changed by the exercise . the latter data are interesting also regarding the role of the pvn which represents an important neuroendocrine and preautonomic output nucleus and is considered as the important central site for integration of sympathetic nerve activity [ 3 , 4 ] . this could be explained , at least in part , assuming that the effect of exercise is mainly acting on those brain areas involved in the sensorial and rewarding processes such as thalamus and cortical areas rather than those brain structures directly involved in the interface between cns and periphery . intriguingly , previous data showed no changes in activated microglia in the pvn and cortex 24 hours after myocardial infarction induction [ 13 , 14 ] . our data demonstrated that after 48 hours postmyocardial infarction there are changes in microglia phenotypes . in particular , we observed the appearance of a dystrophic - like phenotype of microglia mainly in the thalamus and prefrontal cortex . this phenotype of microglia is predictive of a brain malaise that could , in turn , mirror in a malfunction of the neurons . indeed , dystrophic ( senescent ) rather than activated microglia seem to be associated with tau pathology and likely precede neurodegeneration in alzheimer 's disease in human . this microglia phenotype is not well characterized yet in terms of the specific marker expression . recent data in rodents highlighted for the first time dystrophic - like microglia phenotype in transgenic mouse model lacking for the d - aspartate oxidase , the enzyme responsible of the d - aspartic acid degradation , that showed high glutamate levels which , in turn , could be in part responsible of those microglia changes . moreover , a recent report showed a possible microglia change from a reactive to an age - like phenotype with the time in culture . according to the early appearance of dystrophic microglia all these changes could be due to a glutamate spillover possibly induced by an overstimulation of afferent fibers after the surgically - induced mi , rather then to the mi per se . despite this hypothesis , which needs to be verified , it is intriguing that the prolonged exercise prevents these microglia and astrocyte alterations in the prefrontal cortex , thalamus , and prefrontal cortex . the present study suggests that myocardial infarct may be correlated with a supraspinal synaptic reorganization in which microglia and astrocytes might play a role . indeed , in our experiments the scale of infarction was strongly correlated with the proportion of dystrophic or activated microglia and hypertrophic astrocytes in the brain . it is well known that , upon activation , morphological cells rearrangements are correlated with the synthesis and secretion of cytokines , by leading critical neuronal activity modifications [ 14 , 26 ] . thus , one can speculate that supraspinal nonneuronal cells may significantly contribute to the functional as well as behavioral alterations observed following myocardial infarction . however , further analysis will be necessary to more conclusively evaluate the phenotypical cells profiles induced by mi . in this context , the exercise seems to drive down the possible glial / microglial contribution to the functional alterations associated with myocardium infarct .

myocardial infarction ( mi ) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and poor quality of life . recent data showed a possible interaction between infarction and brain inflammation and activity . previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after mi . in this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after mi in pvn , thalamus , prefrontal cortex , and hippocampus through immunofluorescence approach . we found significant changes in specific microglia phenotypes in the brain areas analyzed together with astrocytes activation . prolonged exercise normalized these morphological changes of microglia and astrocytes in the prefrontal cortex , hippocampus , and thalamus but not in the pvn . our data suggest that there is an early brain reaction to myocardial infarction induction , involving nonneuronal cells , that is attenuated by the prolonged exercise .

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

PMC1637246

neurotoxic substances affect the nervous system in a selective manner . one possible basis for this selectivity is blood vessel permeability . in general , the central nervous system and the peripheral nerve trunks have impermeable blood vessels , but in certain parts the capillaries are " leaky , " allowing the passage of a plasma filtrate . intravenously injected protein tracers rapidly reach nerve cells in these regions , with the implication that these nerve cells are also readily accessible to circulating neurotoxic substances . some examples of neurotoxicity in the central nervous system show a selectivity that could be due to capillary permeability . in experimental methylmercury poisoning , cranial nerve v and sensory dorsal root ganglia , which lie in regions of vascular permeability , are particularly susceptible . a number of drug and chemically induced neuropathies are predominantly sensory , and may be due , directly or indirectly , to the accessibility of neurotoxic substances to sensory neurons . examination of areas of potential vulnerability to circulating toxic substances may be of value in the experimental testing of substances for neurotoxicity , where pharmacological tests may be negative and clinical symptoms difficult to assess.imagesfigure 1 . ( a)figure 1 . ( b)figure 1 . ( c)figure 2.figure 3 . ( a)figure 3 . ( b)figure 4.figure 5 . ( a)figure 5 . ( b)figure 6 . ( a)figure 6 . ( b )

Images

PMC3649448

a traumatic right diaphragmatic rupture as the result of blunt trauma is a rare condition . the overall incidence of diaphragmatic rupture after blunt trauma is 0.8 - 3.6% ( 1 ) . most injuries are due to vehicular - related incidents ( 79.5% ) and falling from a height ( 15.9% ) ( 2 ) . right - sided tears are significantly less likely than left - sided tears , 27.3% and 68.2% respectively ( 3 ) . the correct diagnosis of diaphragmatic injury is initially missed in 12%-66% ( 1 ) delayed diagnosis and treatment of diaphragm rupture is associated with increased rates of morbidity and mortality . in our case on our outpatient clinic , a 37-year - old man was presented with 31 years before a fall from a wall of 3 meters on his right side . next to this major event the man was involved in a moped -car accident with only his face wounded , 18 years ago . during his life the patient presented to us with primary complaints of progressive , stabbing pain in the upper right abdomen and right lower thorax with simultaneous periods of dyspnoea . the symptoms worsened over a year , eventually leading to shortness of breath during rest . beside this presentation the patient had right upper abdominal pain while eating spicy food . on admission , his vital signs were stable and colour was fair . pulmonary examination revealed diminished breath sounds at the basal right hemithorax and the diaphragm was determined to be higher on the right by percussion . the diagnosis was confirmed by thoracic - abdominal x - rays and computerised tomography ( ct ) imaging , which revealed intra - thoracic displacement of the liver and also the gallbladder ( figure 1 ) . an elective thoracotomy in the sixth intercostal space was performed with repairing the diaphragmatic hernia ( figure 2 ) . the liver with gallbladder was replaced in the abdominal cavity , after adhesiolysis of the lung from pleura and diaphragm , whereupon the diaphragm was closed tensionless by running technique with non - absorbable sutures . two drains were left behind , one near the diaphragm and one in the right upper thorax . right sided diaphragmatic rupture with consequently hepatothorax is a rare condition . blunt traumatic diaphragm rupture a delay could result in increased rates of morbidity and mortality ( 5 ) . in case of hepatothorax , hypovolemic shock can occur , because of possible kinking of the inferior vena cava with obstruction of the blood . in this case there was a lateral impact on the chest , which distorted the chest wall and sheared the diaphragm . there remains doubt about the cause of delayed diagnosis and treatment of this diaphragm injury , since it could occur as the result of either delayed rupture or delayed detection . even if the herniation did not take place initially it would result in herniation eventually , because of the significant discrepancy between the intra - thoracic pressures and higher intra - abdominal pressure . most of the diaphragmatic ruptures occur on the left side , because the right diaphragm is congenitally stronger than the left . moreover the liver partially protects the right side as the impact can be spread over a large area . pain in the upper abdomen and lower thorax , dyspnoea , cyanosis and hypotension are typical symptoms of diaphragmatic injury , but if the rupture is small the patient can remain symptom free for a long time . the true incidence of traumatic diaphragm rupture is unknown because in 12 - 66% ( 1 ) of major trauma victims , the diagnosis is missed . there is no consensus yet , on the gold standard of imaging technologies to diagnose diaphragm rupture . based on the literature , diagnosis of diaphragmatic injury requires a high index of suspicion regardless whether you make an x - ray or ct . the choice of surgical approach includes thoracotomy , laparotomy , or both if it is necessary . the decision to repair the diaphragm either way is dictated by the stability of the patient and the presence of other associated organ injuries .

a traumatic right diaphragmatic rupture is an uncommon condition , as it occurs in 0.8 - 3.6% after blunt trauma . it is challenging to find the diagnosis immediately and is illustrated by the incidence of 12 - 66% initially missed diagnosis ( 1).most blunt traumatic diaphragm ruptures are an indication for early aggressive surgical intervention by way of thoracotomy , laparotomy , or both if it is necessary . delayed diagnosis and treatment of diaphragm rupture is associated with increased rates of morbidity and mortality . therefore , diagnosis of diaphragmatic injury requires a high index of suspicion . we report a case of a 37-year - old man with right diaphragmatic rupture after blunt injury 31 years prior to admission .

INTRODUCTION CASE REPORT DISCUSSION

PMC3457643

previous research has clearly demonstrated that cognitive change in old age does not occur in a homogenous manner for all individuals [ 13 ] . a number of predictors of cognitive change in old age have been identified , such as education , hypertension , objective indices of health and cardiovascular disease , and apolipoprotein e . regular engagement in different types of activities may also influence cognitive change . more specifically , according to the use it or lose it hypothesis , regular engagement in different activities may buffer age - related decline in cognitive functioning . a number of studies have found that general lifestyle activity engagement ( often operationalized as the combination of intellectual , social , and physical activities ) is associated with cognitive change [ 68 ] and that decline in activity in older age is associated with decline in cognitive functioning . in addition to general activity , other studies have specifically targeted the association of physical activity with cognitive change . indeed , a growing body of the literature highlights the potential benefits of physical activity on the structure and function of the brain [ 9 , 10 ] . the first line of evidence for the relationship between physical activity and cognition comes from a number of cross - sectional studies demonstrating that physically active older adults have higher cognitive performance and functioning compared with less active older adults [ 11 , 12 ] . however , the evidence derived from these cross - sectional studies is limited , as it is not . longitudinal studies may be viewed as the second line of evidence , offering valuable information on the relationship between physical activity and cognition across time . several prospective , longitudinal studies provide evidence for the association of physical activity with cognitive functioning [ 1320 ] . these studies have generally shown that higher physical activity at baseline is associated with less decline in cognitive functioning over time , offering support for the notion that regular physical activity may buffer against future cognitive decline . however , results from these longitudinal studies are inconclusive and several critical questions remain . the longitudinal studies described above may test two different classes of hypotheses regarding the relationship of lifestyle variables , such as physical activity and cognitive change . the first type of hypothesis stipulates that the level of physical activity is related to subsequent cognitive change . the majority of the abovementioned studies have targeted this first class of hypothesis , examining a stable change hypothesis by looking at how physical activity at baseline predicts change in cognitive functioning . the second class of hypothesis instead examines the relationship between concomitant change in activity and change in cognition . in contrast to the baseline effect of activity , this hypothesis deals with the concept of intraindividual change and associations among intraindividual rates of change , providing a more dynamic perspective . for example , positive changes ( increases ) in physical activity across time may be hypothesized to contribute to a less negative change ( less decline ) in cognitive functioning , whereas a negative change in activity ( decreased activity ) would be expected to be related to faster cognitive decline with age . mackinnon and colleagues used a latent growth curve modeling approach to examine how change in overall activity ( defined as a composite of physical activity , rest , interest and hobby related , and planned activities ) , rather than physical activity , was related to change in health and cognitive performance . they found substantial correlations between rates of change in activity and cognitive and health measures , and it was concluded that decline in mental and physical activity in older age is paralleled by decline in cognitive functioning and health . however , decline in cognitive functioning was still evident for participants who were stable in their level of activity across time , suggesting that maintenance of activity may not be enough to protect from cognitive decline . unfortunately , few previous studies have actually targeted the hypothesis of whether there are associations among rates of individual change in physical activity and cognitive functioning in long - term observational studies of aging individuals . van gelder and colleagues found that men who decreased their physical activity duration or intensity also demonstrated a stronger decline in overall cognition ( measured by the mini mental state examination ) compared with men who maintained their activity duration and intensity . first , only change in one global measure of cognitive functioning was used , rather than several different measures that may capture more diverse and complex relationships of cognitive ability with activity change . moreover , in the study , change in physical activity was categorized in terms of quality ( change / no change ) rather than quantity ( how much change ) . finally , the analyses were based on between - group comparisons and therefore did not target relationships of within - person changes in physical activity and cognition . bielak and colleagues , however , used random effects models to examine how level and change in physical activity were related to level ( within - person mean ) and inconsistency ( within person standard deviation across trials ) of cognitive speed at baseline and change in level and inconsistency . although physical activity at baseline was related to mean cognitive speed in some tasks , there were no associations between change in physical activity and change in cognitive speed . moreover , a recent study using bivariate dual - change - score models to analyze data from the victoria longitudinal study found that changes in physical activity influenced changes in verbal speed and episodic memory . however , they also found that changes in cognition influenced changes in activity , thus supporting a dual - coupling model or a reciprocal relationship between physical activity change and change in cognition . although previous longitudinal studies have resulted in increased understanding of how physical activity at one point ( baseline ) may predict future cognitive performance , or change in cognitive performance , they have generally not helped us understand the more complex and dynamic characteristics of the longitudinal relationship between physical activity and cognition . for example , is there an association between within - person change in physical activity and within - person changes in cognitive functioning when taking into account the change in cognition due to time ? or , put differently , do persons demonstrate lower cognitive scores ( relative to their within - person trajectory over time ) on occasions when they also report less physical activity ? relative to a cross - sectional analysis that compares individuals to other similar aged individuals , the answers to these questions afford relevant insight into the more complex and dynamic patterns of associations between changes in physical activity and cognitive functioning within individuals across time . another question that has not been properly addressed by previous longitudinal studies is if physical activity , or change in activity , has similar effects across different cognitive domains and/or tests . from previous experimental work using randomized controlled trial designs , there is support for the notion that physical activity training has the strongest effect on executive control processes and working memory , supporting the selective - improvement hypothesis . however , as the majority of previous longitudinal studies of the relations between physical activity and cognition have included a single measure of cognition ( often mmse ) rather than different tests and domains , the theoretically , as well as practically , important question of whether changes in physical activity may relate more strongly to changes in some cognitive domains relative to others remains unresolved . an essential step for the sound cumulative development of this body of knowledge is the reproduction and extension of research findings across independent longitudinal studies that focus on observed within - person change . although most previous longitudinal studies have found that physical activity is protective against age - related cognitive decline , the findings are disparate and far from clear . moreover , previous studies have typically used data from one population ( e.g. , adults ranged from 5594 in age ) and one design ( e.g. , 3 waves of measurements over a 6-year period ) , leaving the generalization of the results highly contingent on sample specific - characteristics . differences between studies in terms of sample , design , measures , and analytical approach make it difficult to compare results across studies and to derive more general conclusions of the meaning of these results . therefore , there is a clear need for more coordinated integrative data analyses that use data from different samples with different measures , but examine these data with the same research question and the same analytic approach . using a coordinated analysis approach for cross - study comparisons and synthesis of independent results has the potential to bring new relevant information to the field of cognitive change and physical activity . the purpose of the present study is to investigate the longitudinal associations of physical activity with four domains of cognition ( i.e. , reasoning ( executive function ) , episodic memory , fluency , and semantic knowledge ) in older adults using a coordinated approach with data from four independent longitudinal studies : long beach longitudinal study ( lbls ) , the seattle longitudinal study ( sls ) , the victoria longitudinal study ( vls ) , and the origins of variance in the oldest - old : octogenarian twins study ( octo - twin ) . more specifically , the following research questions were examined : is physical activity at baseline associated with cognition at baseline?is baseline physical activity associated with the rate of change in cognition?are occasion - specific changes in physical activity associated with occasion - specific changes in cognition , controlling for change in cognition due to time alone ? is physical activity at baseline associated with cognition at baseline ? is baseline physical activity associated with the rate of change in cognition ? are occasion - specific changes in physical activity associated with occasion - specific changes in cognition , controlling for change in cognition due to time alone ? this research , initiated as a partnership between the advanced psychometric methods workshop series ( mungas et al . , nia conference grant ) and the integrative analysis of longitudinal studies on aging ( ialsa ) network [ 25 , 26 ] , brought workshop participants together with researchers from four ialsa member studies . these studies were specifically selected based on their collection of cognitive , physical , and social activity data along with a range of cognitive functioning measures over multiple occasions held in common across the four studies . while the activity and cognitive functioning variables are not always identical , the subsets of variables in each study were chosen based on the rationale that they tapped similar domains at the construct level ( e.g. , fluid reasoning ( gf ) , crystallized knowledge ( gc ) , short - term memory ( gsm , ) and long - term storage and retrieval ( glr ; category fluency ) . in some cases the measures are the same , but more often they differ , providing opportunities for both strict and conceptual replication . the octo - twin study is comprised of the oldest - cohort of the swedish twin registry aged 80 and older at the time of first examination . beginning in 199193 , the longitudinal design included a maximum of five measurement occasions at 2-year intervals . individuals with a dementia diagnosis at baseline ( n = 98 ) were excluded from the initial sample of 702 participants . approximately 20% of the sample was lost to follow up at each wave ( 10% per year ) , but most of this attrition was due to death . reasoning was assessed using block design . in this task , participants were presented with red and white blocks and instructed to reproduce the design shown on a card using these blocks within a predetermined time limit . memory was assessed using immediate recall of the prose recall test , in which participants were presented with a brief , 100-word story that had a humorous element . amount of information recalled was coded in a manner similar to the scoring of story units in the wechsler memory scale logical memory test . semantic knowledge was assessed using the swedish version of the information task , in which participants provided responses to factual knowledge questions . raw scores were transformed into t - scores with a mean of 50 and standard deviation of 10 to facilitate comparisons across measures . respondents were asked , at each of the five waves , the following : are you presently doing or have you previously done anything special to train your body or keep your body fit ? the possible responses were no ( 0 ) , yes , to some extent ( 1 ) , or the participants gave one reply for their present physical activity status and one in regards to their previous status . physical activity change scores were computed by subtracting baseline activity from each follow - up activity measure . the lbls was initiated in 1978 when participants were recruited from the family health plan health maintenance organization ( hmo ) , mainly including residents of long beach and orange county . the ethnic composition of the older group ( 98% caucasian ) was similar to the 65 + population for the area based on the 1970 census . panel 2 , initiated in 1992 , included 633 individuals from the same hmo ( 64 were excluded due to frank dementia or serious sensory or neurological problems ) . in order to include the same measures as those in the seattle longitudinal study , lbls panel 1 ( n = 106 ) and panel 2 ( n = 631 ) data from 1994 to 2003 , excluding participants younger than age 55 in 1994 ( n = 541 ) , were used in the current analysis . during this period , reasoning was based on a composite score of the letter and number series tests from the schaie - thurstone adult mental abilities test ( stamat ; . in this task , participants viewed a series of letters ( e.g. , a b c c b a d e f f ) and were asked to identify the next letter in the series from alternate choices by extracting the rule that governed the series . participants were given six minutes to complete as many of the 30 items as possible . fluency was assessed using word fluency , in which participants wrote down as many words as possible that begin with the letter s during a five - minute period according to predetermined rules . these rules included no proper names and no addition of endings to words that the participant had already provided ( e.g. , sit , memory was measured using immediate written recall of a 20-item noun list that participants had studied for 3.5 minutes . participants were provided with 50 target words and asked to select the synonym from four choice alternatives . performance was based on total correct responses provided in a five - minute period . a composite score was created by summing the number of physical activities ( e.g. , walking , outdoor hobbies , etc . ) that included one or more hours of these activities per week . activity change variables were computed by subtracting the activity measure in 1994 from activity in 1994 , 1997 , 2000 , and 2003 . the sls is a long - running longitudinal study initiated by k. warner schaie , who first recruited members of a local health maintenance organization in 1956 . current analyses used up to four waves of sls data from 19842005 , which include an expanded set of measures that also overlapped with the long beach study . only participants baseline was defined as each participant 's first study visit , and time was measured in all analyses as years in study ( coded as 0 , 7 , 14 , and 21 ) . see table 3 for sls participant characteristics over the four waves of data analyzed here . reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test ( stamat ; ) . in this task , participants were provided with a printed word series that was ordered according to an inherent rule . the participant 's task was to select , from multiple - choice options , the next word in the series consistent with that rule . total score was based on number of correct responses to the 30 trials completed in 6 minutes . as in lbls , fluency was indexed by performance on the word fluency test from the primary mental abilities test and memory by the verbal list - learning task . semantic knowledge was assessed with the test of advanced vocabulary from the educational testing service ( ets ) , in which participants identified synonyms for printed words from five choices . the total score was derived from the number of correct responses provided within 4 minutes to the 36-itemtest . the methodology described in the lbls method portion of this paper was used in order to generate roughly equivalent indices of physical activity . following this methodology , a composite physical activity measure was created by summing dichotomized test responses from a modified version of the life complexity scale , resulting in a four - item physical activity composite ( playing sports , walking , fitness , and outdoor hobbies ) . activity change was computed by subtracting baseline activity from each follow - up activity measure . the victoria longitudinal study began in 198687 with a sample of 484 community residing volunteers . using a longitudinal sequential design , second and third independent samples began in 1992 - 93 ( n = 530 ) and 2001 - 2002 ( n = 550 ) . to date , sample 1 has been tested on seven occasions ( over 18 years ) , sample 2 on five ( over 12 years ) , and sample 3 on two occasions ( over 6 years ) . participants in all three samples were recruited between the ages of 55 and 85 years . data from seven waves of sample 1 and five waves of sample 2 were included in the current investigation . approximately 20% of the sample was lost to follow up at each wave , or 10% per year . reasoning was indexed by letter series . in this task , participants were presented with a series of letters and asked to identify the next letter in the sequence based on the rule that governed the sequence . fluency was measured by performance on a similarities task , in which participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning during a 6-minute period . memory was indexed based on free recall of a 30-item noun list comprised of five semantic categories . participants were given two minutes to study the words and then five minutes to recall them . semantic knowledge was assessed using a 54-item recognition vocabulary test adapted from the ets kit of factor referenced tests . the physical activity measure was derived from a subset of four items from the vls- activity lifestyle questionnaire ( vls - alq ; ) . these items indexed the physical activities of gardening , jogging , sailing , and tennis . for each item , participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 ( i.e. , never , less than once a year , about once a year , 2 or 3 times a year , about once a month , 2 or 3 times a month , about once a week , 2 or 3 times a week , and daily ) . in order to examine the effects of physical activity on cognition , a series of multilevel models was fit with time varying covariates using multilevel mixed - effects regression in stata ( statacorp , 2011 ) , the restricted maximum likelihood estimator ( reml ) , and an unstructured covariance matrix . separate models were fit for each of the four cognitive measures ( reasoning , fluency ( except octo - twin ) , memory , and semantic knowledge ) and for each of the four studies . in order to improve ease of interpretation of our results , age , education , and activity measures were mean centered to the baseline mean of each measure in the sample so that the intercept and linear slope terms could be interpreted as the expected value for an individual at the mean age , education , and respective activity level at baseline . octo - twin participants were modeled as nested within their twin pair and in vls we controlled for enrolment cohort . our goal was to build a common model for comparisons across all outcomes for the four longitudinal studies . this common model was not necessarily the optimal model for each of the 16 cognition - physical activity combinations . an initial 19-term model included all ten two - way interactions that included activity , change in activity or time , and three 3-way interactions of time and activity with age , sex , or education . however , several terms were not significant for most of the studies and outcomes and so were trimmed to facilitate model interpretation . first , the 3-way interactions were eliminated , then the interactions with change in activity . this resulted in a final model that included 12 terms summarized in table 5 for separate cognitive constructs of reasoning , memory , semantic knowledge , and fluency . our significance criterion of p < 0.05 shaped the familywise alpha rate within each study , however our focus was on the repetition of results across studies , which we used to minimize the potential impact of chance associations . we did not implement formal meta - analytic techniques as they require identical measures and a larger number of studies . between - person age differences are seen at the first occasion of measurement for all memory , reasoning , and fluency tests , with older adults performing less well . semantic knowledge results are less consistent , with older adults performing less well in lbls and octo - twin , but better in vls . at baseline , lbls and sls women scored higher than men of the same age on all measures , except for semantic knowledge . for the reference individuals ( men with sample average baseline age and years of education ) , within - person declines were seen over time in all cognitive abilities and all samples except sls ( the youngest ) , where the relationship , as in lbls and vls , depended on age . older individuals declined faster on all vls , sls , and lbls measures except lbls immediate memory . no clear pattern was identified in regards to differential decline related to sex or education . higher physical activity at baseline was associated with higher scores on reasoning and memory in octo - twin and vls and fluency in sls . the association between physical activity score at baseline and cognitive score did not differ by age . however , there was some indication that the relation between physical activity and cognition differed by education . for semantic knowledge in lbls and sls the association with physical activity at baseline was stronger for people with less education . in terms of associations with cognitive decline , higher physical activity score at baseline was related only to less decline on fluency in vls and sls . however , we found a consistent pattern of positive relationships between time - specific changes in physical activity and time - specific changes in cognition beyond those expected by the estimated linear trajectories in the four studies . more specifically , after controlling for the trend in cognitive functioning over time , time - specific changes in physical activity change were related to cognitive fluctuations in the following cognitive domains : ( a ) reasoning in all four studies ; ( b ) fluency in two ( vls and sls ) of three studies ; ( c ) memory in two studies ( octo - twin and vls ) ; ( d ) semantic knowledge in one study ( octo - twin ) . using data from four longitudinal studies of aging , the present study examined the relationship between physical activity and cognitive functioning at three different levels : ( a ) cross - sectionally ; ( b ) longitudinally using physical activity as predictor of cognitive change ; ( c ) longitudinally using change in physical activity as a time - varying covariate to predict change in cognition , adjusting for the normative development ( effect of time ) in cognition . on the cross - sectional level , higher physical activity at baseline was associated primarily with higher scores on reasoning and fluency , generally supporting previous studies demonstrating that physically active older adults have higher cognitive performance and functioning compared with less active older adults [ 11 , 12 ] . although relevant , these well - known and well - documented findings contribute little to a deeper understanding of the likely very complex relationship between activity and cognition . the second level of analysis addressed more theoretically relevant and interesting longitudinal associations and the question of whether physical activity at baseline is associated with cognitive change . from a broader perspective , this research question is also linked to the use it or lose it hypothesis [ 5 , 21 ] , generally proposing that physical activity may buffer against future cognitive decline . a number of prospective studies have found support for this notion [ 1320 ] , offering preliminary support for the longitudinally beneficial and buffering effect of physical activity on cognitive decline . a general limitation in many of these previous prospective studies , however , is that they have examined the association between physical activity and a broad global measure of cognition ( typically mmse ) , thereby proving an incomplete picture of the potentially diverse longitudinal associations between different cognitive domains and physical activity . as a consequence , these previous studies generally have not answered the theoretically and practically relevant question what cognitive domains most benefit from physical activity ? . in the present study four different domains were examined , representing a broader spectrum of cognitive abilities , ranging from the more crystallized knowledge - based domain of semantic knowledge to more fluid or process - based factors of reasoning , fluency , and memory . higher baseline physical activity was associated with less fluency decline in two of three studies . thus , the preliminary answer to the question what cognitive domain benefits most from physical activity , based on the results of the present study , is fluency , which is one of the more process - based / fluid domains . however , it should be mentioned that for most cognitive domains across the four studies , we did not find support for the protective effect of baseline levels of physical activity on cognitive decline . aside from the more stationary change relationships typically investigated in previous studies ( how level of physical activity at baseline relates to change in cognition ) , we also targeted more dynamic associations between changes in physical activity and changes in cognitive functioning by using change in physical activity from baseline as a time - varying covariate in longitudinal multilevel models . the time - varying covariate model used in this study examined occasion - specific intraindividual relations between physical activity and cognition , after controlling for individual rates of change over time . such time - specific associations between fluctuations in activity and cognition have rarely been examined and may be highly relevant to understanding how to prescribe exercise and how to design and implement interventions including physical activity to optimize effects on cognition . as associations of change and time - specific fluctuations are of key importance in the analyses and interpretation of intervention studies , the results of studies like the present one may offer new relevant information both from a scientific as well as from an applied perspective . variation in physical activity was associated with variation in reasoning in all four studies and in fluency in two of three studies . hence , although evidence for the association of between - person differences in baseline physical activity with subsequent cognitive change was generally weak across domains , aside from fluency , support for the notion that change in physical activity covaries with fluctuations in cognition is much more robust across studies . these results are inline with previous work , indicating that physical activity may specifically moderate the decline in cognitive domains that is typically associated with aging . moreover , from a broader perspective , the stronger associations across time of physical activity with more fluid cognition may be linked to the hypothesis that exercise and aerobic fitness training results in selective improvement in executive control processes and working memory . although the cognitive tests used in the present study to measure reasoning and fluency were derived from a psychometric tradition of psychological testing and do not map well onto more recent conceptualizations of executive processing , they do share some of these features , being a more fluid measure of cognition . an obvious limitation of the current study is the observational nature of the longitudinal designs , making inferences in terms of cause and effect irresolvable . the notion that decline in cognition leads to decline in physical activity is equal in validity to the interpretation that a decline in physical activity leads to a decline in cognition , that the relationship is reciprocal , or that both are a result of some third variable . recent studies provide evidence for not only the reciprocal relationship across time between physical activity and mental health in older adults , but also for the reciprocal relationship between physical activity and cognition . another limitation is the problem associated with using different tests in different studies to tap the same cognitive domains . as noted earlier , the studies were selected because they shared similar measures of activity and cognition . moreover , single cognitive tests are always imperfect markers of a cognitive domain . therefore , a general feature of the integrated analytical approach in the present study , where data and tests from four different studies are used to answer the same research question , is the risk of heterogeneity in terms of how well the different tests indicate the higher order construct they should measure . as a result , when patterns of results are not consistent across studies , additional questions , testable in future research , are raised with respect to the source of these differences . it is , for example , interesting to note that for octo - twin , in which physical activity was operationalized as the extent to which persons saw themselves as purposefully keeping their body fit , baseline activity was associated with cognitive functions more consistently than were the physical activity measures in the other studies . the apparent importance of physical activity , however , may also be due to the more advanced age of this sample . in situations where the studies with identical measures agree with each other , but not the remaining studies , for example , where neither sls nor lbls shows associations between memory performance and either baseline or change in activity , but octo - twin and vls do , we may draw conclusions that something about the measurement is important . in contrast , lbls and sls fluency results do not agree , suggesting instead that some detail relevant to the sampling , retest interval , or other study characteristic may be relevant . on the other hand , when patterns of results do show consistency across studies that have used different measures to tap the same underlying construct , such as the association of change in activity with change in reasoning and fluency , these differences become a major strength , as the reliability of the conclusions drawn is considerably strengthened compared with traditional analysis of a single dataset . moreover , in contrast with the more specific measurement of cognition , the physical activity variables used in the current analyses were broad and self - reported and did not differentiate aerobic from strength or resistance training . combining objective measures of physical activity with more specific multi - item , self - report instruments would likely provide future studies with a more robust base for the analysis of the association of change in activity with change in cognition . the dynamic associations between physical activity and cognitive functioning underscore the broader question of associations between biological and cognitive aging . also , the effect of intraindividual change in physical activity on cognitive functioning ( adjusting for the trend in cognition ) raises the question of what drives , or causes , these relationships across time ? these dynamic associations with the more fluid cognitive domains may be mediated , or explained , by a number of factors , such as physical resources ( sleep , energy / fatigue , appetite , pain , or drug / medication use ) , disease states ( hypertension , diabetes , and cvd ) , and mental resources ( chronic stress , depression , and self - efficacy ) . more specifically , a number of physiological mediators , such as aerobic fitness , hormones , lipid profiles , cerebral blood flow , blood pressure , neurotransmitters , and neurotrophins have all been identified as potential mediators in physical activity - cognition relationships . although intuitively appealing and quite frequently investigated , the hypothesis of physical activity leading to improved cognition via increased aerobic fitness ( the cardiovascular fitness hypothesis ) is not , however , supported by meta - analyses [ 24 , 42 ] . thus , although single mediation models are theoretically attractive , and may fit data to some extent , the more complete pathways explaining why physical activity and cognition seem to change together more likely include multiple mediators and complex micromediational chains , that also may vary in strength and validity across individuals and groups . nevertheless , increasing knowledge about what precise mechanisms are active ingredients in the effects of physical activity on cognition constitutes a vital step towards the development of appropriate physical activity intervention designs to test these specific models of mediation and the effects of physical activity and exercise on cognition in experimentally controlled trials . the major strength of the present study is the ability to elucidate consistent patterns of complex associations across time through coordinated analyses of data from four longitudinal studies . contrary to previous research based on analyses of single samples , which are limited by the specific characteristics of the sample and data , we instead used a coordinated and integrated analytical approach and framework [ 25 , 26 ] to examine the same research question in data from four longitudinal studies , thereby making the conclusions less vulnerable to study specific characteristics . as such , the present study is unique ( in particular considering the choice of analytical approach ) and may pave the way for similar collaborative projects where the same research question and analytical approaches are used to answer relevant questions simultaneously across different studies linked to the association of lifestyle , physical activity , and cognitive functioning . the four studies included afford considerable heterogeneity in terms of age ( ranging from mean age of 67 in sls to 83 in octo - twin ) , number of available waves of measurement ( four in sls and lbls to seven in vls ) , years of followup ( 8 years in octo - twin to 21 years in sls ) , years between measurements ( every 2 years in octo - twin to every 7 year in sls ) , and cultural background ( scandinavia to north - america ) . yet , as discussed above , a surprisingly clear pattern emerged across studies in the relation of change in activity to fluctuations in cognition . thus , in terms of capacity to identify patterns of associations from a larger and broader perspective and to be able to generalize results and conclusions , the present study brings reproduced evidence to the field as well as to practitioners working with health related behavior , lifestyle , and cognition in elderly . based on the results in the present study , the main message is that change in activity , and not only previous or current level of activity , seems to matter and may play a significant role in the pursuit of maintaining benign nondecreasing trajectories of cognition along the path of cognitive aging .

the present study used a coordinated analyses approach to examine the association of physical activity and cognitive change in four longitudinal studies . a series of multilevel growth models with physical activity included both as a fixed ( between - person ) and time - varying ( within - person ) predictor of four domains of cognitive function ( reasoning , memory , fluency , and semantic knowledge ) was used . baseline physical activity predicted fluency , reasoning and memory in two studies . however , there was a consistent pattern of positive relationships between time - specific changes in physical activity and time - specific changes in cognition , controlling for expected linear trajectories over time , across all four studies . this pattern was most evident for the domains of reasoning and fluency .

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

PMC5149473

one of the important risks is the existence of high levels of gasoline vapors including benzene that during work shifts employees are exposed to . employees in functional units are more vulnerable population because of their continuous contact with harmful substances such as benzene . based on usepa ( united state environmental protection agency ) and iarc ( international agency for research cancer ) reports , benzene has been classified to be a group a and class 1 human ( 1 ) . adverse health effects of benzene appear in two different type ; short - term and long - term . short - term effects are associated with high concentration and may involve headaches , dizziness , distraction and defects temporary memory and tremors . whereas , exposure to benzene in a long - term is connected to intricate adverse health effects such as immunological , hemato - toxicity , geno - toxicity , adverse effects on reproductive organs , and as well as various cancers ( 1 , 2 ) . therefore , a powerful method is required to predict existent exposure to harmful substances besides to evaluate probable adverse effects ( 3 ) . although several mathematical models have been carried out so far , there is high complexity related to chemical exposure in terms of human health . due to the lack of information about chemical impacts on human particularly in long - term impacts , some unpredictable factors that called uncertainties can affect health risk assessment ( 4 , 5 ) . health risk assessment through inhalation concerning benzene emission from equipment at the unit of benzene production was accomplished . this project is an example of using a hybrid approach that can incorporate uncertainties to assessment s process . major variables affecting the absorption of chemicals moreover key parameters in the dispersion of chemicals can be reflected in a hybrid system which combining fuzzy logic and neural networks ( 5 , 6 ) . in this research however , breathing rates are affected by many individual characteristics , including age , sex , weight , health , and level of physical activity ( running , jogging , etc . ) ( 5 , 9 ) . perhaps the first and distinguished use of fuzzy sets in health risk assessment was on the application of fuzzy logic in the environmental risk assessment ( 10 ) . one more example which connected to human health risk assessment is the application of fuzzy sets in human health risk assessment . fuzzy sets were employed to estimate carcinogenic risk caused by air pollution in ten russian cities ( 11 ) . the study area was in the northern part of the persian gulf with a distance of 10 miles from the coast . the plant mainly produced about one million tons annually of benzene , paraxylene , orto - xylene used to produce ethylbenzene , styrene , cyclohexane , and nitrobenzene . benzene from refinery streams was typically produced from catalytic reformats pyrolysis , gasoline , and toluene de - alkylation . this study focused on tasks and activities those are exposed to potential contact with benzene during refinery . main workplaces related to workers exposed to benzene such as loading of tankers were listed in table 1 . each workplace maybe had a different group of job and activities . in order to increase the information about jobs , activities , tasks , and main work places for workers the sampling procedure was based on absorption of benzene in an active charcoal tube ( active sampling ) ( 12 ) . skc model 222 pumps have been utilized for gas sampling . the glass tubes with a 6 mm external diameter , 4 mm internal diameter and 70 mm height , containing activated charcoal holder with a restrictive orifice ( separated by a 2-mm part of urethane foam ) were installed . the pump was adjusted to work for 30 min at a flow rate of 100 ml / min ( 12 ) . four measuring procedures were performed lasting one week in the middle of each season ( 12 , 13 ) . consequently , by the end of the week 70 samples were taken and during 15/2/2012 to 21/09/2013 totally 280 samples were collected . for detailed information stickers attached on each tube to tag sampling number , the time of end duration of sampling , the pump number , the humidity , the wet and dry temperature , and the date of sampling ( 13 ) . the human lifetime average daily dose ( ladd ) equation for a single chemical exposure proposed by u.s.epa ( 2011 ) that related to cancer risk model ( presuming that the inhalation is the only route of intake ) is , as shown in eq . ( 14 ) : ladd = ca.ir.ef.ed / bw.at the cancer risk ( cr ) is calculated for exposure to benzene using eq . ( 15 ) : cancer risk = ladd ( g / kg / day)sf ( g / kg / day)1 for equation 1 , ca is the concentration of a chemical in an exposure medium ( g / m ) , ir represents the inhalation rate ( m / h ) , ef is the exposure frequency ( number of working days per year ) , ed is the exposure duration ( working years ) , bw is the body weight ( kg ) , at is averaging time ( at=70 yr 365 d / year for carcinogens ) , ladd shows lifetime average daily dose ( g / kg / day ) and sf in equation 2 , is the cancer slope factor of benzene ( linear low - dose cancer potency factor ) ( 15 ) . the quality of data in equation 1 is uncertain even if definite exposure - related measurements are available for variables ( 16 ) . to explain more , some unpredictable parameters can change the results . as an illustration , movement of contaminants among environmental media changes remarkably the quality of data , therefore , uncertainty plays a crucial role involved in each variable as well as affected by physical and chemical factors ( 11 , 16 ) . these variables ; wind speed , ambient temperature , humidity and rainfall cause fluctuation in concentration of air pollutants ( 17 ) . furthermore , the inhalation rate is also influenced by multiple individual aspects including age , body weight , and amounts of physical activity ( 5 , 11 ) . owing to these numerous variables , the trustworthy approach is required to make a better risk evaluation . therefore , to overcome the problems of uncertainties the hybrid method was prepared ( 5 , 18 ) . in this figure , each tablet represents a sub - system combined ; they determine the chemical absorption rate through inhalation . the receptor description ( inhalation rate ) and dose estimation sections use a set of fuzzy rules and obtain the average daily inhalation dose , based on fuzzy inference . the exposure prediction section consists of a designed neural network using a new back - propagation algorithm . this section created in order to calculate of real ambient concentration . in the following sections , details about the subdivisions will be explained ( 5,18 ) . integrated system of risk assessment ( 5 ) a. artificial neural network b. life lifetime average daily inhalation dose standard quantities technique in health issues has been often vague and unclear adjectives such as a little , too much ; so at the start it is necessary to quantify these adjectives and change them into fuzzy sets ( 19 , 20 ) . in this section age , body weight , and activity tend to be the significant reasons of daily energy expenditure in healthy people for as long as is also in energy balance ( 20 , 21 ) . variables must be delineated with linguistic values rather than numerical values ( 5 , 21 ) . 2 shows one of the prepared fuzzy sets ( fuzzy numbers ) that related to age with three linguistic values . the horizontal vector indicates the size of the parameter and the vertical vector represents the degree of membership ( degree of dependence ) of each value ( 5 ) . membership functions of the variable age ( 5 ) the membership function for age was set to range from 18 to 80 yr ( 22 ) . using census and demographic details , age ( in years ) was divided into three fuzzy linguistic sets : low , ranging from 18 to 30 ; medium , ranging from 25 to 64 ; and high , ranging from 59 to 80 ( 5 , 22 ) . according to brainard and burmaster ( 23 ) , body weight ( 45110 kg ) it has been divided into three linguistic sets : low ( from 45 to 68 kg ) , medium ( from 56 to102 kg ) , and high ( from 90 to110 kg ) . fuzzy set was made for physical activity by means of four linguistic values and follows skewed ( normal ) distribution ( 5 ) . as for long - term physical activity levels ( pals ) , it is ranging from 1.2 to 2.5 times the bmr ( basal metabolic rate ) , where 1.2 represents the minimal intensity of activity and 2.5 shows a very physically active lifestyle . aging is a parameter that can remarkably decline high - intensity activity ( 21 ) . moreover , seven linguistic values ranging from 525 m / day with normal distribution have been provided to show inhalation rate , which is compatible with the human capability ( 14 ) . anns are working based on specified transfer function and made by artificial neurons ( 17 ) . using particular function anns can adjust the value of different weights properly . in order to neural network learning hence , the data set for this project were randomly divided into a ratio of 3:1 between training and testing sets , respectively ( 7 , 10 , 17 ) . as a result , available data ( 280 samples ) arbitrarily fragmented into two subsets . one of those was training groups and included 75% of data . by developing the model this group predicted the concentration of benzene for the second group that contained the 25% of data ( 10 , 17 ) . parameters ; wind speed , temperature , relative humidity , rainfall and measured concentration was chosen as model input data . the predicted benzene concentration was the output parameter . the third layer involved just one neuron which is output parameter and shows actual benzene concentration . a typical three - layer neural network is shown in fig . 3 ( 17 ) . selected three layer neural network ( 17 ) details of the meteorological monitoring devices before conducting the network preparation procedure , a training group ( 75% of the data ) consisting of 210 cases had to be prepared from the environmental data done by the back propagation paradigm . training group itself became divided into training set and testing set that this process conducted randomly . first one is the training set consisted of 90% of the data or 189 cases used to train the model . the second or final 10% contains 21 cases were left out as a test set ( 7 , 17 , 24 ) . output variables from the artificial neural network stage and fuzzy logic section contributed to the last part of the hybrid model and produced a chronic daily intake dose ( 18 ) . the predicted benzene concentrations lie between 0 and 67 ( g / m ) considered very high . ( 25 ) , the peak concentrations in the study area are high and the membership function for output and some fuzzy rules must be adjusted to range 0 and 67 ( g / m ) . the process of adjustment was easily done that indicates a powerful feature of the dose assessment modeling system as well as the adaptability to different scenarios . in different circumstances , the user can change fuzzy rules according to the different situations ( 6 , 9 ) . the outputs from the benzene prediction divided into six linguistic variables based on the frequency distribution of the data . the membership function of variable concentration is shown in fig . 4 ( 18 ) . the output from the receptor block was divided by the body weight to derive m / d / kg then contributed as input to the dose estimation section ( 5 , 18 ) . the daily intake rate for the dose estimation is expressed by four linguistic fuzzy sets and is expressed in m / d / kg ( 18 ) . finally , the life average daily intake dose defined as seven fuzzy linguistic values and ranging from 0 to 6 g / kg / d . input variable predicted concentration ( g / m ) a sample fuzzy inference calculation for the dose estimation block is shown in fig . 5 . where ir denotes inhalation rate normalized for body weight ( m / day / kg ) ; cc indicates chemical concentration ( g / m ) ; ladid = lifetime average daily inhalation dose ( g / kg / d ) . as an illustration , if the inhalation rate were considered 0.35 m / kg / d , the ambient benzene concentration is 15 g / m , lifetime average daily inhalation dose would be 1.63 g / kg / day . fuzzy inference for estimation of ladid ( matlab software ; fuzzy toolbox ) comparison of observed and predicted concentration ( source : author 's calculations ) the output from this last module gives the overall dose through inhalation for each individual around the site , taking into account both the exposure factors and the receptor factors in a quantitative way , based on neural network and fuzzy inference . when the exposure is quantified , combined with integrated risk information system ( iris ) toxicity factors , then as a final point the related risk can be estimated . the study area was in the northern part of the persian gulf with a distance of 10 miles from the coast . the plant mainly produced about one million tons annually of benzene , paraxylene , orto - xylene used to produce ethylbenzene , styrene , cyclohexane , and nitrobenzene . benzene from refinery streams was typically produced from catalytic reformats pyrolysis , gasoline , and toluene de - alkylation . this study focused on tasks and activities those are exposed to potential contact with benzene during refinery . main workplaces related to workers exposed to benzene such as loading of tankers were listed in table 1 . each workplace maybe had a different group of job and activities . in order to increase the information about jobs , activities , tasks , and the sampling procedure was based on absorption of benzene in an active charcoal tube ( active sampling ) ( 12 ) . skc model 222 pumps have been utilized for gas sampling . the glass tubes with a 6 mm external diameter , 4 mm internal diameter and 70 mm height , containing activated charcoal holder with a restrictive orifice ( separated by a 2-mm part of urethane foam ) were installed . the pump was adjusted to work for 30 min at a flow rate of 100 ml / min ( 12 ) . four measuring procedures were performed lasting one week in the middle of each season ( 12 , 13 ) . consequently , by the end of the week 70 samples were taken and during 15/2/2012 to 21/09/2013 totally 280 samples were collected . for detailed information stickers attached on each tube to tag sampling number , the time of end duration of sampling , the pump number , the humidity , the wet and dry temperature , and the date of sampling ( 13 ) . the human lifetime average daily dose ( ladd ) equation for a single chemical exposure proposed by u.s.epa ( 2011 ) that related to cancer risk model ( presuming that the inhalation is the only route of intake ) is , as shown in eq . ( 14 ) : ladd = ca.ir.ef.ed / bw.at the cancer risk ( cr ) is calculated for exposure to benzene using eq . ( 15 ) : cancer risk = ladd ( g / kg / day)sf ( g / kg / day)1 for equation 1 , ca is the concentration of a chemical in an exposure medium ( g / m ) , ir represents the inhalation rate ( m / h ) , ef is the exposure frequency ( number of working days per year ) , ed is the exposure duration ( working years ) , bw is the body weight ( kg ) , at is averaging time ( at=70 yr 365 d / year for carcinogens ) , ladd shows lifetime average daily dose ( g / kg / day ) and sf in equation 2 , is the cancer slope factor of benzene ( linear low - dose cancer potency factor ) ( 15 ) . the quality of data in equation 1 is uncertain even if definite exposure - related measurements are available for variables ( 16 ) . to explain more , some unpredictable parameters can change the results . as an illustration , movement of contaminants among environmental media changes remarkably the quality of data , therefore , uncertainty plays a crucial role involved in each variable as well as affected by physical and chemical factors ( 11 , 16 ) . these variables ; wind speed , ambient temperature , humidity and rainfall cause fluctuation in concentration of air pollutants ( 17 ) . furthermore , the inhalation rate is also influenced by multiple individual aspects including age , body weight , and amounts of physical activity ( 5 , 11 ) . owing to these numerous variables , the trustworthy approach is required to make a better risk evaluation . therefore , to overcome the problems of uncertainties the hybrid method was prepared ( 5 , 18 ) . the construction of the system is displayed in fig . 1 ( 5 ) . in this figure , each tablet represents a sub - system combined ; they determine the chemical absorption rate through inhalation . the receptor description ( inhalation rate ) and dose estimation sections use a set of fuzzy rules and obtain the average daily inhalation dose , based on fuzzy inference . the exposure prediction section consists of a designed neural network using a new back - propagation algorithm . this section created in order to calculate of real ambient concentration . in the following sections , details about the subdivisions will be explained ( 5,18 ) . integrated system of risk assessment ( 5 ) a. artificial neural network b. life lifetime average daily inhalation dose standard quantities technique in health issues has been often vague and unclear adjectives such as a little , too much ; so at the start it is necessary to quantify these adjectives and change them into fuzzy sets ( 19 , 20 ) . in this section age , body weight , and activity tend to be the significant reasons of daily energy expenditure in healthy people for as long as is also in energy balance ( 20 , 21 ) . variables must be delineated with linguistic values rather than numerical values ( 5 , 21 ) . 2 shows one of the prepared fuzzy sets ( fuzzy numbers ) that related to age with three linguistic values . the horizontal vector indicates the size of the parameter and the vertical vector represents the degree of membership ( degree of dependence ) of each value ( 5 ) . membership functions of the variable age ( 5 ) the membership function for age was set to range from 18 to 80 yr ( 22 ) . using census and demographic details , age ( in years ) was divided into three fuzzy linguistic sets : low , ranging from 18 to 30 ; medium , ranging from 25 to 64 ; and high , ranging from 59 to 80 ( 5 , 22 ) . according to brainard and burmaster ( 23 ) , body weight ( 45110 kg ) it has been divided into three linguistic sets : low ( from 45 to 68 kg ) , medium ( from 56 to102 kg ) , and high ( from 90 to110 kg ) . fuzzy set was made for physical activity by means of four linguistic values and follows skewed ( normal ) distribution ( 5 ) . as for long - term physical activity levels ( pals ) , it is ranging from 1.2 to 2.5 times the bmr ( basal metabolic rate ) , where 1.2 represents the minimal intensity of activity and 2.5 shows a very physically active lifestyle . aging is a parameter that can remarkably decline high - intensity activity ( 21 ) . moreover , seven linguistic values ranging from 525 m / day with normal distribution have been provided to show inhalation rate , which is compatible with the human capability ( 14 ) . anns are working based on specified transfer function and made by artificial neurons ( 17 ) . using particular function anns can adjust the value of different weights properly . in order to neural network learning hence , the data set for this project were randomly divided into a ratio of 3:1 between training and testing sets , respectively ( 7 , 10 , 17 ) . as a result , available data ( 280 samples ) arbitrarily fragmented into two subsets . one of those was training groups and included 75% of data . by developing the model this group predicted the concentration of benzene for the second group that contained the 25% of data ( 10 , 17 ) . parameters ; wind speed , temperature , relative humidity , rainfall and measured concentration was chosen as model input data . the third layer involved just one neuron which is output parameter and shows actual benzene concentration . a typical three - layer neural network is shown in fig . 3 ( 17 ) . selected three layer neural network ( 17 ) details of the meteorological monitoring devices before conducting the network preparation procedure , a training group ( 75% of the data ) consisting of 210 cases had to be prepared from the environmental data done by the back propagation paradigm . training group itself became divided into training set and testing set that this process conducted randomly . first one is the training set consisted of 90% of the data or 189 cases used to train the model . the second or final 10% contains 21 cases were left out as a test set ( 7 , 17 , 24 ) . output variables from the artificial neural network stage and fuzzy logic section contributed to the last part of the hybrid model and produced a chronic daily intake dose ( 18 ) . the predicted benzene concentrations lie between 0 and 67 ( g / m ) considered very high . ( 25 ) , the peak concentrations in the study area are high and the membership function for output and some fuzzy rules must be adjusted to range 0 and 67 ( g / m ) . the process of adjustment was easily done that indicates a powerful feature of the dose assessment modeling system as well as the adaptability to different scenarios . in different circumstances , the user can change fuzzy rules according to the different situations ( 6 , 9 ) . the outputs from the benzene prediction divided into six linguistic variables based on the frequency distribution of the data . the membership function of variable concentration is shown in fig . 4 ( 18 ) . the output from the receptor block was divided by the body weight to derive m / d / kg then contributed as input to the dose estimation section ( 5 , 18 ) . the daily intake rate for the dose estimation is expressed by four linguistic fuzzy sets and is expressed in m / d / kg ( 18 ) . finally , the life average daily intake dose defined as seven fuzzy linguistic values and ranging from 0 to 6 g / kg / d . input variable predicted concentration ( g / m ) a sample fuzzy inference calculation for the dose estimation block is shown in fig . 5 . where ir denotes inhalation rate normalized for body weight ( m / day / kg ) ; cc indicates chemical concentration ( g / m ) ; ladid = lifetime average daily inhalation dose ( g / kg / d ) . as an illustration , m / kg / d , the ambient benzene concentration is 15 g / m , lifetime average daily inhalation dose would be 1.63 g / kg / day . fuzzy inference for estimation of ladid ( matlab software ; fuzzy toolbox ) comparison of observed and predicted concentration ( source : author 's calculations ) the output from this last module gives the overall dose through inhalation for each individual around the site , taking into account both the exposure factors and the receptor factors in a quantitative way , based on neural network and fuzzy inference . when the exposure is quantified , combined with integrated risk information system ( iris ) toxicity factors , then as a final point the related risk can be estimated . in this section , leave - one - out cross - validation ( loocv ) was followed with the aim of training and testing the ann model . frequently , one sample is kept for testing while the rest is used for training up to all samples are finally tested ( 26 ) . before the proposed model is applied to the particular application , it must be trained using all available samples ( 27 ) . the training of network continued until maximum correlation within the measured and predicted output was achieved ( table 3 ) . correlation expressed by r - squared that r is coefficient of multiple determinations and relative root mean square error ( rmse ) ( 26 ) . correlation results are perfect when an r - squared value of 1 , a very good fit is next to 1 and a very poor fit less than 0 . on the other side , how much the value of rrmse is smaller ; the performance of the model is better . ambient air temperature affects significantly the exposure levels , as a result , the high temperatures in the summer and spring explains the increased benzene concentrations in exposure values . when temperatures are very low ( winter - time ) , the exposure levels are less than usual for an equivalent quantity of close benzene level ( fig . 8) . the presence of wind reduces exposure levels , especially to employees who are performing outdoor activities . no strong correlation found between wind speed and exposure levels of employees working in laboratory and control room . annually average concentration ( source : author 's calculation ) seasonal comparison of ambient concentration of benzene ( source : author 's calculations ) the measured benzene concentrations and the related exposure levels are not considered as an acute health risk ; meanwhile , this is the possibility of leukemia existence after chronic occupational contact with benzene due to measured concentration . long - term individual risk will be calculated by combining usepa integrated risk information system ( iris ) toxicity features with results from exposure assessment section ( 18 ) . the individual excess cr was calculated for exposure to benzene through inhalation is presented in table 4 . the best estimate for the variable distribution of risk in a population implied 33% of people exposed less than 110 . based on epa clean air act risk range , 110 risk range is considered as the most health protective end of the range ( 27 , 28 ) . whereas 110 is the midpoint of risk range and 67% of the population is ranging from 110 to 9.810 cancer risk probability ( 27 , 28 ) . individual health risk assessment ( based on average exposure obtained by passive sampling ) the average estimated risk for all work areas considering exposure to benzene is equal to 2.410 , ranging from 1.510 to 6.910 . the results counsel potential cancer risk for period exposure to benzene within the numerous areas but at different levels ( fig . these differences result from differences in the employee 's type of activity , age , weight , and breathing rate that in common methods of risk assessment they did not pay attention to them in this section , leave - one - out cross - validation ( loocv ) was followed with the aim of training and testing the ann model . frequently , one sample is kept for testing while the rest is used for training up to all samples are finally tested ( 26 ) . before the proposed model is applied to the particular application , it must be trained using all available samples ( 27 ) . the training of network continued until maximum correlation within the measured and predicted output was achieved ( table 3 ) . correlation expressed by r - squared that r is coefficient of multiple determinations and relative root mean square error ( rmse ) ( 26 ) . correlation results are perfect when an r - squared value of 1 , a very good fit is next to 1 and a very poor fit less than 0 . on the other side , how much the value of rrmse is smaller ; the performance of the model is better . ambient air temperature affects significantly the exposure levels , as a result , the high temperatures in the summer and spring explains the increased benzene concentrations in exposure values . when temperatures are very low ( winter - time ) , the exposure levels are less than usual for an equivalent quantity of close benzene level ( fig . 8) . the presence of wind reduces exposure levels , especially to employees who are performing outdoor activities . no strong correlation found between wind speed and exposure levels of employees working in laboratory and control room . annually average concentration ( source : author 's calculation ) seasonal comparison of ambient concentration of benzene ( source : author 's calculations ) the measured benzene concentrations and the related exposure levels are not considered as an acute health risk ; meanwhile , this is the possibility of leukemia existence after chronic occupational contact with benzene due to measured concentration . long - term individual risk will be calculated by combining usepa integrated risk information system ( iris ) toxicity features with results from exposure assessment section ( 18 ) . the individual excess cr was calculated for exposure to benzene through inhalation is presented in table 4 . the best estimate for the variable distribution of risk in a population implied 33% of people exposed less than 110 . based on epa clean air act risk range , 110 risk range is considered as the most health protective end of the range ( 27 , 28 ) . whereas 110 is the midpoint of risk range and 67% of the population is ranging from 110 to 9.810 cancer risk probability ( 27 , 28 ) . individual health risk assessment ( based on average exposure obtained by passive sampling ) the average estimated risk for all work areas considering exposure to benzene is equal to 2.410 , ranging from 1.510 to 6.910 . the results counsel potential cancer risk for period exposure to benzene within the numerous areas but at different levels ( fig . these differences result from differences in the employee 's type of activity , age , weight , and breathing rate that in common methods of risk assessment they did not pay attention to them . potential cancer risk the complete conception in establishing an integrated product is a dynamic model for risk estimation , able to evaluate probable interactions among the levels of benzene exposure through different task designs , those lead to different levels of benzene metabolic rate and subsequently to discount risk estimations ( 29 , 30 ) . in this model , risk factors that can influence inhalation rate were age , body weight , and physical activity of persons . these parameters considered as the input variable of fuzzy logic in order to estimate exposure level for the various situation of employees . the hybrid system also used artificial neural network approach to predict actual atmospheric benzene concentration . major variables affecting pollutant concentration were wind speed , ambient temperature , humidity , and rainfall . expert viewpoint is used as fuzzy rules and the rules can be changed according to the needs of the user . the produced composite model shows promise as a new tool for chemical exposure and health risk assessment , which it allows multiple uncertainties incorporate into health risk assessment . in this project , the new hybrid dose model has been able to replicate a measured exposure prospering believed to be more representative of actual intake dose than data from previously accepted methods . overall , according to aforementioned reasons this system can produce risk assessment data that appear realistic . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the author .

background : reliable methods are crucial to cope with uncertainties in the risk analysis process . the aim of this study is to develop an integrated approach to assessing risks of benzene in the petrochemical plant that produces benzene . we offer an integrated system to contribute imprecise variables into the health risk calculation.methods:the project was conducted in asaluyeh , southern iran during the years from 2013 to 2014 . integrated method includes fuzzy logic and artificial neural networks . each technique had specific computational properties . fuzzy logic was used for estimation of absorption rate . artificial neural networks can decrease the noise of the data so applied for prediction of benzene concentration . first , the actual exposure was calculated then it combined with integrated risk information system ( iris ) toxicity factors to assess real health risks.results:high correlation between the measured and predicted benzene concentration was achieved ( r2= 0.941 ) . as for variable distribution , the best estimation of risk in a population implied 33% of workers exposed less than 1105 and 67% inserted between 1.0105 to 9.8105 risk levels . the average estimated risk of exposure to benzene for entire work zones is equal to 2.4105 , ranging from 1.5106 to 6.9105.conclusion:the integrated model is highly flexible as well as the rules possibly will be changed according to the necessities of the user in a different circumstance . the measured exposures can be duplicated well through proposed model and realistic risk assessment data will be produced .

Introduction Methods Study area Sample collection The human health risk Model Using fuzzy logic for estimation of inhalation rate Artificial neural network for estimation of exposure to benzene Fuzzy inference calculation for LADD estimation Results Artificial network validation and results Predicted concentration results Health risk assessment results Discussion Conclusion Ethical considerations

PMC3212667

more recently , tewari et al reported this structure as the retrotrigonal layer and emphasized that this tissue may serve as a key surgical anatomical landmark to orient the location of the vasa and seminal vesicles , which are located just beneath this layer ( fig . , we have used this structure to identify the vas deferens and seminal vesicles in the midline at the posterior aspect of the prostato - vesical junction . however , aside from the presence and position of the layer , it seems that there have been no reports about the natural function of the layer . the aim of our study , therefore , was to discover a possible role of this structure . considering this layer 's positioning around the bn , we postulated that it might have some role in the control of urinary continence . to verify this , we assessed whether there was difference in the recovery of postprostatectomy incontinence ( ppi ) according to whether the retrotrigonal layer ( which was incised during posterior dissection of ralp ) was reconstructed or not . from may 2007 to september 2010 , a total of 181 consecutive patients underwent ralp by a single surgeon ( bhc ) at our institution . all of the preoperative , perioperative , and postoperative data were collected through a retrospective review of the patients ' charts . all men were continent for urine before surgery ( no involuntary urine loss of any kind ) and had neither undergone transurethral resection of the prostate nor had a history of neurological disease . to control the learning curve , we divided our patients into two groups depending on whether a retrotrigonal layer backup stitch ( rtbs , as described below ) was performed . these stitches were not used in group 1 ( cases 31 to 124 ) but were used for the patients in group 2 ( cases 125 to 181 ) . all cases were carried out by use of the transperitoneal , six - port technique . after the endopelvic fascia was opened and the dorsal vein complex ( dvc ) ligated , the anterior bn was divided . the posterior bn was then divided at the midline of the prostate - vesical junction . at this stage , after division of the retrotrigonal layer , both the vasa and seminal vesicles were found just beneath the retrotrigonal layer ( fig . the seminal vesicles were then completely dissected as the transected distal ends of the vasa were pulled anteriorly by using the fourth robotic arm . subsequently , posterior dissection was continued as far distally as possible , and the posterior aspect of the prostate was nearly freed from the rectum . then the nerve sparing ( interfascial ) was performed from the apex to the base , if deemed indicated according to the patient 's preoperative potency and tumor characteristics ( table 2 ) . after the prostatic pedicles were ligated , apical dissection and urethral transection were performed . posterior anastomosis was performed in a clockwise direction starting at the 5 o'clock position and ending at the 10 o'clock position . anterior anastomosis was performed with the second arm of the suture in a counterclockwise direction , and both sutures were tied together . here , the only technical difference between our two groups was an incorporation of the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis in group 2 . in group 2 , when we made conventional , continuous running sutures on the posterior bladder wall , this stitch was performed at a minimum of four points on the posterior bladder wall . after completing the anastomosis , routine postoperative care was administered , and the urethral catheter was removed 5 to 7 days after surgery . follow - up for all patients was conducted at 1 week , 1 month , 3 months , and then every 3 months for up to 2 years . continence recovery was determined by direct interview with the patients at each visit to our outpatient clinic . the two groups were statistically compared for patient age , body mass index ( bmi ) , prostate volume , pre - ralp prostate - specific antigen ( psa ) level and international prostate symptom score ( ipss ) , length of membranous urethra , number of nerve - sparing procedures , pathologic gleason score , pathologic stage , margin - positive rates , biochemical recurrence , rates of adjuvant radiation therapy , overall operative time , robotic console time , and postoperative continence rate . the student 's t - test or the mann - whitney test was used to analyze numerical variables , and the chi - square test or the fisher 's exact test was used to analyze categorical variables . to compare the interval before the return of urinary continence between the two groups , we used the kaplan - meier method with the log - rank test to analyze the differences between the curves . to minimize and control for selection bias there were no significant differences between the groups with respect to patient age , bmi , prostate volume , preoperative psa level , or preoperative obstructive and irritative ipss ( p>0.05 ) ( table 1 ) . there were also no significant differences between the groups in the number of nerve - sparing procedures , pathologic gleason score , pathologic stage , positive surgical margin , biochemical recurrence , or rates of adjuvant radiation therapy ( p>0.05 ) ( table 2 ) . for overall operative time and robotic console time , times were considerably shorter in group 2 ( cases 125 to 181 ) than in group 1 ( p<0.0001 ) ( table 2 ) . according to our definition , group 1 showed continence rates of 40.4% , 70.2% , and 90.4% at 3 , 6 , and 12 months , respectively ; in group 2 , the continence rates were 42.1% , 70.1% , and 89.7% , respectively . the median ( 95% confidence interval ) time to continence recovery was four months ( range , 1 to 12 months ) in group 1 and four months ( range , 1 to 9 months ) in group 2 . kaplan - meier curves showed no significant difference in the recovery of continence between the two groups ( log rank test , p=0.629 ) ( fig . prostate volume , serum psa , preoperative total ipss , pathologic gleason score , length of membranous urethra , and rtbs technique were not significant risk factors for the recovery of continence after ralp ( p>0.05 ) ( table 3 ) . in patients with no preoperative erectile dysfunction ( international index of erectile function-5 score > 21 ) , intercourse was reported in 72.5% and 76.2% of the patients undergoing bilateral nerve - sparing surgery at 12 and 24 months of follow - up , respectively . various intraoperative techniques for improving ppi have been introduced for use in open radical prostatectomy [ 6 - 14 ] , lrp , and ralp series [ 16 - 18 ] . although many of these techniques showed continence outcomes that were superior to those of procedures performed without the techniques , some controversies regarding the efficacy or limitations of these techniques persist [ 19 - 24 ] . besides , we had found that our initial experiences with ralp ( cases 1 to 124 ) showed acceptable continence outcomes without the use of such techniques . for these reasons , we did not fully trust the efficacy of the current and past intraoperative techniques for improving ppi until now . for techniques focusing on the bn , which is regarded as an internal sphincter a bn preservation technique was introduced to theoretically improve ppi on the basis of the idea that sparing as much bn as possible during rp might result in a sphincter mechanism more closely resembling that of the preoperative state . however , srougi et al found that the technique did not improve ppi and in fact might compromise cancer control because of the marginal positivity of the bn . intussusception of the bn , a technique introduced by walsh and marschke , was another modification intended to result in earlier return of urinary control . concerning the anatomy around the bn , there are some newly visited aspects ( especially posterior to the bn ) in addition to the well - known anatomy that was described by myers . such trends have resulted from the evolution of rp from an open technique to lrp or ralp . that is to say , the anatomy around the posterior bn has been examined under the enhanced and magnified vision of laparoscopic surgery and , more importantly , as a result of the different ( antegrade ) dissection of lrp / ralp . for example , in 2001 and 2002 , myers described the extension of the longitudinal detrusor muscle of the bladder anteriorly , in front of the anterior commissure of the prostate . he termed this the detrusor apron and explained that this structure had generally been unappreciated during rp by many urologists . in another example , in 2006 , secin et al described some longitudinal muscle fibers extending from the bn to the base of the prostate during the dissection of the posterior bn in lrp . they proposed that these fibers actually correspond to the posterior longitudinal fascia of the detrusor muscle . an earlier report regarding this structure could be found . when describing the ' montsouri ' technique of lrp , guillonneau and vallancien described the presence of a fascial structure with cephalocaudal striations that needed to be incised horizontally during the posterior bn dissection . however , this terminology was challenged by several authors , because , according to our current anatomical knowledge , the dnf does not extend anterior to the seminal vesicle . instead , the term posterior layer of the detrusor apron was suggested , in view of the similarity between this structure and the detrusor apron that myers had described . more recently , tewari et al used cadaveric dissections and real - time videos from ralps to describe a tissue layer posterior to the bn that extended from the posterior aspect of the trigone to the base of the prostate , which is consistently encountered after division of the posterior bn . , they emphasized that this tissue may serve as a key surgical anatomical landmark to locate the vasa and seminal vesicles , which are located just beneath this layer . aside from its presence or accurate termination , however , it seems that the natural role or function of the layer is not clearly known . therefore , we postulated that this layer might have some role in the control of urinary continence , considering its position around the bn . to verify this , we analyzed the outcomes in continence recovery after dividing our ralp cases into two groups ( with or without rtbs ) . accordingly , we started to perform rtbs techniques from the 125th case of our ralp series . we compared the data of the patients with rtbs ( cases 125 to 181 ) with those of our initial ralp cases ( without rtbs , cases 31 to 124 ) , in which the retrotrigonal layer had been used only as a landmark for posterior dissection of the bn . our ralp technique in all 151 cases did not include any of the current or past techniques for improving ppi , including puboprostatic ligament ( ppl ) preservation , additional anterior support of the ppl / dvc , bn preservation , membranous urethral lengthening , or posterior reconstruction of denonvilliers ' musculofascial plate ( such as the rocco stitch ) . moreover , there was no significant difference in the number of nerve - sparing procedures between our two groups . accordingly , we think that our comparison of the two groups was quite reliable in terms of both surgical aspects and baseline patient characteristics . during posterior dissection of the bn , the retrotrigonal layer was clearly identified in all but four of our patients , although the thickness varied from one case to another . from our experience , we fully agree with tewari et al that this tissue may be a key landmark for use in the dissection of the posterior bn . although some authors have reported the use of an " ultradissection technique " or modified ultradissecction ( in asians with relatively small body sizes ) during bn dissection , we felt that the approach via the retrotrigonal layer was easier . also , incorporating the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis presented no difficulty . however , upon analysis of our results , our rtbs technique did not show any role in the improvement of ppi . we think that this conclusion is bolstered by the fact that the surgeries in group 2 ( with rtbs ) were performed when our procedures for ralp were far more stabilized in every other step that might affect the continence outcome ( e.g. , nerve sparing , etc ) , and yet there was no significant difference in continence recovery between the two groups . the finding of significant differences in operative time and console time between the groups ( table 2 ) also supports this conclusion . our study would have more power if verified knowledge existed of the innate , accurate point at which the retrotrigonal layer has its distal insertion . such knowledge will be essential to put our conclusions in context , because the residual retrotrigonal layer was sutured to the membranous urethra ( fig . the limitations of the present study include the retrospective comparison with a historical cohort and the lack of randomization . as such , the results should be considered exploratory or observational , not definitive . to address this concern , we are planning a prospective randomized trial with validated continence measures to more rigorously assess the effectiveness of rtbs . . a verified description of the innate , accurate point at which the retrotrigonal layer has its distal insertion and prospective randomized studies with larger numbers of patients will be essential to confirm our results .

purposeto evaluate the impact of a retrotrigonal layer backup stitch ( rtbs ) during robot - assisted laparoscopic radical prostatectomy ( ralp ) on post - prostatectomy incontinence.materials and methodswe compared the difference in continence recovery between 94 patients ( group 1 , as historical controls ) and 57 patients ( group 2 ) . the only technical difference between our two groups was the incorporation of the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis ( group 1 : without rtbs ; group 2 : with rtbs ) . postoperative continence recovery was defined as the use of no absorbent pads.resultsin group 1 , the continence rate at 3 , 6 , and 12 months postoperatively was 40.4% , 70.2% , and 90.4% , respectively;in group 2 , the continence rate was 42.1% , 70.1% , and 89.7% , respectively . the median ( 95% confidence interval ) time to continence recovery was four months ( range , 1 to 12 months ) in group 1 and four months ( range , 1 to 9 months ) in group 2 . kaplan - meier curves showed no significant difference in the recovery of continence between the two groups ( log rank test , p=0.629).conclusionsa rtbs does not appear to improve urinary incontinence after ralp . further anatomical study and prospective randomized studies will be needed to confirm this .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC5088333

diabetic high blood glucose or hyperglycemia causes mortality and morbidity through dn and dr via disrupting the vascular function in the kidney and retina . these pathologies are the major cause of death associated with renal failure and blindness among the type 1 and type 2 diabetes patients [ 13 ] . similar molecular pathways appear to govern the development of diabetic renal and retinal microvascular injury . this speculation arises from higher coincidence rates of dn and dr ; that is , patients with dn have already developed dr and patients with dr are vulnerable to develop dn [ 4 , 5 ] . according to the similarities in pathologic background affecting the retina and kidney , it was hypothesized that dn and dr could arise by injuries to perivascular supporting cells in glomeruli and retina . the vulnerable vascular cell types affected by diabetic hyperglycemia are retinal pericytes ( rpc ) and their analogs within glomeruli are the mesangial cells . in dr , the rpc undergo cell death and disengage from the retinal vasculature , predisposing the retina to neoangiogenesis , vascular leakage , and tractional retinal detachment culminating in reduced vision or terminal blindness [ 68 ] . dn is characterized by mesangial matrix expansion and the obstruction of the glomerular capillaries within the renal filtration units , the glomeruli . the glomerulopathy in dn is associated with reduced efficiency of renal filtration , and in chronically established cases it triggers end - stage renal failure and mortality . several biochemical mechanisms and pathways have been described in speculation of the pathogenesis caused by either dn or dr . these mechanisms include enhanced oxidative stress , enhanced polyol pathway , pkc activation , inflammation , and advanced glycation end product formation [ 3 , 1113 ] . the most extensively investigated mechanisms associated with the development of both dn and dr are enhanced oxidative stress and inflammation caused by metabolic alterations . however , a plethora of evidence indicates that immunological and inflammatory mechanisms are important factors in development and progression of dr and dn [ 1417 ] . the recruitment of the activated macrophages and increased generation of inflammatory and proinflammatory mediators ( tnf- , il-1 , il-6 , mcp-1 , and opn ) in retinal and renal milieu is linked to the progression and exacerbation of the dn and dr [ 1722 ] . however , the exact molecular and cellular mechanisms involved have remained elusive . oxidative stress is a phenomenon in which the balance between the generation of free oxidizing radicals and the system responsible for their removal in the cell is disturbed leading to enhanced formation of free reactive ions including reactive oxygen species ( ros ) . the increased ros levels in cells subjected to diabetic hyperglycemia drive the antioxidant gene expression to protect the cells from oxidative injuries . one of the target genes to be induced following oxidative stress is the nf - e2-related transcription factor 2 ( nrf2 ) . two of the recently characterized genes that are regulated by the nrf-2 and provide protection against oxidative stress and stress - adaptation are proteasome activator genes , pa28 and pa28 . it is speculated that pa28 proteins increase the proteasomal degradation activity to clear the oxidized or misfolded proteins [ 2426 ] . the pa28/ genes were initially identified as components of immunoproteasomes which are induced in response to interferon-. pa28 and pa28 proteins form a heptameric complex ( 4/3 ) acting as the gate opener for the 20s proteasomes , hence stimulating the degradation of the nonubiquitinated short peptides . the well - characterized function for the pa28 proteins is the generation of the antigenic peptides to be presented by the mhc class i molecules [ 27 , 28 ] . the pa28/ are upregulated in the cultured rpc under high glucose conditions and also in the intraglomerular capillaries of older type 1 diabetic akita mice . to understand the role of pa28/ genes in development of dn and dr , pa28dko mice were tested and their physiological and biometric indices were compared with stz - induced diabetic wild type mice . the diabetic pa28dko mice provided higher survival rate , higher body weight , more efficient renal filtration function , and reduced microvascular damage in their retinae compared to diabetic wild type mice . the glomeruli , mesangial cells , and the rpc isolated from pa28dko mice had lower levels of opn and mcp-1 under high glucose conditions . opn is known as a proinflammatory protein associated with progression of diabetic microvascular injury [ 19 , 22 ] . the pa28-dependent regulation of opn expression was stimulated by high glucose and abrogated by synthetic peptides that block the binding of pa28 to 20s proteasomes . therefore , the findings of this study provide novel insights into the role of the ubiquitin proteasome system ( ups ) and specially the pa28 proteins , in regulating the microvascular injury in diabetes . animal maintenance , genotyping , treatments , and analytical procedures followed the guidelines accredited by institutional animal care and use committee of the university of wisconsin , school of medicine and public health . all the mice used for the experiments were in c57bl/6 background . for cell isolation , 6-week old male immorto mice ( stock number 006553 ) and to study diabetes , pa28dko male mice ( stock number 021202 ) were used . diabetes was induced in animals with a single injection of streptozotocin ( stz ; 180 mg / kg in citrate buffer , ph 4.2 by ip injection ) . three days following the treatment , the blood glucose of all stz - injected animals was above 400 mg / dl . for acr analysis the urine samples were collected by housing each individual mouse in a metabolic cage ( tecniplast , italy ) for 24 hours . urinary albumin and creatinine levels were measured by elisa ( albuwell m , exocell ) and the acr measurements were conducted according to the guidelines provided by the manufacturer . mouse kidneys were sliced and immersion - fixed in a solution of 2% paraformaldehyde ( pfa ) and 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.4 , overnight at 4c . the tissue was postfixed at room temperature for 2 hours in 1% osmium tetroxide in the same buffer . subsequently the samples were dehydrated in a graded ethanol series , then dehydrated in propylene oxide , and embedded in epon epoxy resin . ultrathin sections were prepared using leica uc6 ultramicrotome and mounted on 200-mesh carbon - coated copper grids . tissue sections were observed with a philips cm120 electron microscope , and images were captured with a megaview iii side - mounted digital camera . for pas and jms - h&e staining the formalin fixed , paraffin embedded , 5 - 6 m tissue sections were deparaffinized in xylene and rehydrated in descending graded percentage of ethanol ( 100% , 95% , 80% , and 70% ) and routinely stained with recommended reagents . digital images were taken with na pl apo objectives ( 10x/0.25 na , 40x/0.95 na , and 63x/1.4 na oil ) on a scanscope xt system using imagescope version 10 software ( aperio technologies inc . ) . briefly , the retinae from one litter ( 6 - 7 pups , 6-week old ) immorto mice were collected under a dissecting microscope . the collected retinae were rinsed with serum - free dulbecco 's modified eagle 's medium ( dmem ) , pooled , minced , and digested for 45 min with collagenase type ii ( 1 mg / ml , worthington ) with 0.1% bsa in serum - free dmem at 37c . cells were resuspended in equal amount of dmem containing 10% fetal bovine serum ( fbs ) and spun for 5 min at 400 g . the pelleted cells were resuspended in 4 ml dmem containing 10% fbs , 2 mm l - glutamine , 100 g / ml streptomycin , 100 u / ml penicillin , and recombinant murine ifn- ( r&d systems ) at 44 u / ml . cells were evenly divided into 4 wells of a 24-well tissue culture plate and maintained at 33c with 5% co2 . cells were progressively passed to larger plates and maintained and propagated in 60 mm dishes . the retinae from one litter ( 6 to 7 pups , 6-week old ) of immorto mice were dissected out aseptically under a dissecting microscope and kept in hbss buffer containing penicillin / streptomycin . retinae were pooled together , rinsed with hbss buffer ( life technologies ) , minced into small pieces in a 60 mm tissue culture dish using sterile razor blades , and digested in 5 ml of collagenase type i ( 1 mg / ml in serum - free dmem , worthington ) for 45 min at 37c . following digestion , dmem with 10% the cells were passed through a sterile 40 m nylon strainer ( bd falcon ) and spun at 400 g for 10 min to pellet cells , and cells were washed twice with dmem containing 10% fbs . the cells were resuspended in 1.5 ml medium ( dmem with 10% fbs ) and incubated with anti - pecam-1 antibody - conjugated dynabeads ( life technologies ) . after affinity binding , magnetic beads were rinsed six times with dmem with 10% fbs and bound cells in endothelial cell growth medium were plated into a single well of a 24-well plate precoated with 2 endothelial cells were grown in dmem containing 20% fbs , 2 mm l - glutamine , 2 mm sodium pyruvate , 20 mm hepes , 1% nonessential amino acids , 100 g / ml streptomycin , 100 u / ml penicillin , freshly added heparin at 55 u / ml ( sigma ) , endothelial growth supplement 100 g / ml ( sigma ) , and recombinant murine ifn- ( r&d systems ) at 44 u / ml . cells were progressively passed to larger plates , maintained , and propagated in 1% gelatin - coated 60 mm dishes . briefly , 8 10 dynabeads ( life technologies ) were diluted in 40 ml of phosphate - buffered saline and perfused through the heart of the adult mice . the kidneys were extracted , mechanically minced , digested with type 1 collagenase , and filtered through 100 m strainer . the glomeruli were collected with a magnet and washed three times to eliminate the nonglomerular cells and debris . the glomeruli were grown in either pericytes growth medium to establish the mesangial cell culture . the kgec were isolated from the glomeruli similar to mesangial cell isolation following a procedure described before . the purified glomeruli were seeded and allowed to grow in rec medium on coated 35 mm plates . upon 8085% confluency , the cells were incubated with anti - pecam-1 antibody - conjugated dynabeads , washed , and enriched similar to rec cultures . the bpc , bec , kpc , hpc , and lpc were isolated by following a procedure similar to rpc isolation . the peptides ( cpc scientific ) and the chariot transfection reagent ( active motif ) were reconstituted and used according to the manufacturer guidelines . the enucleated eyes were fixed in 4% paraformaldehyde for at least 24 h. the dissected retinae were washed overnight in deionized water and incubated in 3% trypsin ( difco trypsin 250 , becton dickinson and company ) prepared in 0.1 m tris , 0.1 m maleic acid , and ph 7.8 containing 0.2 m naf for 2 h at 37c . the neuroretinal tissue was gently brushed away , and the resultant isolated vascular tree was air dried onto a glass microscope slide . total rna from cells was extracted by total rna isolation kit ( norgenbiotek ) according to the manufacturer 's instructions . cells were allowed to reach 8590% confluence , rinsed twice with pbs , scraped off the plates , and transferred to rnase - free microfuge tubes . one microliter of 1/10-diluted cdna was used as real - time pcr template in triplicate groups and reaction was completed using sybr green master mix in mastercycler realplex ( eppendorf ) with the specified primers in supplemental experimental procedures . thermal cycles were programmed as 95c for 2 min ; 40 cycles of amplification ( 95c for 15 s and 60c for 40 s ) ; and a dissociation curve step ( 95c for 15 s , 60c for 15 s , and 95c for 15 s ) . standard curves were generated from known quantities for each target gene of linearized plasmid dna . tenfold diluted series were used for each known target to be amplified by sybr green qpcr premix . the linear regression line for ng of dna was calculated from relative fluorescent units at a threshold fluorescence value ( ct ) to quantify amplification targets from cell extracts by comparing the relative fluorescent units at the ct to the standard curve , normalized by the simultaneous amplification of rpl13a ( a housekeeping gene ) for all samples . for protein isolation , the cells in 80% confluent dishes were harvested following adding the lysis buffer ( 150 mm nacl , 1% triton x-100 , 20 mm tris - hcl ( ph 7.4 ) , protease inhibitor cocktail ( 1 mm pmsf , 10 mm leupeptin , 10 mm pepstatin ) ) . for western blot analysis , 50 g cell lysate or conditioned medium proteins were mixed with laemmli sample buffer and the reduced or nonreduced samples were boiled for 10 min . the boiled samples were loaded into 420% tris glycine acrylamide gels ( life technologies ) and electrophoresis was performed using sds running buffer ( 25 mm tris , 190 mm glycine , 0.1% sds , ph 8.3 ) . mouse cdna encoding the pa28 and pa28 were cloned into plvx - ires - hyg and plvx - ires - neo plasmids ( clontech ) , respectively . the lentiviral particles were prepared by transfecting the lenti - x 293 t cells ( clontech ) and collecting the conditioned media . the pbabe - opn / puro plasmid and the packaging cells are described elsewhere . cultured cells were transfected using the fugene6 reagent ( promega ) according to recommended protocol . after 48 h , hygromycin b ( 500 g / ml ; sigma - aldrich ) , neomycin ( 750 g / ml ; life technologies ) , and puromycin ( 5 g / ml ; sigma - aldrich ) were added to cell cultures . the data are represented as the mean sem . statistical significance was determined by analysis of variance ( anova ) and tukey - kramer post hoc analysis for multiple comparisons using p value of 0.05 in graphpad prism software ( san diego , ca , usa ) . the pa28 proteasome regulators were previously shown to be dramatically upregulated in the glomerular capillaries of akita mouse model with chronic hyperglycemia for 8 months but not in the same age wild type nondiabetic mice . in this study the role of pa28/ in modulating diabetic microvascular disorders was investigated via streptozotocin ( stz ) injection to both wild type and pa28dko mice . stz has cytotoxic effects on insulin - secreting pancreatic -cells and induces type i diabetes in experimental animals . all the pa28dko / stz and wild type / stz mice had blood glucose levels above 450 mg / dl three days after stz injection and on the day of sacrifice ( table 1 ) . in comparison with pa28dko / stz mice , the wild type / stz mice exhibited significantly reduced survival rates and weight loss by four months of stz injection . the pa28dko / stz mice were protected and had no obvious weight loss or reduced survival after six months of stz injection ( figures 1(a ) and 1(b ) ) . it was assumed that improved renal function was responsible for the observed improvement in the health of the pa28dko / stz mice . to test this , acr is a cornerstone assay for the diagnosis of renal filtration disorders [ 36 , 37 ] and reflects the efficiency of the kidney filtration function . the measured albuminuria was significantly higher in wild type / stz ( 291 8.54 g / mg ) compared to that of non - stz wild type ( 25.61 2.18 g / mg ) mice and 28dko / stz mice ( 119.6 5.61 g / mg ) ( figure 1(c ) ) . next the histological and ultrastructural properties of the glomeruli in the non - stz and stz - injected mice were assessed . renal histology in kidney sections was evaluated using the combined jones methenamine silver- ( jms- ) hematoxylin and eosin ( h&e ) staining and also periodic acid schiff ( pas ) staining . the jms and pas staining is routinely used to detect the mesangial matrix increase and changes in the glomerular basement membrane ( gbm ) . there were no discernible alterations in the appearance of gbms , the adjacent tubules , and also the mesangial or endothelial cells in different animal groups ( figure 2(a ) ) . similar to light microscopic analysis , tem analysis did not show any significant difference in the thickness of gbm in wild type / stz and pa28dko / stz mice . the lack of visible diabetic glomerular pathology in tem test despite the reduced renal filtration efficiency ( determined by acr test ) in mice studied here is likely due to the resistance of the c57bl/6 mouse line against morphological changes in gbm ( figures 2(b ) and 2(c ) ) . however , there was a significant reduction in the relative number of intraglomerular endothelial fenestrae in four - month stz - injected wild type mice compared to those of pa28dko / stz mice ( 47.5 1.74 versus 60.3 0.67 ) ( figure 2(d ) ) . similarly , the six - month stz - injected pa28dko mice provided higher endothelial fenestrae compared with four - month stz - injected wild type mice ( 56 0.93 versus 47.5 1.74 ) ( figure 2(d ) ) . since the inflammatory mediators opn and mcp-1 have been shown to be closely associated with the development and progression of the dn and dr , we investigated their levels using real - time pcr in the glomerular mrna isolated from stz - injected and nondiabetic wild type and pa28dko mice . the qpcr analyses did not provide any differences in the levels of il-1 and tnf- mrna among wild type and pa28dko mice under both normal and stz- injected conditions ( not shown ) . however , the mrna for opn and mcp-1 showed significant increase in wild type / stz versus nondiabetic wild type glomeruli ( figures 3(a ) and 3(b ) ) . interestingly , unlike the wild type / stz glomeruli , in pa28dko / stz glomeruli the expression of opn and mcp-1 was not altered significantly . these data demonstrate that pa28 and pa28 regulate the diabetic glomerular vascular injuries most likely through regulating the expression of proinflammatory agents . to understand how pa28 and pa28 genes promote glomerular injuries during diabetes , as an in vitro model , the mesangial cells from wild type and pa28dko mice were isolated for further characterizations [ 29 , 31 , 39 ] . since the mrna levels for opn and mcp-1 in pa28dko / stz glomeruli were lower than those of wild type / stz mice and the expression of these proteins has been linked to the development of dn and dr , we investigated whether higher glucose concentrations in vitro could induce the expression of opn and mcp-1 genes in isolated mesangial cells . western blot and qpcr analysis of these genes were performed following three days of treatment with either normal ( 5 mm d - glucose ) , high glucose ( 25 mm or 40 mm d - glucose ) , or osmolarity control ( 5 mm d - glucose plus 20 mm l - glucose ) . western blot analysis of the conditioned medium provided robust induction of opn expression in wild type but not in pa28dko mesangial cells following treatment with high glucose ( figure 4(a ) ) . the qpcr analysis of wild type and pa28dko cells also confirmed the upregulation of opn expression in wild type but not in pa28dko mesangial cells treated with high glucose ( figure 4(b ) ) . because mcp-1 protein could not be detected by western blot ( not shown ) , we used qpcr to evaluate changes in mcp-1 transcript levels in cultured mesangial cells under normal and high glucose conditions . consistent with the qpcr data for opn , the mcp-1 transcripts were significantly increased in the wild type mesangial cells grown under high glucose conditions , whereas the pa28dko cells had markedly lower levels of mcp-1 transcripts at the same conditions ( figure 4(c ) ) . it was next tested whether lentiviral - mediated reexpression of the pa28 and pa28 individually or simultaneously restored the opn expression in pa28dko mesangial cells . the reexpression of pa28 alone or pa28 and pa28 together restored the opn release from pa28dko cells under both normal and high glucose conditions ( figure 4(d ) ) . the overexpression of pa28 resulted in less vigorous expression of opn compared to simultaneous overexpression of both pa28 and pa28. this effect could be attributed to the ability of the pa28 subunits to form an active homoheptameric proteasome regulatory complex that has not been reported for pa28 monomers . dr is another microvascular complication of diabetes attributed to the diminution of pericytes in retinal microvessels and subsequent death of endothelial cells . in retina therefore , we investigated to test in respect of protection against glomerular microvascular injury in pa28dko / stz mice whether these mice were similarly protected against microvascular injury in the retina . compared with four - month stz - injected wild type mice , the prepared retinal flat mounted microvessels of pa28dko mice showed remarkable reduction in the number of acellular capillaries after four months ( 8.71 0.43 versus 4.43 0.45 ) and six months of stz injection ( 8.71 0.43 versus 5.07 0.28 ) , respectively ( figures 5(a ) and 5(b ) ) . reduction in the number of the acellular capillaries in pa28dko / stz retinae demonstrated that loss of pa28/ genes protected the rpc against cell death triggered by hyperglycemia . to assay the effect of high glucose treatment on cultured rpc and investigate whether they respond to high glucose the same as to mesangial cells , rpc were isolated and cultured from wild type and pa28dko mice . high glucose treatment of wild type rpc robustly induced the expression of opn and mcp-1 while the pa28dko rpc treated with high glucose did not manifest such increase in opn and mcp-1 expression ( figures 5(c)5(e ) ) . next it was tested whether increased expression of opn in response to high glucose was uniquely restricted to rpc and mesangial cells or other vascular cell types also responded similarly . for this purpose , different mouse endothelial and perivascular cell types were isolated from various organs and exposed to high glucose similar to rpc and mesangial cells . these cells included retinal endothelial cells ( rec ) , kidney glomerular endothelial cells ( kgec ) , total kidney nonglomerular pericytes ( kpc ) , brain pericytes ( bpc ) , brain endothelial cells ( bec ) , heart pericytes ( hpc ) , and lung pericytes ( lpc ) . western blot analyses of opn demonstrated that any of these vascular cell types did not contain elevated opn production under high glucose conditions ( figure 5(f ) ) . hence , increased production of opn under high glucose conditions is a unique characteristic of rpc and mesangial cells . to investigate if the opn release from mesangial cells exposed to high glucose was caused by the binding of pa28/ proteins to the 20s proteasomes , we examined peptides capable of disrupting the association of pa28/ with the 20s proteasome . the pa28-inhibitory peptides fused to tat domain of the hiv virus were used for this assay . tat sequence was added to the xapc7 and hbx peptides to promote the transduction of the cultured cells . the xapc7-tat and hbx - tat peptides as well as scrambled control peptides fused to tat were used to transfect the cultured mesangial cells for three days under high glucose condition to promote opn release . western blot analyses of conditioned media provided strong inhibition of opn release after treatment with xapc7-tat and hbx - tat at 5 m and 10 m concentrations , respectively ( figures 6(a ) and 6(b ) ) . treatment with the liver x receptor ( lxr ) agonists , t0901317 and gw3965 , was shown to inhibit opn release and ameliorate the severity of dn . hence , as positive control for our peptide treatment experiments and suppression of opn release , we used lxr agonists in cotreatment of the mesangial cells with high glucose . as a result , almost both t0901317 and gw3965 completely inhibited the opn release at 10 m concentrations ( figures 6(a ) and 6(b ) ) . interestingly , the tat - fused scrambled peptides also suppressed opn release in response to high glucose . tat peptide had been previously demonstrated to inhibit the pa28 binding to the 20s proteasomes [ 43 , 44 ] . therefore , the inhibitory effect of the tat - scrambled peptides could be attributed to the tat domain of the peptides . however , treatment of the mesangial cells with tat - only peptide even at 200 m concentration did not inhibit the opn release ( figure 6(c ) ) . the failure of the tat - only peptide in inhibiting the opn release under high glucose conditions suggests that tat requires fusion to a juxtaposing peptide to inhibit pa28 binding to the 20s proteasome . in order to exclude the inhibitory effect of the tat domain , xapc7 and hbx peptides without tat domain treatment with xapc7 and its scrambled control resulted in complete dose - dependent inhibition of the opn release ( figure 6(d ) ) . this effect of control peptide is possibly caused by the shorter size and positive charge of these peptides compared with hbx peptide . the hbx showed marked reduction in opn release at various concentrations but this inhibition was less potent than xapc7 . the scrambled hbx peptide did not markedly affect the opn release from the mesangial cells ( figure 6(d ) ) . collectively , these findings demonstrate that high glucose - induced binding of pa28 and pa28 to 20s proteasomes in rpc and mesangial cells regulates opn production . taking advantage of the pa28dko mice , the mesangial cells , and rpc isolated from pa28dko mice it was demonstrated in this study that pa28 and pa28 genes are key determinants of diabetic microvascular injuries . evidence is provided for the involvement of pa28 proteasome regulators in exacerbating the pathogenesis of dn by demonstrating that , compared with wild type / stz mice , the pa28dko / stz mice do not develop severe albuminuria . further testing provided that , compared to wild type , the opn and mcp-1 inflammatory mediators are markedly suppressed in the pa28dko / stz glomeruli , mesangial cells , and rpc under high glucose conditions . opn secretion was regulated by pa28/ and , among different vascular cell types , was only detectable in cultured rpc and mesangial cells exposed to high glucose . this is consistent with the higher sensitivity of rpc and mesangial cells to the cytotoxic effects of high glucose compared to other vascular cell types including the rec , kgec , bpc , and bec . these findings have strong implications in addressing why the trigger for the development of diabetic microvascular complications lies in the rpc and mesangial cells . the cell injury and death of these perivascular supporting cells in the retina and kidney caused by hyperglycemia precede the development of dr and dn . the increased opn expression is reportedly associated with the development of dn and dr and pharmacological reagents capable of inhibiting opn release provide protection against dn and dr [ 21 , 42 , 45 ] . opn has macrophage chemoattractive function and the progression of dn and dr is facilitated by the recruitment of the macrophages and monocytes into retina and kidney . opn release by perivascular cells in diabetic kidney and retina might regulate the local inflammatory cues to increase macrophage recruitment and activation . the novel function of pa28 and pa28 proteins in regulating opn expression and diabetic microvascular injury demonstrates that , apart from their antigen processing properties , these proteins possess unexplored functions in sensing and responding to metabolic changes in a certain subset of vascular cells . importantly , the increased expression of opn following exposure to high glucose exclusively in rpc and mesangial cells that are main targets of diabetic hyperglycemia injury demonstrates a cell - specific function for pa28 and pa28 in sensing and responding to altered glucose levels . similar to lxr agonists , t0901317 and gw3965 , that inhibit opn expression and the severity of dn in stz diabetic mice , the delivery of peptides that inhibit pa28 binding to 20s proteasomes , the xapc7 , hbx , and tat - conjugated peptides inhibited opn release from mesangial cells under high glucose conditions . this suggests that opn expression under high glucose conditions from mesangial cells is regulated by pa28 and pa28 binding to the 20s proteasome . in agreement with the role of pa28 and pa28 in regulating the opn expression , the reexpression of pa28 alone or pa28 and interestingly the expression of pa28 alone also stimulated the opn release from the pa28dko mesangial cells . this observation can be explained by the exceptional ability of the pa28 monomers to form an active regulatory complex capable of binding and activating the 20s proteasomes . presently , it is unclear whether the reduced expression of opn or mcp-1 alone is accountable for the ameliorated diabetic microvascular injury in pa28dko mice or other pathways are also involved . nevertheless , since other groups showed that proteasome inhibition in mice attenuates the development of dn [ 4648 ] , the relative inhibition of the proteasomes in pa28dko rpc and mesangial cells would also provide a suitable explanation for their protection against diabetic renal and retinal injury . this can be supported by previous study in which the pa28 proteins were shown to be upregulated in rpc and also in diabetic glomeruli where expression level of pa28 proteins correlated with the severity of diabetic renal injury [ 1 , 49 ] . it therefore appears that excessive activation or deregulation of the proteasomes by upregulated pa28 proteins could possibly result in microvascular damage in the retinal and glomerular vasculature . other investigators have shown that high glucose treatment of rpc and mesangial cells induces oxidative stress [ 50 , 51 ] . the pa28 and pa28 were demonstrated to play key role in the clearance of misfolded and oxidized proteins and adaptation to oxidative stress in cultured cardiomyocytes . similarly , in rpc and mesangial cells , the pa28 and pa28 proteins could assist the rpc and mesangial cells in their adaptation to oxidative stress caused by high glucose . for instance , it was shown that rpc have lower proteasomal degradational capacity compared to other vascular cell types such as rec . therefore , pa28-/- proteins could assist the degradation of proteins damaged by exposure of rpc to high glucose but overt activation of the proteasomes in rpc and mesangial cells provokes tragic consequences . this study provides major insights into the role of pa28 and pa28 genes in promoting diabetic renal and retinal microvascular injury . first of all , deletion of pa28 and pa28 genes protected diabetic animals against dn . secondly , the expression of mcp-1 and opn as important mediators of dn or dr was shown to be regulated by pa28 and pa28 in rpc and mesangial cells grown under high glucose . thirdly , pa28dko mice showed reduced severity of dr reflected by reduced number of acellular capillaries in their retinae . lastly , the suppressive effect of pa28-inhibitory peptides on opn release by mesangial cells grown in high glucose was demonstrated . therefore , modulating the degradational activity of ubiquitin proteasome system mediated by pa28 and pa28 proteins offers a suitable candidate for the development of future treatments for microvascular complications of diabetes particularly in the retina and kidney .

diabetic nephropathy ( dn ) and diabetic retinopathy ( dr ) are major complications of type 1 and type 2 diabetes . dn and dr are mainly caused by injury to the perivascular supporting cells , the mesangial cells within the glomerulus , and the pericytes in the retina . the genes and molecular mechanisms predisposing retinal and glomerular pericytes to diabetic injury are poorly characterized . in this study , the genetic deletion of proteasome activator genes , pa28 and pa28 genes , protected the diabetic mice in the experimental stz - induced diabetes model against renal injury and retinal microvascular injury and prolonged their survival compared with wild type stz diabetic mice . the improved wellbeing and reduced renal damage was associated with diminished expression of osteopontin ( opn ) and monocyte chemoattractant protein-1 ( mcp-1 ) in the glomeruli of stz - injected pa28/pa28 double knockout ( pa28dko ) mice and also in cultured mesangial cells and retinal pericytes isolated from pa28dko mice that were grown in high glucose . the mesangial pa28-mediated expression of opn under high glucose conditions was suppressed by peptides capable of inhibiting the binding of pa28 to the 20s proteasome . collectively , our findings demonstrate that diabetic hyperglycemia promotes pa28-mediated alteration of proteasome activity in vulnerable perivascular cells resulting in microvascular injury and development of dn and dr .

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

PMC3015953

adenocarcinoma of the vermiform appendix is a rare neoplasm of the gastrointestinal tract with an incidence of about 0.1% to 0.2% . age - adjusted incidence of cancer of the appendix is 0.12 cases per 1 000 000 per year . these rare tumors are seldom suspected before surgery , and < 1/2 are diagnosed intraoperatively . the most common presentation of appendiceal malignancy is right lower abdominal pain that often mimics acute appendicitis . right iliac fossa mass and intestinal obstruction have also been reported ; these presentations reflect various stages of a locally expanding tumor causing luminal obstruction of the appendix . there are other clinical presentations , and we report herein a case of appendicular adenocarcinoma found unexpectedly in a patient who presented to the gastroenterology unit with per - rectal bleeding . the patient is a 55-year - old lady admitted with rectal bleeding due to the penetration of mucinous adenocarcinoma of the appendix into the sigmoid colon . based on a review of the literature , this is the first reported case of an appendiceal malignancy presenting and being treated in this manner . we diagnosed a case of appendicular mucinous cystadenocarcinoma found unexpectedly in 55-year - old lady admitted to the gastroenterology unit because of per rectal bleeding and a rectal polyp suspected on an outpatient colonoscopy . colonoscopy revealed evidence of external compression in the rectosigmoid junction with tumor infiltration of the sigmoid mucosa . in the rest of the colon , there was a small polyp in the ascending colon that was removed by snaring . gastrografin enhanced ct - scan after per rectal filling and maximal distension of the rectum and sigmoid colon revealed a tightly stenosed 6.5-cm lumen in the distal part of the sigmoid colon . specifically , this segment of the sigmoid colon was infiltrated by a 6 cm 6 cm large hypodense irregular tumor mass that also infiltrated part of the adjacent ileum . this tumor mass was connected to the end of the appendix , which had a diameter of 1.2 cm ( figure 1 ) . computed tomographic scan showing sigmoid colon infiltrated by 6 cm 6 cm large hypodense irregular tumor mass connected to the end of the appendix . the clinical investigations , ct and colonoscopy , indicated the possibility of appendicular carcinoma with an infiltrated sigmoid colon . a laparoscopic sigmoid colectomy was performed first . with the patient in the supine position , pneumoperitoneum was established with the so - called open technique , and 4 trocars were placed in the upper , lower , left , and right abdomen . the chief surgeon operated from the right side via the first monitor placed at the left side of the patient , and the assistant was on the left side . exploration of the abdomen showed a conglomerate tumor in the pelvis with involvement of the cecum and sigmoid colon . for sigmoid and right colon resection , we mobilized , devascularized , divided , and resected the colon intracorporeally . the retroperitoneum was incised medial to the left ureter , and the left hypogastric nerve was cautiously identified . after the sigmoid colon had been mobilized , 2 mesenteric windows were created at the sigmoid mesentery , one at the level of the rectosigmoid junction , and the second at the midsigmoid level . the inferior mesenteric vessel was divided with a vascular stapler , and the mesosigmoid was dissected up to the proximal part of the sigmoid colon . after that , the distal part of the sigmoid colon and the proximal rectum were mobilized under the tumor mass attachment . following this , we mobilized the right colon as described previously . the chief surgeon operated from the left side of the patient , and the endoscope was placed into the left paraumbilical trocar . to carry out the lymphadenectomy simultaneously with the resection of the vascular stem , primarily the laterally running ileocolic / right colic arteries and the branches of the superior mesenteric artery were sought out , exposed , and ligated using a window technique . the laterocaudal mobilization extended to the cecal pole and the terminal ileum , cranially to the ascending and meso - ascending colon until the mesenteric root in the outflow area of the lateral colonic vessels . after the terminal ileum was mobilized , the ileum was divided with a stapler . at this stage , the tumor mass including the sigmoid colon and fully mobilized cecum and distal part of the sigmoid colon under the tumor were well visualized and easily divided . with this action as the preparation was advanced cranially , the lateral abdominal wall ties were released , at first , using the ultrasonic dissector or scissors step - by - step until the flexure . then , the ascending colon was released of its retroperitoneal ties . with a properly layered dissection towards the aorta between the posterior side of the meso ascending colon and the renal fascia , the preparation was continued until the horizontal part of the duodenum came into view and the stump was detached from its mesenteric ties . a dissection rod was indispensable for the traumatic dissection , because the embryonically preformed tissue strands were best detached from one another by blunt dissection . with detachment of the right colonic flexure , the status of the already mobilized colomesenteric segment changed in such a way that a caudal dissection of the flexure could become complicated , eg , at the vascular stems , due to visual obstruction by the falling intestinal convolution . after complete mobilization of the right - sided colon intended for resection , the pneumoperitoneum was released , the trocar removed , and the incision extended for minilaparotomy . externalization of the prepared sigmoid and right colon was carried out through a foil ring introduced into the minilaparotomy over the symphysis pubis for wound protection . after the right transverse colon was divided extracorporeally and the specimen removed , the distal ileum was exteriorized ( figure 2 ) . after the anvil was placed in the descending colon , then retained into the abdomen , the incision was closed . we placed the stapler through the rectum and mated it with the anvil laparoscopically to complete the colorectal anastomosis . first flatus was recognized on the second postoperative day , a solid diet was started on the third postoperative day , and the patient was discharged and went directly home on the eighth postoperative day ( figure 4 ) . histopathological examination showed mucinous adenocarcinoma of the appendix with infiltration of the sigmoid colon of about 6 cm ( figure 3 ) . the patient seen 2 months after surgery was in good general condition with no complications . a laparoscopic sigmoid colectomy was performed first . with the patient in the supine position , pneumoperitoneum was established with the so - called open technique , and 4 trocars were placed in the upper , lower , left , and right abdomen . the chief surgeon operated from the right side via the first monitor placed at the left side of the patient , and the assistant was on the left side . exploration of the abdomen showed a conglomerate tumor in the pelvis with involvement of the cecum and sigmoid colon . for sigmoid and right colon resection , we mobilized , devascularized , divided , and resected the colon intracorporeally . the retroperitoneum was incised medial to the left ureter , and the left hypogastric nerve was cautiously identified . after the sigmoid colon had been mobilized , 2 mesenteric windows were created at the sigmoid mesentery , one at the level of the rectosigmoid junction , and the second at the midsigmoid level . the inferior mesenteric vessel was divided with a vascular stapler , and the mesosigmoid was dissected up to the proximal part of the sigmoid colon . after that , the distal part of the sigmoid colon and the proximal rectum were mobilized under the tumor mass attachment . following this , we mobilized the right colon as described previously . the chief surgeon operated from the left side of the patient , and the endoscope was placed into the left paraumbilical trocar . to carry out the lymphadenectomy simultaneously with the resection of the vascular stem , primarily the laterally running ileocolic / right colic arteries and the branches of the superior mesenteric artery were sought out , exposed , and ligated using a window technique . the laterocaudal mobilization extended to the cecal pole and the terminal ileum , cranially to the ascending and meso - ascending colon until the mesenteric root in the outflow area of the lateral colonic vessels . after the terminal ileum was mobilized , the ileum was divided with a stapler . at this stage , the tumor mass including the sigmoid colon and fully mobilized cecum and distal part of the sigmoid colon under the tumor were well visualized and easily divided . with this action as the preparation was advanced cranially , the lateral abdominal wall ties were released , at first , using the ultrasonic dissector or scissors step - by - step until the flexure . then , the ascending colon was released of its retroperitoneal ties . with a properly layered dissection towards the aorta between the posterior side of the meso ascending colon and the renal fascia , the preparation was continued until the horizontal part of the duodenum came into view and the stump was detached from its mesenteric ties . a dissection rod was indispensable for the traumatic dissection , because the embryonically preformed tissue strands were best detached from one another by blunt dissection . with detachment of the right colonic flexure , the status of the already mobilized colomesenteric segment changed in such a way that a caudal dissection of the flexure could become complicated , eg , at the vascular stems , due to visual obstruction by the falling intestinal convolution . after complete mobilization of the right - sided colon intended for resection , the pneumoperitoneum was released , the trocar removed , and the incision extended for minilaparotomy . externalization of the prepared sigmoid and right colon was carried out through a foil ring introduced into the minilaparotomy over the symphysis pubis for wound protection . after the right transverse colon was divided extracorporeally and the specimen removed , the distal ileum was exteriorized ( figure 2 ) . side - to - side ileotransverse anastomosis was performed with a linear stapler . en bloc resection of the sigmoid and right colon . after the anvil was placed in the descending colon , then retained into the abdomen , the incision was closed . we placed the stapler through the rectum and mated it with the anvil laparoscopically to complete the colorectal anastomosis . first flatus was recognized on the second postoperative day , a solid diet was started on the third postoperative day , and the patient was discharged and went directly home on the eighth postoperative day ( figure 4 ) . histopathological examination showed mucinous adenocarcinoma of the appendix with infiltration of the sigmoid colon of about 6 cm ( figure 3 ) . the patient seen 2 months after surgery was in good general condition with no complications . primary adenocarcinoma of the appendix is a rare neoplasm of the gastrointestinal tract and constitutes about 0.5% of all gastrointestinal tract tumors . these rare tumors are seldom suspected before surgery , and less than one - half are diagnosed intraoperatively . several studies describing primary tumors of the appendix have appeared in the literature since 1903 , when elting published a review and case series . there are 4 major histological subtypes : cystic , colonic , carcinoid , and adenocarcinoid . carcinoids are most common , constituting nearly 90% of all the primary tumors of the appendix . macroscopically , they produce mucin - filled cystic dilatation of the appendix indistinguishable from that associated with benign tumors . diagnosis of malignancy is made using 2 features : invasion of the appendicular wall and identification of epithelial cells in peritoneal mucous collections . spread of the neoplasm above the diaphragm or invasion of the abdominal viscera is exceptional , and lymphatic and hematogenous spread is also very rarely seen . sometimes this tumor may present in an unusual manner like polyploidy infiltration of the abdominal wall , bladder infiltration mimicking bladder carcinoma , cecocecal intussusceptions , retroperitoneal abscess , vaginal hydronephrosis , or per - rectal bleeding . sometimes they may present with distension of the abdomen due to pseudomyxoma peritoneum and obstruction of the gut due to involvement of the colon . even if an appendicectomy has been performed previously , cystadenocarcinoma of the appendicular stump may develop ; therefore , the possibility of carcinoma in the appendicular stump that increases in size should be born in mind . accurate and complete preoperative diagnosis has been rare in the past , but modern imaging techniques today allow us to recognize most of the complications and associated conditions . also in such advanced cases with clear preoperative examination , it should not to be considered a contraindication to performing laparoscopic surgery . due to intraperitoneal spread of the appendicular carcinoma , invasion of the abdominal viscera , as in our case , and the poor prognosis of this tumor , an aggressive approach should be taken when treating patients with this type of tumor . further development of instruments and techniques has made it possible to apply laparoscopic surgery to malignant diseases in such complex cases as we have described . compared with conventional surgery , laparoscopic surgery is beneficial with respect to short - term outcomes , including earlier recovery and less need for postoperative analgesia . radiotherapy , chemotherapy , and new therapeutic modalities like radioimmunotherapy and matrix metalloproteinase inhibitors have still to be proven by prospective analysis .

adenocarcinoma of the vermiform appendix is a rare neoplasm of the gastrointestinal tract that most commonly presents as right lower abdominal pain , mimicking acute appendicitis . presentation caused by loco - regional spread with involvement of adjacent structures is rare . an accurate and complete preoperative diagnosis has been rare in the past ; however , modern imaging techniques allow recognition of most complications and associated conditions . the diagnosis is confirmed postoperatively . aggressive surgical management is the treatment of choice in appendicular adenocarcinoma . we report the case of appendicular mucinous cystadenocarcinoma in a 55-year - old lady with penetration of the sigmoid colon treated with laparoscopic - assisted sigmoid and en block right hemicolectomy.it was possible to manage this complex case by using a laparoscopic procedure with all the known benefits of minimally invasive surgery .

INTRODUCTION CASE REPORT Procedure DISCUSSION CONCLUSION

PMC3389436

it is generally established that the harmful effects of communicable disease and malnutrition on public health are of the utmost importance notably in the poor countries . consequently , the priority of the health institutions and care workers has been focusing on control and preventive measures of the infectious diseases.[13 ] however , in many cases , health care promotion and enhancement of medical insurance have tackled these issues . on the other hand , rapid changes in lifestyle , nutrition , and physical activity , especially among the youngsters , have been accompanied by long - term change in disease patterns which is no longer allocated to industrialized countries . as a direct result of this , chronic noncommunicable disease ( ncd ) is becoming epidemic and global issue as two out of three deaths in the world are related to chronic diseases . these are affecting an abundance of people of all ages , nationalities , and socioeconomic classes[68 ] in addition to developed countries , the prevalence of ncds is now increasing in developing countries , which would lead to increased morbidity and mortality , as well as to a high financial burden . a growing body of literature documented changes in lifestyle habits and the global increase in childhood overweight . given the long - term consequences of childhood obesity on morbidity and mortality , health care in the pediatrics group deserves further consideration . similar to the children and adolescents of other developing countries , iranian youths are experiencing lifestyle changes , making them prone to risk factors of chronic diseases.[1419 ] considering the tracking of ncd - related risk behaviors and risk factors from childhood to adult life , surveillance of such factors , e.g. , blood pressure , avoidance of smoking , and overweight as well as encouraging regular physical exercise in children and adolescents , can provide useful information for long - term national planning . some international organizations have implemented surveillance systems for tracking such factors . the global school - based student health survey ( gshs ) has developed and supported by the world health organization ( who ) in cooperation with united nations unicef , unesco , unaids , and centers for disease control and prevention ( cdc ) . the youth risk behavior surveillance system ( yrbss ) aims to monitor those health - risk behaviors contributing to the young and adults morbidity and mortality . they include behaviors related to unintentional injuries and violence , tobacco use , alcohol and other drug use , sexual risk behaviors , unhealthy dietary behaviors , and physical inactivity , as well as obesity and asthma . in iran , a school - based surveillance system entitled the childhood and adolescence surveillance and prevention of adult noncommunicable disease ( caspian ) study is implemented from 20032004 . the surveys are repeated every 2 years , with blood sampling for biochemical factors every 4 years . this paper presents the methods and primary findings of the third survey of this school - based surveillance system . this school - based nationwide health survey was conducted in iran as the national survey of school student high risk behaviors ( 20092010 ) . it was conducted as the third study of the school - based surveillance system entitled caspian - iii study . it was performed with corporation of the ministry of health and medical education ; ministry of education and training , child health promotion research center , isfahan university of medical sciences ; and endocrinology and metabolism research institute of tehran university of medical sciences . the survey was performed among 5570 students aged 1018 years , who were selected by multistage random cluster sampling from urban and rural areas of 27 provinces of iran . eligible schools for our study were stratified according to information bank of ministry of education , and then , they were selected randomly . in selected schools , study protocols were reviewed and approved by ethics committees and other relevant national regulatory organizations was obtained after complete explanation of the study objectives and protocols for students and their parents , written informed consent was obtained from parents and oral assent from students , sampling and examinations and were begun . we prepared the questionnaires in farsi based on and the who global school health survey , and added some more questions to the questionnaires of parents . questions were about family dietary habits , students past history , and familial history of chronic diseases . the validity of their content was affirmed based on observations of an experts panel and item analysis . reliability measures were assessed based on a pilot study . under the supervision of expert health care professionals , the students filled out the self - administered questionnaire at school . a team of trained health care professionals recorded information in a checklist and conducted the examinations under standard protocol by using calibrated instruments . weight was measured to the nearest 200 g in barefoot and lightly dressed condition . body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) . waist circumference ( wc ) was measured by a nonelastic tape to the nearest 0.2 cm at the end of expiration at the midpoint between the top of iliac crest and the lowest rib in standing position . we used the who growth curves to define bmi categories , i.e. , underweight as age and sex - specific bmi < -2 z - score , overweight as sex - specific bmi for age of > + 1 z - score , and obesity as sex - specific bmi for > + 2 z - score . we used the spss for windows software ( version 16.0 , spss , chicago , il ) for statistical analyses , and p < 0.05 was considered as statistically significant . this school - based nationwide health survey was conducted in iran as the national survey of school student high risk behaviors ( 20092010 ) . it was conducted as the third study of the school - based surveillance system entitled caspian - iii study . it was performed with corporation of the ministry of health and medical education ; ministry of education and training , child health promotion research center , isfahan university of medical sciences ; and endocrinology and metabolism research institute of tehran university of medical sciences . the survey was performed among 5570 students aged 1018 years , who were selected by multistage random cluster sampling from urban and rural areas of 27 provinces of iran . eligible schools for our study were stratified according to information bank of ministry of education , and then , they were selected randomly . in selected schools , study protocols were reviewed and approved by ethics committees and other relevant national regulatory organizations was obtained after complete explanation of the study objectives and protocols for students and their parents , written informed consent was obtained from parents and oral assent from students , sampling and examinations and were begun . we prepared the questionnaires in farsi based on and the who global school health survey , and added some more questions to the questionnaires of parents . questions were about family dietary habits , students past history , and familial history of chronic diseases . the validity of their content was affirmed based on observations of an experts panel and item analysis . reliability measures were assessed based on a pilot study . under the supervision of expert health care professionals , the students filled out the self - administered questionnaire at school . a team of trained health care professionals recorded information in a checklist and conducted the examinations under standard protocol by using calibrated instruments . weight was measured to the nearest 200 g in barefoot and lightly dressed condition . body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) . waist circumference ( wc ) was measured by a nonelastic tape to the nearest 0.2 cm at the end of expiration at the midpoint between the top of iliac crest and the lowest rib in standing position . we used the who growth curves to define bmi categories , i.e. , underweight as age and sex - specific bmi < -2 z - score , overweight as sex - specific bmi for age of > + 1 z - score , and obesity as sex - specific bmi for > + 2 z - score . we used the spss for windows software ( version 16.0 , spss , chicago , il ) for statistical analyses , and p < 0.05 was considered as statistically significant . this national survey was conducted among 5570 school students ( 2784 girls and 2786 boys ) and one of their parents . data of 5528 students ( 2726 girls and 2802 boys ) were complete and are reported . the mean age of participants was 14.7 2.4 years without significant difference in terms of gender . more than 90% of students were from public schools and the rest from private schools . most students parents were literate ; 97.8% of fathers and 98.2% of mothers had at least elementary literacy . the large number of students father was worker or employee ( 45.35% ) , whereas 90.7% of mothers were housewives . characteristics of study participants : caspian - iii study overall , 17.3% ( 17.3% of girls and 17.5% of boys ) were underweight , and 17.7% ( 15.5% of girls and 19.9% of boys ) were overweight or obese . abdominal obesity was documented in 16.3% of students ( 17.8% of girls and 15% of boys ; 18.6% in urban and 11.2% in rural areas ) . bread is the staple food for iranians , 42.4% of families consumed whole wheat bread , and the rest used breads prepared with white flour . most families , i.e. , 43.8% in urban areas and 58.6% in rural areas used solid fats for food preparation at home . some dietary habits of study participants : caspian - iii study according to the students report , 14.2% of them had a regular daily physical activity for at least 30-min a day ; 19.7% reported not to have any physical activity . of students studied , 45% used computer in week days and 56.5% in weekends ; these frequencies were higher in urban areas and among girls [ table 3 ] . physical activity pattern of study participants : caspian - iii study regarding passive smoking , 48.5% of students reported to have at least one cigarette smoker in their relatives , the corresponding figure was 33.9% for waterpipe . overall , 10.4% of students ( 11.7% in urban areas and 7.3% in rural areas ) reported that they used tobacco products , often waterpipe . overall , 32.8% of students asserted that in the 3 months before the study , they experienced at least three times of bullying at school . bullying was more frequent among girls than boys and decreased from elementary to high - school levels . regarding injuries , 14.46% of students ( 14.8% elementary , 15.2% middle , and 14% in high school ) reported that during the year prior to the survey , they had suffered an injury needing the interference by school health providers . the prevalence of these injuries was higher in girls than in boys , and in rural than in urban areas . in the most cases , the injury type has been cuts and scratching , and 42.4% of injuries occurred when students were exercising , running , and/or jogging . the findings of this national survey can serve for action - oriented policy making for health promotion of children and adolescents , as well as for primordial / primary prevention of chronic diseases . most students were in a good health condition ; however , in most of the time , the lifestyle habits of families exposed them to chronic diseases in their future adulthood . , 80% of children and adolescents mentioned that they often add salt to their table food . despite that youths taste have changed recently , and the preference for salty food has been escalated , our findings were about double in comparison with australian ( 10%39% ) or greek children ( 39% ) . unhealthy eating habits among children and their families are considered as a problem for both developed and developing countries , which necessitate taking heed of needs for improvements in family and school food environments.[2729 ] in addition to some unhealthy dietary habits , the frequency and intensity of physical activity were low among children and adolescents . furthermore , physical activity level was lesser in students of higher grades than in lower grades . overall , the screen time , watching tv or computer games , was about 4 h a day , i.e. , twice than the recommended time . our findings are consistent with some other studies in showing low time allocated to physical activity among children and adolescents . similar to the populations of most developing countries , in the current survey , we documented a dual burden of nutritional disorders in terms of underweight and overweight among iranian children and adolescents . the epidemiologic transition with rapid changes in the habitual diet along with physical inactivity makes young individuals more susceptible to weight disorders.[3234 ] our findings also demonstrated a considerable prevalence of abdominal obesity , which even needs more attention for future chronic diseases . principally , one of the leading causes of hypertension in this age group is the frequency of consuming fast food and solid hydrogenated fat . many developing countries have encountered with dyslipidemia among children and adolescences , which can be justified by their sedentary lifestyle and high consumption of hydrogenated fat.[4042 ] thus , according to the association of dyslipidemia in childhood and its relation to the future cardiovascular morbidity and mortality , encouraging healthy lifestyle from childhood is of crucial importance . a large number of students were exposed to secondhand smoking ; families should be aware of the hazards of their smoking habits on the health and the tendency of their children to begin smoking in the near future . although the prevalence of self - reported smoking was lower than some other countries,[4547 ] but these statistics deserve more consideration for preventive measures in this vulnerable age group , particularly because of the young age of begging to smoke at about 11 years . needless to say that exposure to violence and bullying is extremely likely to impose detrimental effects to the overall well - being and development of all youths . moreover , to establish successful socioeconomic life and subsequently prosperous society , having a healthy educational system is essential . our findings were consistent with studies conducted in some other countries.[4850 ] barriers to healthy lifestyles , e.g. , physical activity , should be recognized in each community , and facilities should be provided accordingly . the current study documented important and preventable unhealthy lifestyle behaviors , which need proper attention both within the family and at school . living a healthier life is every society 's right , but it will not become true without amending the young 's lifestyle and behavior . this survey is confirmatory evidence on the importance of establishing surveillance systems for risk behaviors to implement action - oriented interventions for primordial and primary prevention of chronic diseases .

background : a school - based surveillance system entitled the childhood and adolescence surveillance and prevention of adult noncommunicable disease ( caspian ) study is implemented at national level in iran . this paper presents the methods and primary findings of the third survey of this surveillance system.methods:this national survey was performed in 20092010 in 27 provinces of iran among 5570 students and one of their parents . in addition to physical examination , fasting serum was obtained . body mass index was categorized based on the world health organization growth charts.findings:data of 5528 students ( 2726 girls , 69.37% urban , mean age 14.7 2.4 years ) were complete and are reported . overall , 17.3% ( 17.3% of girls and 17.5% of boys ) were underweight , and 17.7% ( 15.5% of girls and 19.9% of boys ) were overweight or obese . abdominal obesity was documented in 16.3% of students ( 17.8% of girls and 15% of boys ) . 57.6% of families consumed breads , the staple food for iranians , prepared with white flour . most families ( 43.8% in urban areas and 58.6% in rural areas ) used solid hydrogenated fats . 22.7% of students did not add salt to the table food . 14.2% of students reported to have a regular daily physical activity for at least 30 min a day . overall , 10.4% of students ( 11.7% in urban areas and 7.3% in rural areas ) reported that they used tobacco products , often waterpipe . 32.8% of students experienced at least three times of bullying in the previous 3 months . during the year prior to the survey , 14.46% of students had an injury needing the interference by school health providers.conclusion:this survey is confirmatory evidence on the importance of establishing surveillance systems for risk behaviors to implement action - oriented interventions .

INTRODUCTION METHODS Study population Ethical concerns Procedure and measurements Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC3975998

as altitude increases , the weight of air decreases , and the atmospheric pressure decreases . low atmospheric pressure means there is more space between air molecules . as a result , the number of oxygen molecules existing in the same volume of air deceases , so the amount of oxygen intake decreases in the uplands , even if the same amount of air is respired . that is , an increase in altitude reduces oxygen partial pressure in the pulmonary alveoli as well as the blood s ability to carry oxygen due to reduced atmospheric pressure , causing hypoxia . therefore , for an individual to consume the same amount of oxygen in the uplands as is consumed in the lowlands , the volume of air respired needs to increase1 , 2 . exposure to low oxygen due to altitude change acts as a powerful stimulus to induce diverse changes in the lungs3 . when exposed to upland environments , the densities of erythrocytes , hemoglobin , capillary vessels , and myoglobin in the skeletal muscles increase , which may improve the body s ability to transport oxygen4 . since cardiopulmonary function is improved and athletic performance is affected as a result of processes adapting to the characteristics of upland environments1 , upland training is widely used as a method to intensively reinforce physiological functions centering on cardiopulmonary function6 . since respiration supplies oxygen for the performance of activities of daily living ( adl ) , breathing disorders affect the performance of adl6 . that is , the efficiency of the respiratory function is closely related to that of the circulatory function , and is important not only for the performance of athletes , but also for the health of the general population . upland - related studies have mostly dealt with athletes training1 , 7,8,9,10 , and studies conducted among the general population living in the uplands are lacking . in korea , where approximately 70% of the country s area is mountainous , methods for improving respiratory function utilizing upland environments should be particularly useful . accordingly , the authors of this study examined the effects of alternate living experiences in the uplands and the lowlands on respiratory function and oxygen saturation in order to provide basic data for improving cardiopulmonary function exploiting altitude . the study subjects were 100 female students in the 1st through 4th grades of k university , which is located at an altitude of approximately 850 m. the study took place from march 27 to april 10 , 2013 . the mean age and height of the study subjects , comprising 25 freshmen ( 18.920.57 years , 163.044.25 cm ) , 25 sophomores ( 19.960.54 years , 161.525.45 cm ) , 25 juniors ( 21.040.35 years , 159.844.40 cm ) , and 25 seniors ( 22.120.67 years , 160.80 4.17 cm ) , were 20.51 years and 161.30 cm , respectively . the study subjects were selected from among those who had no history of smoking , and had no respiratory organ disease , or symptoms that would have affected their lung capacity . the study s objectives and procedure were explained to the subjects and individual consent to participate was obtained from all of them . the study was approved by the ethical committee of the kangwon national university hospital institutional review board . in korea , one school year is divided into two semesters and each semester consists of 15 or 16 weeks . except for the freshmen , the students who were the subjects of this study took classes at the school located at an altitude above sea level of approximately 850 m for approximately 30 weeks per year . these students spend at least 15 weeks per semester at the school located in the uplands ; they then go home to the lowlands during vacations . when this study was conducted , freshmen had been attending the school for approximately one month , and the students in the more senior grades had already experienced living arrangements alternating between the uplands and the lowlands . in this study , oxygen saturation , heart rate , and cardiopulmonary function were measured at the same time on the 8 floor of a building ( altitude at least 850 m ) located at the highest level of k university , and in the student life center located in the lowlands . oxygen saturation and heart rates ( beats per minute , bpm ) were measured using a heart rate and arterial blood oxygen saturation measuring instrument ( pulse oximeter , mp110p mek - ics co. , korea ) . the pulse oximeter was attached to the subject s middle finger with subjects and the values were measured in a stable state 68 times during five minutes . pulse oximetry is a non - invasive method , using an instrument that can monitor oxygen saturation easily and quickly , and indicate the measured values as spo2 . in this study , forced vital capacity ( fvc ) , forced expiration volume of 1 sec ( fev1 ) , peak forced expiratory flow ( pef ) , forced vital capacity of 1 sec were measured using cardiopulmonary measuring instruments ( microplus spirometer , uk carefusion co. ) , and the fev1/fvc ratio was calculated . when a subject arrived at the location where she was to be measured , she was instructed to rest for approximately five minutes to ensure that she was stabilized . then , oxygen saturation and heart rate were measured using the right index finger . demonstrations and explanations about the posture required were provided before measuring the cardiopulmonary functions , which were measured three times . vital capacity was measured using the method recommended in the chronic obstructive pulmonary disease treatment guidelines11 by referring to the standardized guidelines11 for the implementation and reading of pulmonary function tests published by the american thoracic society and the european respiratory society . when pulmonary functions were measured , the subject was instructed to look forward , standing in an upright position . the subject was instructed to maximally inhale and then exhale as quickly and strongly as possible for the longest time possible , while holding the nose firmly and biting the mouthpiece properly . to obtain better measurement results , the subject was encouraged by the researcher saying , more , more , more , hoo~. the subject was instructed to rest for approximately two seconds after each measurement so as not to record rebound measurements . when measured values could not be properly obtained , because the subject coughed or otherwise , the subject was instructed to rest , after which the measurement was repeated . the data were analyzed using spss 12.0 for windows by conducting one - way anova to test respiratory functions and oxygen saturation according to grade . this study was conducted to examine the effects of living in the uplands and the lowlands on respiratory functions and oxygen saturation . oxygen saturation , heart rate , and respiratory functions of students in different grades at different altitudes were examined . the freshmen showed statistically significant differences between the uplands and the lowlands in all items except pef and fev . heart rates were higher in the uplands , oxygen saturation was higher in the lowlands , fvc was higher in the lowlands , and fev1/fvc was higher in the uplands . significant differences in oxygen saturation were noted in sophomores and significant differences in oxygen saturation and pef were noted in juniors . on the other hand , there were no statistically significant differences in any of the items measured in the uplands and the lowlands among the seniors . therefore , it can be said that alternating living experiences in the uplands and the lowlands affects respiratory functions ( table 1table 1 . differences in oxygen saturation and pulmonary functions with altitudefreshmensophomoresjuniorsseniorsmsdupland hr82.8812.02 * 81.1611.0480.649.83**79.689.78lowland hr78.0410.23 * 76.8411.8575.927.63**80.1211.31upland saturation97.800.96**97.41.04**98.201.0097.881.05lowland saturation98.440.77**98.320.80**98.321.1897.961.24upland pef293.2074.61321.6067.01345.3559.70**356.7757.35lowland pef283.1084.11307.0462.84326.2762.70**356.1557.56upland fvc2.730.48**2.930.502.700.312.890.26lowland fvc2.980.48**2.980.532.660.392.860.38uplandfev2.590.512.690.422.620.282.750.23lowland fev2.650.462.690.402.570.352.700.30upland ratio92.136.60 * 92.097.6897.013.8295.375.40lowland ratio88.928.51 * 91.178.1596.735.3294.935.60*p < 0.05 , * * p < 0.01 ) . to examine the effects of alternating living experiences in the uplands and the lowlands on oxygen saturation , heart rate , and respiratory functions , differences in the values between grades in the uplands and the lowlands were examined separately ( table 2table 2 . differences in vital capacity according to gradeplaceitemgradenmsduplandoxygen saturation ( % ) 12597.800.96297.401.04398.201.00497.881.05hr ( bpm)12582.8812.02281.1611.04380.649.83479.689.78fev1 ( ml)1252.590.5122.690.4232.620.2842.750.23fvc ( ml)1252.730.4822.930.5032.700.3142.890.26pef ( ml)**125293.1874.602321.6067.003345.3559.704356.7757.40fev1/fvc ( % ) * * 12592.136.60292.097.68397.013.82495.375.40lowlandoxygen saturation ( % ) 12598.440.77298.320.80398.321.18497.961.24hr ( bpm)12578.0410.23276.8411.85375.927.63480.1211.31fev1 ( ml)1252.650.4622.690.3932.570.3542.700.30fvc ( ml)*1252.980.4822.980.5332.660.3942.860.38pef ( ml)**125283.0984.102307.0462.803326.2762.704356.1557.60fev1/fvc ( % ) * * 12588.928.51291.178.15396.735.32494.935.60*p < 0.05 , * * p < 0.01 , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume of 1 sec to forced vital capacity . ) . in the uplands , there were no significant differences in oxygen saturation and heart rate between grades . there were statistically significant differences in pef and fev1/fvc ( p < 0.05 ) . pef was shown to be the highest in seniors , followed by juniors , sophomores , and freshmen , in order of precedence , and fev1/fvc was the highest among juniors ( 97.013.82 ) and the lowest among sophomores ( 92.097.64 ) . in the lowlands , there were no significant differences in oxygen saturation and heart rate between grades . there were significant differences in fvc , pef , and fev1/fvc between the grades . fvc was shown to be the lowest among juniors ( 2.660.39 ) , pef was the highest among seniors ( 356.1557.60 ) , and fev1/fvc was the highest among juniors ( 96.735.32 ) and the lowest among freshmen ( 88.928.51 ) ( table 2 ) . * p < 0.05 , * * p < 0.01 * p < 0.05 , * * p < 0.01 , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume of 1 sec to forced vital capacity . cardiopulmonary functions by grade in the uplands and the lowlands were judged after dividing the subjects into lower grade students ( freshmen , sophomores ) , who had less experience of alternating living between the lowlands and the uplands , and upper grade students ( juniors , seniors ) , who had more experience . in the uplands , the cardiopulmonary functions of lower grade students and those of upper grade students showed statistically significant differences ( x = 0.014 ) . upper grade students showed only normal spirometry ( ns ) ( 70% ) and mild restriction ( mr ) ( 30% ) , and some lower grade students showed moderate restriction ( mor ) , mild obstruction ( mo ) , and moderate severe restriction ( mosr ) , indicating that they were experiencing cardiopulmonary function disorders ( table 3table 3 . assessment of on breathing disorders n(%)nsmrmormosrmomoototalupland*a29 ( 58%)10 ( 20%)5 ( 10%)2 ( 4%)4 ( 8%)0 ( 0%)50 ( 100%)b35 ( 70%)15 ( 30%)0 ( 0%)0 ( 0%)0 ( 0%)0 ( 0%)50 ( 100%)lowland**a30 ( 60%)7 ( 14%)0 ( 0%)2 ( 4%)10 ( 20%)1 ( 2%)50 ( 100%)b29 ( 58%)19 ( 38%)1 ( 2%)0 ( 0%)1 ( 2%)0 ( 0%)50 ( 100%)a : freshmen+sophomore , b : juniors+seniors , ns : normal spirometry , mr : mild restriction , mor : moderate restriction , mosr : moderate severe restriction , mo : mild obstruction , moo : moderate obstrction ) . in the lowlands , the cardiopulmonary functions of lower grade students and those of upper grade students showed statistically significant differences ( x = 0.005 ) . ns showed the highest proportion in both lower grade and upper grade students . in the lowlands , lower grade students showed ns , mo , mr , mosr , and moderate obstruction ( moo ) , while upper grade students showed ns , mor , mo , and mr ( table 3 ) . a : freshmen+sophomore , b : juniors+seniors , ns : normal spirometry , mr : mild restriction , mor : moderate restriction , mosr : moderate severe restriction , mo : mild obstruction , moo : moderate obstrction this study was conducted to examine the effects of alternating living experiences of the uplands and the lowlands on oxygen saturation and respiratory function in order to provide basic data for improving cardiopulmonary function exploiting altitude . in this study , there were differences in oxygen saturation , heart rate , and most respiratory function items between the uplands and the lowlands among the freshmen ; however , there were no differences in any of the items among the seniors . to take in the same amount of oxygen taken at sea level at 850 m in the uplands , the amount of ventilation would have to increase two - fold . to compensate for this therefore , long - term life in the uplands improves the blood s oxygen carrying capacity2 . this can explain the phenomenon that was apparent in the present study in which freshmen who had just began living in the uplands showed higher oxygen saturation and heart rates , increased fvc , and decreased fev1/fvc . seniors who had long - term experience of alternating life between the uplands and the lowlands did not show differences in any of the items . therefore , it can be said that alternating living experiences in the uplands and the lowlands affects pulmonary functions , even without continuous exposure to the upland environment . when exposed to the upland environment , and the associated low - pressure , low - oxygen environment , the heart rate increases to at a slightly higher level than in the lowlands due to the action of the sympathetic nerve and an increased energy metabolic rate . the heasr rate it returns almost to the original level , after adapting to the upland environment , after approximately one week12 . therefore , the reason why differences in oxygen saturation and heart rate between the grades did not appear in both the upland - and lowland - living students was that even the freshmen had started school at least three weeks earlier , and thus , all of the students had already adapted to their environment . in the uplands , pef and fev1/fvc were shown to be higher among upper grade students , who indicated smooth expiration . this indicates that upper grade students had better adaptability to the uplands than lower grade students . in the lowlands , fvc was shown to be higher among lower grade students , and pef and fev1/fvc were shown to be higher among upper grade students , compared to those from the uplands , indicating that the pulmonary functions of the upper grade students were better . therefore , even alternating living between the uplands ( during semesters ) and the lowlands ( during vacations ) , as opposed to continuous life in the uplands , appears to improve respiratory function . when an individual returns to the lowlands after training in the uplands , his / her maximum oxygen intake increases , aerobic enzymes are activated , mitochondria count increases , glycogen storage ability improves , capillary vessel density increases , hemoglobin concentration increases , and heart functions are activated13 . however , in this study , although there were differences in respiratory function tests between grades , there were no differences in oxygen saturation and heart rate , unlike the findings of the study conducted by bartsch and saltin13 . the degrees of respiratory functional disorders by grade in the uplands and the lowlands were judged after dividing the subjects into the lower grade students ( freshmen , sophomores ) who had less experience of alternating living between the lowlands and the uplands , and the upper grade students ( juniors , seniors ) who had more experience of doing so . in the uplands , the upper grade students showed only ns ( 70% ) and mr ( 30% ) , indicating that their breathing was normal or had slight limitations . on the other hand , some lower grade students showed moderate breathing restrictions , severe breathing restrictions , and mild breathing disorders . in the lowlands , lower grade students showed fewer breathing disorders compared to the uplands , although some had breathing disorders , and upper grade students showed more cases of slight restriction . this was due to large lung capacity , as living in the uplands did not seem to inhibit breathing . the maximum oxygen intake at 1,800 2,300 m corresponds to approximately 8085% of that at sea level and increases by approximately 25% after a 2- to 3-week adaptation periods . the fact that increases in the maximum oxygen intake in the uplands is not shown to be the same as those at sea level indicates the human body s adaptability to low oxygen environments . therefore , upper grade students having longer experience of exposure to upland environments are thought to have shown more breathing disorders in the lowlands than in the uplands because their bodies had adapted to the upland environments . kim et al.5 reported that cardiopulmonary functions improved after upland training . in that study , upper grade students who had been exposed to upland environments for a longer time generally showed better respiratory functions , as in our study . currently , physical strength and the body s abilities based on cardiopulmonary and respiratory functions are known to be insufficient among students as well as in the general population . both athletes and the general population could benefit from upland training . periodic living in the uplands is considered to be an effective method for improving respiratory function ; however , most people can not move on a regular basis . in this study , we were unable to determine the period of upland living required to influence an improvement in respiratory function ; therefore , further studies are necessary . through such studies , researchers may discover methods for improving cardiopulmonary function or breathing disorders exploiting natural environmental conditions , for athletes as well as for the general population .

[ purpose ] the purpose of this study is to examine the effects on respiratory function and oxygen saturation of intermittent life in the uplands at an altitude above sea level of approximately 850 m. [ subjects and methods ] the study subjects were on 100 female student subjects attending a university located in the uplands . the subjects oxygen saturation , heart rates and respiratory functions were measured at the university , which has campuses both in the uplands and the lowlands . [ results ] freshmen showed differences in oxygen saturation , heart rate , and most respiratory function items between the different altitudes ; however , seniors did not exhibit any differences . there were no differences in oxygen saturation and heart rate between the uplands and the lowlands either group . in the uplands , peak forced expiratory flow was shown to be high in the seniors , who also had better cardiopulmonary function . [ conclusion ] senior students , who had been exposed to the upland environment for a longer period of time , generally showed better respiratory function . therefore , alternating living between the uplands and the lowlands can be said to improve an individual s respiratory function .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4868236

triathlon is a challenging individual sport that involves the disciplines of cycling , swimming , and running ; these disciplines are combined in a continuous manner through two transitions1 . the ability to sustain high metabolic power for long periods is very important for athletes competing in triathlons . several field - based studies have shown that physiological and morphological functional characteristics change negatively with age2 , 3 . it is very important that triathletes have suitable anthropometric and physiological characteristics to advance performance in the three disciplines involved . some studies have found that anthropometric characteristics are associated with better performance , especially during the swimming and running components of triathlon competitions4 . the triathletes anthropometry was found to account for 47% of the variance in triathlon performance landers5 . it is also well known that endurance runners have low levels of body mass and that it is related to improved running performance6 , 7 . thus , success in a competitive sport is determined not by anthropometric characteristics alone , but by a combination of other characteristics such as physiological variables . a high maximal oxygen uptake ( vo2max ) is a physiological characteristic that plays an important role in the performance of triathletes in competitions . however , vo2max has not been shown to be a good predictor of triathlon performance in elite triathletes8 . in fact , the vo2max plateau duration has more correlation to triathlon performance than the vo2max value9 . other important physiological characteristics , such as anaerobic power and capacity , which are required during to perform sprints , overtake , break away , or follow with the group , are very important for the performance of triathletes , particularly at the end of the triathlon competitions10 . heart rate recordings have been used to assess maximal heart rate , minimum heart rate , and training intensity among sportsmen , particularly in endurance sports11 . heart rate variability ( hrv ) , which is analyzed from the heart rate recordings , provides information about the training load and psychophysiological status in endurance sports such as triathlons and cycling . a reduction in the high frequency component and an increase in the low frequency component of the power spectrum of hrv have been shown to relate with fatigue and training load12 , and it has been demonstrated that continued exposure to prolonged periods of intense cycling exercise ( about 3-week ) can cause a marked reduction in hrv indices in cyclists . thus , anthropometric characteristics may not be the only determinants of competitive athletic performance ; a combination of other physiological characteristics such as maximal oxygen uptake and anaerobic power and capacity may also play significant roles . the objective of the study was to compare of body composition , heart rate variability , aerobic and anaerobic performance between competitive cyclists and triathletes . after a 2-weeks pre - season training period for the participants in the aerobic and anaerobic performance tests , the anthropometric , body composition , and heart rate variability measurements were performed at a temperature - controlled performance laboratory in a public university . six cyclists ( age 32.33.0 yrs ; height 175.55.5 cm ; body mass 75.47.4 kg ; training experience 9.74.8 yrs ) and eight triathletes ( age 36.25.6 yrs ; height 181.64.1 cm ; body mass 74.33.8 kg ; training experience 10.62.9 yrs ) ( 14 males athletes in total ) who had participated for more than three years in regional and national competitions were recruited for the study . all the athletes were notified of the research procedures , requirements , benefits , and risks before obtaining informed consent . the study was approved by the research ethics committee of the local university and was conducted in a manner consistent with the institutional ethical requirements for human experimentation in accordance with the declaration of helsinki . standardized testing procedures were followed as defined in the american college of sports medicine guidelines ( acsm)13 . all measurements were performed by the same researchers after overnight fasting , at a similar time of the day - in order to have similar chronobiological characteristics14 . the temperature ( 2022 c ) and relative air humidity ( < 60% ) of the room were consistent throughout all the steps of the study . the subjects were asked not to exercise exhaustively on the day prior to assessment . the height and weight were measured for each volunteer while they were wearing light clothing and no shoes and socks . weight was measured to the nearest 0.1 kg using calibrated scales ( seca , germany ) , while the height was measured to the nearest 0.1 cm using a calibrated stadiometer ( holtain ltd , england ) . a gulick anthropometric tape ( holtain ) with an accuracy of 1 mm was used to measure the circumferences of the waist , hip , abdominal , thighs , and calves . to estimate the body fat percentage , a seven site skinfold thickness technique was used with a scientific skinfold caliper ( holtain , uk ) to the nearest 0.1 mm . the anatomical sites used were : the chest , abdominal , thigh , triceps , suprailium , subscapular , and iliac crest . body density was estimated using the equation that has been validated for males aged 18 to 61 years15 . the body density estimate was in turn used to estimate the body fat percentage using the siri equation : [ % body fat = ( 495 / body density ) 450 ] . fat mass was calculated by the transformation of the percent body fat values [ fat mass = ( body mass % body fat)/100 ] . lean body mass was determined by the fractionation of body mass into two components : lean body mass = body mass fat mass ) . body mass and body fat percentage were measured using bioelectrical impedance analysis ( tbf 401 a , tanita , tokyo , japan ) . two measurements were taken for each of these variables , and then the average values were used to perform the statistical analysis . all of the athletes completed an incremental treadmill test on a motorized treadmill ( cosmed , italy ) according to the procedures suggested by rossato et al.11 . during the test , the expired gases were analyzed using a breath - by - breath automated gas - analysis system ( oxycon mobile ; viasys healthcare , hoechberg , germany ) and heart rate was monitored continuously throughout the test with a polar s610 heart rate monitor ( polar , finland ) . mean hr and vo2 values measured during the test were used as maximum values ( hrmax and vo2max ) . heart rate variability ( hrv ) was measured using the omegawave sport technology system ( omegawave technologies , llc , portland , or , usa ) as described in the manufacturer s reference manual and standardized guidelines for the measurement of hrv . hrv was tested , the athletes voided their bladders and lay in a supine position for 15 minutes in silence with the lights dimmed ; the actual hrv recording took 67 min . before any strenuous activity16 . after hrv test , the wingate anaerobic test ( want ) was conducted using a mechanically braked cycle ergometer ( 834 e , monark , vansbro , sweden ) according to the procedures suggested by inbar et al.17 . during the test , the athletes were verbally encouraged to give the maximum effort possible . at the end of the test , the peak power and mean power were calculated automatically by the want test computer program . a fatigue index ( fi ) was calculated using the following equation17 : fi = [ ( peak power output - minimum power output ) / peak power output ] 100 . the data are reported as means and standard deviations . before using the parametric tests , the assumption of normality differences in the all tests performance variables between the cyclists and the triathletes were tested by the nonparametric wilcoxon signed rank tests for independent samples . effect sizes ( ) values of < 0.20 , 0.200.60 , 0.601.2 , 1.22.0 , 2.04.0 were considered to represent small , moderate , large , very large and extremely large differences , respectively18 . all statistical analyses were performed with the spss version 16.0 , and the level of statistical significance was set at p<0.05 . table 1table 1.anthropometric characteristics and body composition of the cyclists and triathletesskinfold thicknesstriathletes ( n=8)cyclists ( n=6)chest ( mm)10.14.310.33.6abdominal ( mm)17.92.719.23.2thigh ( mm)12.73.614.92.3triceps ( mm)9.91.912.63.7supraspinal ( mm)11.83.912 . 84.4subscapular ( mm)12.84.812.94.3iliac crest ( mm)7.91.99.72.6sum 7sf ( mm)83.223.292.524.1body fat ( % ) 17.73.219.33.2fat mass ( kg)13.22.814.63.2lean body mass ( kg)61.12.760.83.7 shows the anthropometric characteristics and body composition of the cyclists and triathletes . there were no significant differences between the anthropometric characteristics and body composition of the cyclists and triathletes . however , cyclists had a higher body fat percentage ( 17.719.3% , p=0.44 , =0.23 moderate effect ) and fat mass ( 13.214.6 kg , p=0.44 , =0.22 moderate effect ) compared to the triathletes . in addition , lean body mass was higher in the triathletes ; however , no significant difference was found ( 61.160.8 kg , p=0.89 , = 0.03 small effect ) . table 2table 2.aerobic performance of cyclists and triathletesincremental treadmill testtriathletes ( n=8)cyclists ( n=6)vo2max ( ml.kg.min)58.55.757.75.8hrmax ( b.min)187.18.1193.56.7hrresting ( b.min)59.67.360.79.5maximum power ( w)527.564.8516.662.1maximum power ( w.kg)7.10.96.80.8 shows the aerobic performance of the cyclists and triathletes studied . there were no significant differences in the aerobic performances responses of the cyclists and triathletes . however , triathletes demonstrated higher vo2max ( 58.557.7 ml.kg.min , p=0.85 , =0.07 small effect ) and maximum power ( 7.16.8 w.kg , p=0.21 , =0.14 small effect ) compared to the cyclists . in addition , hrmax was higher in the cyclists , but no significant difference was found ( 187.1193.5 b.min , p=0.09 , =0.39 moderate effect ) . table 3table 3.anaerobic performance of cyclists and triathleteswanttriathletes ( n=8)cyclists ( n=6)peak power ( w)796.474.6933.3189.5peak power ( w.kg)10.71.312.42.3average power ( w)586.945.8702.5139.3average power ( w.kg)7.90.89.31.7minimum power ( w)377.529.0471.790.9*minimum power ( w.kg)5.10.46.31.2*fatigue index ( % ) 52.44.149.32.2 * p the cyclists group showed statistically significant differences in minimum power ( 377.5471.7 w , p=0.02 , =0.57 moderate effect ) and relative minimum power ( 5.16.3 w.kg , p=0.02 , =0.55 moderate effect ) compared to the triathletes . table 4table 4.heart rate variability responses of cyclists and triathleteshrvtriathletes ( n=8)cyclists ( n=6)sdnn ( ms)50.216.857.220.9rmssd ( ms)35.718.842.224.2tp ( ms)1066.0860.7804.7756.4lf ( ms)643.4545.5537.0411.1hf ( ms)279.1314.8456.2420.6lf / hf4.33.72.43.0lfnu72.115.158.719.1hfnu27.915.141.219.1vlf ( ms)161.9202.2131.570.1sdnn : standard deviation of rr interval ; rmssd : root mean square of successive differences in rr intervals ; tp : total power ; lf : low frequency ; hf : high frequency components ; nu : normalized units ; vlf : very low frequency components shows the heart rate variability responses of the cyclists and triathletes studied . there were no significant differences in any heart rate variability responses between cyclists and triathletes . sdnn : standard deviation of rr interval ; rmssd : root mean square of successive differences in rr intervals ; tp : total power ; lf : low frequency ; hf : high frequency components ; nu : normalized units ; vlf : very low frequency components the aim of this study was to compare the body composition , heart rate variability , aerobic and anaerobic performances between competitive cyclists and triathletes . the study revealed no significant differences in the seven sites skinfold thicknesses , body fat percentage , fat mass , and lean body mass between cyclists and triathletes . moro et al.19 also found no differences in seven sites skinfold thicknesses in cyclists and triathletes , except for medial calf skinfold thickness . in addition , laursen et al.20 observed no significant differences in body mass or the sum of five skinfolds between cyclists and triathletes . furthermore , rst et al.4 found no differences in anthropometric characteristics and body composition between triathletes and cyclists . in contrast , millet et al.21 , found significant differences in height and body mass between competitive cyclists and triathletes . brunkhorst and kielstein7 , found that cyclists who were males had a higher bmi and larger thighs and were taller when compared to the male triathletes . in addition , landers et al.5 reported a strong and positive correlation between height and mass in elite triathletes who were males . these reported findings are similar to the results of the present study and indicate that anthropometric characteristics are similar in competitive cyclists and triathletes . previous researches have shown that the physical characteristics of athletes may play a role in determining performance . the participant s anthropometry was found to account for 47% of the variance in triathlon performances5 , and low levels of adiposity positively influenced swimming and running performance during triathlons4 , 22 . other performance and anthropometric characteristics studies have revealed that endurance runners have low levels of body mass and reduced skinfold thickness , which are related to improved running performance6 . given these findings , successful performance may not be determined only by physiological characteristics , but it may also be affected by a combination of anthropometric and physiological characteristics . knechtle et al.22 suggested that the anthropometry of triathletes is associated with training volume . the seven sites skinfold thickness , body fat percentage , and fat mass were lower in triathletes than in cyclists ( table 1 ) . these finding can be explained by the nature of triathlon training , because running training and overall training volume cause a reduction in the skinfold thickness of the lower limbs . the present study also showed that triathletes had higher relative vo2max values compared to cyclists . similarly , triathletes generally possess high vo2max values in studies , compared with cyclists in studies19 . we believe this might be because triathletes divide their training time into three disciplines ( swimming , cycling , and running ) and their training volume and overall training time are higher than that of cyclists . generally , vo2max values are reported as relative because of extra body mass affects running performance negatively . for example , costill et al.23 reported a relationship between both absolute and relative vo2max and running performance , and a stronger relationship was found for relative compared to absolute vo2max ( r=0.83 and 0.59 respectively ) . the specificity of each discipline can influence the aerobic performance ; cyclists achieve a higher performance than triathletes in cycling tests , whereas the opposite is observed in running tests24 . numerous studies have demonstrated that vo2max and wpeak value measured by treadmill are higher for triathletes compared to cycle ergometer25 . the present study also showed that triathletes had higher maximum power measured with treadmill compared to the cyclists maximum power measured by cycle ergometer . in similar with our result , mujika and padilla26 showed that wpeak value of 4403.3 w was reported for 14 elite cyclists , compared to a wpeak of 439 w for 24 professional cyclists . we believe that this difference might be because of the specific features of the individual sports , their requirements , and the different measurement tools . the wingate anaerobic test ( want ) is the gold standard for evaluating anaerobic capacity17 . there were no significant differences in absolute and relative peak and average power and fatigue index obtained on the want between the cyclists and triathletes , except for minimum power . in a similar study showed that triathletes and cyclists had lower peak power and mean power than we found in our results . furthermore , higher values of peak power and average power have been reported for cyclists , although similar statistical results to the present study have been obtained in the literature19 . according to the sport - specific requirements , anaerobic power and capacity are more important for cyclists than triathletes . a cycling race is characterized by power maintained for prolonged periods of time and high peak power for short periods of time for sprints8 . in contrast , triathletes perform fewer sprints during the cycling race than cyclists , since a higher demand on the anaerobic energy system during cycling may accelerate the process of muscle fatigue , and reduce the performance of the triathletes during running . however , it is believed that anaerobic fitness is important for the performance of triathletes , since the intervals in short - distance triathlon races permit the athletes to perform sprints when they overtake , break away , or follow with the group and when they pick up speed again1 . in addition , a previous study demonstrated a relationship between running speed and heart rate from low to submaximal speeds27 . one limitation of the present study is the fact that we monitored hrv for 7 min . prolonged recordings ( 24-hour ) for hrv is a better assessment to reflect training load and to collect psychophysiological information for this study . in conclusion , the anthropometric characteristics , aerobic and anaerobic profile , and heart rate variability were similar in triathletes and cyclists . triathletes demonstrated higher vo2max , power outputs and fatigue indexes , suggesting that the specificity of their training caused different anthropometric , psychophysiological , and physiological adaptations in triathlon and cycling . these parameters could provide further information about the anthropometric and psychophysiological profile of cyclists and triathletes .

[ purpose ] the aim of this study was to compare the body composition , heart rate variability , and aerobic and anaerobic performance between competitive cyclists and triathletes . [ subjects ] six cyclists and eight triathletes with experience in competitions voluntarily participated in this study . [ methods ] the subjects body composition was measured with an anthropometric tape and skinfold caliper . maximal oxygen consumption and maximum heart rate were determined using the incremental treadmill test . heart rate variability was measured by 7 min electrocardiographic recording . the wingate test was conducted to determine anaerobic physical performance . [ results ] there were significant differences in minimum power and relative minimum power between the triathletes and cyclists . anthropometric characteristics and heart rate variability responses were similar among the triathletes and cyclists . however , triathletes had higher maximal oxygen consumption and lower resting heart rates . this study demonstrated that athletes in both sports have similar body composition and aerobic performance characteristics .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4259491

obesity increases the risk for hypertension and is a major driver of morbidity and mortality due to cardiovascular diseases ( dustan , 1983 ; poirier et al . , 2006 ) . studies in rodents with diet - induced obesity ( dio ) suggest that increased sympathetic nerve activity ( sna ) is an important mediator of obesity - induced hypertension as and adrenergic receptor antagonists and renal denervation significantly blunt the rise in blood pressure ( bp ) associated with weight gain ( carlyle et al . , 2002 ; however , the precise molecular and neural mechanisms that link changes in weight with changes in bp have not been fully elucidated . circulating concentrations of the adipocyte - derived hormone leptin increase in proportion to adipose tissue mass and fall with weight loss ( considine et al . , we hypothesized that leptin may be involved in coupling changes in body weight ( bw ) to changes in bp . leptin regulates energy homeostasis by acting on hypothalamic neuronal circuits expressing the signaling isoform of the leptin receptor ( lepr ) to reduce calorie intake and increase energy expenditure ( halaas et al . , 1997 ; leptin can increase sna , leading to increases in bp and heart rate ( hr ) ( haynes , 2000 ; mark et al . , 1999 ) . in the arcuate nucleus of the hypothalamus ( arh ) , leptin stimulates the expression of pro - opiomelanocortin ( pomc ) and increases the activity of pomc neurons , which release the pomc peptides ( , , and melanocyte - stimulating hormones [ mshs ] ) that act on melanocortin 4-receptor ( mc4r)-expressing neurons in the paraventricular nucleus of the hypothalamus ( pvh ) and other brain regions to increase sna ( cone , 2005 ; cowley et al . , 1999 , 2001 ; however , pomc neurons become unresponsive to leptin in obesity , and leptin can act independently of mc4r signaling ( enriori et al . , 2011 ; patterson et al . , 2011 ; scott et al . , 2009 ) . therefore , leptin s effects beyond the melanocortin circuits need to be investigated . the dorsomedial hypothalamus ( dmh ) is critical for leptin s ability to regulate brown adipose tissue ( bat ) temperature and the cardiovascular system ( enriori et al . , 2011 ; ; rezai - zadeh et al . , 2014 ) . here , we investigated the development of obesity - induced hypertension . we demonstrate that , in dio mice , increasing levels of leptin directly lead to an increase in hr and bp and that blocking the actions of leptin reverses these effects via neural circuits originating in the dmh . furthermore , humans with loss - of - function mutations in leptin and its receptor have normal bp despite severe obesity , suggesting that these mechanisms are likely to be preserved in humans . the temporal association between weight gain , changes in circulating leptin levels , hr , and bp were first examined . four - week - old c57bl/6j mice on a chow diet were implanted with radiotelemetric bp probes ; baseline measurements were recorded at 6 weeks . as bw increased ( figure 1a ) , plasma leptin levels also progressively increased ( figure 1b ) . after 4 weeks of hfd , hr became significantly elevated ( figure 1c ) and remained elevated throughout the 20 week recording period compared to chow fed mice . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were significantly elevated after 12 weeks of hfd ( figures 1d and 1e ) , subsequent to the rise in plasma leptin concentration . when hfd - fed mice were returned to a chow diet following 20 weeks of hfd feeding , mice lost 3.9 g in the first week , 3.9 g in the second week , 3.4 g in the third week , and 0.4 g in the fourth week ( figure 1a ) . dbp reduced after 1 week ( figure 1e ) , and sbp ( figure 1d ) and hr ( figure 1c ) reduced after 2 weeks of chow feeding . these findings suggest that leptin appears to increase before hr and bp increases in dio , effects that are reversed with weight loss . we examined whether the effects seen in dio mice are dependent upon leptin signaling , using mice lacking leptin ( ob / ob ) or the lepr ( db / db ) . hfd - fed ob / ob and db / db mice were significantly ( p < 0.001 ) heavier than hfd - fed dio and chow - fed lean , ob / ob , and db / db mice ( figure 2a ) . despite the increased severity of obesity in the ob / ob and db / db mice , only dio mice exhibited elevated hr , sbp , and dbp ( figures 2b2 g ) , demonstrating that increased bw alone is not the cause of the increase in bp in obesity . there was a greater difference in hr and bp between dio mice to other mice during the dark period compared to the light period ( figures s1a s1c available online ) . the increase in cardiovascular parameters in dio mice was not due to increased activity ( figures s1d and s1e ) . strong correlations were found in c57bl/6j mice between plasma leptin concentration and hr ( figure 2h ) , sbp ( figure 2i ) , and dbp ( figure 2j ) . no correlation was found between plasma insulin concentration and hr , sbp , or dbp ( figures s1g s1i ) . to examine the direct effect of leptin on bp , leptin - deficient ob / ob mice were treated for 11 days with either leptin or vehicle . after 11 days of treatment , the average plasma leptin concentration of ob / ob mice treated with leptin was 27.7 2.9 ng / ml compared to 36.3 4.9 ng / ml in dio mice and 6.6 1.1 ng / ml in lean mice ( figure s1f ) . exogenous leptin treatment in ob / ob mice progressively decreased bw ( figure s1 m ) and food intake ( fi ) ( figure s1n ) compared to leptin - treated db / db mice and vehicle - treated ob / ob mice . despite this decrease in bw , leptin treatment increased hr ( figures 2k and 2l ) and sbp ( figures 2 m and 2n ) in leptin - treated ob / ob mice compared to leptin - treated db / db mice and vehicle - treated ob / ob mice . dbp also increased in ob / ob leptin - treated mice , however , not to the same extent as sbp ( figures 2o and 2p ) . the magnitude of the increase in hr and bp in leptin - treated ob / ob mice decreased with time , coincident with their substantial reduction in bw . the 24 hr circadian rhythm of hr ( figure s2j ) , sbp ( figure s2k ) , and dbp ( figure s1l ) of leptin - treated ob / ob mice was increased toward the baseline recordings of dio mice . peripheral administration of a leptin antibody into dio mice for 5 days caused a significant reduction in hr ( figure 3a ) , sbp ( figure 3b ) , and dbp ( figure 3c ) , although no change in bw was observed ( figure 3d ) . central administration of a leptin receptor antagonist ( lra ) into the lateral ventricle ( lv ) of hypertensive dio mice over a 7 day period significantly reduced the elevated hr ( p < 0.001 ; figure 3e ) . by the seventh day of lra treatment , hr was comparable to the average hr of lean animals ( figures 3f and s2a ) . systolic bp also progressively decreased when dio mice were treated with the lra , and by day 5 of lra treatment , sbp was significantly ( p 0.05 ) decreased compared to vehicle - treated dio mice ( figure 3 g ) . by the seventh day of lra treatment , sbp was significantly ( p < 0.05 ) lower compared to the sbp at baseline in dio mice and was comparable to the baseline sbp of lean mice ( figures 3h and s2b ) . dbp also decreased throughout the lra treatment period ( figures 3i , 3j , and s2c ) . these changes were seen despite the absence of a significant change in fi ( figure 3k ) , indicating that these changes are mediated by blocking the effects of leptin signaling rather than through changes in fi and bw . to determine whether leptin s effects on bp and hr were mediated by neurons in the dmh , the lra was injected directly into the dmh of hypertensive dio mice daily over 7 days . lra treatment in the dmh decreased hr ( figures 4a , 4b , and s3a ) and sbp ( figures 4c , 4d , and s3b ) by day 7 of treatment . no significant change in dbp ( figures 4e , 4f , and s3c ) was observed . furthermore , injection of an aav expressing a short hairpin rna directed against the lepr ( hommel et al . , 2006 ) in the dmh of dio mice led to a sustained decrease in sbp ( figures 4 g , 4h , and s3d ) after 4 weeks . utilizing mice in which the lepr is flanked by loxp sites and can be deleted by an aav cre , hfd for 20 weeks induced increased sbp at baseline , whereas aav cre administration into the dmh ( hence deleting the lepr in only the dmh regions ) decreased sbp ( figure 4i ) . additionally , we examined the effects of reactivation of the lepr by injection of an aav - expressing cre recombinase into the dmh of normotensive morbidly obese lepr null mice ( expressing a loxp flanked stop codon in front of the lepr , called lepr transcriptional blocker or both hr ( figure 4j ) and sbp ( figure 4k ) increased rapidly after lepr expression was reactivated in just the dmh of obese lepr - deficient mice . to determine the electrophysiological effect of leptin on dmh neurons , we recorded the electrical activity of neurons expressing lepr in lepr - cre - yfp mice ( leshan et al . , 2012 ) . application of 100 nm leptin induced membrane depolarization and/or an increase in spontaneous action potential firing rate in 38.2% of recorded neurons ( figure 5a ) . leptin induced a mean peak amplitude depolarization of 4.9 1.0 mv from a mean resting potential of 49.9 3.1 mv to a new steady - state membrane potential of 45.0 2.7 mv ( p = 0.0004 ) . leptin - induced excitation was associated with an increase in firing rate from a mean control basal level of 0.48 0.28 hz to 0.64 0.34 hz in the presence of leptin ( n = 5 ) , effects that were at least in part reversible after washout of leptin ( p = 0.59 ) . in two neurons , voltage - current relations , generated in response to a range of depolarizing and hyperpolarizing rectangular - wave current pulses ( 150 to + 100 pa , 1,000 ms duration ) revealed that leptin - induced excitation was principally associated with a trend for a decrease ( p = 0.86 ) in neuronal input resistance from 841 111 m in the absence to 743 138 m in the presence of leptin . plots of the voltage - current relations revealed extrapolated reversal potentials for leptin - induced excitation around 35 mv ( figure 5b ) . taken together , these data suggest that leptin - induced excitation is mediated via activation of one or more nonselective cation conductances . in addition to these effects on membrane input conductance , leptin also induced modulation of intrinsic subthreshold active conductance in a subpopulation of dmh neurons . in dmh neurons , membrane depolarization from negative holding potentials ( < 65 mv ) or depolarization at the offset of the membrane response to hyperpolarization from potentials close to resting potential ( 45 to 50 mv ) evoked a transient depolarizing potential consistent with activation of a low - threshold t - type calcium conductance . in the presence of leptin , this potential was prolonged ( figure 5b ) , the half - time to decay increasing from 132 59 ms in the absence to 179 61 ms in the presence of leptin . these data are consistent with leptin modulating intrinsic active conductances in a subpopulation of dmh neurons . in addition to these postsynaptic effects , leptin induced an increase in spontaneous excitatory postsynaptic potentials ( epsps ) in a subpopulation ( n = 2 ) of dmh neurons ( figure s4b ) . the mean frequency of spontaneous epsps increased from 0.07 0.02 hz to 0.31 0.11 hz in the presence of leptin . these epsps could be of sufficient magnitude to reach threshold for firing , suggesting that indirect presynaptic effects of leptin on dmh neurons can contribute to leptin - induced increases in neuronal excitability . in vivo , the effects of directly altering the neuronal activity of lepr - expressing dmh neurons were assessed using engineered pharmacologically selective chimeric ion channels for activating and silencing neuron activity ( magnus et al . , 2011 ) . briefly , this technology requires the injection of a virus , which only infects and replicates in a cre - dependent manner , into cre - expressing mice . the virus drives the cre - dependent expression of a modified ion channel , containing a psam element . after injection ( intraperitoneal injection ) , the otherwise inert molecule psem binds to an ion channel that contains a psam element , which opens the ion pore and allows ion flux across the cell membrane , which depolarizes or hyperpolarizes virus - infected , cre - expressing neurons . using male 20-week - old chow - fed lean lepr - cre - yfp transgenic mice ( leshan et al . , 2012 ) , we investigated whether activation of dmh lepr - expressing neurons could increase hr and bp . lean lepr - cre - yfp mice were injected bilaterally with activator virus , an engineered ionotropic serotonin receptor packaged in an aav2 ( psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ) into the dmh . in lean mice 21 days after virus injection , twice daily intraperitoneal ( i.p . ) administration of the receptor ligand psem ( i.p . 2x daily [ 5 mg / kg ] ) for 3 days significantly increased hr and bp ( p 0.05 ; figures s4d lepr - cre - yfp mice , an inhibitory virus , an engineered ionotropic glycine receptor packaged into an aav2 ( aav2 : psyn::flex - rev - psam l141f : glyr - ires - gfp ) was administered bilaterally into the dmh . compared to vehicle treated mice , psem acutely decreased hr ( figures 5c and 5d ) and sbp ( figures 5e and 5f ) , but no significant change in dbp was observed ( figures 5 g and 5h ) . sub - chronic daily treatment with psem reduced hr ( by 72.6 5.6 bpm ) , sbp ( by 6.1 1.7 mmhg ) and dbp over 3 days ( figures 5i , 5j , and s4c ) , effects which were reversed once psem treatment ceased . treatment of mice with psem , prior to virus administration had no effect on hr or bp ( figures s4 g and s4h ) . cumulatively , these findings indicate that leptin signaling is required for the changes in bp seen in dio and that lepr expressing neurons in the dmh are necessary and sufficient to , cause these effects . these data also support the premise that the hypertension induced by leptin in the dmh is due to leptin - induced depolarization of dmh neurons . homozygous complete loss - of - function mutations in the gene encoding leptin ( lep ) are associated with undetectable leptin levels and severe early - onset obesity in humans ( montague et al . , 1997 ) . we measured bp in the fasted rested state in eight leptin - deficient children and 42 equally obese controls of the same age with normal leptin levels for the degree of obesity studied in the same conditions ( table s1 ) . sbp was significantly lower in leptin - deficient children compared to age- and bmi - matched controls ( figure 6a ; p < 0.05 ) ; there was no difference in dbp . a statistically significant attenuation of sbp was also seen in severely obese children who were homozygous for complete loss - of - function mutations in the lepr gene ( n = 12 ) compared to 48 age- and bmi - matched controls ( figure 6b ; p < there were no significant differences in resting hr between the groups ( data not shown ) . administration of recombinant methionyl human leptin to individuals with congenital leptin deficiency leads to significant weight loss ( farooqi et al . , 1999 ; ozata et al . , 1999 ) . in a previously reported experimental paradigm ( galgani et al . , 2010 ) , three leptin - deficient adults were studied before and after treatment with recombinant leptin for 19 weeks , which was sufficient to cause 15.5 0.5 kg weight loss . in parallel , three age- and bmi - matched controls were studied before and after a diet and exercise intervention , which achieved a comparable degree of weight loss ( 14.8 1.76 kg ) . the three adult leptin - deficient individuals were found to have normal bp despite severe obesity ( figure 6c ) . whereas the map of the obese control group decreased as expected ( figure 6d ) , the bp of the leptin - deficient adults did not change . similarly , there was no statistically significant change in bp after recombinant leptin administration to leptin - deficient children ( data not shown ) . these studies in rare individuals completely lacking leptin or functioning lepr support the assertion that leptin is necessary for the increased bp associated with obesity in humans . cumulatively , these studies demonstrate that leptin signaling is necessary for obesity - induced increased bp . we have used multiple convergent methods , including making lean mice obese , by adding leptin systemically and by restoring lepr to the dmh of lepr - deficient mice . in all these studies , bp increased . similarly , we have studied reduced leptin signaling in leptin- and lepr - deficient mice , neutralized circulating leptin with a systemic antibody , infused lepr antagonist icv and intra - dmh ; used shrna to knock down lepr expression ; selectively deleted lepr ; and inhibited lepr - expressing neurons in the dmh of obese hypertensive mice . in all of these experiments , reducing leptin signaling reduced bp , even in the presence of obesity . clinical studies in severely obese humans with two different forms of defective leptin signaling show that these observations are relevant to human physiology and pathophysiology . leptin can acutely increase hr , bp , and bat , even in anesthetized animals , presumably through activation of the sns ( enriori et al . intracerebroventricular ( icv ) leptin increases sna to numerous organs , including the lumbar , kidney , and bat regions ( haynes et al . , 1999 ) . central antagonism of lepr caused a reduction in bp and hr in dio hypertensive mice . although early lesion studies confirm the importance of the dmh for the control of energy homeostasis , little is known about the neurochemical phenotype of the neurons present in the dmh ( elmquist et al . , 1999 ; the dmh appears to have a critical ability to control thermogenesis and is involved in elevated bat - mediated thermogenesis found in obese mice ( enriori et al . , 2011 ; fan et al . , 2005 ; morrison et al . , 2008 ) . in the studies conducted with antagonism or knocking down of the lepr in dio mice these results suggest that the dmh lepr - expressing neurons are not essential for leptin s effects on bw because the mice lost no additional weight when leprs were inactivated . although lesion studies have previously demonstrated a critical role of the dmh in the control of bw , we have shown that long - term knockdown of the lepr does not significantly affect bw , despite the significant reduction in bp and previously reported increase in bat temperature in obesity ( elmquist et al . , 1999 ; enriori et al . , 2011 ) . additionally , acute activation or inhibition of lepr - expressing neurons did not change fi or bw . contrary to our expectations , we did not observe an increase in bp with the administration of leptin to leptin - deficient patients . first , the magnitude of leptin s effect on bp may be too small to be detected given the sensitivity of the tools for measurement of bp in humans and the small number of individuals studied . notably , despite weight loss with continued leptin administration , we did not see a significant decrease in bp in these patients as would be expected in more common forms of obesity . these effects are comparable to the effects of leptin on total energy expenditure ( tee ) , another phenotype mediated by sympathetic tone . we have previously shown that , in contrast to weight loss in obese controls where tee decreases , tee does not change with leptin administration in leptin deficiency ( galgani et al . , 2010 ) . whether leptin increases bp ( to a small degree ) or leptin - deficient individuals respond differently to weight loss can not be deduced from our data . however , our results are in line with the suggestion that the effect of leptin administration may be to prevent the reduction in bp expected in congenitally leptin - deficient individuals as they lose weight . in previous trials of leptin in common obesity , no effects on bp were observed ( heymsfield et al . , 1999 ) . rosenbaum and leibel have shown that controlled 10% weight loss in an experimental setting is sufficient to reduce leptin levels and is associated with decreased sna ( rosenbaum et al . , 2005 ) . it is possible that leptin could be producing some of its cardiovascular effects by acting peripherally . lepr are expressed throughout the brain , including the brainstem ; additionally , lepr are expressed on cardiac myocytes , the kidney , and arteries , including the coronary arteries ( knudson et al . we treated dio mice peripherally with a leptin antibody and then in a separate experiment , dio mice were treated centrally with the lepr antagonist , and in both experiments , similar responses were observed . in mice lacking the lepr , re - expression of the lepr in only the dmh caused a dramatic increase in bp and hr . the opposite effects occurred when the leptin receptors were deleted from only the dmh region in hypertensive mice . thus , although lepr expression in peripheral regions may play a role , the results presented here clearly demonstrate a key role for the dmh lepr - expressing neurons in mediating the changes in bp in obesity . experiments in rodents and humans support a role for melanocortin signaling in the regulation of bp . central administration of -msh increases bp and hr in wild - type mice , but not in mc4r mice , which maintain a normal bp despite severe obesity ( kuo et al . , 2003 heterozygous loss - of - function mutations in mc4r are associated with a reduced prevalence of hypertension , low sbp , lower urinary noradrenaline excretion , and reduced peripheral nerve sna ( greenfield et al . , 2009 ; sayk et al . , 2010 ) . moreover , systemic administration of a centrally active melanocortin receptor agonist acutely increased bp in obese volunteers ( greenfield et al . , 2009 ) . as such , some of leptin s effects on bp may be mediated by the melanocortin system . however , there are some indications that , in obesity , the pomc neurons in the arh become nonresponsive to leptin ( this has been termed leptin resistance ) , and this may limit how much the actions of leptin can be transduced by the pomc neurons in the arh ( enriori et al . , 2007 ; knight et al . , 2010 ; previous research has implicated the pomc neurons of the arh in the regulation of obesity - induced hypertension via activation of the ikk/nf-b pathway ( purkayastha et al . , 2011 ; zhang et al . , how pomc neurons interact with the dmh lepr - expressing neurons has not been addressed in these studies , but the dmh lepr - expressing neurons do send direct efferent projections to arh neurons ( gautron et al . it is possible that the lepr - expressing neurons in the dmh express mc4r or that the dmh lepr - expressing neurons act through other neurons that express mc4r ( liu et al . , 2003 ) . the degree to which leptin s effects on bp in obesity are dependent upon intact melanocortin signaling remains to be determined ( do carmo et al . , 2011 ; harlan et al . dmh lepr - expressing neurons are known to project to numerous brain regions , including the pvh ( elmquist et al . , 1998 ; gautron et al . dmh neurons also project to other nuclei , including the raphe pallidus nucleus ( rpa ) and rostral ventrolateral medualla ( rvlm ) ( cao and morrison , 2003 ; horiuchi et al . microinjection of leptin into the dmh of anesthetized rats can induce an acute increase in hr and bp ( marsh et al . , 2003 ) . the dmh - rpa connection has already been recognized to be important in the regulation of bat thermogenesis in response to leptin ( zhang et al . , 2011 ) . the rvlm , however , appears to have a greater control over the regulation of bp , compared to regulation of thermogenesis and hr ( horiuchi et al . the neurochemical phenotype of the lepr - expressing neuron populations in the dmh is still debated ( lee et al . , 2012 ) . further studies will be necessary to characterize the dmh circuits that contribute to leptin s effects on blood pressure and to characterize the mechanisms by which these neurons modulate sympathetic outputs to the heart and peripheral blood vessels . although results here strongly demonstrate the role of leptin in contributing to elevated bp in obesity , a number of other hormones that change in response to weight gain could contribute . as expected , we found that , compared to lean mice , plasma insulin was elevated in all obese models . despite this increase in plasma insulin , it was only dio mice that exhibited significantly elevated bp , and insulin levels were not correlated with hr or bp , suggesting that insulin contributes little to the chronic elevation of bp seen in obesity . also , insulin sensitivity as measured by hyperinsulinemic euglycemic clamps is comparable in human mc4r deficiency versus obese controls despite a lower bp and reduced urinary catecholamine excretion in mc4r - deficient subjects ( greenfield et al . , 2009 ) . therefore , our data do not support a role for insulin in mediating obesity - induced increases in bp . in conclusion , these observations suggest that pharmacological approaches based on the modulation of leptin s effects on specific subpopulations of neurons could represent a potentially useful therapeutic strategy for the treatment of obesity - associated hypertension and for the prevention of some obesity - associated cardiovascular disease . all mice were housed in a controlled environment in which lights were on a 12 hr light/12 hr dark cycle ; temperature and humidity remained constant . in experiments to examine the development of hypertension , 4-week - old male c57bl/6j mice were implanted with radiotelemetry probes ( dsi , usa , model ta11pa - c10 ) . these mice were allowed 2 weeks recovery postsurgery and , following baseline recordings , mice were spilt onto either chow ( 4.8% of fat , mouse and rat rodent chow diet , specialty feeds , glen forrest , australia ) or hfd ( 43% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 20 weeks ( 140 days ) in which recordings were taken every 13 to 15 days . after 20 weeks ( 140 days ) , the hfd fed mice were swapped onto a chow diet ( 4.8% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 4 weeks . in all other experiments , male c57bl/6j , leptin - deficient ( ob / ob ) , lepr - deficient ( db / db ) , lepr - deficient ( leprtb ( berglund et al . , 2012 ) , lepr flox , and lepr - cre - yfp mice ( leshan et al . , 2012 ) were placed on either a chow diet or hfd diet at 4 weeks of age and continuing for 20 consecutive weeks , after which mice were used for experiments . specific experimental procedures for radiotelemetry , pharmacological studies , genetic manipulations , and electrophysiological studies are detailed in the supplemental information . all human studies were conducted according to the principles outlined in the declaration of helsinki and after approval by local ethical committees . systolic and diastolic bp were measured in the rested , fasted state using wrist bp monitors ( omron healthcare , hamburg , germany ) in leptin - deficient ( n = 8) and leptin - receptor - deficient children ( n = 12 ) ( farooqi et al . , 2002 , 2007 ) . these control subjects had been tested for mutations in leptin , leptin receptor , and mc4r . control subjects were age and bmi matched to leptin - deficient and leptin - receptor - deficient subjects ( n = 42 and 48 , respectively ) ( wheeler et al . , 2013 ) . leptin - deficient adult patients received pretreatment testing at the pennington center , along with weight- and bp - matched control subjects . the leptin - deficient patients then received a 3 month treatment of recombinant leptin ( leptin [ amgen inc . , thousand oaks ; galgani et al . , 2010 ] ) . leptin levels were administered a low - calorie diet ( galgani et al . , 2010 ) for 920 weeks to cause the same weight loss and also returned to the pennington center for posttesting . the study design was approved by the ethics committee of the pennington biomedical research center . approval for leptin replacement included ucla irb approval ( april 9 , 2001 ) and fda approval ( ind application number 61690 ) . p values were calculated by either unpaired or paired two - tailed student s t test , one - way anova with bonferroni post hoc test or two - way anova with bonferroni post hoc test . p < 0.05 , p < 0.01 , and p < 0.001 . extended experimental proceduresradiotelemetryradiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia . all mice received 2 weeks recovery before any baseline recordings were measured.chronic leptin infusionchow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made . alzet 14 day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30 g / per day in 6 l of solution [ 1.25 g/0.25 l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period . pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation . pumps where removed after 11 days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11).immunohistochemistry and radioimmunoassayimmunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et al . , 2007).peripheral neutralization of endogenous leptinto neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period . injections of 24 g in 90 l of antibody solution or 90 l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et al . , 2004 ) . all mice where randomly treated.central blockade of endogenous leptinto reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5 l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et al . , 2011 ) . in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2 mm long 22 gauge , implanted 0.5 mm posterior and 1 mm lateral to bregma , 2 mm below the surface of the skull ) or dmh cannula ( plastics one , 4 mm long , 26 gauge , implanted 1.9 mm posterior , 0.4 mm lateral to bregma , 4 mm below the surface of the skull ) as described previously ( enriori et al . , 2011 ) . following a 7 day recovery period , mice where all injected for 5 days twice daily with sham acsf injection ( 0.5 l ) into cannulas . mice then received acsf or the lra , injected directly into cannulas , twice daily for 7 days , immediately following the dark period and immediately prior to the dark period . following the 7 day injection period mice where monitored daily for a 7 day recovery period.chronic knockdown of leptin receptors in the dorsomedial hypothalamusdio mice were implanted with radiotelemetry probes and after 14 days of recovery baseline recordings mice were injected bilaterally into the dmh region ( coordinated 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) 0.5 l of either an aav scrambled shrna or aav leprshrna over a 20 min period each side ( hommel et al . , 2006 ) . bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period.reactivation and deletion of leptin receptor in the dmhlepr loxtb mice and lepr flox mice ( berglund et al . , 2012 ) after which mice were implanted with radiotelemetry probes . following the surgery ( 2 weeks ) , baseline recordings where measured . mice where then injected into the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with an aav cre virus . mice where then monitored and cardiovascular parameters measured throughout.neuron activation and silencing with psam / psem systemsto examine the effect of activation or inhibition of dmh lepr expressing neurons , we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et al . , 2012 ) . after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons . the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et al . , 2011 ) . these mice where placed on either the chow or hfd for 20 weeks then implanted with radiotelemtry probes and 2 weeks later bp radiotelemetry baseline recordings were made . following this the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) . injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons . after a 21 day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5 mg / kg , i.p . , twice daily ; apex scientific inc . stony brook , ny ) or vehicle , randomized , crossover design ( magnus et al . , 2011).electrophysiologyexperiments were undertaken on lepr - yfp - cre mice ( leshan et al . , 2012 ) following 20 weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250 m thick ) cut using a vibratome ( leica , vt1000s , uk ) . slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording . for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3 7.4 . lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier . patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate , the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose . data were filtered at 2 5 khz , digitized at 2 10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software . analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) . was determined using unpaired , non - parametric student s t test with p < 0.05 considered as significant . leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording . radiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia . chow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made . alzet 14 day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30 g / per day in 6 l of solution [ 1.25 g/0.25 l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period . pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation . pumps where removed after 11 days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11 ) . immunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et al . , 2007 ) . to neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period . injections of 24 g in 90 l of antibody solution or 90 l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et al . , 2004 ) . all mice where randomly treated . to reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5 l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et al . , 2011 ) . in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2 mm long 22 gauge , implanted 0.5 mm posterior and 1 mm lateral to bregma , 2 mm below the surface of the skull ) or dmh cannula ( plastics one , 4 mm long , 26 gauge , implanted 1.9 mm posterior , 0.4 mm lateral to bregma , 4 mm below the surface of the skull ) as described previously ( enriori et al . , 2011 ) . following a 7 day recovery period , mice where all injected for 5 days twice daily with sham acsf injection ( 0.5 l ) into cannulas . mice then received acsf or the lra , injected directly into cannulas , twice daily for 7 days , immediately following the dark period and immediately prior to the dark period . following the 7 day injection period mice where monitored daily for a 7 day recovery period . dio mice were implanted with radiotelemetry probes and after 14 days of recovery baseline recordings were measured . in a stereotaxic apparatus ( stoelting , usa ) mice were injected bilaterally into the dmh region ( coordinated 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) 0.5 l of either an aav scrambled shrna or aav leprshrna over a 20 min period each side ( hommel et al . , 2006 ) . bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period . lepr loxtb mice and lepr flox mice ( berglund et al . , 2012 ) after which mice were implanted with radiotelemetry probes . following the surgery ( 2 weeks ) , baseline recordings where measured . mice where then injected into the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with an aav cre virus . mice where then monitored and cardiovascular parameters measured throughout . to examine the effect of activation or inhibition of dmh lepr expressing neurons , we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et al . , 2012 ) . after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons . the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et al . , 2011 ) . these mice where placed on either the chow or hfd for 20 weeks then implanted with radiotelemtry probes and 2 weeks later bp radiotelemetry baseline recordings were made . following this the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) . injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons . after a 21 day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5 mg / kg , i.p . , twice daily ; apex scientific inc . stony brook , ny ) or vehicle , randomized , crossover design ( magnus et al . , 2011 ) . experiments were undertaken on lepr - yfp - cre mice ( leshan et al . , 2012 ) following 20 weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250 m thick ) cut using a vibratome ( leica , vt1000s , uk ) . slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording . for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3 7.4 . lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier . patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate , the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose . data were filtered at 2 5 khz , digitized at 2 10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software . analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) . was determined using unpaired , non - parametric student s t test with p < 0.05 considered as significant . leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording .

summaryobesity is associated with increased blood pressure ( bp ) , which in turn increases the risk of cardiovascular diseases . we found that the increase in leptin levels seen in diet - induced obesity ( dio ) drives an increase in bp in rodents , an effect that was not seen in animals deficient in leptin or leptin receptors ( lepr ) . furthermore , humans with loss - of - function mutations in leptin and the lepr have low bp despite severe obesity . leptin s effects on bp are mediated by neuronal circuits in the dorsomedial hypothalamus ( dmh ) , as blocking leptin with a specific antibody , antagonist , or inhibition of the activity of lepr - expressing neurons in the dmh caused a rapid reduction of bp in dio mice , independent of changes in weight . re - expression of leprs in the dmh of dio lepr - deficient mice caused an increase in bp . these studies demonstrate that leptin couples changes in weight to changes in bp in mammalian species .

Introduction Results Discussion Experimental Procedures Author Contributions

PMC5177562

although the exact embryological mechanism for this condition remains unclear , it is thought to relate to a disruption of development of the lateral folds of rathke during hindgut development.1 several anatomical variants have been described , and the classification system proposed by effman , lebowitz , and colodny has been widely adopted2 ( fig . type 1 : blind ending channels or incomplete duplication type 1a : a blind ending channel opening on the dorsal or ventral surface of the penis in the midline , without communicating with either the bladder or urethra . type 2 : patent and complete duplication type 2a : two urethral meati ( which may open anywhere along the midline ) . 2a - ii : the accessory urethra divides from the main urethra and maintains a separate course 2a- ii y - type : the ventral urethra opens in the perineum type 2b : the two urethrae unite and form a single channel before opening at the skin type 3 : urethral duplication associated with caudal duplication ( i.e. , duplication of the bladder ) some of these lesions may be totally asymptomatic ; obviating the need for surgery . however , the more complex variants may require multiple operations to obtain functionality and these interventions entrain future risks of incontinence and stricture.3 4 5 in this case we describe the management of a urethral duplication with two hypospadic meati in a 21-month - old boy . a 27-week premature boy was referred to the pediatric urology clinic at the age of 9 months for assessment of a proximal hypospadias . he had a complicated neonatal course ; spending 11 weeks in the neonatal intensive care unit , initially requiring a ventilator . he had neonatal complications of jaundice requiring phototherapy , and necrotizing enterocolitis , which was managed medically . however , closer inspection revealed what appeared to be a second urethral meatus opening within the glans ( fig . ( a ) preoperative picture ( the arrows show the position of the two urethral meati ) . ( b ) ( d ) follow - up 21 months postoperatively . a preoperative contrast study ( fig . 3 ) revealed two distinct urethral channels originating as a single channel from the bladder neck and then running separately by definition an effman type 2a - ii . surgery was performed at the age of 21 months : the challenge was the presence of a hypospadic rudimentary dorsal urethra , chordee , and a hooded foreskin ( fig . 2b ) , the dorsal urethra was opened in the midline from the meatus to the level of the proximal urethral ending . a micturating cystourethrogram demonstrating a single distinct channel arising from the bladder , splitting into two ( arrowed ) . the bridge between proximal urethra and duplicated urethra was divided and a urethra - urethroplasty was performed a complete distal urethral tubularization , layered closure , and glanuloplasty resulted in a single , glanular urethral opening ( fig . 2c ) . a urinary catheter was left in place for 1 week and then removed without complication . at follow - up 21 months postoperatively , there was a good cosmetic result with no postsurgical complications ( fig . 2d ) . the mother reported that her son was passing urine with a thick and straight stream with no evidence of fistula . various theories to explain the origin of a urethral duplication have been suggested , with different types of classification proposed,6 7 8 the most widely adopted being that proposed by effman.2 of the cases of urethral duplication reported in the literature , type i lesions are thought to be the most common , although since these are generally asymptomatic , their true incidence is unknown.2 there have been cases reported that are associated with a single hypospadic urethra , commonly associated to an atretic accessory channel distally , such as in type 1a lesions.9 10 the type 2 a - ii group is characterized by the presence of a second urethra which divides from the main urethra , and maintains a separate course . this group includes a subgroup of patients in whom the ventral urethra opens in the perineum ( y - type duplication ) ( fig . the ventral urethra opened at the base of the shaft , thus representing a variant that could be considered an intermediate between the classic type 2 a - ii and the y - type duplication . the ventral urethra was the more functional , while the dorsal urethra was less developed . several established techniques have been described for the correction of a duplicated urethra.5 11 12 a general consensus is that each patient should be considered individually , with no standard fit - all approach really being suitable for all cases of a particular lesion.13 some authors would tend toward using the orthotopic urethra , even if it is hypoplastic ; ortolano and nasrallah first proposed progressive urethral dilation to achieve adequate caliber.14 we believe , as suggested by salle et al , that after dilatation of the accessory urethra the risk of inadequate urine flow is high.10 therefore in the case described , we opened the dorsal urethra and performed a tubularization over a 8 fr catheter , as in the classic repair for hypospadias . the use of a voiding cystourethrogram is typically adequate to trace the course and caliber of the two channels , however , retrograde contrast may be required in cases ( as the one presented ) with a hypoplastic urethral opening.15 postsurgical complications tend to manifest as stricture at the anastomosis or related to the hypoplastic channel . there have been reported cases of fistula formation much like that documented in penoscrotal hypospadias repair.5 15 we believe this case of duplicated urethra is unique for the particular anatomy of the two urethral channels , with the main urethra being the one opening at the base of the shaft . a distal blind pit is common in hypospadias and should always prompt thorough examination for accessory urethra , in particular , in proximal hypospadias cases . this report demonstrates a new variant of urethral duplication of which pediatric surgeons should be aware . in cases of proximal hypospadias , the surgeon should consider the possibility of a distal accessory urethra , which may require consideration when planning surgery . this case highlights that this unusual variant can be successfully managed in a single - stage procedure with good functional and cosmetic results .

duplication of the urethra is a rare congenital anomaly , with approximately 300 cases reported in the literature . we report a unique case of this condition in a male infant . this case differs from the classical effman type ii - a2 duplication because of the presence of two hypospadic urethral meati , as opposed to a ventral or dorsal accessory meatus with a normally positioned distal urethra . the patient underwent a single - stage repair consisting of a proximal urethra - urethral anastomosis and distal urethral tubularization at 21 months of age with excellent results in terms of both function and cosmesis .

Introduction Case Report Discussion New Insights and Importance for the Pediatric Surgeon

PMC3870652

the ageing process is accompanied by an increased prevalence of the signs and symptoms of physical fragility , including sarcopenia , a decrease in exercise tolerance , osteopenia , an increase in visceral adiposity and a worsening of quality of life . adults with growth hormone deficiency ( ghd ) exhibit similar clinical signs , but it is possible to treat them with gh to ameliorate these signs and symptoms . the gh secretion declines gradually with age , whereby studies demonstrate a progressive reduction of 14% secretion per decade of life beginning in the second decade [ 2 , 3 ] . these findings suggest a possible association between ghd and the ageing process [ 4 , 5 ] . the association between the physiological alterations of the ageing process and the reduction of gh secretion is called somatopause . gh replacement is well known to improve body composition , leading to a decrease in total body fat and an increase in lean body mass . however , gh replacement has only shown an effect on muscle strength in gh - deficient adults subjected to long - term gh therapy . few studies have evaluated the effect of gh replacement on muscle strength in elderly people engaged in a program of exercise training [ 2 , 710 ] . specific training that aims to increase muscle strength can potentiate the anabolic effect of gh , which leads to a greater impact on body composition in comparison to gh replacement alone [ 11 , 12 ] . these subjects showed increased exercise capacity with improvements in oxygen uptake and ventilatory threshold after gh therapy . this improvement was most likely due to some combination of increased muscle strength , improved body composition , and improved thermoregulation . gh has been speculated to improve physical capacity in subjects without ghd through stimulation of collagen synthesis in the tendon and skeletal muscle , which leads to better exercise training and increased muscle strength . administration of supraphysiological doses of gh to athletes increases fatty acid availability , reduces oxidative protein loss , particularly during exercise , and increases lean body mass . our purpose was to evaluate the effect of gh therapy on muscle strength in healthy , nonsedentary men over 50 years old . healthy and nonsedentary men ageing 5070 years were recruited by newspaper advertisements with information that patients were needed for a study of hormonal evaluation . exclusion criteria were pituitary disease , gh use in the last 12 months , severe acute disease , hepatic and/or renal chronic disease , uncontrolled systemic arterial hypertension , diabetes mellitus , psychiatric disorders , history of cancer , nontreated hypogonadism , and the presence of any other disease that could interfere with the somatotrophic axis . all patients practiced some kind of exercise ( minimum of three times per week , aerobic and resistance exercise ) to be eligible to be included in the study . twenty - nine subjects were screened for a project about somatotrophic profile in healthy men over 50 years old ( which includes this paper ) and have resulted in other published studies [ 15 , 16 ] . after that , fourteen from the 29 initial patients agreed to participate in this part of the project that included the evaluation of muscle strength in gh x placebo replacement ; those subjects who gave up had personal problems , such as work compromises and address change . all patients signed the informed written consent approved by the ethics committee of the clementino fraga filho hospital / ufrj . at baseline , subjects were submitted to evaluation of gh secretion , testosterone level , body composition , and muscle strength . absence of pituitary disease and normal testosterone levels were necessary for the inclusion in the study . gh secretion was evaluated through insulin tolerance test ( itt ) or glucagon stimulation test ( gst ) and igf - i levels . the itt was performed by intravenous injection of regular insulin at a dose of 0.10.15 iu / kg . blood samples were drawn every 30 minutes from baseline until 120 minutes for the determination of glucose and gh levels . all patients had hypoglycemia ( glucose nadir lower than 40 mg / dl ) during the test . severe ghd was considered when gh peak was lower than 3 g / dl , and it was an exclusion criterion . g / l and the intra - assay coefficient of variation ( cv ) was 5.3% at 1.7 g / l . serum igf - i concentrations were determined by an immunoradiometric assay ( kit dsl-5600 ) , with acid - ethanol extraction . the minimal detection limit was 0.80 ng / ml and the intra - assay was 3.4% at 9.38 ng / ml and 3.7% at 255.8 ng / ml . all subjects had gh peak at gst higher than 3 g / l and had igf - i levels normal according to their age ; that is , none of them had severe ghd ( table 1 ) . subjects were then randomised according to their arrival to the medical evaluation : one patient to each group ( gh and placebo groups ) , sequentially . the gh group received an initial dose of 0.5 ui / day ( 0.2 mg / day ) , with readjustments to 1.0 ui / day ( 0.4 mg / day ) and 1.5 ui / day ( 0.6 mg / day ) after 1 and 2 months of treatment , respectively . these gh dosages were based on the dose used for adults of the same age with ghd at the time of this research . subjects were reevaluated after 6 months of gh therapy or placebo according to the parameters above . pfizer laboratory also provided the placebo . during treatment , every month the patients should take their used gh flask to the medical consult in order to realize a counting flask for control of the correct use of gh . body mass index was calculated using quetelet 's formula ( weight / height ) and the percentage of body fat was calculated using the adipometer on seven cutaneous folds ( tricipital , subscapular , suprailiac , pectoral , axillary , abdominal , and thigh ) according to the pollock and jackson protocol . abdominal circumference was measured using a nonelastic tape at the greatest diameter between the last ribs and the iliac crests . muscle strength was evaluated for the maximum strength based on the concept of maximum repetition ( 1 mr ) ( i.e. , the maximum load that can be performed using the correct technique for an exercise ) . the exercises evaluated were bench press and leg press using a righetto machine ( so paulo , brazil ) , which are dynamic exercises . the bench press focuses on the strengthening of the pectoralis major muscle as well as other muscles including anterior deltoids , serratus anterior , coracobrachialis , scapulae fixers , trapezzi , and the triceps . the bench press was performed according to the following technique : ( a ) initial position : dorsal decubitus , elbows in extension with hands sustaining the barbell , knees and hips semiflexed , and feet on the equipment base ; ( b ) exercise development : elbows flexion , to form a 90 degree angle with the arm and forearm , followed by complete extension of the elbows and horizontal flexion of the shoulders , returning to the initial position . the leg press works the following muscles groups : quadriceps , hamstring , gluteus maximus , and calves ( partially ) . the leg press was performed according to the following technique : ( a ) initial position : hips and knees flexed at a 90 degree angle , feet on the platform , and inferior members slightly separated and lined up within the limits of the iliac crests ; ( b ) exercise development : complete extension of the knees then returning to the initial position . before the use of the equipment , subjects could familiarise themselves with the exercises and the equipment without the load . to determine the load associated with 10 mr , each subject initially performed 10 repetitions of each exercise with a submaximum load , which was considered a load that was possible to mobilise . then , the loads were progressively increased and , with a maximum of 3 trials , the load of 10 mr was reached . if the 10 mr load was not reached after 3 attempts , the subject was asked to come to the laboratory for another opportunity . to reduce the error margin , the following strategies were adopted : ( a ) standardised instructions were given before the test to make the subjects aware of the data collection routine ; ( b ) subjects were instructed on the execution technique of the exercises ; ( c ) the examiner was attentive to the correct position of the patients ' joints at the time of measurement to avoid small variations in joint positioning that could put action on other muscles and lead to erroneous interpretations of the obtained scores ; and ( d ) verbal stimuli were given during the exercise to maintain a high level of motivation . during the 10 mr test , a 25 minute interval was provided between the attempts of each exercise . after the load was obtained for a determined exercise , intervals comparisons between the groups and timepoints were performed using two - way anova for repetitive measures , followed by fischer post - hoc evaluation if necessary . the differences between the baseline and the last measure ( delta ) were compared by student 's t - test for independent variables . thirteen subjects completed the study ( 6 subjects in the placebo group and 7 subjects in the gh group ) . one subject in the placebo group dropped out of the study because of the development of arthralgia . subjects of both groups were similar in age , weight , height , and bmi at baseline ; they were overweight according to bmi ( between 2530 kg / m ) , except one subject who was obese ( bmi 38.5 kg / m)table 1 . all subjects of both groups were nonsedentary ; that is , they performed planned physical activity for more than 150 minutes per week . after 6 months of therapy , the evaluation of the gh and placebo groups did not show evidence of significant differences in weight , bmi , waist , and the sum of the cutaneous folds ( triceps , biceps , subscapular , and iliac folds ) . furthermore , there were no differences on the delta ( 6 months baseline ) for these parameters between the groups ( table 2 ) . muscle strength in the upper body part , as evaluated by supine horizontal rising , did not increase after 6 months in both groups . muscle strength in the lower body part , as measured by the leg press , showed a statistically significant increase in the gh group when compared to placebo ( table 2 ) . the effects of ageing cause undesirable changes in body composition , a reduction of bone mineral density and muscle strength ( with greater falling risk ) , and worsening exercise capacity , which is associated with a decrease in gh and igf - i levels . however , gh replacement to minimise the adverse effects of ageing must be based on cost - benefit and risk - benefit analysis . furthermore , randomised studies demonstrated that the effectiveness of gh replacement is modest , either on its own or in combination with sex steroids or exercises [ 2 , 4 , 7 ] . our study evaluated muscle strength in men over 50 years of age after 6 months of gh therapy , and we observed a statistically significant increase in leg press responsive muscles ( quadriceps being the mainly muscle evaluated ) when compared to placebo . there was no evidence of alterations in body composition in either group ( gh therapy or placebo ) . the gh group had a bmi and body fat percentage ( represented by the sum of the cutaneous folds ) slightly greater than the placebo group but without statistical significance . therefore , our results differed from those found in the literature , in which an improvement in body composition and no difference on muscle strength during gh replacement are described [ 7 , 9 ] . the effects of gh therapy on body composition ( increased lean body mass and decreased fat mass ) are very well reported [ 1 , 1924 ] , but the effects on muscle strength are unclear , which was one of the objectives of our study . it has already been reported that recombinant human gh administered for 6 months to healthy older men decreases the percentage of body fat and increases lean body mass and igf - i levels to values observed in young adult males . a recent study using a low - dose sustained - release recombinant human gh administered during 26 weeks to subjects with the so - called somatopause showed improvements in body composition and quality of life . many studies in ghd adults demonstrated the normalisation of muscle strength after 10 years of gh replacement [ 6 , 26 , 27 ] but did not show an increase in muscle strength with the use of gh for short periods of time . however , a study of 18 elderly men submitted to progressive weight training for 14 weeks and then randomised to receive either gh or placebo during a further 10 weeks of strength training showed that gh had no effect on muscle strength at any time , but lean body mass increased and fat mass decreased in the gh group . frail older people subjected to gh treatment with or without a structured resistance exercise program had a significantly increase in muscle strength in both gh / exercise as well as the exercise alone groups , but there was a significant increase in the proportion of type 2 fibers at the end of the study in the combined gh / exercise treated subjects . there are few studies in the elderly that combine gh replacement and specific training for muscle strength [ 7 , 8 ] . this type of training could potentiate the anabolic effects of gh , which would cause a greater impact on body composition than gh replacement alone as well as greater impact on muscle strength . gh has been speculated to improve physical capacity in subjects without ghd through its stimulation of collagen synthesis in the tendon and skeletal muscle . furthermore , gh appears to be more important for the reinforcement of the tissue matrix than for muscle cell hypertrophy . thus , the gh use could attenuate or prevent muscle fibre injuries , causing a lower frequency of training disruption and leading to greater training in athletes or nonathletes with the use of gh , which leads to a muscle fibre hypertrophy and consequently an increase in strength . this could explain the increase in leg press responsive muscles in the gh group of the present study . although no participants of our study were athletes or performed supervised physical training before and during the gh x placebo use , none of the subjects were considered sedentary and all practiced some type of exercise ( minimum of three times per week150 minutes per week , aerobic and resistance exercises ) . we can postulate that this group , even without supervised training , had fewer injuries and consequently performed more exercises to increase muscle strength . moreover , the majority of subjects realized walk and running as exercise , which prioritizes the lower body part muscles . another point that corroborates the gain of muscle strength in this group is the documented evidence that adults with ghd have lower fatigue sensation when subjected to gh replacement , which increases the adherence to exercise and leads to cardiopulmonary benefits . although there are conflicting results regarding adults without ghd , it could be another fact that increased the disposition to exercise and consequently to an increase in lower body part strength in subjects in the gh group . the median age of the subjects in our study was lower than the studies cited above . although our objective did not include evaluation of the gonadic axis , subjects were required to have normal serum levels of testosterone to be included in the study . thus , we can attribute the observed amelioration of strength in leg press responsive muscles to the gh therapy , independent of gonadic function . another point is that the majority of the studies were performed for periods less than 6 months , and this could be an explanation for the different results obtained in our study . another consideration is the gh dosage used in our study ; we used a fixed dosage with increases in the first and second months , independent of the patient 's weight . the final gh dosage ( 0.6 mg / day ) was the median dosage used for adults ( men with an average age of 50 years ) with ghd at the time that our study began . however , at the present data , gh dosages are lower in ghd adult replacement ( initial dose of 0.10.3 mg / day ) . we can not discard a pharmacologic effect of this higher gh dosage on muscle strength . although anthropometry is an important method of nutritional evaluation in subjects over 50 years of age , one limitation of our study is that we did not use a more precise method of body composition evaluation . fat tissue redistribution is more prominent in women , and reduction in body water content is more common in men . in addition , the lack of references for anthropometric measures in the brazilian population can reduce the precision of such evaluations . the use of dual energy x - ray absorptiometry ( dexa ) would improve evaluation of modifications in body composition , mainly lean mass . even still , the results could still be vulnerable to error because dexa is not able to estimate body water accumulation caused by gh therapy . the bmi was demonstrated to be well correlated with anthropometric indicators of nonvisceral fat ( tricipital and subscapular folds ) and abdominal fat ( abdominal circumference ) , in addition to being in direct relation with total body fat mass [ 33 , 34 ] . the fact that we did not find a change in weight in the study group despite an improvement of strength in leg press responsive muscles might suggest that there was an increase in muscle mass and a decrease in fat mass . our study differs from the literature in the following parameters : ( 1 ) age ( our population was younger ) ; ( 2 ) exercise training ( subjects in our study were not considered sedentary and did some type of exercise but not a specific training ) ; ( 3 ) gh dose ( similar to the dose used for ghd adult men at the time of the study ) ; and ( 4 ) longer duration of gh use ( 6 months ) . our study demonstrated an increase in leg press responsive muscles in men over 50 years old after gh therapy , which is important for reducing falls and fractures in aged populations . this finding must be considered and tested in frail older populations , whose physical incapacity is caused primarily by proximal muscle weakness . considering that ageing is related to loss of strength in lower body part and a worsening quality of life , our results can be taken into account for future studies with higher numbers of subjects . however , we must emphasise that gh therapy has been absolutely contraindicated for aesthetic or antiageing reasons until now .

growth hormone ( gh ) use has been speculated to improve physical capacity in subjects without gh deficiency ( ghd ) through stimulation of collagen synthesis in the tendon and skeletal muscle , which leads to better exercise training and increased muscle strength . in this context , the use of gh in healthy elderly should be an option for increasing muscle strength . our aim was to evaluate the effect of gh therapy on muscle strength in healthy men over 50 years old . fourteen healthy men aged 5070 years were evaluated at baseline for body composition and muscle strength ( evaluated by leg press and bench press exercises , which focus primarily on quadriceps lower body part and pectoralis major upper body part muscles , resp . ) . subjects were randomised into 2 groups : gh therapy ( 7 subjects ) and placebo ( 7 subjects ) and reevaluated after 6 months of therapy . thirteen subjects completed the study ( 6 subjects in the placebo group and 7 subjects in the gh group ) . subjects of both groups were not different at baseline . after 6 months of therapy , muscle strength in the bench press responsive muscles did not increase in both groups and showed a statistically significant increase in the leg press responsive muscles in the gh group . our study demonstrated an increase in muscle strength in the lower body part after gh therapy in healthy men . this finding must be considered and tested in frail older populations , whose physical incapacity is primarily caused by proximal muscle weakness . the trial was registered with nct01853566 .

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

PMC3062949

humans ( indeed all biological multicellular organisms ) are made of multiscale hierarchy of structures ranging from subcellular structures ( 10 m ) to cells ( 10 m ) , basic functional units ( the smallest aggregation of diverse cells that behaves like the parent organ 10 m ) , organs ( 1010 m ) , and bodies ( 10 m ) . in addition to the local scale variation , biological structures are also characterized by shape . for example , the blood vessels are tubular objects interconnected into a complex network and have a range of structural scale ( 5 m diameter capillaries to 3 cm aorta ) . this large - scale range presents two major problems ; one is just making the measurements , and the other is the exponential increase of component numbers with decreasing scale . quantitative analysis of such systems , for example , blood vessel trees and networks of neurological dendrites and axons , seem to be best measured from 3d images of those structures . with the remarkable increase in the volume imaged by , and resolution of , modern day imagers , the practical problem now is the extraction of this multiscale data from those large , detailed image data sets . thus , there is a need for an automatic tool which mines data across both space and scale to capture local information about the objects which describes the feature and now becomes associated with its appropriate position and scale . without prior information for a scale description of the image content , an image has to be studied at all scales . the basis for design of an automatic tool for such description could be derived from human perception model . the human eye comprises a large number of individual receptors ( over 150 million rods and cones ) . such imager has no prior information about input , and therefore , it is designed to extract the information by applying sampling apertures at a wide range of sizes simultaneously [ 1 , 2 ] . since the information from a single individual sensor is almost meaningless , the sampling should be done not by the individual rods and cones in a human eye or detectors in imagers but by the sensor neighborhoods . such sensor neighborhoods implement fundamental multiscale perception at different scales simultaneously but at this point no memory or analysis is involved yet . the very first level of analysis starts when grouping and storing the information from the local neighborhoods into meaningful sets ( so - called blurring signal ) and establishing interconnections between neighborhoods . since a priori size of the features in the object ( signal ) is unknown , the blurring is used by both humans and machines for adaptive multiscale representation of meaningful signal features of a different size . within the next step when we extract an object , certain properties have been already attributed to it , and the rest of the information is now considered as nonobject and often called noise or as both noise and object are always parts of the perception process , there is no way to separate the noise from the object if models of the object and of the noise are absent . to distinguish noise from object , we need to model the image content . that could be achieved by using certain mathematical operators , feature detectors , interacting with the data followed by analysis of the results of feature extraction . since objects in images comprise features with varying size , it is quite logical to perform feature extraction at the scale which matches the local feature size , its native scale . a recipe for automatic feature extraction in multiscale framework can be given as follows : ( i ) build multiscale representation by smoothing the image at each scale ; ( ii ) choose an appropriate feature detector to compute local structural properties ( e.g. , gradients , curvatures , flows ) ; ( iii ) compute local extrema of a feature detector response function ; ( iv ) find the strongest local response of the structural properties which is considered as feature identification . to build the multiscale representation of the image , the proper aperture ( windowing ) function as an operator should be chosen . formalism for the scale - space representation was introduced by witkin and further developed by koenderink . the idea of this approach is to generate a one parameter family of smoothed images i(x , y ; t ) obtained by convolving the original image i0(x , y ) with a gaussian kernel g(x , y ; t ) of size t ( 1)i(x , y;t)=i0(x , y)g(x , y;t),where g(x , y;t)=12te(x2+y2)/2 t . in case of gaussian kernels , the kernel size t is called variance , which is related to standard deviation as t = . the parameter t in this family represents the scale at which the finer image structures are still perceptible whereas the spatial structures with size smaller than t= will be smoothed out as shown in figure 1 . as pointed out by koenderink and hummel , this family of smoothed images may also be derived as the solutions of the diffusion equation ( 2)i(x , y;t)t=[c(x , y;t)i(x , y;t)]=c(x , y;t)i(x , y;t)+c(x , y;t)i(x , y;t ) . if c(x , y ; t ) is constant , it reduces to the isotropic diffusion equation i(x , y ; t)/t = ci(x , y ; t ) , and the linear spatial scale - space representation can be generated using gaussian ( continuous ) or binomial ( discrete ) kernels [ 24 , 610 ] . data sampled in the temporal domain ( e.g. , the movie frames are samples taken at regular intervals ) can also be scaled in similar to the spatial domain fashion . to treat a multiscale context over the temporal domain , perona and malik extended the scale - space concept to nonlinear scale - spaces based on nonlinear diffusion formulation with nonconstant diffusion coefficient c(x , y ; t ) ; ( see ( 2 ) ) . comprehensive overview of nonlinear scale - spaces based on parabolic partial differential equations could be found in [ 15 , 16 ] . for such nonlinear cases , the bessel scale - space can be built . recently , wavelets and their applications to signal and image processing have attracted attention of the scientists in many fields . a very good collection of papers on the wavelet theory and its applications can be found in the book by heil and walnut . the relationship between the wavelets and the scale spaces was demonstrated by mallat and hwang . in [ 2022 ] , methods for generating scale - space representations based on wavelets were suggested , and the results of application of wavelets were very promising . as an extension of the gaussian and wavelet approaches , wang and lee proposed the scale - space representation derived from b - splines . even though different kernel functions have been proposed to generate the scale - space representations , the gaussian kernels remain the best candidates so far [ 110 ] . such uniqueness of the gaussian kernels was shown by babaud et al . and is based on a priori scale - space constraint formulated by witkin , koenderink , and yuille and poggio : no new feature points ( no spurious detail ) should be created with increasing scale . [ 25 , 26 ] extended constraints and formulated the mathematical requirements known as axioms for an uncommitted visual front - end which should be satisfied for the systems without any a priori knowledge about inputs : ( i ) linearity : no feedback from the system ; ( ii ) spatial shift invariance : no preferred spatial or temporal location ; ( iii ) isotropy : no preferred spatial or temporal orientation ; ( iv ) scale invariance : no preferred size or scale of the aperture . another motivation to use the gaussian scale spaces has some support from neurophysiological and psychological experiments which has shown that receptive field profiles in the mammalian retina and visual cortex can be well modeled by sums of gaussian and gaussian derivative components [ 2931 ] . all these properties make the linear gaussian scale spaces the best choice for development of the automatic unsupervised systems for multiscale signal and image analysis , when there is no in advanced information available concerning preferable scales . the recipe which allows adaptively choosing the proper local scale parameter at every geometrical location was suggested by lindeberg [ 32 , 33 ] . this recipe comprises a two - step procedure : ( i ) convolving the original image i0(x , y ) with a gaussian kernel g(x , y ; ) of size ; ( see ( 1 ) ) and ( ii ) analyzing a response r(x , y ; ) function from derivatives or some ( possibly nonlinear ) combination of derivatives of that convolution ( 3)r(x , y;)=m+ni(x , y;)xmyn , where m and n are orders of derivatives . the strongest response of such function ( with respect to ) over scales then indicates the proper gaussian scale probe ( gaussian observation kernel ) probe with width corresponding to object feature size obj has been found . due to the commutative properties of the convolution and taking derivative operations , the order of operations in the procedure above could be changed to convolving the original image i0(x , y ) with the operators constructed from the derivatives of gaussian ( so called gaussian derivatives ) . the distinguishing characteristic of such operators is a combination of the opposing properties , localization , and optimal response to noise [ 3537 ] . to demonstrate this principle , we modeled the original image with an object with gaussian intensity profile of kernel size obj = 5 and convolved this image with the first- ( figure 2 ) and second- ( figure 3 ) order gaussian derivatives with kernel sizes varying over the range probe = 115 ( in the figures only probe = 1 , 5 , 10 are shown ) . then , we measured intensity values of response functions ( see ( 3 ) ) , across the object and extracted the strongest responses for all scales of the gaussian probe . the strongest responses to both first and second gaussian derivatives with kernel probe varied over the range probe = 115 and convolved with the original image with feature size obj = 5 are shown in figure 4 . these plots demonstrate the property of the response functions such that it has the strongest response when gaussian probe size approaches the object size . unfortunately , the amplitude of gaussian derivative operators tends to decrease with increasing scale due to the fact that with increasing scale , the response is increasingly smoothed . this gives more preference to smaller scales . to compensate such increase and , thus , improve accuracy of the automatic scale selection , lindeberg [ 10 , 32 , 34 ] suggested using the so - called -parameterized normalized derivatives ( 4)m+numvn=(m+n)m+nxmyn . this method of scale selection allows feature detectors to find such points in the image that the -normalized operator response has an extremum with respect to both position and scale . in the case of tubular - like structures in an image , ridge detection with automatic scale selection can be done using a second derivative of gaussian kernel function [ 38 , 39 ] . depending on values of the -parameter , the detected object features can be quite different . analyzing the influence of the -parameter on feature detection with automatic scale selection , lorenz et al . chose to be 1.5 which worked well for variety of intensity line profiles ( e.g. , gaussian , bar- , triangle - like ) . however , for elongated structures with bar - like intensity profiles ( such intensity profiles can be found in high - quality imagers with narrow point spread functions [ 41 , 42 ] or imagers that use deconvolution preprocessing algorithms [ 43 , 44 ] ) , the ridge detector creates false responses at small scales ( basically these are edge responses at small scales ) as depicted in figure 5 . the line intensity profiles demonstrating the problem are shown in figure 6 . since in automatic approaches there is no the preferred scale ( all scales should be treated equally ) in an image , a number of solutions have been proposed to avoid or suppress these false responses in scale space . koller et al . suggested applying a nonlinear operator that combines the response of two edge - detectors on both sides of a hypothetical ridge . lorenz et al . used an edge - indicator to suppress the response to edges . lindeberg used a hybrid approach taking the useful properties from both the scale - space height ridge and the second derivative scale - space ridge . while studying the problem of the influence of the -parameter on feature detection with automatic scale selection , majer [ 38 , 39 ] derived the g - normalization parameter value from the statistical approach based on a white noise sampling model . in these studies , he concluded that the ridges generated by a second - order gaussian derivative operator do not suffer from the false responses to edges if the value = 1.25 is used . early vision perception occurs at all scale simultaneously and can be modeled by generating the image scale - space representation that introduces an additional variable , spatial scale size [ 14 ] . at this point , as soon as the local scales are established , the early analysis of an image starts with analysis of intensity variations and directions by means of spatial derivatives to reveal local image structure . for example , for elongated objects , the derivative value along the object is close to zero whereas the derivatives across the object are large negative values and their ratio is close to unity . following the scale - space ideas developed so far , a complete hierarchical set of scaled differential operators has to be used . the gaussian derivative operators described earlier in this paper constitute the natural differential operations on a given scale . thus , a set of gaussian derivatives and their combinations could be used for very complex object models and analysis . some well - known combinations of the high - order derivatives have special names like hessian , laplacian , and so forth and are used to build special functions for identification of certain shape patterns in images . such mathematical functions model human and machine perception and are ultimately used in unsupervised object tracking systems [ 1 , 2 , 35 , 36 ] . for this purpose , the hessian - based multiscale object enhancement filters were developed [ 4 , 25 , 4648 ] . in these anisotropic filters , the pixel intensity transformation is locally governed by the objectness measure functions which are built using the combination of local hessian eigenvalues and calculated in multiscale framework [ 45 , 4954 ] . the responses of the filters based on these functions are computed at different scales and are expected to have a maximum value at a scale corresponding to the width of the object [ 32 , 54 ] . such selectivity to the object shape along with capability to adaptively choose the optimal scale allows these filters to extract the looked - for objects at their native local scales . as noted by others [ 32 , 55 ] , the hessian matrix h(f ) ( or simply hessian ) is the square matrix composed of second - order partial derivatives of some scalar - valued multivariable function f(x1 , x2 , , xn ) , and it describes the local curvatures of this function f(x1 , x2 , , xn ) . assuming continuity of the second - order derivatives , the mixed derivatives do not depend on the order of differentiation ( e.g. , f/x1x2 = f/x2x1 , etc . ) . the hessian is then a symmetric matrix which for a 3d image i(x , y , z ) can be written a 3 3 matrix ( see ( 5 ) ) ( 5)h(i)=[ixxixyixziyxiyyiyzizxizyizz ] . in ( 5 ) , ixx = ( /xx)i(x , y , z ) , ixy = ( /xy)i(x , y , z ) , ixy = iyx , and so on . due to symmetric property of the hessian , for 3d images only , six out of nine values have to be calculated . let the eigenvalues of the hessian h(i ) be 1 , 2 , and 3 with their corresponding eigenvectors e1 , e2 , and e3 . if the eigenvalues ordered as 1 > 2 > 3 , then the eigenvector e1 gives the direction of the maximum of the second derivative . following the scale - space ideas described earlier , the partial second derivatives of the image i(x , y , z ) in the hessian h(i ) have to be replaced by the -parameterized normalized gaussian derivatives ( see ( 4 ) ) , convolved with the image which results in that now the eigenvalues 1 , 2 , and 3 become adjusted to the local size of the tubular object in an image . various algorithms for multiscale tubular object tracking and enhancement were developed depending on the way hessian eigenvalues are combined in the objectness measure function . for instance , sato et al . [ 4951 ] suggested the objectness measure function which used only two out of three values of hessian eigenvalues ( 6)f(1;c)={exp ( 122(1c)2 ) 10 , c0exp ( 122(2c)2 ) 1>0 , c00 c=0 . in ( 6 ) , 1 0 and 2 3 0 ( for a bright line on dark background ) , c = min ( 2 , 3 ) , and 1 < 2 . however , it did not have a parameter to control noise ( background ) suppression . extended the objectness measure function so that it includes the combination of all three hessian eigenvalues and a factor with a parameter which controls noise suppression ( see more details later in this paper ) . that function reflects shape and scale of the objects , has a single maximum on the center of the vessels segments , and has bell - shape close to gaussian with width proportional to object size . de facto become a basis for building even more sophisticated hybrid filters . manniesing et al . used this response function to develop an effective denoising filter , where the image intensity transformation is based on anisotropic diffusion governed by the . such an approach , which incorporates the shape pattern analysis along with multiscale data representation , would give us an extremely powerful tool to model artificial system learning . for neuron network reconstruction from 3d confocal microscope images , the tubularity measure function was used to design a statistical learning system for training a classifier and generating the probability that a given structure belongs to the tubular - like object [ 56 , 57 ] . in pulmonology , the algorithms based on tube detectors were effectively used for airway and lung vascular tree reconstruction from 3d ct images [ 58 , 59 ] . if properly normalized , the multiscale tube detectors could be used to build various cost and propagation functions required in the level set and fast marching segmentation algorithms [ 60 , 61 ] . to take the full advantage of the power of the multiscale shape detector filter in object tracking algorithms applied for a large variety of medical applications , in this work we focus on the process of automation of this filter . the filter itself has many control parameters which can be separated into several groups : brightness measure ( objects are bright relative to background ) ; objectness measure ( shape description ) , scale description ( range plus scale step function ) , and background noise suppression parameter ( frobenius norm scale factor ) . parameters in all groups , except the last one , describe general properties of the object itself so they do not depend on the imaging system characteristics . since in vascular studies the object brightness , tubularity , and range of diameters hence , the only parameter which prevents the algorithm to be fully automated is control of the noise suppression . this parameter depends on the acquisition system and imaging conditions ; therefore , it has to be experimentally found for each image set . we present our development of the multiscale hessian - based tubular object - tracking filter with automatic selection of the parameter used for suppression of background noise . that finalizes the automation of the filter . in our approach , the information required for the parameter calculation is acquired from the image being processed thus it automatically takes into account all the individual properties of the particular image such as voxel size and noise level . this allows for increased automation as well as parallel processing thereby greatly decreasing processing time . for our studies , we used both gray - scale images numerically derived and acquired by scanners . tubular objects with various widths were placed amid different background : gaussian random noise , nontubular objects , background with noise , and polynomial varying intensity . for simulations of images degraded by noise , we used the c++ classes contributed to the itk insight - journal by lehmann . these noise simulation classes are implemented with multithread support and are based on the mersenne twister uniform pseudorandom number generator which has a period ( 21 ) , 632-dimensional equidistribution , and up to 32-bit precision . thus , this generator could be considered as a true random which results in that generated noise does not produce any the noise was generated with various standard deviations sd = 25.0 , 50.0 , 100.0 , and mean m = 0.0 . we also used biomedical images of the heart acquired by the micro - ct scanner , cerebellar climbing fibers [ 6567 ] , and hippocampal ca3 interneuron . specimen h61 ( coronary artery branch within a human heart wall ) was a methyl methacrylate cast prepared as described previously . a cast of that coronary arterial tree was scanned with an isotropic voxel size of 0.018 mm and 500 500 541 voxel ct image volume . the custom - made micro - ct scanners generate images up to 2048 2048 1000 isotropic voxels down to 4 m on a side . to be able to process large images using the developed algorithms , we built a specialized server with four 64 bit amd opteron 8350 quad core 2.0 ghz cpus and 128 gb memory . the server is located in a server room and it is accessible in multiuser mode through the local network using remote clients . for our software development , we used the library of c++ classes from the national library of medicine insight segmentation and registration toolkit ( itk ) [ 6971 ] . the library was compiled with multithread support based on the posix thread ( pthreads ) model using 64 bit c++ compiler gcc 4.3.2 [ 7375 ] and installed on 64 bit debian linux 2.6.26 [ 76 , 77 ] . the developed multiscale shape detector filter is based on the objectness measure function suggested by frangi et al . after thoroughly conducted studies and tests , we found that the c++ classes contributed by antiga satisfy our purposes the best ; therefore , our further developments are based on those classes . let k be the eigenvalue of the hessian matrix at voxel x ordered such that |1 | |2 | |3| ( we drop the dependency to x ) . in the case of the ideal bright tubular structure , the voxels should satisfy the following relation for eigenvalues |1 | 0 ; |1 | |2| ; 2 3 and for bright objects both 2 and 3 must be negative . proposed to use the eigenvalues to define vesselness measure (x ) as below : ( 7)(x)={0 if 2>0 or 3>0,(1exp ( ra22a2))exp ( rb22b2)(1exp ( s22c2 ) ) , where a , b , and c are the parameters that control the sensitivity of the filter to the measures ra , rb , and s. the measures have the following meaning . ra = |2 | /|3| is used to distinguish between plate - like and line - like patterns . the measures ra and rb are gray intensity level invariant and capture only the topological information of the objects in the image . the choice of the control parameters a and b defines the object pattern to be studied . for example , if tubular - like objects are chosen , the parameters are fixed to a = 0.5 , b = 0.5 . the parameter c ( see ( 7 ) ) , controls background noise suppression in the hessian - based object enhancement filter . the frobenius hessian matrix norm is chosen as a measure s = sqrt(1 + 2 + 3 ) to distinguish background noisy pixels . since the parameter c strongly depends on the individual properties of particular image such as voxel size and noise level , for every new study , the optimal parameter value should be experimentally found again by trial . the very wide search range of the optimal parameter value in concert with a highly time - expensive calculation to derive the hessian taken in multiscale framework makes this algorithm very labor - inefficient especially for large 3d biomedical images with high resolution . for example , one trial run to process the 16 bit 500 500 514 gray - scale micro - ct image on our server took about an hour ; thus , the interactive search for the parameter c might take hours for the user who operates the program interactively . the method for automating the selection of the parameter for suppression of background noise uses a scalar function ( nondirectional ) of the image voxels the laplacian of the image . the laplacian is a well known operator in image processing which is easy to calculate [ 8183 ] . ultimately , the laplacian calculates the trace of hessian matrix or , equivalently , the sum of its eigenvalues ( 1 + 2 + 3 ) making it invariant with respect to a change of tensor basis . the schematic description of complete algorithm is below . for each voxel in the image , calculate the laplacian . in the calculated laplacian array , find the maximum value of laplacian , that is , ( 1+2+3)max . take one tenth of that maximum value of laplacian , that is , ( 1+2+3)max /10 . assign the calculated value to parameter c. in this approach , the information required for parameter calculation is acquired solely from the image being processed ; thus , it automatically takes into account all the individual properties of the particular image such as a voxel size and noise level . there are two measures commonly used for objective evaluation of the perceptual quality of images : mean square error ( mse ) and peak signal to noise ratio ( psnr ) [ 8490 ] . consider two images being compared x = { xi | i = 1,2 , , n } and y = { yi | i = 1,2 , , n } , where n is the number of points ( pixels ) in the data sets and xi and yi are intensity levels in the images . let x be an ideal image and y be a degraded image . the mse measure is then defined as mse = ( 1/n)i=1(xi yi ) and psnr is , respectively , psnr = 10 log10(l / mse ) where l is the dynamic range of allowable pixel intensities . as follows from the definitions above , the lower the values of mse , the lower the error and the higher the psnr , the better quality of processed image . the mse and psnr measures were developed using c++ classes from the itk library and contribution . in manual mode , the control parameters have to be provided by the operator before running the code . if the result is not acceptable , the operator has to change parameters and rerun the code again . the mse and psnr measures were used for objective quality evaluation of the processed image . as an ideal image , there was used a modeled image comprised three tube - like objects with gaussian intensity profiles of different width . then , the ideal image was degraded by the random gaussian noise with standard deviation sd = 100.0 and mean m = 0.0 and processed in manual mode by the algorithm . the background suppression parameter value was sampled over a wide range 10500 to surely cover prospective optimal control parameter . it can be seen that both measures indicate the optimal value is about c = 200 which corresponds to mse value 0.00214 and psnr value 26.69 . if run in automatic mode , the algorithm finds even more precise the optimal value c = 194.3 which corresponds to mse value 0.00203 and psnr value 26.93 . these results demonstrate capability of the developed algorithm in automatically finding the optimal control parameter for background suppression . to test efficiency of the algorithm in fully automatic mode , we used the modeled images described above . the control parameters were chosen and fixed : brightness on , objectness measure tubular , scale range 130 , scale steps 20 , step function logarithmic , noise suppression mode first , we processed the images with curved tubular objects with various widths which were placed amid nontubular objects . the images were degraded by gaussian random noise with sd = 25.0 , 50.0 , and m = 0.0 . as can be seen from figure 8 , the algorithm automatically finds the optimal parameters and successfully tracks tubular objects . we also tested performance of the algorithm for neurological confocal microscopy image processing affected by tiling , shading , gaussian noise , nonlinear background , and so forth ( see , e.g. , figure 9 ) . the modeled images were programmed so to present three parallel tubular objects aligned in the z - direction with different diameters and distance between objects along with gray - scale distribution described by gaussian intensity profiles . the polynomial background was added with and without gaussian random noise ( figure 10 ) . in figure 11 , there are depicted intensity profiles across the tubular objects with gaussian intensity profiles in the processed images with various polynomial noisy backgrounds added sd = 0.0 , 25.0 , 50.0 , and mean m = 0.0 . as could be seen , the algorithm effectively suppresses the background and successfully extracts the tubular images . to evaluate time efficiency of our automatic method , we processed the 500 500 514 ( 16 bits per voxel ) micro - ct image with 0.018 cubic mm size in multithreaded mode using a linux - based server with the 16 processors and 128 gb memory as described above . first , we ran the automatic algorithm to find the parameter c. then , we ran nonautomatic ( the found parameter was manually plugged into a program ) version . the amount of cpu time spent for a single run in non - automatic mode is 58 minutes whereas for automatic mode is 64 minutes , which is just about 10% longer . if the server is able to allocate enough memory for the run , the time spent by the algorithm in fully automated mode is comparable with time for the nonautomatic mode . the result of applying our automatic algorithm to the micro - ct image of the coronary arteries in a heart in concert with the nonprocessed image is shown in figure 12 using maximum intensity projection ( mip ) images . before preprocessing ( a ) there are the blobs of white material due to a contrast medium accumulation in a cardiac chamber . those blobs are selectively removed by the algorithm as can be seen in right panel ( postprocessing ) . in figure 13 , there are the not processed ( a ) and processed ( b ) mip images of cerebellar climbing fibers . as expected , the tubular measure filter effectively suppresses the fixed pattern background and noise and delineates the tubular objects . we also explored the efficiency of the algorithm as an initial filter in the central line extraction pipe line . the original image sample h61 was processed with the developed filter and then segmented using the region growing connected threshold algorithm [ 6971 ] . the seed for segmentation was allocated at the beginning of the main tree root thus the side trees were excluded . afterwards , a center line was extracted using the 3d thinning approach based on the c++ classes submitted to the insight journal by homann . as could be seen from the figure , the algorithm effectively suppresses background and delineates the tree . we have presented a method for automation of adaptive nonsupervised system for tracking tubular objects that is based on analysis of local structures performed in multiscale framework . the designed filter has demonstrated a great potential for complete automation and showed very good performance in both background noise suppression and tubular object tracking . the developed approach can be used in the reconstruction pipeline right after image deconvolution operation . even though the convolution operator will reconstruct the object features at finer scales , those features will appear in increased noise environment which in return might require additional postprocessing for noise suppression yet to preserve extracted features . this filter can be used as a preprocessing filter for vessel enhancement and background noise suppression right before segmentation or immediately in the segmentation algorithms itself , for instance , in the family of segmentation algorithms which require distributed seeds [ 70 , 71 ] . by using the thresholded output of the vessel enhancement filter as a seeder , it can increase the speed efficiency of the segmentation process considerably . since the response function is built using exponents , with proper normalization this function can be considered as a probability function with values distributed over the interval 0.0 - 1.0 . in this case , after processing , the output image holds voxels with values of probability of the event that a voxel belongs to the object with tubular shape . these probabilities can be used in many ways . the most traditional way is to rescale it back to a gray - scale image . although such images do not keep a proper intensity calibration , they still can be used for morphometric analysis . if calibration is of concern , the probabilities could be converted to a mask for sampling the original micro - ct image from which the calibration could be recovered . since the filter generates the response function with only one maximum across scale space at a scale that is proportional to the diameter of the tubular object and that maximum is located at the center of the object , the probability image is more suitable to construct various cost functions . the images with cost functions can further be used as the feature image in various image processing pipelines , for instance , such as in flux - driven centerline extraction algorithms [ 92 , 93 ] , level - set and fast - marching segmentation algorithms [ 61 , 6971 ] , and so forth . as the multiscale vessel enhancement filter is very robust against noise , it can be superior over traditional approaches like , for example , in a filter pipeline segmented image , distance map , and cost function , since it can directly generate the cost function avoiding steps for producing segmentation and distance map . in addition , the multiscale space feature can be used to build the cost function in multiscale representation and use it for multiscale vessel tracking as suggested in .

the blood vessels and nerve trees consist of tubular objects interconnected into a complex tree- or web - like structure that has a range of structural scale 5 m diameter capillaries to 3 cm aorta . this large - scale range presents two major problems ; one is just making the measurements , and the other is the exponential increase of component numbers with decreasing scale . with the remarkable increase in the volume imaged by , and resolution of , modern day 3d imagers , it is almost impossible to make manual tracking of the complex multiscale parameters from those large image data sets . in addition , the manual tracking is quite subjective and unreliable . we propose a solution for automation of an adaptive nonsupervised system for tracking tubular objects based on multiscale framework and use of hessian - based object shape detector incorporating national library of medicine insight segmentation and registration toolkit ( itk ) image processing libraries .

1. Introduction 2. Background and Principle 3. Methods 4. Results 5. Discussion and Future Work

PMC1933179

this is because it can provide site - specific information about protein motions over a large range of time scales . over the past decade , n nmr relaxation experiments employing model - free analysis ( 1,2 ) have become the de facto standard used to characterize protein motions on a picosecond to nanosecond time scale . however , as with any scientific method , this approach has certain limitations ( 3,4 ) . perhaps the most obvious difficulty lies in the fact that n relaxation measurements are inherently time - consuming and tedious , often requiring many hours of data collection , processing and spectral analysis . a second problem is that relaxation measurements are often seriously compromised by peak overlap , poor signal intensity or peak broadening . a third problem is that the precision and accuracy of relaxation - derived measurements tends to deteriorate rapidly as the frequency of internal nanosecond motions approach that of the protein 's overall tumbling rate ( 5,6 ) . this is because n relaxation rates become insensitive to internal fluctuations that are much slower than overall tumbling . since calculations of relaxation rates are based on measuring peak intensities , their accuracy can be severely compromised by low signal - to - noise ratios . this is especially true when dealing with larger ( > 150 residues ) proteins or proteins undergoing s ms conformational exchange . another complication to the model - free formalism lies in the fact that its proper application often requires information about anisotropy of protein overall diffusion and , as a result , the method can not be used when the 3d structure is not known or when it is largely disordered . these limitations with traditional relaxation measurements prompted us to develop a new , chemical - shift - based technique to characterize protein mobility from nmr data . specifically , we wanted to develop an easy - to - use , robust approach that would not be affected by protein tumbling rates , uncertainties in peak intensities or lack of knowledge about the protein 's 3d structure . this method is called the random coil index or rci ( 7 ) . as described in our previous publications ( 7,19 ) , the rci method exploits the fact that there is a remarkable amount of dynamic information intrinsic to nmr chemical shifts . the connection between chemical shifts , especially random coil chemical shifts , and protein flexibility has been known for quite some time ( 811 ) . random coil chemical shifts can be defined as shifts that result from a fast exchange among energy - weighted populations of all theoretically possible conformations of an unfolded polypeptide chain ( 12,13 ) . the difference between an observed chemical shift for a given amino acid in a given protein , and its corresponding random coil value is called the secondary chemical shift . secondary chemical shifts have been used for many years to qualitatively estimate the level of protein structural disorder ( 1418 ) . however , until recently , no quantitative relationships between secondary chemical shifts and protein dynamic parameters had been derived . the rci is able to combine the chemical shift data from six different nuclei ( c , c , co , n , hn and hor any combinations thereof ) into a single parameter that closely correlates with amplitudes of backbone protein motions such as order parameters ( s ) and root mean square fluctuations ( rmsfs ) of structural ensembles . previous descriptions of the rci method focused on explaining the algorithm , rationalizing its utility and assessing its accuracy . as a result , only a modest effort went into making the rci - based software user - friendly and flexible . in an attempt to make the rci approach more accessible to the nmr community and much more robust , we decided to create an rci web server and to optimize the rci protocol for nmr assignments with different degrees of completeness and mis - referencing . the rci server is a unique server , designed to support rapid ( 510 s ) , residue - specific determination of protein flexibility and protein mobility using only chemical shift assignment data as input . it accepts almost any combination of nmr chemical shift assignments as its input and it outputs the expected values of the rmfs of md and nmr ensembles as well as model - free order parameters ( 1,2 ) . to improve the accuracy and reproducibility of the calculations , assessments of the rci server performance show an agreement between the values it calculates and experimentally obtained amplitudes of motions range from r = 0.770.82 depending on the type of experimental method . the rci server is composed of two parts , a front - end web - interface ( written in python and html ) and a back - end consisting of several programs including rci ( 7 ) , csi ( 11 ) , refcor [ based on ( 19 ) ] , as well as several parsing and conversion utilities for handling different input files . the csi program is written in ansi standard c , while rci , refcor , the input parsing and the conversion utilities are written in python . the rci server accepts protein chemical shift assignments in standard bmrb nmr - star ( 20 ) and shifty ( 21 ) formats as input . users can either upload an input file into the web server ( via a browse button ) or paste the data in a standard text box ( figure 1 ) . users are also offered several options to adjust program operations to suit their specific needs . to begin : ( 1 ) experimental chemical shifts are first uploaded by the user . ( 2 ) the input chemical shifts are then re - referenced ( if necessary ) by refcor . ( 3 ) the protein sequence is extracted from the nmr assignments and used to ( 4 ) determine the appropriate random coil chemical shifts ( 22 ) and ( 5 ) determine the neighboring residue correction factors for the i 1 and i 2 residues ( 23 ) . the correction factors are applied to the random coil chemical shifts to obtain reference chemical shifts . ( 6 ) reference chemical shifts are then subtracted from the corrected experimental chemical shifts to obtain the secondary chemical shifts for the c , c , co , n , hn and h nuclei . ( 7 ) optionally , gaps in the chemical shift assignments , if any , are filled in by averaging the chemical shifts of neighboring residues . ( 9 ) secondary chemical shifts of are scaled to account for differences in their resonance frequencies , and , if below a certain floor limit ( currently 0.5 p.p.m . ) , replaced with this floor value . ( 10 ) initial rci values are calculated using the following expression . 1 where |c| , |co| , |c| , |n| , |nh| and |h| are the absolute values of the secondary chemical shifts ( in p.p.m . ) of c , co , c , a , b , c , d , e and f are weighting coefficients ( table 1 in the rci online help ) . left angle and right angle brackets ( < > ) indicate that the average is being calculated . ( 12 ) if the rci values are above a certain ceiling limit ( currently 0.6 ) , they are replaced with this ceiling value . ( 13 ) the final rci values are obtained after a second smoothing by three - point averaging . ( 14 ) finally , in the last step the expected values of model - free order parameters ( s ) , rmfs of md and nmr ensembles are calculated using the following empirical expressions . 2 3 4 a more detailed description of the aforementioned steps has been published elsewhere ( 19 ) . figure 2.a flow chart describing the rci protocol . a flow chart describing the rci protocol . by default , the rci web server uses random coil reference chemical shifts and neighboring residue correction values originally published by schwarzinger and co - authors ( 22,23 ) . this is the only set of random coil values and neighboring residue correction for residues i 1 and i 2 that were obtained under similar experimental conditions and , therefore , are expected to be quite consistent with each other . however , users can also select different sets of random coil chemical shifts , including those values published by wang and jardetzky ( 24 ) , wishart et al . users are also offered an option to select i 1 neighboring residue correction values published by wang and jardetzky ( 24 ) instead of the default values . while these options do affect rci - based flexibility profiles , the differences in mean correlation coefficients for different combinations of random coil values and neighboring residue corrections ( table 2 of the rci online help ) are relatively small and may be considered statistically insignificant . by default , however , if unassigned residues are located in the middle of a well - defined secondary structure region ( e.g. -helix , -sheet ) , it is not unreasonable to expect that their flexibility will be similar to that of neighboring assigned resides . in these cases , the rci web server allows users to predict the flexibility of unassigned residues based dynamic properties of adjacent regions . the rci web server also has a somewhat similar option to fill small gaps in the per - residue distributions of secondary chemical shifts . such gaps happen more often than a lack of assignments for all the six nuclei and should be dealt with separately . the use of incomplete assignments generally reduces the quality of rci - based flexibility predictions ( see table 1 of the rci online help ) . by default , the rci web server fills small gaps in per - residue distributions of secondary chemical shifts by averaging secondary chemical shifts of residues i + 1 and i 1 , and , if not available , residues i + 2 and i 2 . end effects. these are poorly understood phenomena that alter the chemical shifts of the n- and c - termini of proteins in unpredictable ways . the rci web server offers an option to apply an end - effect correction to the rci values . this end - effect correction is turned on by default . a detailed protocol describing how the end - effect correction is handled was published elsewhere ( 27 ) . users should be aware that the improvement of rci predictions due to the end - effect correction is not related to the dynamic averaging of chemical shifts . rather , it originates from the indirect correlation between the severity of the end effects and protein flexibility due to their shared dependence on the proximity to a terminal residue . therefore , if the correction is applied , the rci profile at the terminal regions should be interpreted as a commonly observed trend of protein flexibility at termini of proteins . the rci server offers users the option to disable the end effect correction or to exclude the first and the last three residues from predictions . a particularly useful and important feature of the rci web server is its capacity to correct mis - referenced chemical shifts . this automated reference correction is done using a local implementation of the refcor program . about 20% of newly deposited assignments in the bmrb database were found mis - referenced by a recent survey ( 28 ) . given the relatively high level of mis - referencing in biomolecular nmr and given the fact that mis - referenced shifts can substantially reduce the performance of chemical - shift - based methods such as rci ( 7 ) , we believed that implementing this reference correction protocol was absolutely vital to maintaining the server 's performance . the refcor reference correction protocol is based on the secondary structure predictions from the chemical shift index ( csi ) ( 29 ) and was published elsewhere ( 27 ) . briefly , refcor uses csi - based predictions of secondary structure along with typical chemical shift values observed in different secondary structures are used to calculate the necessary reference correction for each nucleus . since csi predictions are also affected by shift mis - referencing , the protocol is repeated several times using newly generated re - referenced shifts until the csi predictions become stable . the reference correction option is always turned on by default although it can be switched off , if necessary . an average rci run takes between 5 cpu s ( without chemical shift reference correction ) and 20 s ( with reference correction ) . as might be expected , the rci web server displays the name of the input file , the options selected and a plot of the per - residue rci distribution . users may download per - residue distributions of the rci , predicted model - free order parameters , predicted rmsfs of md and nmr ensembles as both text files and graphical images . these files may also be obtained individually or as a single linked file containing all the results . re - referenced chemical shifts and csi - based predictions of secondary structure are also available for download or direct viewing on the website . in addition to its extensive data output , the rci web server also offers a comprehensive list of help pages to assist users in preparing their input files , in understanding the rci method and in interpreting the web server output . this information is provided to make the rci protocol as transparent as possible and to facilitate protocol troubleshooting if required . the rci web server was optimized and evaluated on a set of 18 proteins ranging in length from 56 to 283 residues ( 1585 residues in total ) , for which complete or nearly complete h , c and n chemical shifts were known ( table 3 of the rci online help ) . a subset of 14 proteins was used to generate molecular dynamic ( md ) ensembles and optimize weighting coefficients in the rci expression [ equation ( 1 ) ] . this was done by using a simple grid search to maximize rci correlation with rmsf values determined by molecular dynamics . the weighting coefficients were obtained for all 63 possible assignment scenarios ( table 1 of the rci online help ) . to evaluate the performance of the algorithm for the training set , we used a leave - one - out procedure by removing the query protein from rci training set prior to running the program . the average correlation coefficients for the full training set and for all leave - one - out runs were identical ( r = 0.82 ) . to ensure that the rci algorithm had not been over - trained , four additional proteins , which were not previously included in the training set , were tested . the average correlation coefficient between the rci determined md rmsf and the calculated md rmsf for these proteins was identical ( r = 0.82 ) to that of the training set ( see table 3 of the rci online help for information about the tested proteins ) . to validate the relationship between rci predictions and the amplitudes of protein motions , the correlation of rci with model - free order parameters [ observed and predicted from the structure ( 30 ) ] and per - residue rmsf values of nmr ensembles were calculated . the average correlation coefficients for the eighteen sets of order parameters and sixteen sets of nmr rmsfs were 0.77 and 0.81 , respectively . the performance of the conversion expressions shown in equations ( 24 ) were assessed by calculating the average error between rci - predicted motional amplitudes and the corresponding experimentally and theoretically obtained parameters . the average errors for the order parameters ( 18 proteins ) , the nmr rmsfs ( 16 proteins ) and the md rmsfs ( 18 proteins ) were 0.05 , 0.44 and 0.50 , respectively . figure 1 in the rci online help shows examples of the good correlations obtained between rci and other measures of protein motional amplitudes ( e.g. nmr rmsf , md rmsf , s ) for such pharmaceutically important proteins as interleukin-4 and the hiv-1 gag protein . additional information about optimizing and testing the rci method ( i.e. details of md simulations , pdb ids and bmrb codes of proteins , etc . ) can be found in the original papers on the rci method ( 7 ) as well as on the rci online help pages . in summary , we have described a web server that is capable of rapidly and accurately predicting protein flexibility using only chemical shift assignments as the input . comparisons suggest that these predictions correlate well with other measures of protein mobility , such as model - free order parameters and root - mean square fluctuations ( rmsfs ) of nmr and md ensembles . the approach is generally applicable to proteins of any size for which h , c and n shift assignments are available . the web server is unique in its ability to extract dynamic information from nmr data without the prior knowledge of tertiary structure , without the need for favorable rates of rotational diffusion and without the need for exceptionally good spectral sensitivity . we have already used the rci approach to monitor a number of interesting dynamic processes in proteins , such as the ph - induced conversion of the prion protein ( prp ) into its scrapie form ( prp ) and the dynamic response of picornaviral protease active sites to inhibitor binding ( manuscripts in preparation ) . we believe the rci method may lead to important changes in the ways backbone protein flexibility is measured and reported by the scientific community .

protein motions play important roles in numerous biological processes such as enzyme catalysis , muscle contractions , antigen antibody interactions , gene regulation and virus assembly . knowledge of protein flexibility is also important in rational drug design , protein docking and protein engineering . however , the experimental measurement of protein motions is often difficult , requiring sophisticated experiments , complex data analysis and detailed information about the protein 's tertiary structure . as a result , there is a considerable interest in developing simpler , more effective ways of quantifying protein flexibility . recently , we described a method , called the random coil index ( rci ) , which is able to quantitatively estimate backbone root mean square fluctuations ( rmsfs ) of structural ensembles and order parameters using only chemical shifts . the rci method is very fast ( < 5 s ) and exceedingly robust . it also offers an excellent alternative to traditional methods of measuring protein flexibility . we have recently extended the rci concept and implemented it as a web server . this server allows facile , accurate and fully automated predictions of md rmsf values , nmr rmsf values and model - free order parameters ( s2 ) directly from chemical shift assignments . it also performs automatic chemical shift re - referencing to ensure consistency and reproducibility . on average , the correlation between rci predictions and experimentally obtained motional amplitudes is within the range from 0.77 to 0.82 . the server is available at http://wishart.biology.ualberta.ca/rci .

INTRODUCTION SERVER AND PROGRAM DESCRIPTION PERFORMANCE AND VALIDATION CONCLUSION

PMC4713780

it is believed that physical activity for the prevention of falls should primarily aim to increase the strength of large groups of muscles , mainly in the lower limbs , as well as improve gait , balance , and coordination parameters1 . among the organized exercise systems only tai - chi has been mentioned in this context , and the importance of physiotherapy is emphasized2 . however , it has not been determined which specific forms of rehabilitation activities should be undertaken in order to achieve satisfactory results in this field . it involves marching using poles , and is an adaptation of cross - country skiing . the main purpose of using the poles is to involve the muscles that are not used during normal walking . this enables high intensity exercises to be performed at a relatively low level of perceived exertion . nw is also a form of physical activity which is recommended for the elderly . there is evidence of its importance in the prevention and physiotherapy of musculoskeletal disorders , vascular diseases , cardiovascular diseases and parkinson s disease3 . reported that nw improved all health - related components of fitness of the elderly based on functional tests , however , no improvement of gait parameters was observed4 . takeshima et al . have confirmed the efficacy of a 12-week nw training program and its positive impact on selected fitness parameters on the basis of a physical fitness test and a test on a force platform5 . however , gait parameters were not assessed . in another study it was demonstrated that nw training improved both the strength and walking speed of the elderly , compared to the traditional walking training6 . exercise is recommended to improve abnormal posture control during walking , and the use of exercises focused on performing two independent tasks has a particular impact on the improvement of gait parameters and postural control7 . in this study , a set of objective tests were used to evaluate the fitness elements related specifically to postural control , especially those used during daily routines , such as reaching in various directions or bending the body and gait parameters . this study used a force platform to evaluate performance in specific functional tests the forward reach test ( frt ) and the upward reach test ( urt ) to evaluate the changes in postural control during reaching . moreover , kinematic gait analysis was performed in order to assess the potential impact of nw training on selected gait parameters . our hypothesis was that a long - term nw training would improve gait symmetry , lengthen the gait cycle , and decrease the frequency of the strides made eliciting greater economy of energy and improvement in incorrect gait patterns of the elderly . gait analysis of this study mainly focused on assessment of coordination and symmetry of gait . the aim of the study was to evaluate the effects of 12 weeks nordic walking training on selected gait parameters and elements of fitness related to postural control . the subjects were randomly assigned to 2 groups : the experimental group ( nwg ) and the control group ( cg ) ( fig . the tests were conducted by blinded assessors , in a single blind experiment ; the person making the measurements did not know to which study group a given subject belonged . simple random sampling was used , with unmarked envelopes and a 1:1 chance of drawing a group . the flow of participants through the trial sixty - seven volunteers who met the inclusion criteria , were enrolled in the study . the inclusion criteria were : age between 65 and 74 years , no regular physical activity in leisure time , and a willingness to participate in the study . the exclusion criteria were : age , regular forms of physical activity undertaken in leisure time , professional activity , musculoskeletal dysfunctions preventing exercise and walking , acute inflammations possibly associated with a disease , previous episodes of cardiac arrest , uncontrolled arrhythmias , chronic heart failure ( nyha 3 and 4 ) , uncontrolled and untreated high blood pressure ( over 140/90 mm / hg at rest ) , uncontrolled asthma , diabetes with insulin treatment , liver or renal failure , neoplastic disease or medical advice against taking exercise . the level of physical activity of all subjects was assessed using a physical activity questionnaires the 7-day physical activity recall ( 7par)8 , in order to compare the initial level of physical activity between the groups at baseline . all the patients were informed about the purpose and procedures of the study in detail and gave their consent to participation in the study . the study received the approval of the local bioethical committee of university of medical sciences no . the subjects of the experimental group and the control group performed the same set of tests at the beginning and at the end of the 12-week training period . the functional equilibrium tests performed on the force platform aimed to evaluate postural control while reaching . the tests were performed with the subjects standing on a force platform ( cq stab poland ) placed against a wall . measuring tapes were stuck to the wall , in accordance with the diagram presented in the study by row et al9 . the assessed parameters included the length of forward reach ( measured tape affixed horizontally ) and upward reach ( centimetre tape affixed at a 45-degree angle ) , and the forward displacement of the centre of pressure ( cop ) in relation to the initial position of the patients , labeled point 0 . point 0 was determined as the vertical projection from the centimetre tape stuck to the wall onto the furthest point of the foot on the force platform . these points were marked with a special tape , which the subjects were not allowed to cross , and were identical for all measurements and subjects . the position of the platform in the baseline and post - intervention tests was identical ( marked with a tape stuck to the floor ) , the position of the feet and the upper limbs also remained unchanged ( the tape stuck to the platform , the measuring tapes stuck to the wall ) . the evaluated parameters included the forward displacement of the cop ( the furthest point in the computer diagram ) , and the forward and upward reach ( forward or upward movement of the fingers on the centimetre tape ) . both in the baseline and post - intervention tests , the patients performed the test 3 times and the best result , in which the patient reached the furthest , was chosen for use in the analysis . gait parameters were evaluated on a zebris fdm - t treadmill ( zebris medical gmbh , germany ) . this treadmill has often been used as a research tool to examine the elderly , and patients with neurological deficits10 , 11 . the subjects performed two trials at baseline and post - intervention at a rate of 4 km / h , which reflected their average walking speed . the first trial lasted for 30 seconds and was supposed to prepare the patient for walking on the treadmill , the second lasted for 5 min and the results were used in the analysis . a training session lasted for 75 minutes : 10 minutes of warm - up activities with stretching , 60 minutes of walking and 5 minutes of cool - down activities with stretching . each session was performed in a group and was supervised by two instructors . the walking distance and pace of the groups were gradually increased by the instructors who took into consideration both the period of the intervention and the level of perceived exertion of the participants ( mean pace : 1st week : 4.20.2 km / h ; 12th week : 5.60.2 km / h ) . the pace of walking was monitored by the instructors , using handheld equipment ( polar gps sensor g5 , polar electro oy , kempele ) . in each session , when participants reached half the distance , they had short break during which they performed breathing exercises . the participants in both groups received the recommendation , that they should perform exercise , no less than 12 and no more than 14 points on the perceived exertion scale12 . the subjects of cg received detailed instructions regarding walking training , which they performed unsupervised over the next 12 weeks . moreover , each cg subject was given a gps sensor with instructions to turn it on before starting a walk , and to turn it off after finishing the walk . once a month the data concerning the amount of training and the times of individual training were checked . the subjects of both groups ( nwg and cg ) were told not to take up any additional forms of physical activity , and not to change their current eating and motor habits during the course of the study . before the start of the training , the subjects in nwg were taught to the proper technique of walking with poles ( a 2-hour instruction ) . as data were not normally distributed , comparisons between the two study groups were performed using the mann - whitney test . for paired variables , the subjects of both groups were similar in terms of the basic descriptive characteristics and the level of physical activity as assessed by 7par ( table 1table 1.the descriptive characteristics of the subjects ( mean sd)featurenordic walking group ( n=28)control group ( n=29)age ( years)70.5 3.771 2.9genderfemale 100%female 100%weight ( kg)67 9.966.4 11.2bmi ( kg / m)27.13 3.726.9 4.5height ( cm)157.2 5.9159.5 5.8the time spent in physical activity during leisure in the week preceding the study ( 7par hours : min)rest : 45:52rest : 46:00light pa : 84:12light pa : 80:16average pa : 31:02average pa : 30:01high pa : 05:51high pa : 06:45very high pa : 0:00very high pa : 0:00there were no significant differences in any of the parameters between the nordic walking group and the control group . ) . there were no significant differences in any of the parameters between the nordic walking group and the control group . the results obtained in the urt and frt tests demonstrate that after nw training , the nwg subjects showed significant improvement in the range of reach in both tests , compared to the baseline . also the difference between the urt results at the baseline and post - intervention tests proved to be significantly different from that of the control group . neither group showed a statistically significant improvement in the maximum forward cop displacement ( table 2table 2.the results of the forward reach test and the upward reach test performed on a stabilometric platform ( mean sd)testnordic walking groupcontrol groupbaseline test frt ( cm)114.83.4115.93.8end test frt ( cm)115.84.6116.74.1baseline test frt cop ( mm)89.0510.384.813.2end test frt cop ( mm)85.109.480.513.2baseline test urt ( cm)105.55.9105.23.7end test urt ( cm)107.85.5105.94.4baseline test urt cop ( mm)87.1310.384.613.8end test urt cop ( mm)82.012.281.912.8frt : forward reach test , urt : upward reach test , cop : centre of pressure . p < 0.05 in the wilcoxon test.p<0.05 for differences between the groups in the changes from baseline to post - intervention ) . frt : forward reach test , urt : upward reach test , cop : centre of pressure . p < 0.05 in the wilcoxon test.p<0.05 for differences between the groups in the changes from baseline to post - intervention gait analysis failed to identify any significant changes during the evaluation of a single stride and any of its phases . the only parameter which showed a noticeable change was the slightly reduced percentage difference from baseline in the swing phase of the control group at post - intervention . however , the analysis of the full gait cycle showed it was longer ( cm ) at post - intervention in nwg , and this was accompanied by prolongation of the time of strides and a reduction in their frequency compared to cg ( table 3table 3.the treadmill walking parameters ( mean sd)gait parametersnordic walking groupcontrol groupbaselineendbaselineendwidth ( cm)6.21.65.91.65.91.85.81.8the difference in stride length between the left and the right leg ( cm)2.42.32.91.82.41.63.11.3 the difference in stride - making time between the right and the left leg ( sec)0.010.010.010.010.090.010.010.01the percentage difference in the total time of the stance phase between the left and the right leg ( % ) 0.80.50.70.71.10.70.70.4 load response ( % ) 0.90.70.80.91,00.50.70.6 single support ( % ) 0.81.30.60.61.11.10.70.4 pre - swing ( % ) 0.90.70.840.91.00.60.70.5the percentage difference in swing phase time between the left and the right leg ( % ) 0.81.30.70.61.11.10.70.4the percentage of double support time ( % ) 25.02.324.12.425.52.324.82.0the length of the cycle ( cm)101.89.01112.411.2106.212.4108.910.5the time of the cycle ( sec)1.000.11.10.11.00.11.00.1cadence ( strides / min)60.66.955.67.157.75.256.94.3p < 0.05 in the wilcoxon test . p<0.05 for differences between the groups in the changes from baseline to post - intervention ) . p<0.05 for differences between the groups in the changes from baseline to post - intervention the study attempted to evaluate the impact of nordic walking training , on some parameters of functional fitness of women over 65 years of age . the evaluated parameters were associated with postural control , balance and gait , i.e. the elements of fitness which may play important roles in the prevention of falls . in most cases , the obtained results were consistent with the research hypothesis and showed that nordic walking has potential as a form of exercise for the elderly . postural control is defined as the ability to control the body s position in space for the purposes of stability and orientation , and is critical during standing balance and walking tasks13 . postural control was analyzed using a stabilometric platform , which is often used in studies of the elderly and in neurology14 , 15 . however , in the present study it was used as an auxiliary tool to evaluate the displacement of the centre of pressure ( cop ) during two functional tests , urt and frt . moreover , urt has been defined as the more demanding test , reflecting everyday - life activities better than frt9 . drawing on the experience of the authors , the measurements were performed in a similar way , but facilitated by the placement of coloured sticky tapes on the floor and the stabilometric platform , and measuring tapes on the wall , which were not removed throughout the study in order to maintain a uniform standard of research . the results suggest that only nwg subjects improved their range of reach the both in the frt and urt tests , however only significant found between nwg and cg was in the post - intervention changes from baseline in the urt test . no significant changes in the forward displacement of the centre of pressure moreover , the average results indicate that there was a decreasing trend in this result , which may suggest that during trials , the nwg subjects moved cop to their heels and at the same time tried to reach forward and upward more boldly . it is possible that nw training did not increase body control while moving cop forward during the urt and frt tests , but it could have improved muscle strength and trunk stability , thereby improving postural control . the urt or frt tests help to evaluate the ability to maintain balance during a functional task16 , 17 , and perhaps , in the future , these tests should be incorporated into a comprehensive assessment of dynamic balance and postural control . parkatti et al.4 , demonstrated a statistically significant improvement in all the components of the fitness test for the elderly , apart from the backscratch test . similar results were reported by lee et al.18 , and by kukkonen - harjula et al . in a study in which the tested women showed improvements in most tests evaluating physical fitness , but each time on the basis of a different set of tests19 . in that study measurements of postural control were not taken into account , and balance was assessed using the standing on one - leg time and walking backwards . it seems that the strength of the trunk muscles plays a vital role in maintaining balance when crossing the circumference of the support base , and during each phase of walking the erector spinae and multifidus muscles are engaged20 . these muscles probably work in a similar way regardless of whether we use poles or not , especially since opinions about the reduced burden on the legs and the trunk during walking with poles were premature21 , 22 . the evaluation of the involvement of individual trunk muscles during nw calls for further investigation . studies suggest that gait parameters should be evaluated at least once a year23 , since selected gait parameters may become an important index for determining the risk of falling . our study results are promising , because they demonstrate that in the nordic walking group the length ( cm ) and time ( sec ) of the gait cycle were increased and the frequency of strides decreased . earlier studies have demonstrated that walking training performed once a week , can improve gait speed and performance during the timed up & go test24 . moreover , the results of our study are consistent with other findings that show that the use of different forms of unconventional walking ( e.g. with music or with exaggerated upper - body involvement ) improves gait parameters25 . the assumptions of the present study seem similar to those made by takeshima et al5 . in their study , it was suggested that nw should be the recommended form of physical activity for improving fitness and reducing the risk of fall in the elderly . in our study however , it seems that the intervention had a greater efficacy among elderly persons with high risk of falls or a history of falls . the subjects of this study were only women ( no man volunteered ) , therefore , it is difficult to draw conclusions for the entire population of the elderly and , in order to draw definitive conclusions , the size of the experimental group needs to be increased . for this reason , an objective and universally recognized functional test should be used at the later stage of the study for the comparison of results . at this stage of the study there was no supervised control group and it was difficult to determine whether walking would not affect the results in a way similar to the nordic walking training . therefore , at a further stage of the study , a group will be created which , instead of the nordic walking training , will perform traditional walking in a supervised form . finally , it is in our opinion that 12-week nw training has a beneficial impact on selected gait parameters and may improve postural control , on the basis of results of selected functional tests performed by women between the ages of 65 and 74 .

[ purpose ] to assess the effect of 12-weeks nordic walking training on gait parameters and some elements of postural control . [ subjects and methods ] sixty - seven women aged 65 to 74 years were enrolled in this study . the subjects were divided into a nordic walking group ( 12 weeks of nordic walking training , 3 times a week for 75 minutes ) and a control group . in both study groups , a set of functional tests were conducted at the beginning and at the end of the study : the forward reach test ( frt ) and the upward reach test ( urt ) on a stabilometric platform , and the analysis of gait parameters on a treadmill . [ results ] the nw group showed improvements in : the range of reach in the frt test and the urt test in compared to the control group . the length of the gait cycle and gait cycle frequency also showed changes in the nw group compared to the control group . [ conclusion ] a 12-week nw training program had a positive impact on selected gait parameters and may improve the postural control of women aged over 65 according to the results selected functional tests .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4757398

a 79-year - old woman with a history of hypertension was transferred in a near - shock state from a regional hospital where she had presented 10 days previously , complaining of one week of nausea , diarrhea , and nocturnal fever . despite various conservative treatments including antibiotics , dyspnea with palpitations she denied any history of trauma , recent invasive procedures such as dental care , infections , or tuberculosis . one year previously , she refused to undergo recommended surgical treatment for symptomatic severe aortic stenosis at st . her vital signs at arrival were a blood pressure of 70/30 mmhg , an irregular heart rate of 127 beats / min , a respiratory rate of 26 cycles / min , and a body temperature of 36.9c . laboratory testing revealed a white blood cell count of 3,730/mm , a hemoglobin level of 9.0 g / dl , an aspartate transaminase / alanine transaminase ratio of 168/89 iu / l , and a c - reactive protein level of 23.7 mg / dl , while other parameters were within their normal limits . severe cardiomegaly and bilateral pulmonary edema were apparent in chest radiography , and electrocardiography showed atrial fibrillation with rapid ventricular response . portable transthoracic echocardiography ( tte ) revealed a massive pericardial effusion , which prompted emergency pericardiocentesis with the drainage of large amounts of serous pericardial fluid . the findings of tte and trans - esophageal echocardiography ( tee ) after pericardiocentesis were suspicious for a 1.2-cm hypermobile vegetation on the anterior mitral valve leaflet with calcification through the posterior mitral annulus and severe aortic valve stenosis . brain magnetic resonance imaging revealed focal acute embolic infarction of the left basal ganglia , without focal neurological signs . chest computed tomography showed extensive mitral valve calcification ( mac ) through the posterior mitral annulus , pulmonary edema , and pleural effusion around both lungs ( fig . emergency surgery was performed with a diagnosis of mitral infective endocarditis with extensive posterior mac and aortic valve stenosis . after aortic cross - clamping , exposure of the mitral valve was achieved through a longitudinal left atriotomy via the interatrial groove , revealing multiple friable vegetations attached to the anterolateral commissure and leaflets , as well as extensive mac through the posterior annulus . the anterior mitral valve leaflet was nearly completely necrotized with inflammation , but the annulus and subvalvular apparatus were preserved with only degenerative changes . an abscess pocket containing dirty yellowish pus was found on the posterior annulus between p2 and p3 , extending into the endocardium of the left ventricular posterior wall , without an intracavitary connection to the left ventricle ( fig . after complete debridement and resection of the infected tissue leaving the mac intact , multiple 2 - 0 polyester pledgeted mattress sutures were placed on the left atrial wall and the remaining posterior leaflet for plication . we passed the valve sutures from the atrial wall to the posterior leaflets , while avoiding passing through the mac and obliterating the abscess pocket site . the sutures were sequentially passed through bovine pericardium , and then through the sewing ring of a prosthetic valve ( 27-mm carpentier - edwards perimount magna valve ; edwards lifesciences inc . , irvine , ca , usa ) ( fig . the bovine pericardium was sutured to the left atrial wall with 4 - 0 polypropylene running stitches to cover the abscess pocket site and mac . after repairing the left atriotomy , the aortic valve was also replaced with a prosthetic valve ( 21-mm carpentier - edwards perimount magna valve ; edwards lifesciences inc . ) . preoperative blood culture and tissue culture were negative due to the use of preoperative antibiotics , and the patient was treated with broad - spectrum antibiotics ( ampicillin - sulbactam , 8 g / day ; gentamicin , 3 mg / kg / day ) for six weeks . follow - up tte on postoperative day 19 showed well positioned and functioning prosthetic valves without paravalvular leakage or dehiscence ( fig . although mitral valve repair is the preferred surgical approach if circumstances allow , the complete debridement of infectious necrotic lesions on the mitral valve may often hinder valve repair , inevitably requiring replacement . in particular , the existence of extensive mac renders mitral valve replacement a risky procedure because aggressive annular de - calcification is associated with an increased risk of fatal complications , including intractable hemorrhage from atrioventricular disruption or ventricular rupture , as well as acute myocardial infarction secondary to circumflex artery injury . partial annular decalcification can lead to a postoperative paravalvular leak and/or dehiscence and fracture of the remaining calcified annulus . several surgical techniques have been reported for addressing the presence of an unsuitable mitral annulus during mitral valve replacement , and can be divided into two categories : those for mac and those for the management of conditions involving destruction of the annulus , such as infective endocarditis or redo valve replacement . one approach involves leaving the mac intact and building a new annulus , securing the prosthesis to the leaflet tissue with intra - atrial implantation of a mitral prosthesis . some surgeons have also used the cavitron ultrasonic surgical aspirator ( cavitron surgical systems inc . , stanford , ct , usa ) to decalcify mac . despite their variety , the proposed surgical techniques must be modified in practice for cases combining an extensive endocarditis lesion and mac , as in the case presented here . in this case , we planned to avoid decalcification of the mac and to reconstruct a stronger annulus with the hope of minimizing the risk of grave postoperative complications . we used bovine pericardium to reinforce the new anchoring site after plicating both the mitral posterior leaflet and the left atrial wall to secure the mitral prosthesis without decalcifying the mac . the bovine pericardium not only reinforced the suture on the new annulus , but also acted as a barrier to prevent perivalvular leakage , and may have strengthened the vulnerable left atrial wall by covering the fragile site after debridement of the abscess pocket . in conclusion , the presence of an unsuitable mitral annulus remains a challenge for current surgical techniques . we report a surgical modification used to successfully treat a patient with extensive endocarditis complicated by subendocardial abscess and posterior mac by reconstructing the new annulus with plication between the left atrial wall and posterior mitral leaflet , reinforcing the bovine pericardium without decalcifying the mac . further evaluation and extended follow - up are warranted to assess the effectiveness of this surgical technique .

the concomitant presence of posterior mitral annular calcification and infectious mitral valve lesions poses a technical challenge with considerable perioperative risk when using previously proposed techniques for mitral valve surgery . herein , we report a case of the use of a modified surgical technique to successfully treat a patient with mitral infective endocarditis complicated by a subendocardial abscess and extensive posterior mitral annular calcification .

CASE REPORT DISCUSSION

PMC4784630

adolescent obesity is a major public health problem in the united states , with prevalence rates disproportionately high among certain youth populations . non - hispanic black and hispanic children and adolescents are consistently more likely to be obese ( body mass index [ bmi ] 95% ) compared with non - hispanic white youth . this pattern is seen in both adolescent males ( hispanic 26.5% , non - hispanic black 22.6% , and non - hispanic white 17.5% ) and females ( hispanic 19.8% , non - hispanic black 24.8% , and non - hispanic white 14.7% ) aged 12 to 19 years . obesity prevalence is overall significantly higher among males ( 16.1% ) than among females ( 11.7% ) , although this difference by sex is not significant in hispanic or non - hispanic black children and adolescents . the lack of daily - recommended levels of adolescent physical activity ( pa ) , particularly among girls and members of ethnic populations , has been seen . a comprehensive review of 108 studies revealed that ethnic minority children were as active as non - hispanic whites ; however , non - hispanic whites were more physically active as adolescents than other ethnic groups . despite the health benefits of pa , levels of inactivity are increasing among all youth over time . this trend is disturbing because youth pa is a predictor of sport participation and physical fitness activities during early adulthood . while youth from all backgrounds generally fail to meet national pa standards , low - income adolescents are less likely to adhere to these guidelines than their counterparts . understanding the impact of sports participation is essential since time for school physical education declines in sport participation are particularly significant among girls and boys during the transition from middle school to high school . youth from higher income families are more likely to participate in out - of - school sport programs than youth from lower income families , leaving lower income children particularly vulnerable to reduction in pa during school . since physical inactivity and sedentary behavior are important risk factors for overweight and obesity in adolescence , considerable research attention is focused on creating policies that encourage pa and sport participation . studies demonstrate that pa is positively associated with healthy weight , but the relationship between adolescent sport participation and obesity can be influenced by factors such as sociodemographic or the built environment ; thus , further research is necessary . identifying associations between sport participation , pa , and patterns of obesity in adolescents is vital for the development of effective intervention strategies . research on sport participation that explored activity involvement among early adolescents includes team sports , school / community clubs , and organizational activities . students involved in organized sports alone or in combination with other activities had significantly higher odds for exercise and healthy self - image . although the benefits of sport participation are clear , few studies report on sports program participation by weight status for adolescents . furthermore , race , gender , and socioeconomic status ( ses ) are rarely compared in sport participation studies of overweight and obese adolescents . for example , in a study of australian adolescents , obese adolescents spent considerably less time than their normal - weight peers on team sports . over the past 3 decades , scholars and public health officials have been challenged to understand the pronounced reduction in pa once children reach adolescence . consequently , examining sport participation in this population is important , especially for adolescent males , a group whose obesity prevalence has steadily risen in recent years despite girls having reached a plateau . notwithstanding these differences among girls and boys , national health and nutrition examination survey ( nhanes ) data over the past 2 decades show a dramatic increase in overweight and obesity prevalence across all population groups and a declining disparity of obesity across ses . this article uses a nationally representative sample of 12- to 19-year - old us adolescents to identify the relationship between obesity and leisure time sport participation , with a focus on differences by race , ethnicity , household income , and sex . we used the nhanes from years 1999 to 2006 ; the activity portion of nhanes was discontinued in 2006 . nhanes is a stratified , multistage probability sample of the civilian , noninstitutionalized us population . it includes an in - home questionnaire on several demographic and health - related topics , a computer - assisted interview , and an examination component consisting of a thorough physical exam including measured height and weight . we included 9152 adolescents age 12 to 19 years . because our goal was to identify adolescents in middle or high school whose activities were most likely to be related to leisure time sport participation , we excluded 1220 who were not enrolled in school ( most were 18 - 19 years ) , 754 enrolled in college , and 513 in elementary school , for a final sample of 6675 ( n = 3345 females and 3330 males ) . children aged 12 years and older were eligible for the survey portion that collected data on their participation in specific activities . they self - reported all activities during the previous 30 days , the number of times they participated in the activity , the average duration , and average intensity . nhanes does not provide information on whether the activity was part of an organized sports team . so we chose to identify adolescents who listed their most frequent activity as 1 of the 5 most popular sports for high school athletes . although some adolescents may have been classified as participating in a sport that were not in a school - based team or organized program outside of school , we can capture differences based on their activities using the frequency described below . to operationalize participation in leisure time sports , we first identified the most common sports according to the national federation of state high school associations , based on total number of participants . for girls these are running / track , * basketball , volleyball , soccer , and softball ; and for boys , football , running / track , basketball , baseball , and soccer . in order to create mutually exclusive categorizing , and to maximize the likelihood that reported pa was part of sport participation , we then assigned each individual to their most frequent common sport based on this hierarchy : ( a ) most amount of total time spent playing the sport , ( b ) most amount of vigorous time ( defined in nhanes as activity that causes heavy sweating or large increases in breathing or heart rate ) spent playing the sport , and ( c ) number of unique occasions playing the sport . for the small number of adolescents who participated in 2 sports equally using all criteria ( n = 84 , 1.2% of the sample ) , we assigned their sport based on the overall popularity ( eg , a boy playing equal amounts of football and basketball was assigned to football ) . nonathlete / participant - in - other - activities , although it is possible they participated in another , less common , sport . adolescents were categorized as obese , overweight , or healthy weight based on the bmi percentiles and current centers for disease control and prevention recommendations . we used height and weight as measured during the examination component to calculate bmi percentile , using sas code developed for that purpose . because of concerns that athletes with relatively low adiposity may be considered overweight based on standard definitions , we compared those who were obese to the combined group of overweight and healthy weight adolescents . this is consistent with previous research that suggests participants in sports like american football and rugby tend to be more muscular , and thus a high bmi in these players does not necessarily reflect excessive adiposity . race and ethnicity were categorized as non - hispanic white , non - hispanic black , hispanic , or other race . income was based on the percentage of federal poverty level ( fpl ) of household income , from < 100% ( most deprived ) to 500% ( most affluent ) . grade was categorized as middle school ( grades 6 - 8 ) and high school ( grades 9 - 12 ) . we used adjusted wald tests to examine the differences in the prevalence of obesity by organized sport participation for girls and boys separately . because demographic factors such as race , ethnicity , and ses likely affect sports participation , we used logistic regression analyses to examine the relationship between sport participation and obesity , controlling for child s school level , race / ethnicity , and income . all analyses were adjusted for the complex survey design of nhanes , as recommended by nchs . analyses were performed using the survey estimation routines in stata 12.0 ( college station , tx ) . because this study used only de - identified secondary data , it was deemed exempt from further review by the institutional review board under federal regulation 45 cfr table 1 shows the demographic characteristics of the sample , stratified by sex . there were no significant differences by sex for race , income , grade , or age . for girls , 48% did not participate in the 5 most common female high school sports ( table 2 ) , whereas only 29% of boys did not participate in the 5 most common male high school sports ( table 3 ) . white girls ( table 2 ) were more likely to play softball or volleyball , while black girls were more likely to play basketball , and hispanic females more likely to play soccer . higher income girls were more likely to play softball , and lower income girls were more likely to play basketball ; participation did not otherwise differ by income . percentage of girls ( n = 3345 ) playing each sport , by demographic characteristics . percentage of boys ( n = 3330 ) playing each sport , by demographic characteristics . p value represents adjusted wald test for difference in participation by demographics . overall , black males ( table 3 ) were more likely than white males to participate in 1 of the 5 most common high school sports and were most likely to play basketball . white males were more likely to play baseball or run track , and hispanic males were more likely to play soccer . the only differences in income were that higher income boys were more likely to play baseball , while lower income boys were more likely to play basketball . although leisure time sport participation declined from middle school to high school , football was the only sport for which this decline was statistically significant . there were few differences in leisure time sport participation by weight for girls ( table 4 ) . the prevalence of obesity did not differ overall among those who played organized sports compared to those who did not . girls who reported they participate in running / track had a significantly lower prevalence of obesity than girls who did not participate in any leisure time sport activity . in contrast , the prevalence of obesity among male nonathletes / participants - in - other - activities was significantly higher than among boys who did . this finding was consistent for male baseball , basketball , running / track , and soccer athletes , although the prevalence of obesity among football players did not differ from nonathletes / participants - in - other - activities . percentage of girls and boys who are obese ( body mass index > 95th percentile ) , by sport . p value represents adjusted wald test comparing each sport to the population playing no sport / participant - in - other - activities . girls were no more likely to be obese when playing any common sport , compared to female nonathletes / participants - in - other - activities . girls participating in running / track were less likely to be obese , controlling for grade , race , and income . however , lower income was significantly associated with greater likelihood of being obese for girls ( or = 3.17 for < 100% fpl ) . male athletes were significantly less likely to be obese compared to male nonathletes / participants - in - other - activities , except for football players . in contrast to girls , income was not significantly associated with greater odds of obesity among boys ( see table 5 ) . the results of this study are consistent with national trends for declining levels of pa among adolescent youth entering high school . while an estimated 30 to 45 million american youth play recreational and competitive organized sports , nearly two thirds of youth are estimated to withdraw from sport participation each year , with attrition rates being particularly high among adolescents . additionally , the results of this study correspond with national trends for sport participation among adolescent girls . while pa is important for the health of youth , adolescent girls reduce participation in sports more than adolescent boys in high school . our analysis shows that rates of participation in the 5 most popular sports was 16.6% lower for girls in high school compared to middle school . one potential explanation for the drop in sport participation among adolescent girls may be related to the timing of puberty and menarche . prior research found that advanced pubertal girls had significantly lower self - reported pa and accumulated fewer minutes of moderate - to - vigorous and vigorous pa and accelerometer counts per day at age 13 years than girls that physically mature later . this may be particularly important for girls who are overweight or obese , as they are more likely to begin puberty earlier than their healthy - weight peers . however , we found virtually no differences in obesity prevalence between most common sports and nonathletes / participants - in - other - activities in girls . our analysis does show that runners are significantly less likely to be obese than their peers who play the most popular organized female team sports . the differences in obesity rates among runners and 4 other organized team sport athletes are particularly important since previous research indicates that adolescent girls achieve pa primarily through sports . it is difficult to surmise why obesity prevalence among adolescent girls who play basketball , softball , soccer , and volleyball is similar to nonathletes / participants - in - other - activities . one explanation may be that these sports do not provide each participant with the sufficient level of daily moderate to vigorous pa . the us department of health and human services recommends children and adolescents aged 6 to 17 should have 60 minutes of activity or more of pa each day , which includes moderate or vigorous intense aerobic exercise at least 3 days per week . by grade 12 only 32% of females meet the recommended levels of pa , and less when using objectively measured activity . whereas running / track can be an individual activity within an organized team , the 4 remaining sports examined in this study are group - based activities , which might not offer adequate levels of vigorous pa for each team member . perhaps core team members receive significantly higher levels of moderate to vigorous pa than injured athletes or teammates who are afforded less practice or game time . another consideration may be that females with lower bmi percentile select to participate in track rather than the 4 remaining popular sports . compared with income and racial differences , the type of leisure time sports adolescent boys played had the strongest , most consistent inverse relationship with obesity , except for football . consistent with other research , obesity prevalence for football players was comparable to obesity prevalence of nonathletes / participants - in - other - activities . the self - selection of heavier adolescents into playing football may contribute to the findings that their risk of obesity is equal to male nonathletes / participants - in - other - activities . . a higher than normal bmi could potentially represent greater muscle density rather than fat . an examination of 71 males from different high school football players determined the use of age - adjusted bmi percentile can lead to misclassification of overweight and obese status especially for bigger lineman do have muscular body composition . further studies conclude that bmi calculations from body mass and weight may not accurately portray the modern nfl athlete , division - i ncaa , and high school football players regarding health status because bmi calculations do not distinguish between fat and lean tissue . we are unaware of any existing longitudinal research or studies consisting of a larger sample size that examines the accuracy of bmi for high school aged or younger football players and our dichotomy of healthy weight versus obese ( as opposed to overweight ) should help with bmi accuracy . as expected , boys who played baseball , basketball , soccer , and running / track had significantly lower obesity . aside from football , the obesity among male athletes is significantly lower than nonathletes / participants - in - other - activities . similar to the female population in this study , 7% fewer boys played leisure time sport activity in high school , compared to middle school . researchers estimate that 3.5 million kids play football in youth leagues ; that number dwindles to 1.3 million in high school . boys who spent their childhood playing in soccer leagues may find other activities more appealing as teenagers . other reasons for attrition cited by soccer players include a lack of free time and the need for more time to study . consistent with studies measuring pa among adolescent females , household income was not associated with a decline in leisure time sport activity . as family income levels increased , the girls in our study were more likely to participate in basketball , running / track , softball , and soccer . however , differences in family income did not have a significant impact on volleyball participation . among the factors cited for influencing girls sport participation is a lack of access , decreased quality of experience including poor facilities and nonoptimal playing times , and a shortage of positive role models . perhaps any one or all of these factors combined with other environmental issues may have a greater impact on participation rates for certain sports such as volleyball . the present study proposes that household income has a greater impact on differences in female adolescent obesity than sport participation or race . previous research suggests the prevalence of obesity among black girls was not related to household income , while the prevalence of obesity among white females was significantly higher in lowest income category than at higher income levels . conversely , participation in sport participation has a bigger impact on obesity for boys than race or family income . this income paradox may be explained by school - level income ( schools with median incomes that are substantially lower than the national or state median incomes ) or other environmental characteristics . while previous studies have shown a robust association between household income and pa , richmond and colleagues found that after taking into account characteristics of the school that adolescent attended , individual household income was no longer significantly associated with adolescent pa in either males or females . our data show that a greater proportion of male youth reporting leisure time sport participation are healthy weight than are female sport participants . obesity prevalence is significantly lower among male athletes than nonathletes / participants - in - other - activities in all sports except football . we are unaware of the reasons why differences in obesity among athletes persist along gender lines . possibly , healthy weight boys self - select to play common sports at a higher rate than their female peers . previous research has determined that certain women do not report weight management to be significantly important for sport and exercise participation . it is also possible that overweight or obese girls may be encouraged to exercise more than overweight or obese boys . our findings do not corroborate previous reports of an association between race / ethnicity , sex , sports participation , and the prevalence of obesity . as expected , our results did indicate that football players and black and hispanic males were at greater odds of being obese than their white male counterparts who played the other popular sports , but these results were not statistically significant . one significant limitation of these findings is that we are unable to determine if reports of sport participation were based on organized teams and leagues , or if they represent unorganized activities . it is possible that levels of pa ( low , moderate , and vigorous ) differ when adolescents play unstructured games with friends rather than compete in organized leagues . a second limitation is that the study relies on self - reports of pa , which suffer from significant bias . estimating pa frequency and duration is believed to be particularly challenging for children . also , given that with nhanes one can only examine relationships cross - sectionally , it is not clear whether weight status influences organized sports participation or vice versa . to our knowledge , this is the first study to use multivariate analysis to examine the impact of leisure time sport activity , race , and household income on the likelihood of obesity . our findings are that , on average , across a nationally representative sample , male and female adolescences who participate in common sports have lower rates of obesity than nonathletes / participants - in - other - activities . however , the effect of leisure time sport activity on obesity for female adolescents is not significant except for the negative relationship between obesity and running / track . these results suggest adolescent athletes who participate in the 4 most popular organized female team sports may benefit from increased levels of vigorous activity . physicians who counsel parents and girls toward sport participation should recognize that while leisure time sport activity may have significant benefits , the relationship between participation and healthy weight is not as clear . additionally , nationally represented surveys such as nhanes will benefit by collecting future data on pa , recreational sport participation , and organized sport participation among adolescents .

objective . to understand the relationships between participation in different types of leisure time sport activity and adolescent obesity , and how those relationships might differ based on race , gender , and household income . methods . data consisted of 6667 students that took part in the 1999 to 2006 national health and nutrition examination survey . the authors used adjusted wald tests to examine differences in the prevalence of obesity ( body mass index > 95th percentile for age and sex ) by sport for boys and girls separately . results . among adolescent youth age 12 to 19 years , 16.6% of male leisure time sport participants and 15.3% of female sport participants were obese , compared with 23.6% for male nonathlete participant - in - other - activities and 17.0% obesity rate for female nonathlete / participant - in - other - activities . for both males and females , reported participation in leisure time sports decreased between middle school and high school , and this reduction was associated with higher body mass index .

Introduction Methods Results Discussion

PMC4210175

computational methodologies utilized for in silico high throughput screening ( hts ) are a critical component of drug discovery approaches . within the available in silico hts approaches , methodologies that combine ligand- and structure - based screening procedures find the widest application . the challenge in any hts virtual screening ( vs ) platform is to develop an algorithm that is sufficiently fast and robust to evaluate many compounds while maintaining sufficient accuracy to identify a subset of biological active compounds ( i.e. , hits ) that have diverse structural scaffolds ( i.e. , scaffold - hopping ) . we sought to employ in silico screening to evaluate the repurposing of current drugs for a new therapeutic target . drug - repurposing maximizes the potential value of each hit by screening well - known compounds that have minimal toxicity and/or few side - effects . comparative studies of well - established ligand- and docking - based approaches concluded that shape - based ligand screening yielded markedly better outcomes than protein docking schemes . a ligand - based computational method involved two essential elements : an efficient similarity measure and a reliable scoring method . the similarity measure varied among different methods and focused on three factors : pharmacophores , molecular shapes , and molecular fields . the molecular - shape approaches maximized the overlap of shapes and determined a similarity value based on the degree of shape overlap . over the years , despite the investment made in developing scoring functions for molecular - shape approaches , none possessed accuracy and general applicability . consequently , investigators turned to the consensus - scoring technique that improved the probability of finding solutions by combining the scores from multiple scoring functions or using different reference molecules . we recently developed an efficient 3d shape - based similarity algorithm encoding the consensus molecular shape pattern of a set of active ligands into one descriptor , called the 4d fingerprint ( figure 1 ) . the 4d fingerprint formalism was originally proposed by hopfinger and co - workers and developed the quantitative structure the 4d - qsar model estimates molecular similarity measures as a function of conformation , alignment , and atom type . the resulting descriptors values were the occupancy measures for the atoms in the investigated set of bioactive molecules . while the similarity measures achieved excellent predictions for a variety of enzyme inhibitors , the weakness of this approach lies with the occupancy measures for the atoms ( or pharmacophoric groups ) which may also be present in similar , the resulting 4d fingerprint encoded in the 3d shape of the candidate ligand b is docked and ranked using the hwk scoring function . the application of the 4d fingerprint to the ligand b decreases the interaction ( purple arrow ) with the receptor . the 4d fingerprint approach implemented in the shape - approach - based routines enhanced or sabre program possessed a number of attractive advantages over other vs methods . first , it depended explicitly on 3d shape , not on the underlying chemical structure , and thus it excelled in identifying novel chemical scaffolds based on a set of known active ligands ( scaffold - hopping ) . the iterative 4d fingerprint approach was particularly robust for several reasons : ( i ) the 4d fingerprint descriptors were very sensitive to the details of molecular shape of active ligands , reducing the need to use multiple conformers of multiple query structures ; ( ii ) the method excel by the incorporation of the spatial distributions of chemical features of similar inactive ligands during the optimization and screening procedures ; ( iii ) the algorithm was fast and had the ability to scan a library of millions of compounds in a matter of hours . the method unified ligand- and structure - based 4d fingerprint vs approaches by docking the shape filtered ligand structures into the receptor - binding cavity . finally , running searches using this methodology was remarkably easy and required only that the end - user supply a query structure and runtime parameters to control the number of hits that were returned . despite these advantages , the 4d fingerprint method , as previously reported , suffered from a weakness in the empirical hwz scoring function for ranking and selecting the active ligands from large databases . to remedy this deficiency , we modified the shape - based vs algorithm of the sabre program and implemented a new , robust scoring function that accommodated the diversity of ligand scaffolds with an accuracy that exceeded our prior efforts . tuberculosis ( tb ) is a chronic and complex disease resulting from infection with the bacterium mycobacterium tuberculosis . tb remains an important public health problem worldwide , with 8.6 million estimated cases and 1.3 million deaths attributed to the disease in 2012 . in order to combat the spread of tb it is necessary to identify new molecular targets for tb drugs and develop new , more efficient , methods for screening ligands as potential drug candidates than methods used in the past . historically , high - throughput screening ( hts ) approaches coupled with in vitro testing served to identify promising hits with anti - tb activity . while successful in some cases , the hts approach frequently failed in the antibacterial drug discovery area due to the poor admet properties and insufficient or improper molecular diversity of the compounds screened . the mycobacterial esx secretion system , also referred to as the type vii secretion system , represents a promising , new target for tb drug development . the esx secretion system is a specialized system unique to mycobacteria that secrete a large number of proteins necessary for m. tuberculosis virulence . each esx secretion system includes a membrane - associated subtilisin - like protease , called the mycosin : mycp1mycp5 ( numbered according to gene cluster ) . mycp1 from the esx-1 system hydrolyzes the esx associated protein b ( espb ) during secretion , and this processing affects virulence in a mouse model of tb infection . the recent description of the molecular structure and substrate specificity of mycp1 prompted interest in mycp1 as a promising target for structure - based drug design . recently , we applied the combined ligand- and structure - based virtual screening procedure and 4d fingerprint algorithm to identify new inhibitors for mycp1 protease . the study reported here extends our previous work and reports a rational approach for ranking the ligand databases and demonstrates the performance of the novel hwk scoring function using 81 targets from the dekois database . validation of the efficiency of the vs method for scaffold - hopping and drug repurposing involved the application of this methodology for the identification of diverse inhibitor scaffolds against mycp1 protease and experimental testing of some of these scaffolds in an in vitro enzyme assay . we have recently developed a ligand and structure shape - based vs algorithm implemented within the sabre program . unlike other ligand - based shape overlapping methods , our approach efficiently detected the key pharmacophore groups of the active ligands responsible for binding to the target . the main advance of our methodology resided in the consideration of virtual but similar inactive structures ( decoys ) during the consensus molecular - shape detection process ( figure 1 ) . after similarity scoring , the selected structures were ranked according to their shape complementarity in the receptor - binding site . this report highlights the major steps of the algorithm and describes the approach used for the development of this scoring function . the molecular shape density function (r ) of a ligand is expressed in terms of the shape density functions of individual atoms and their overlap1 in which each atom i with coordinates ri = ( xi , yi , zi ) is described by a spherical gaussian:2where i is the van der waals radius of the atom i. the molecular volume v of the ligand is defined as3the volume vi of an atom i is4the intersection volume of atom pairs is defined as5the overlap volume of two molecules a and b is defined as6 in the sabre algorithm , the shape - density model is enhanced and defined as a linear combination of weighted atomic gaussian functions . thus , the molecular shape - density is the sum of all individual weighted pharmacophore densities , and the molecular volume is defined as7where vkpharm k = idr ki(r ) is the partial volume of the pharmacophore group k and is defined as a linear combination of atomic gaussian functions . the optimal coefficients { ck } are determined by iteratively adjusting the coefficients of the set of known active ligands { a } in the presence of virtual decoy structures { b } ( virtual decoys are inactive similar compounds that are not necessarily synthetically feasible or identified in the previous vs rounds ) until they satisfy these two criteria:7athis algorithm quickly builds a consensus molecular - shape pattern in which the optimal coefficients { ck } define a 4d fingerprint of the entire set of active ligands that also effectively excludes structurally similar , but inactive ligands ( decoys ) ( figure 1 ) . given a set of known active ligands { ai } with volumes v{ai } and a query structure a with the volume va , we effect the shape - filtering and ranking the candidate molecule b with volume vb . during the vs process , we observe two trends : ( i ) vbi va or ( ii ) vbi va . as a result , we can rank the structure b according to either the condition ( i ) , ( ii ) , or the combination of ( i ) and ( ii ) for two different ligands b and b. thus , we have two possible outcomes:(1)the volume of the candidate structure b is smaller than the volume of the query a : vbi va . the maximal overlap volume vabi for the two structures is restricted as vabi vbi and rewritten as8(2)the volume of the structure b is larger than the volume of the query a : vbi va and vabi va is rewritten as 9(3)we illustrate this case by considering two candidate structures with vbi va and vbj va . for clarity we consider that they have the same overlap volume with the query structure and consequently , vabi vabj . adding eqs 8 and 9 givesand we obtain the tanimoto scoring function:10we rewrote eqs 8 and 9 as smooth gaussian distributions and defined the scoring function hwk ( hamza wei korotkov ) that converges to one for optimal similarity:1112the tanimoto function and eqs 11 and 12 clearly reveal that the ranking of the candidate structure b is determined by several inhomogeneous criteria . for a fixed overlap volume , eq 11 gives the highest score for the ligand b with a smallest size even if it possess less similar chemical features than other ligands with larger volume sizes . second , the vabi term takes into account the overlap volume of the full ligand size instead the volume of the key chemical features present in both the query a , and candidate ligand b. this result in a higher ranking for the ligand b with largest size since the overlap volume varies with the full size of the ligand . third , we recently demonstrated the weakness of the tanimoto scoring function when used for filtering the 3d shape of the ligands and found that the tanimoto function only efficiently ranks the ligands with comparable volume size to the query . the volume of the candidate structure b is smaller than the volume of the query a : vbi va . the maximal overlap volume vabi for the two structures is restricted as vabi vbi and rewritten as8 the volume of the structure b is larger than the volume of the query a : vbi va and vabi va is rewritten as 9 we illustrate this case by considering two candidate structures with vbi va and vbj va . for clarity we consider that they have the same overlap volume with the query structure and consequently , vabi vabj . adding eqs 8 and 9 gives these drawbacks can be overcome by taking into account the specific atom - type information , such as the consensus molecular shape pattern or according to the 4d fingerprint approach ( eq 7 ) , the volume of the query a and the set of active ligands { ai } are defined as13the optimization of the coefficients { ck } leads to the residual volume v vapharm , and the value of the vapharm term in eq 7 ranges across the interval [ min v{a}pharm ; maxv{a}pharm ] . in the following demonstration , we assumed that the candidate b is an active ligand and similar to the query a in the set of active ligands { ai } , since if b is dissimilar the overlap volume converges to a small value . we haveusing the computed 4d fingerprint coefficients , the volume of b is written as14the volume of b is defined by eq 7 and is equals to the sum of the weighted partial volumes of the key pharmacophore groups and its volume size is in the interval of the set of active ligand volumes v{a}*. three possible scenarios exist:(1)vb * va*in this case , vb * [ min(v{a } * ) ; va * ] and the optimal similarity value is reached for(2)vb * va*in this case , vb * [ va * ; max(v{a } * ) ] and the optimal similarity is reached for(3)according to the eqs 13 and 14 and the 4d fingerprint coefficients , the tanimoto scoring function is written as t = vab/(vb * + va * vab ) withthe optimal similarity is reached for t = vb*/va * t = va*/vb * 1 . in this case , vb * [ min(v{a } * ) ; va * ] and the optimal similarity value is reached for in this case , vb * [ va * ; max(v{a } * ) ] and the optimal similarity is reached for according to the eqs 13 and 14 and the 4d fingerprint coefficients , the tanimoto scoring function is written as t = vab/(vb * + va * vab ) with as a result , the candidate ligands with a volume size slightly smaller or larger than the query volume are ranked equivalently when using the 4d fingerprint method with the tanimoto equation . finally , we observe that the weighting coefficients of the 4d fingerprint adjust and group the unknown active candidate structures with miscellaneous volume sizes and scaffolds into three classes relative to the query size . this confers the advantage of ranking more effectively these three types of shape using the hwk scoring function ( hwk , hwk , hwk ) . the docking approach described in our previous work combined the performance and speed of the ligand - based 4d fingerprint method with the shape characteristic of the receptor binding site . the current sabre docking algorithm encodes both the 4d fingerprint and the novel hwk scoring function , and it generates alignments where patterns with similar binding character are oriented in a similar fashion in the binding site of the receptor ( figure 1 ) . during the rigid docking process , the sabre program takes into account only the pharmacophore groups present in the vb(vb * ) that interact in designated ways with key receptor atoms . five important chemical features were assigned to an atom type : hydrogen bond donor , hydrogen bond acceptor , acidic center ( negatively charged at physiological ph 7 ) , basic center ( positively charged at ph 7 ) , and metal - chelation . the main novelty of the sabre docking approach is that the pairwise interaction between the key pharmacophoric groups ( defined by the 4d fingerprint , figure 1 ) of the ligand and the receptor atoms are calculated using the gaussian function gij . the pairwise interaction of atoms i ( ligand atoms ) and j ( receptor atoms ) is defined as15where deqtype is the standard distance between the heavy atoms i and j for each type of interaction ( i.e. , hydrogen bond interaction , electrostatic interactions ) and di , j is the distance between the two atom centers . the parameter is a freely adjustable parameter and controls the distribution of the gaussian function . the parameter controls the weight of the interaction type and depends on the 4d fingerprint coefficients . the pairwise interaction is attractive for > 0 and repulsive for < 0 . the total of n pairwise interactions between the pharmacophoric groups of the ligand and the key receptor atoms is defined by the geometric mean gi , jtotal(di , j ) as16thus , the combination of the total pairwise interactions gi , jtotal(di , j ) and eqs 1012 takes into account both the 4d fingerprint and the key interacting pharmacophoric groups of the ligand and leads to improved enrichment of the vs process . the hwkdock scoring function of the sabre docking method is summarized by the three equations:171819it is interesting to note that a ligand with high similarity score ( eqs 1012 ) is reranked with a lower score if its chemical features are close to repulsive receptor atoms . therefore , our scoring strategy developed in the docking method combines the fast and efficient ligand - shape - based 4d fingerprint vs with an extremely quick calculation of the interactions between the ligand pharmacophoric groups and the key receptor atoms . in addition , we observe in figure 1 that the interaction ( purple arrow ) involving the pharmacophore groups present in both active and decoy structures become negligible . analysis of the hwkdock function ( eqs 1719 ) highlights a new strategy for improving scaffold - hopping and drug repurposing performances . during the vs campaign , the shape size of the query is fundamental and orients the choice of the scoring equations . thus , if the shape of the query is small and does not completely fill the receptor binding cavity , the hwkdock+ is appropriate to identify structural hits with either comparable or larger volume sizes than the query volume . however , the hits with volume sizes ranging in the interval [ min(v{a } * ) ; va * ) ] of the set of active ligands ( eq 14 ) are also ranked with a high hwkdock+ score . in contrast , if the shape of the query complement the receptor binding cavity , the hwkdock is better suited to identify hits with smaller volume sizes while keeping high overlap volume with the pharmacophore groups of the query . finally , the hwkdocktanimoto is effective to identify hits with comparable query shape ( i.e. , rather smaller or larger structural sizes that fit into the receptor binding cavity while retaining structural diversity ) . consequently , three different classes of hits emerge based on the equations of the hwk function that selected them . in each list , the compounds are first ranked according to query similarity using the 4d fingerprint approach , and the diversity is achieved by selecting compounds ranked highly using one of these scoring equations . the dekois ( version 2.0 ) database of annotated active compounds and decoys was used to validate the hwk scoring function . the dekois database is a publicly available vs test database consisting of 81 targets . for our purposes , the ratio of the number of decoys to the number of active ligands was fixed at 30 . we used the dekois database instead of the 40 targets of the dud database for our vs test in order to measure the robustness of the scoring function when screening a large number of targets . this is one of the most commonly encountered measures for estimating prediction accuracy of vs algorithms . the effectiveness of the sabre program was evaluated using the enrichment factor ( ef ) metric at a given percentage of the database screened , and the area under the roc ( receiver operator characteristics ) . to test the efficiency of the hwk scoring function defined by eqs 1012 , we screened each target 10 times using a different set of five randomly selected active ligands ( as templates ) and reported both the highest performance ( roc auc value ) and enrichment factor ef at 1% for the 81 targets . screening results using the empirical hwz scoring function were reported for comparison . for each screening test , the five template structures where first removed from the list of active ligands . the detailed vs procedure was described in our previous work and summarized in the supporting information ( supplementary figure si-1 ) . briefly , the 4d fingerprint algorithm defined in the 3d - shape - based similarity method of sabre was used as the first filter of the nci ( national cancer institute ) from the nci open database compounds ( release 4 , 265 000 structures ) and fda ( food and drug administration , 1217 compounds ) database downloaded from the zinc database . it is important to note that the 4d fingerprint was generated using both the previously identified leads ( active ligands ) and inactive compounds ( considered as decoys ) . the multiple conformation states of each ligand in the database were generated using omega ( openeye scientific software ) . thereafter , we utilized the docking option of the sabre program to place the filtered conformations of each ligand into the active site of the mycp1 ( pdb i d : 4hvl ) and ranked them using the hwk scoring function . according to eq 14 , the candidate b structure is similar to the set of known active ligands { ai } if vb * [ min(v{a } * ) ; max(v{a } * ) ] and vb * the volume vapharm is defined by the 4d fingerprint coefficients { ck } that encoded the chemical features of the consensus molecular shape pattern of known active ligands ( eq 7 ) for the specific binding target . therefore , this suggests that the best fitted and most highly ranked ligands from the vs of the database have similar 4d fingerprint coefficients and thus should interact with the receptor of the known active ligands ( concept of the drug - target profile ) . it is important to note that this approach considers only the 4d fingerprint and the fast - fitting method implemented in the sabre program . to validate the effectiveness of sabre for drug repurposing , we conducted a ligand- and structure - based vs procedure using the fda database . recombinant mycobacterium thermoresistibile mycp1 was expressed and purified as reported previously . a quenched , fluorescent peptide assay was used to measure the activity of mycp1 in the presence of inhibitors . mycp1 was used to digest 20 m of the fluorescent substrate , abzavkaaslgk(dnp)oh ( genscript inc . ) . potential mycp1 inhibitors identified by sabre were diluted to a concentration of 150 m , and assays were measured in 96-well format . compounds that were considered hits showed less than 50% activity compared to controls ( dmso - buffer blank ) . the same in vitro assay was used to measure the inhibitory concentration 50% ( ic50 ) of the most promising hits . for ic50 measurements , inhibitors were added at 0 , 5 , 10 , 50 , 100 , 200 , 350 , and 500 m concentrations . we evaluated the accuracy of the hwk scoring function and compared the current values to those obtained with the empirical hwz scoring function using the multi conformational states of the decoys and active ligands of the 81 targets from the dekois database . the merits of scoring function became clear as it accurately ranked compounds with subtle structural changes . in the present vs trial , we selected query molecules according to the procedures presented in kirchmair et al . and used to describe the performance of the algorithms . as shown in figure 2 , we evaluated the auc for each target of the dekois database and the average auc using the sabre and sabre scoring functions . the average auc value of the best performing query for the 81 dekois targets using hwk and hwz scoring functions was 0.875 0.054 and 0.851 0.054 , respectively . the two scoring functions had similar overall performance for some of the 81 targets based on the auc metric ; however , an analysis of the complete set of targets revealed that sabre performed more consistently in terms of auc with an average auc 0.9 for 26 targets and 0.9 > auc 0.8 for 51 targets than sabre . moreover , sabre did not fail for any of the 81 targets screened . in comparison , sabre ranked the screening results with an auc > 0.9 for only 17 of the 81 dekois targets ( 11 targets out of 81 have auc 0.8 ) . one of the advantages of the sabre approach is that the vs performance combining the 4d fingerprint and the novel hwk scoring function depended less on the screened targets , as already observed in our previous benchmark tests using the hwz function with the 40 dud targets . comparison of the areas under the roc curves ( auc ) of the 81 dekois databases using the sabre and sabre scoring functions . the efficiency of the sabre scoring function was evaluated using the enrichment factor at 1% ( ef ) , and the results were also compared to those using the sabre function ( figure 3 and table si-1 ) . the average ef values for the 81 targets using the novel hwk and empirical hwz score - based virtual screening were 21.8 5.0 and 15.5 5.9 , respectively . the sabre method performed more consistently resulting in an ef less than 10% for only two targets , whereas the results using the sabre method provided enrichment factors below 10% for 24 targets . thus , the enrichments achieved with sabre are considerably better than those obtained with the empirical hwz scoring function , indicating that the novel scoring function was more efficient in identifying hits with notably different scaffolds compared to the query structure . therefore , on the basis of the auc and enrichment factor ef values , these results indicated that the novel hwk score demonstrated an improved and robust vs performance , albeit with the caveat that we used only 81 targets in this study . comparison of the enrichment factor ef at 1% of the 81 dekois databases using the sabre and sabre scoring functions . the sabre program was generally applicable for ranking any bioactive scaffold classes with the exception of inactive decoys . the recognition of a wide variety of structurally different ligand classes was an important goal of our virtual screening strategy . the mycp1 protease represented a challenge for both ligand- and structure - based virtual - screening approaches . indeed , only the crystal structure of the apo form of the enzyme was available , and the protein active site is relatively large , which decreased the probability of successfully identifying and ranking the correct pose of the screened ligands . the structures of mycp1 inhibitors that we previously reported were available , and visual analysis of their putative poses in the active site revealed that the binding mode of compound 1 was reasonable . these compounds have chemical features that enabled sar ( structure activity relationship ) studies and generated a novel 4d fingerprint . for the purpose of sar , an intuitive strategy for scaffold - hopping used the hit compound 1 as query and the hwkdock+ scoring function to rank hits with larger volume sizes from the nci database . we constructed such a ranking of the best 1000 structures ( top-1000 ) according to the docking - score function . the pharmacophore model reduced the number of these structures to a small subset of promising mycp1 lead candidates . the 135 hits derived from the nci database were superimposed with the binding query ( compound 1 ) and visually inspected . forty molecules were selected from the hits and tested in vitro for inhibitory activity against mycp1 . notably , 9 compounds out of the 40 were able to inhibit mycp1 by more than 50% when added at 150 m ( table 1 and figure 4 ) and one compound showed an ic50 less than 100 m . compound 2 inhibited mycp1 activity in the low micromolar range with an ic50 of 76.8 m and does not includes substructures described as pan assay interference compounds ( pains ) . structural scaffold of mycp1 inhibitors identified during the vs of the nci database . pan assay interference compounds ( pains ) remover , see ref ( 64 ) . the vs procedure identified these compounds based on their common chemical features ( 4d fingerprint ) present in the subset of known active ligand structures and their fit in the binding pocket . the coefficients of the 4d fingerprint efficiently encoded the spatial distributions of pharmacophoric points providing the alignment of compounds relative to the binding site surfaces . each point accounted for an important chemical feature such as hydrogen bond donors / acceptors and negative / positive charged groups . the basic physicochemical features of the known mycp1 compounds included the potential to establish hydrogen bonds as donors with thr156 and ser202 , glu203 and thr333 residues . furthermore , analysis of the docking results revealed that the lead compound 2 fit well within the binding site cavity . the compound 2 formed hydrogen bonds with the thr156 , thr333 , ser202 , and glu203 residues of mycp1 . the results were in agreement with our previous docking studies pointing out ser202 and thr156 as key residues to stabilize the ligand scaffold in the mycp1 catalytic binding site . a detailed analysis of the docking mode of the 11 compounds ( figure 4 ) revealed a close match between the pattern of hydrophobic and hydrogen bond donor pharmacophoric points of these hits compared to the pharmacophore model defined in our previous study . stick view of the binding compound 2 ( nsc-67021 ) and 11 ( hydroxystilbamidine ) in the mycp1 active site . as shown in table 2 , the vs procedure ranked 9 lead compounds at different cut - offs among the initial 1000 docked structures . among the top-30 , one lead compound was present , and this outcome corresponded to 11% coverage . furthermore , the 9 lead compounds were among the top-300 of the filtered nci database . these results highlight the merits of our 4d fingerprint vs approach when combined with the novel hwk scoring function . we also compared this simple approach to a complex approach including other likely query conformations . we modeled three plausible binding poses of the compound 1 ( query ) with different conformations in mycp1 cavity and redocked the top-1000 ligands using these three conformations , as shown in table 2 . the fusion approach markedly improved the percentage of retrieved lead compounds in the top-75 and further underscored the potential of the 4d fingerprint and hwkdock scoring vs procedure in the identification of lead compounds using the structure of unliganded receptor . % coverage of leads = ( number of leads in the top / total lead ) 100 . the integration of this newly generated computational method , which combined the 4d fingerprint and the hwk scoring function with in vitro enzyme inhibition studies , was a useful approach for evaluating current drugs , already on the market for a particular therapeutic purpose as potential agents for treating tb . to demonstrate the applicability of this integrated virtual and experimental screening for drug - repurposing , we undertook the virtual screening of the fda - approved drug database consisting of 1,217 compounds ( corresponding to 3358 structures including tautomers ) using the 4d fingerprints previously generated during nci database screening . hits were evaluated using the aforementioned mycp1 enzyme assay . in order to increase the structural diversity of the compounds identified by this process , we conducted the vs procedure three times using compounds 1 , 8 , and 9 as query for each vs round . the choice of the structural query was critical to the success of this approach . as described in methods , the scaffold diversity depended on the selected hwk or hwk or hwk ( tanimoto ) scoring equations , which also depended on the query size . thus , compound 1 , discovered in our previous work , was used as query since it has the highest affinity to mycp1 . the compounds 8 ( larger volume than that of compound 1 ) and 9 ( smaller volume than that of compound 1 ) were selected based on their differential volumes and structural diversity compared to the structure of 1 . the goal of our screen was to find hits with diverse structural scaffolds and comparable volume sizes to the queries . thus , the resulting docked ligands of the fda database were ranked using the hwkdocktanimoto scoring function ( eq 19 ) . since the screening process of the nci database and the benchmark test using the 4d fingerprint of sabre program demonstrated high enrichment factor at 1% of the screened database , we visually inspected the binding mode of the best 30 structures ( 1% ) identified within the fda database . we focused , in particular , on four compounds based on their high hwkdocktanimoto docking score and their interactions with the key residues ( thr156 and ser202 ) of the mycp1 binding cavity . these four compounds were chosen for in vitro inhibition assays , and three out of the four selected compounds exhibited more than 50% inhibition of mycp1 when used at 150 m ( table 1 ) , which validated the merits of our vs approach . finally , the active compounds were filtered for pan assay interference compounds ( pains ) ( table 1 ) and showed that the hydroxystilbamidine scaffold may be used as starting structure for further optimization . an analysis of the three ranked fda approved drugs showed that the compounds 11 , 12 , and 13 were ranked by sabre near the top at positions 2 , 24 , and 3 out of 1217 . the high score for compounds 11 and 13 was attributable to the hwkdocktanimoto scoring function , which took into account the ligand similarity as well as the optimal ligand / receptor pharmacophore model ( eq 19 ) . out of the three , compound 11 had the greatest effect , with an ic50 of 85.6 m , whereas the two other leads had ic50 > 100 m . in addition , we noted the low structural similarity between the three identified fda compounds ( figure 6 ) . as observed for the other leads , compound 11 formed hydrogen bonds with the thr156 , glu203 , and thr333 residues of the mycp1 active site ( figure 5 ) . interestingly , compound 11 is typically used as a histochemical stain to understand the distribution and localization of biomarkers , and these results suggested that it or its analogs may be repurposed for inhibition of mycp1 . more importantly , these preliminary findings show that the sabre algorithm with hwk scoring provides an efficient means for the identification of new uses for current drugs and encourages us to pursue the applicability of methodology in drug repurposing strategy for other medically relevant drug targets . structural scaffolds of mycp1 inhibitors identified during the vs of the fda database . the percentage of inhibition and ic50 are displayed . published data suggested counting hits only when the chemotype of a molecule is not equal to a template chemotype or any other chemotype that already exists in the hit list . this approach resulted in a chemotype enrichment that emphasizes discovery of ligands with different chemotype properties . we assessed the novelty of the confirmed 12 hits by comparing their structural similarities with a we computed the pairwise similarity index using the molecular access system maccs structural keys ( maccs , 166 bits ) of our 13 compounds ( query + 12 leads ) and represented the structural diversity using the heat map ( figure 7 ) . the map visualizes 15 15 = 225 pairwise comparisons and was color - coded by similarity values ranging from red ( low similarity value ) to dark blue ( high similarity value ) . we observed only two lobes in dark blue consistent with high similarity between the compounds ( maccs index > 0.8 ) and most of the compounds were dissimilar . this result supported the increased structural diversity ( maccs index < 0.6 ) of the new lead compounds using the combined 4d fingerprint and hwk scoring function . considering the high degree of substructure encoded in each vs round , it was not surprising that the 4d fingerprint algorithm performed well at finding diverse chemotypes . heat map of the maccs similarity index for the 13 compounds ( 12 leads + query ) . we report a rational method for the design of novel scoring function hwk and validated its performance using a large number of targets from the dekois database . the vs approach test using the 4d fingerprint and the hwk scoring function provided high enrichment factors in detecting active compounds at early stage of the 81 screened databases . we validated the efficiency of the combined 4d fingerprint and hwk scoring function in scaffold - hopping strategy through the identification of nine novel lead compounds in a short hit list from the vs of the nci database . the result of the vs round ranked these compounds in the top-300 of the database , and one of them displayed an ic50 comparable to that of the reference structure . in the absence of new drugs for infectious diseases like tb , it made sense to develop a vs strategy capable of exploring databases of current drugs used to treat diseases other than infectious diseases and potentially repurpose some of them for tb treatment . the merit of this approach lies in the obvious point that these commercially available drugs lack significant toxicity or side - effects . to test this notion , the screening of the fda database using our screening approach identified three fda - approved compounds as potential lead structures . the distributions of pairwise structural similarities presented in the heat map revealed that the 13 lead compounds resulting from the vs of nci and fda databases were structurally diverse . in summary , this study represents the comprehensive quantification of vs approach for scaffold - hopping and drug repurposing and provides a solid strategy for the discovery of new classes of mycp1 inhibitors .

two factors contribute to the inefficiency associated with screening pharmaceutical library collections as a means of identifying new drugs : [ 1 ] the limited success of virtual screening ( vs ) methods in identifying new scaffolds ; [ 2 ] the limited accuracy of computational methods in predicting off - target effects . we recently introduced a 3d shape - based similarity algorithm of the sabre program , which encodes a consensus molecular shape pattern of a set of active ligands into a 4d fingerprint descriptor . here , we report a mathematical model for shape similarity comparisons and ligand database filtering using this 4d fingerprint method and benchmarked the scoring function hwk ( hamza wei korotkov ) , using the 81 targets of the dekois database . subsequently , we applied our combined 4d fingerprint and hwk scoring function vs approach in scaffold - hopping and drug repurposing using the national cancer institute ( nci ) and food and drug administration ( fda ) databases , and we identified new inhibitors with different scaffolds of mycp1 protease from the mycobacterial esx-1 secretion system . experimental evaluation of nine compounds from the nci database and three from the fda database displayed ic50 values ranging from 70 to 100 m against mycp1 and possessed high structural diversity , which provides departure points for further structure activity relationship ( sar ) optimization . in addition , this study demonstrates that the combination of our 4d fingerprint algorithm and the hwk scoring function may provide a means for identifying repurposed drugs for the treatment of infectious diseases and may be used in the drug - target profile strategy .

Introduction Methods Results and Discussion Conclusion

PMC2864897

odor information is known to be of vital importance for most animals , and especially for insects . to achieve optimal fitness insects need to survive and reproduce in an optimal way . odors are known to play vital roles as cues for both these functions . to survive , optimally an insect needs to eat ( in most cases ) and to avoid becoming victim to predators or parasites . to reproduce optimally it needs to find a good mate and a suitable oviposition site . for one of the world s foremost model organisms , the fruit fly drosophila melanogaster ( dm ) , odor information is known to be heavily involved in location and quality determination of mates , food and oviposition sites . drosophila first appeared as a study organism for genetics when castle 1906 investigated effects of inbreeding . in the following two decades it was further established as a real model organism by morgan ( 1910 ) who used crossing experiments in order to map genes onto different chromosomes . due to its small size , short generation time , and easy rearing , today , dm is the most well - developed model animal when it comes to genetic manipulations and presence of mutants for more or less any trait . here , we describe our present state of knowledge regarding plant - odor - dependent neuroethology in dm . we discuss which odors are important , how different odor molecules are detected and transduced , how peripheral information is processed and integrated in the antennal lobe , and , finally , which types of behavior that have been studied . in reviewing scientific progress in a single model organism , it is inevitable that decisive information from other systems is omitted . to get an impression of all the important studies performed in insect olfaction outside drosophila , we refer the reader to the following books and reviews : stengl et al . ( 1992 ) , hildebrand and shepherd ( 1997 ) , hansson ( 1999 ) , hallem et al . ( 2006 ) , de bruyne and baker ( 2008 ) , galizia ( 2008 ) , and spehr and munger ( 2009 ) . the preferred food and oviposition substrate of dm is decaying fruit ( lachaise and tsacas 1983 ) and most key ligands identified can be connected either directly to the fruit , or to microorganisms living in and of the fruit ( stensmyr et al . typical examples of directly fruit - related odors are a number of esters like ethyl hexanoate , geranyl acetate , and pentyl acetate . in addition , more green odors like e-2-hexenal and hexanol are detected . of the bacteria - associated odors 2,3-butanedione seems to play an important role . a full account of the key stimuli identified so far is provided in table 1.table 1odorant receptors ( ors ) of the drosophila olfactory organs , their sensillumassociation , their central nervous target glomeruli , and their , so far , identified key ligandscolor labeling represents different sensillum types [ red basiconic ( antenna ) , yellow basiconic ( maxillary palp ) , green trichoid , blue coeloconic ] ors with the same number in brackets are expressed in the same sensillum odorant receptors ( ors ) of the drosophila olfactory organs , their sensillumassociation , their central nervous target glomeruli , and their , so far , identified key ligands color labeling represents different sensillum types [ red basiconic ( antenna ) , yellow basiconic ( maxillary palp ) , green trichoid , blue coeloconic ] ors with the same number in brackets are expressed in the same sensillum in the fruit fly , volatile chemical information is detected by two pairs of olfactory organs ; the third antennal segment and the maxillary palps . more specifically , odorants are detected by odorant receptors ( or ) , which are localized in the dendritic membrane of olfactory sensory neurons ( osn ) . each antenna carries ca 1200 osns , which are housed in about 420 olfactory sensilla , whereas the maxillary palp has about 120 osns and 60 olfactory sensilla . the olfactory sensilla come in three morphologically distinct types , namely sensilla basiconica , trichodea , and coeloconica that in turn can be divided into further subtypes ( shanbhag et al . each osn typically expresses only a single or ( beyond the ubiquitous or83b , see below ) , which confers a unique odorant response profile on the osn . in dm , apart from the ors , a number of gustatory receptors ( gr ) is expressed in the antennae , e.g. gr21a and gr63a that detect carbon dioxide ( kwon et al . the insect or genes are quite peculiar ; they show an inverted membrane insertion ( benton et al . 2006 ; lundin et al . 2007 ) , form heteromultimers ( neuhaus et al . 2005 , benton et al . 2006 ) and show no sequence homology to any other gpcrs . another peculiarity is that , contrary to what has been found in vertebrates , most insect osns express two ors . one is unique for each osn type , while one is expressed ubiquitously in all basiconic and trichoid - associated osns . contrary to the osn - type - specific ors , or83b is highly conserved among most insects ( jones et al . the initial coding of volatile chemical information is a consequence of the tuning spectra of the expressed ors . how the peripheral olfactory system of the fly decodes odors has been rather well studied ( e.g. clyne et al . 2004 , 2006 ) . that the gene family encoding ors indeed act as chemosensors was first shown by strtkuhl et al . ( 2001 ) . taking a cue from work done in the mouse ( araneda et al . subsequent electroantennogram recordings from the transgenic flies revealed an increased response to the ligand of the misexpressed or versus control flies . further evidence showing that the or family encodes bona fide odor detectors was provided by dobritsa et al . ( 2003 ) , who elegantly exploited the genotype of the halo mutant , which lack ( among other things ) the or22a gene . re - expression of or22a in the empty neuron created by the halo mutation fully rescued the response to ethyl butyrate , one of the ligands for or22a . the empty neuron system of halo flies has since been used extensively to examine the molecular receptive range of a large variety of ors ( e.g. hallem et al . the strength of this approach is that it allows for deorphanization in a more natural setting as compared to the alien environments provided by e.g. xenopus oocytes or human embryonic kidney cells , two frequently used systems for heterologous analysis of protein function . the drawback to the halo approach is that it is both time and labor intensive compared to heterologous systems , which can be incorporated into high throughput screening pipelines , and that the environment surrounding the osns ( e.g. obps , see below ) remains the same , even if the ors are exchanged . . however , those ors that respond to multiple compounds , typically respond to chemicals of structural proximity ( e.g. stensmyr et al . available data accordingly suggest that insect ors are optimally configured for specific chemical properties , or even specific chemicals . a problem though with any data regarding receptive range of ors is that the obtained tuning spectra are always dependent on the number of odor ligands screened , their relevance to the system as well as the concentrations tested . most ors are capable of detecting almost any chemical as long as this compound is tested in high enough concentrations , a feat that extends even to notoriously selective pheromone receptors . likewise , perceived notions of , for example , extreme selectivity might simply be a consequence of missing key stimuli from the test panel . thus , although a wide battery of odorants were used to deorphan the fly ors in the above - mentioned studies , the chosen stimuli only represent a minute fraction of all chemical volatiles that a fly might encounter in its daily life ( flies living in the wild that is ) . for example , a banana alone contains well over 500 volatile compounds , most fruits contain similar numbers of volatiles ( stensmyr et al . hence , it is quite possible that for many of the ors there exist better ligand matches that would differentiate the system further . the use of gas chromatography linked with electrophysiology and/or optical imaging is a way of identifying potent new ligands for the system , which would increase our understanding of how odorants are decoded . in the case of dm , odor sources should preferably be looked for among afrotropical fruit from the ancestral home range . the or and gr families are not the only chemosensory - specific gene families expressed in the peripheral olfactory system . the odorant binding protein family , first discovered in the moth ( vogt and riddiford 1981 ) comprises another large gene family [ 51 members in dm ( hekmat - scafe et al . 2002 ) ] encoding olfactory specific proteins . the obps are produced and secreted by auxillary cells surrounding the osns . within the sensillium lymph , the obps bind the hydrophobic odor molecules and presumably guide them through the aqueous sensillum lymph to the ors in the dendritic membrane of the osns ( vogt and riddiford 1981 ; vogt 2003 ; xu et al . 2005 ) . the obp family member lush nicely illustrates the importance of obps . lush mutants fail to respond to the fly pheromone cis - vaccenyl acetate , both physiologically as well as behaviorally ( xu et al . although the importance of obps for pheromone detection has been clearly demonstrated , their role in the detection of plant odors remains unclear . ors misexpressed in the empty neurons of the halo mutant retain their odorant specificity in their new environment , in spite of a differing obp set - up . recent work done on lush actually suggests that what the or is detecting is the conformationally altered lush molecule , rather than the pheromone itself ( laughlin et al . 2008 ) . if this is a unique feature of the pheromone system or also holds true for the part of the system detecting , e.g. food - related volatiles remains to be elucidated . detection of pheromones is furthermore dependent on the presence of another class of molecules , namely sensory neuron membrane proteins ( snmp ) ( rogers et al . snmps constitute an insect - specific sub - group of the cd36 family , that comprises transmembrane receptors involved in lipid binding and transport . the snmps act as co - receptors with ors in the pheromone detection subsystem of dm ( benton et 2008 ) . whether snmps are also of importance outside pheromone detection remains to be investigated . the osns found in coeloconica sensilla do not conform to the norm , as they do not express the ubiquitous or83b ( with the exception of coeloconica sensilla type 3 , that express or35a as well as or83b ) , suggesting that odorants in these sensilla are detected and/or transduced differently . the identity of the coeloconica ors was recently revealed to be divergent members of the ionotropic glutamate receptor family ( benton et al . the receptors , called ionotropic receptors ( irs ) form a large and divergent gene family comprising 63 members ( 61 putatively functional ) , of which 15 are expressed in the antennae . contrary to the ors , the irs seem to be expressed in a combinatorial manner and also display a slightly different ligand specificity compared to the ors , responding to nitrogen - containing volatiles , such as ammonia and 1,4-diaminobutane . in vertebrate olfactory orns the stimulated ors couple to golf proteins that stimulate like gs proteins adenylyl cyclase and raise the level of camp . these messenger molecules diffuse to and bind on ion channels , the cyclic - nucleotide - gated ( cng ) channels , which they activate . cng channels are non - selective cation channels that conduct na , k , and ca . the rise in intracellular ca activates ca - dependent cl channels that cause a robust depolarization and is the step in the signal transduction cascade providing the strongest amplification of the odor signal ( kaupp et al . the present knowledge regarding how odorant molecule binding to its or is converted into an electrical signal in insects arises from two sources . first , from manipulating parts of putative signal transduction cascades and observing the effect on odor response , and second , from studies of or function in heterologous expression systems . dm mutants with disturbed camp signaling ( dunce and rutabaga ) show abnormal olfactory behavior ( martin et al . 2001 ) . this was especially the case when the phosphodiesterase dunce was overexpressed in orn subsets , and thereby decreasing the camp level in these neurons ( gomez - diaz et al . there is also evidence that dag / ip3 signaling may play a role in odorant signal transduction ( krieger and breer 1999 ) . dm norpa mutants show impaired olfaction , which indicates that plc activity is required for appropriate processing of odor information ( riesgo - escovar et al . furthermore , mutations in the dgq gene , which encodes the gq alpha subunit result in reduced responses to odor stimulation ( kain et al . the responses were further attenuated by additional mutations in plc21c , a gene encoding for a plcb . interestingly , the dgq mutant phenotype was rescued by mutation of the dag kinase rdga ( kain et al . this finding indicates that the efficiency of olfactory signal transduction is controlled by a phospholipid messenger . support for this hypothesis comes from a study on mutants in stmbha , a gene encoding a putative pip2-dag lipase . these mutants show a markedly reduced electroantennogram response to odor stimulation ( kain et al . taken together , any disturbance of the pip2 cleavage and regeneration cycle seems to impair an appropriate processing of the olfactory signal in insect orns . thus , both a gs- and a gq - mediated pathway seem to be involved in dm olfactory transduction . expression of ors in heterologous systems allows us either to perform functional tests by using endogeneous parts of the signal transduction machinery or to coexpress useful reporters . to identify ligands for vertebrate ors a variety of assays has been developed , such as detection of camp production and ca imaging ( katada et al . for example , by coexpression of drosophila or43a with the promiscous g protein gaa15 in xenopus ooytes receptor activation could be monitored by measuring cl currents activated by intracellular ca release ( wetzel et al . elevation of free ca could also be obtained in hek293 cells when or22a was coexpressed with the ubiquitous receptor protein or83b ( neuhaus et al . 2005 ) . further investigations of the or22a / or83b complex in hek293 cells ( wicher et al . 2008 ) or or47a / or83b in hela cells , hek293 t cells and xenopus oocytes ( sato et al . 2008 ) revealed that these heterodimers form non - selective cation channels permeable to na , k , and ca . even in the absence of any stimulation , these channels show some constitutive activity leading to elevated levels of free ca . short odor stimulation was seen to produce an immediate transient response that was independent of g proteins , i.e. the ors act as ionotropic receptors . the fast ionotropic response is followed by a slower developing , but more sensitive , metabotropic response ( wicher et al . it was shown that stimulation of or22a , either expressed alone or coexpressed with or83b , lead to camp production . on the other hand , or83b , either expressed alone or coexpressed with or22a , responded to camp elevation with current production . mutagenesis in the or83b protein caused a change in the permeability ratio of ions , which demonstrates that or83b is responsible for the channel activity of dm ors ( wicher et al . 2008 ) . taken together , the odorant - specific part of the or dimere seems to couple to stimulatory g proteins to stimulate camp production . this g - protein - dependent pathway provides a highly sensitive odor detection , while at higher odor concentrations there is a very fast , ionotropic mechanism of or activation . it remains to be seen what role gq signaling may play ( kain et al . the insect odorant receptor complex is composed of an odorant - specific receptor protein orx and the non - selective cation channel or83 that conducts na , k and ca . the direct activation of or83b by orx ( red flash ) leads to a fast and transient cation flow . the adenylyl cyclase ( ac ) activity ( violet flash ) and thus the camp production . camp in turn activates or83b ( blue - green flash ) . the ionotropic pathway ensures a very rapid recognition of high odor concentrations while the metabotropic pathway allows highly sensitive odor detection scheme of odorant signal transduction . the insect odorant receptor complex is composed of an odorant - specific receptor protein orx and the non - selective cation channel or83 that conducts na , k and ca . the direct activation of or83b by orx ( red flash ) leads to a fast and transient cation flow . the adenylyl cyclase ( ac ) activity ( violet flash ) and thus the camp production . camp in turn activates or83b ( blue - green flash ) . the ionotropic pathway ensures a very rapid recognition of high odor concentrations while the metabotropic pathway allows highly sensitive odor detection the neural components , mediating olfactory information in the brain of the fruit fly , have been intensively studied and are well described ( stocker 1994 ) . the olfactory system of the dm adult is organized according to similar principles as the vertebrate olfactory system , but with vastly reduced numerical complexity ( hildebrand and shepherd 1997 ; hallem and carlson 2004 ) ( fig . as mentioned in the two previous sections , odors are recognized by osns ( not shown in fig . the osns send their axons to the first olfactory neuropil in the insect brain , the antennal lobe ( al ) , which processes the olfactory information . the al is a structure common to all insects except some anosmic species that have lost it secondarily ( strausfeld and hildebrand 1999 ) . the insect al represents an analogous structure to the olfactory bulb in vertebrates but has evolved independently ( strausfeld and hildebrand 1999 ) . the drososphila al comprises 49 olfactory glomeruli , which can be identified individually due to their specific position and size ( laissue et al . however , in dm , osns form an axonal commissure between the two als and individual axons innervate homologous glomeruli in the two lobes ( stocker 1994 ) . interestingly , a few glomeruli ( as v , vl1 , vp1 , vp2 and vp3 ) are innervated only unilaterally ( stocker et al . as mentioned above , each osn expresses only a single or gene ( besides or83b ) . all the osns expressing the same or converge onto a common glomerulus , which thus collects osn input from only one functional type . the glomerulus acts as a collecting basket of osns with identical odor response profiles . 1995 ) , but was finally proven by molecular techniques in dm ( gao et al . 2000 ) . this condition is also assumed to hold true for other insect species , but has not yet been experimentally proven . however , a few aberrant cases of 1 osn type to 2 glomeruli and of 2 to 1 innervation ratios in dm have been described as well ( couto et al . 2schematic of the drosophila olfactory pathway . a antennal olfactory sensory neurons ( osn , blue ) converge in specific glomeruli of the antennal lobe . some of them send an axonal branch through the antennal commissure to the other hemisphere . local interneurons ( ln , green ) branch in all glomeruli and interconnect these with each other . projections neurons ( pn , red ) collect the olfactory information within the antennal lobe and send their axons to higher processing centers as the calyx and the lateral protocerebrum . the three principal populations of neurons and their synaptic connections within the glomeruli ( gray circles ) are represented . , the existence of both inhibitory local interneurons ( iln ) as well as excitatory local interneurons ( eln ) has been shown schematic of the drosophila olfactory pathway . a antennal olfactory sensory neurons ( osn , blue ) converge in specific glomeruli of the antennal lobe . some of them send an axonal branch through the antennal commissure to the other hemisphere . local interneurons ( ln , green ) branch in all glomeruli and interconnect these with each other . projections neurons ( pn , red ) collect the olfactory information within the antennal lobe and send their axons to higher processing centers as the calyx and the lateral protocerebrum . the three principal populations of neurons and their synaptic connections within the glomeruli ( gray circles ) are represented . , the existence of both inhibitory local interneurons ( iln ) as well as excitatory local interneurons ( eln ) has been shown a glomerulus receives not only the input from osns , but contains a highly ordered synaptic organization including two types of interneurons ( fig . 2b ) : the local interneurons ( lns ; approximately 100 , probably analogous to periglomerular cells in mammals ) and projection neurons ( pns ; approximately 150 , analogous to mitral / tufted cells in mammals ) . lns of the al represent an enigmatic element of early olfactory coding in the cns of insects . they have all their projections restricted to the al , where most form wide - field arborizations within most glomeruli . the majority of lns appears to be inhibitory ( ilns ) and release -aminobutyric acid ( gaba ) as a neurotransmitter ( wilson and laurent 2005 ) , indicating that they form a lateral multi - level inhibitory network modulating the olfactory signal within the al . however , a recently described population of excitatory cholinergic lns ( elns ) forms a dense network of lateral excitatory connections between different glomeruli assumed to boost the al output ( olsen et al . 2007 ; shang et al . pns have dendritic arborizations within the glomeruli and convey the olfactory signals through axonal projections to higher olfactory processing centers such as the lateral protocerebrum and the mushroom bodies . a great majority of the pns is uniglomerular with dendrites confined to a single glomerulus , but a few have multiglomerular dendritic fields . several imaging studies in different insect species using either calcium - sensitive dyes ( e.g. joerges et al . 1997 ; galizia et al . 1999 ; sachse et al . 1999 ; carlsson et al . 2002 ) , genetically encoded reporters to measure intracellular calcium ( fiala et al . 2002 ; wang et al . 2003 ; silbering et al . 2008 ) or synaptic vesicle release ( ng et al . 2002 ; yu et al . 2004 ) have shown that odors are encoded as specific spatio - temporal across - glomeruli patterns . each odor evokes activity in several glomeruli , whereas each glomerulus participates in the patterns of several odors ( fig . the olfactory system has in this way developed a strategy to encode a huge number of odors with a limited number of coding units . although the different studies agree on how odors are encoded , different publications report contradicting results regarding the transformation of odor representations at the different processing levels within the al ( i.e. osn versus pn responses , fig . 3 ) . these results reach from sharpening and contrast - enhancement of the olfactory input due to inhibitory processing in honeybees ( sachse and galizia 2002 ) to either not processing at all ( ng et al . 2002 ; wang et al . 2003 ) or a broadening of the output pattern in comparison to the input pattern in dm ( wilson et al . 2004 ) . adding to the already existing complexity of the al network , a recently published study in dm shows that presynaptic inhibition onto osn terminals leads to an inhibitory network activity ( olsen and wilson 2008 ) . odor information processing mechanisms underlying olfactory coding are thus highly diverse and appear to be specific for particular glomerulus - odor combinations ( silbering et al . 2008).fig . the calcium - sensitive protein g - camp has been genetically expressed in either projection neurons ( above ) or sensory neurons ( below ) . calcium signals to two different odors have been superimposed onto the morphological image of the antennal lobe . comparison of the activity patterns between the sensory and the projection neurons to the same odor reveals similar but not identical responses odors evoke specific patterns of glomerular activity in the drosophila antennal lobe . the calcium - sensitive protein g - camp has been genetically expressed in either projection neurons ( above ) or sensory neurons ( below ) . calcium signals to two different odors have been superimposed onto the morphological image of the antennal lobe . comparison of the activity patterns between the sensory and the projection neurons to the same odor reveals similar but not identical responses the al output responses are further processed on their way to higher processing centers . there is a massive divergence between pns and the intrinsic neurons of the mushroom body , the kenyon cells . while most pns have a relatively broad response profile with various levels of activity , a large number of units encode that same information with very few spikes , in an almost binary fashion , with only few units active at any time among kenyon cells ( perez - orive et al . this coding strategy is in strong contrast to the one found in the al , where each pn covers a wide dynamic range . deciphering the coding of odors on all processing levels is crucial for understanding the remarkable capacity of the olfactory system in recognizing and discriminating the vast array of odor molecules . when investigating the sense of smell the first question that comes up is , do flies have to smell ? in an elegant set of experiments , asahina et al . ( 2008 ) demonstrated that the sense of smell is important at least for the survival of fruit fly larvae : when being reared under food ad libitum , anosmic or83b mutants developed as successfully as mutants whose or83b system had been rescued . however , when food was limited , i.e. when larvae had to search for new food sources , the anosmic flies were less successful compared to the rescued flies . in a direct competition experiment anosmic mutants were selectively eliminated in competition with fly larvae whose or83b had been rescued . interestingly , partial rescue of the olfactory system ( only one of 61 receptors was functioning ) led to survival rates that were intermediate between the anosmic and the fully rescued mutants . although a simple sense of smell ( partially rescued mutants ) already increased the fly larvae s survival rates , a more powerful smell ( fully rescued mutants ) was even more efficient . the efficiency of the adults sense of smell can be demonstrated easily by putting about 50 flies in an enclosed arena that is equipped with two traps . when one trap is empty and the other is filled , e.g. with rotten banana , one of drosophila s favorite food sources , the first flies will enter the smelling trap already after a few minutes and after 24 h between 90 and 100% of the total number is usually captured . ( 2004 ) and was recently used , e.g. to demonstrate that different dm strains like wildtype berlin , wildtype canton s , etc . differ substantially in their odor preferences and in their response latencies ( ruebenbauer et al . however , as a dm fly can live for more than 2 weeks , it has ample time to experience diverse and varying qualities of different food sources . although the flies are equipped with some innate odor preferences , i.e. every inexperienced drosophila will head for a plume of rotten banana or balsamico vinegar , they can learn associations of odors with good or bad experiences . a substantial amount of work has been done on olfactory learning , mainly using pavlovian conditioning procedures , where one odor ( conditioned stimulus ) is temporally paired with a positive ( i.e. sugar water ) or negative ( i.e. electric shock ) reinforcement , the so - called unconditioned stimulus ( tully and quinn 1985 ) . this procedure provided knowledge regarding the formation and capacity of short - term and long - term memory in fruit flies . ( 2007 ) could map which cells in the brain are necessary for olfactory memory retrieval ( kenyon cells in the mushroom body whose axons form the /-lobes ) , and which are needed for acquisition and consolidation of memory ( /-kenyon cells ) . however , apart from helping to understand learning in flies , conditioning experiments provide a solution to another problem : only few odors provoke strong innate behavioral responses in dm , e.g. the main component of banana odor isoamylacetate is highly attractive , while benzaldehyde the typical smell of bitter almond is a repellent . in order to check the flies olfactory threshold for innately neutral odors these have to be provided with a meaning by positive or negative reinforcement . after conditioning , it is possible to test , for example , the minimum concentration the flies react to . however , to tell the truth , for an ethologist interested in the sense of smell drosophila would not have been the first choice . while trap assays and mass learning experiments often yield valuable results , the investigation of individual flies is often frustrating . in order to study the sense of smell , one would like to make predictions on how a specific fly responds before and after some kind of manipulation . however , to make any predictions about the fly s response to an odor you need to know the fly s motivational state . contrary to , for example , workers of eusocial insects that have no sex but are dedicated just to foraging , fruit flies are sometimes in the mood for sex , sleeping , ovipositioning or foraging . each motivational stage can provoke different responses to different stimuli , which makes the prediction how a fly will respond rather difficult . despite these problems , experiments using individual flies are necessary in order to investigate , for example , the speed of odor processing or the response to minute differences in an odor s concentration . to illustrate the difficulties in establishing good bioassays for single dm one of us invested considerable time in establishing an experiment where individual flies were exposed to a continuous airflow within 1 cm - wide glass tubes . the flies were tracked automatically and an elaborate stimulus controller allowed exposure of the flies to odor pulses well - defined in timing , blend composition , and concentration . we expected the flies to show upwind runs whenever facing a positive odor , e.g. banana or aceto balsamico . by coupling the stimulus controller to the tracking device , we were able to compare the upwind speed directly before odor contact with the one afterwards . however , despite a comprehensive analysis until now we never found any consistent responses of the flies to different kinds of stimuli ( fig . 4 ) . the picture changes when flies are not tested while sitting or running but during flying . budick and dickinson ( 2006 ) successfully investigated individual drosophila flies in a free - flight wind tunnel . under these conditions , the contact to an odor plume provoked directed upwind flights , while the loss lead to a so - called casting behavior , where the trajectory of the flies becomes more and more perpendicular to the wind direction ( a strategy that is well known from moths and that under natural conditions increases the chance to re - enter the odor plume , fig . like in our walking assay , the use of a tracking device allowed the authors to measure the response latencies after the flies entered ( < 250 ms ) and lost the plume ( ca . this experiment thus provides the tool to investigate fine scale temporal dynamics of the flies sense of smell . these experiments use innate responses of tethered , flying individuals to odor stimuli provided in a constant airflow ( duistermars and frye 2008 ) . the development of admittedly rather complicated experimental procedures gives a vantage point to unravel more details about the fine structure of the dm s sense of smell.fig . c a computer - controlled stimulus device generates odor stimuli , that meet the flies at a predictable time . a 3-d tracking system detects the time of plume contact and plume loss and calculates the flight s directionality and upwind speed before and afterwards steck - knaden assay . c a computer - controlled stimulus device generates odor stimuli , that meet the flies at a predictable time . a 3-d tracking system detects the time of plume contact and plume loss and calculates the flight s directionality and upwind speed before and afterwards considering the progress reported above , we are presently in an extremely interesting time regarding dm olfactory neuroethology . so much background data have been produced on neural and molecular function at different levels that we are approaching stages where behavioral questions can be attacked with a full arsenal of neurogenetic tools . by using the knowledge regarding receptors , transduction mechanisms , and primary and higher integration we can dissect the system , but only when we have relevant and preferably nature - based bioassays and stimuli will we start to really understand the olfactory neuroethology of drosophila melanogaster .

drosophila melanogaster is today one of the three foremost models in olfactory research , paralleled only by the mouse and the nematode . in the last years , immense progress has been achieved by combining neurogenetic tools with neurophysiology , anatomy , chemistry , and behavioral assays . one of the most important tasks for a fruit fly is to find a substrate for eating and laying eggs . to perform this task the fly is dependent on olfactory cues emitted by suitable substrates as e.g. decaying fruit . in addition , in this area , considerable progress has been made during the last years , and more and more natural and behaviorally active ligands have been identified . the future challenge is to tie the progress in different fields together to give us a better understanding of how a fly really behaves . not in a test tube , but in nature . here , we review our present state of knowledge regarding drosophila plant - odor - related olfactory neuroethology to provide a basis for new progress .

Introduction Which plant-related odors are important to Detecting the molecules Chemical to electrical Primary information processingthe antennal lobe The resultbehavior Conclusion

PMC5361002

age - related changes in skeletal muscle result in loss of muscle mass and weakness . this leads to a decrease in daily physical activity in older adults , and can result in poor health and a need for professional care . muscle loss and weakness cause serious health problems , such as decreased strength and aerobic capacity1 . the world health organization ( who ) ranked lack of physical activity as the fourth leading cause of mortality worldwide2 . according to who recommendations , older adults should aim to maintain or increase their daily amount of moderately or vigorously intense physical activity3 . many older people , particularly those 65 years of age or older , who are interested in enhancing their health do perform some kind of exercise training4 ; however , the chosen exercise often has a low intensity level ( e.g. , walking ) . this is not strenuous enough to effectively improve either muscle strength and mass or aerobic capacity5,6,7,8 . hatamoto et al.9 showed that turning during sports increased the energy cost of running and that the magnitude of the increase depended on both the turn frequency and running velocity . in this study , we examined a new exercise pattern called slow walking with turns ( swt ) , which incorporates turns to increase the work load of walking . we have already shown that swt increases the exercise intensity relative to normal walking at the same velocity and hypothesized that the reason for this increase was extra muscle contraction resulting from the turns . the walking velocity and turn frequency is easily adjusted to provide an exercise intensity of 3 to 10 metabolic equivalents ( mets ) over a 3-m distance , and it is thus easy to prescribe an exercise program tailored to individual needs10 . in the current study , the swt ( tempo , turn frequency , and method of turning ) was adjusted to reduce individual differences and make it easy to perform even for older people . the purpose of this study was to confirm the exercise intensity of swt and examine the muscle activity during turns . eight recreationally active volunteers participated in order to confirm the exercise intensity of swt . the participants mean age was 24.9 ( standard deviation ( sd ) , 5.0 ) years , mean stature was 1.66 ( 0.10 ) m , and mean mass was 58.0 ( 6.8 ) kg . none of the participants had any injury that might have influenced their athletic performance . all participants performed swt over five fixed distances and treadmill walking ( tw ) at five different velocities in random order . oxygen consumption ( vo2 ) , rating of perceived exertion ( rpe ) , and heart rate ( hr ) were measured during each exercise . during swt , the participants walked back and forth over several fixed distances and performed 20 turns per minute . the distances were 1.5 , 2.0 , 2.5 , 3.0 , and 3.5 m ; the average velocities for walking back and forth were 2.7 , 3.6 , 4.5 , 5.4 , and 6.3 km / h ; and the cadence was 180 steps per minute . the participants took six steps for each distance during walking and three steps per turn . the tw velocities were set at 2.7 , 3.6 , 4.5 , 5.4 , and 6.3 km / h , which were the same velocities as during the walking portion of the swt . all sessions consisted of 4 minutes of exercise at each velocity with 1 minute of rest between different velocities ( fig . 1fig . swt involved shuttle walking for a short distance , including 20 turns per minute . the distance in each stage was 1.5 , 2.0 , 2.5 , 3.0 and 3.5 m. ) . when participants could not maintain the cadence , the trials were stopped and restarted . swt involved shuttle walking for a short distance , including 20 turns per minute . the distance in each stage was 1.5 , 2.0 , 2.5 , 3.0 and 3.5 m. all participants completed a familiarization session prior to the actual trial session . the experiments were conducted in an indoor facility with hard flooring , and the participants were instructed to wear the same indoor sports shoes for all trials . turning consisted of three steps , a deceleration step , a change of direction step , and an acceleration step out of the turn . the turning technique was adapted from the cross - step turn , and participants turned towards the striding foot during the deceleration step . the participants practiced the cross - step turn technique until they could repeat it consistently . all participants were instructed to avoid food , caffeine , tobacco products , and alcohol for 3 hours prior to the exercise sessions . during the exercise , the participants wore a face mask to collect exhaled gas , which was measured at 12-second intervals using an mass spectrometer ( arco 2000 ; arco system , chiba , japan ) . the mean vo2 of the last minute of each stage was used in the data analysis . the hr ( polar ce0537 hr monitor ; polar electro , kempele , finland ) was measured just before the end of each 4-minute stage , and subjective exercise intensity was assessed using the borg rpe scale11 . measured vo2 was converted into mets ( 1 met=3.5 ml / kg / min ) . second , the participants for measurement of muscle activity were six healthy men with a mean age of 25.0 ( 4.4 ) years , mean stature of 1.74 ( 0.05 ) m , and mean mass of 68.3 ( 5.0 ) kg . the swt protocol was the same as above - mentioned , except the walking distances were 1.5 , 2.0 , and 2.5 m. the tw velocities were 2.7 , 3.6 , and 4.5 km / h ; these were chosen to be the same as the walking velocities during swt . the activity of 11 muscles during swt , tw , and isometric maximum voluntary contractions ( mvc ) was measured using electromyography ( emg ) ( lp - ws1221 ; logical product corp . , fukuoka , japan ) to compare muscle activity during the stance phase of tw and the turning phase of swt . the 11 muscles selected for emg were the rectus abdominis ( ra ) , erector spinae ( es ) , gluteus maximus ( gmax ) , gluteus medius ( gmed ) , hip adductor ( ha ) , vastus lateralis ( vl ) , rectus femoris ( rf ) , vastus medialis ( vm ) , biceps femoris ( bf ) , tibialis anterior ( ta ) and soleus ( sol ) . all measurements were taken on the right side using wireless emg sensors attached over the muscle belly . a foot sensor was attached to the right heel . before application of the sensors , the skin area was shaved , cleaned with alcohol , and abraded with coarse tape to reduce skin impedance and ensure good adhesion of the sensors . the researcher manually fixed the posture of the participant , applying resistance against the direction of mvc . the participant then exerted an isometric mvc against the resistance while emg was performed on the muscle being tested . the mvc for each muscle was measured as follows : ra : the participant lay in a supine position with his knees positioned at 90 flexion and ankles restrained by an assistant . he then raised his shoulders and exerted enough force to further elevate their upper body while the researcher applied resistance against the direction of force . es : the participant lay face down with his hands on the back of his head and raised his upper body ( trunk extension ) . gmax : the participant lay face down with his knee flexed to 90 and extended his hip joint . gmed : the participant lay on his side with the test leg uppermost and abducted his mildly extended hip joint . ha : the participant lay on his side with the test leg down and resting on the bed . the researcher then applied resistance while the participant attempted to elevate ( adduct ) the test leg . vl , rf , and vm : the participant sat on a chair with back support . his trunk was secured with a belt and his feet did not touch the floor . his hip and knee joints were fixed at 90 angles by fastening a belt around his lower legs . he then extended his knees against the resistance of the belt to allow for measurement of the mvc . bf : the participant lay face down on the floor with his lower legs held in an upright position perpendicular to the floor , knees flexed at 90 and hips flexed at 0. he then tried to further flex his knees against resistance applied by the researcher . ta and sol : the participant sat horizontally on a tilt table with his knees fixed to the table and his ankles in a neutral position ( 0 dorsi / plantar flexion ) and his feet fastened to a foot cradle with a belt . he then made maximal attempts to dorsiflex and plantar flex his ankle against the resistance of the belt . two trials were conducted for each muscle , with a rest of 2 minutes or more between trials . after the trials , the emg data were downloaded onto personal computer ( vaio ; sony , tokyo , japan ) . the emg data were full - wave rectified , and the integrated emgs data were averaged during mvc over 1 second including the point at which the torque was maximal . the muscle activities during the stance phase of tw and the turn phase of swt were calculated by integrating the emg value in the reaction time of the foot sensor . each emg level was averaged for five times of turns in swt and five steps in tw . the emg data in swt were adopted from five turns at the beginning of the exercise . in tw , the measurement was started after walking had become stable , and the data were adopted from first five steps . the averaged integrated emg data during the actions were quantified and normalized as the values relative to those during mvc ( i.e. , % mvc ) . all statistical analyses were conducted using spss software ( v21 ; ibm corporation , armonk , ny , usa ) . differences between swt and tw were assessed using t - tests in experiment 1 and one - way analysis of variance in experiment 2 . this study was approved by the ethical committee of fukuoka university , fukuoka , japan ( number 10 - 02 - 02 ) , and informed consent was obtained from all participants . this study followed the guidelines of the world medical association declaration of helsinki : ethical principles for medical research involving human subjects , as well as the ethical guidelines for epidemiological research outlined by the ministry of education , culture , sports , science and technology and the ministry of health , labour and welfare , japan . oxygen consumption during the last minute of each stage remained in a steady state with no difference between the 3rd and 4th minute from the start of the exercise . the results of mets , hr and rpe were shown in table 1table 1.mets , rpe and hr for swt and twtwswt2.7 km / h3.6 km / h4.5 km / h5.4 km / h6.3 km / h1.5 m2.0 m2.5 m3.0 m3.5 mmets2.6 0.23.0 0.23.5 0.24.2 0.35.2 0.24.0 0.4***4.4 0.5***4.8 0.4***5.3 0.6**6.3 0.6**rpe7.8 1.08.1 1.09.3 0.910.4 0.711.6 0.78.5 1.69.0 1.59.4 1.510.5 1.311.6 1.1hr84 1689 1493 12102 8112 1089 1598 17 * 102 18 * 107 22119 19mean sd . distances of 1.5 , 2.0 , 2.5 , 3.0 and 3.5 m during swt correspond to average walking velocities of 2.7 , 3.6 , 4.5 , 5.4 , and 6.3 km / h , respectively . * p<0.05 , * * p<0.01 and * * * p<0.001 , comparison between swt and tw at a velocity corresponding to walking of swt .. the mets of swt were significantly higher than those of tw at a velocity corresponding to walking of swt ( p<0.01 ) . the difference in mets between swt and tw appeared to be larger at slower speeds , but not significantly . the hr and rpe values were measured just before the end of each stage . there was no significant difference in hr and rpe except for the hr at distances of 3.6 and 4.5 km / h ( p<0.05 ) , despite the significantly higher exercise intensity in the swt . mean sd . distances of 1.5 , 2.0 , 2.5 , 3.0 and 3.5 m during swt correspond to average walking velocities of 2.7 , 3.6 , 4.5 , 5.4 , and 6.3 km / h , respectively . * p<0.05 , * * p<0.01 and * * * p<0.001 , comparison between swt and tw at a velocity corresponding to walking of swt . muscle activity was significantly greater in the es , vl and vm during the turn phase of swt relative to the stance phase of tw at all stages ( table 2table 2.% mvc at the turn phase in swt and the stance phase in tw of each muscletwswt2.7 km / h3.6 km / h4.5 km / h1.5 m2.0 m2.5 mra6.86.87.28.79.09.5es5.56.27.514.5**17.7**15.5*gmax5.57.210.18.5 * 9.39.3gmed10.710.611.311.512.712.6ha10.910.811.313.013.213.9*vl7.29.410.913.5**13.5**13.7**rf9.39.410.610.411.011.5vm4.75.05.710.3**9.9**10.9**bf11.312.312.415.9 * 16.618.0ta10.011.813.714.213.313.6sol13.315.517.517.620.321.9*p<0.05 , * * p<0.01 , comparison of muscle activity levels for swt and tw at a velocity corresponding to walking of swt . ) . the activity of the es , vl , and vm during the turn phase of swt was about twice the activity level during the stance phase of tw . further , the activity of gmax and bf at 1.5 m and ha at 2.5 m during the turn phase of swt was significantly greater than during the stance phase of tw . increases in walking velocity in tw did not increase the activity levels of any of the assessed muscles . * p<0.05 , * * p<0.01 , comparison of muscle activity levels for swt and tw at a velocity corresponding to walking of swt . the primary findings of this study were that swt training increased vo2 and the activity of the quadriceps and es muscles relative to tw at the same velocity . the addition of turns caused more muscle contraction due to the acceleration and deceleration associated with the change of direction . we previously reported that vo2 was increased by turns10 , and our current results confirm the results of our previous study . a turn involves deceleration , a change of direction , and acceleration , and this can be difficult for older adults who often suffer from balance disorders . therefore , in this study we divided the turn phase into three separate steps ( acceleration , change of direction , and deceleration ) to make swt a simpler and easier exercise . this prolonged the turn time and reduced the walking distance per hour ; however , the oxygen consumption during swt was still significantly higher than during tw at the same velocity . because of this , performing swt at the fastest natural walking pace resulted in an exercise intensity of up to 6 mets . the physical activity of older people is significantly lower than the recommended amount12 , which results in decreased aerobic capacity , muscle mass and muscle strength , further reducing the amount of physical activity1 . because muscle weakness and loss are associated with physical disability13,14,15 , older adults must perform adequate physical activity to maintain or improve aerobic capacity and muscle mass and strength . the popularity of walking is due to its safety and simplicity , the fact that it does not generally cause hemodynamic stress and the lack of requirement for special equipment . however , it is known that walking training ( excluding race walking ) has little effect on aerobic capacity or muscle strength and volume5,6,7,8 . the regular walking speed of older adults is reportedly slow16 , 17 , and thus walking is not a sufficiently intense exercise to result in aerobic or strength improvements . however , the exercise intensity required to maintain and improve physical fitness in older adults is 3.0 to 6.0 mets18 . adding turns while walking , as in swt , increases the exercise intensity from light to moderate . furthermore , because swt did not result in significant increases in hr or rpe relative to tw in the present study , many older people should be able to perform swt easily and safely . we focused on muscle activity during the turn phase of swt and compared it with the muscle activity during the stance phase of tw . the activity levels of the es , vl , and vm were significantly greater while turning during swt than during tw . this indicates that it is possible to increase the stimulation provided by regular walking by adding turns . performing swt instead of tw or simple straight walking may increase the strength of the knee and trunk extensors and muscle mass in the thigh and back . only a few studies have examined the effect of aerobic training on muscle strength and mass in older adults . several cross - sectional studies have reported no significant differences between the leg strength and power of chronically aerobically trained and sedentary older people19 , 20 . longitudinal studies have shown no effect of regular walking training on leg strength and muscle mass5,6,7,8 . the few studies that have shown positive effects of exercise on the mass and strength of the thigh muscles used brisk walking , jogging at 85% hr reserve21 , or interval training at 70% to 85% vo2 peak6 , 22 as exercises . cycling at 70% vo2 max or 80% hr reserve has been shown to increase quadriceps muscle strength and power and thigh muscle volume23 , 24 . aerobic exercise can increase muscle strength and mass depending on the intensity , frequency , and duration of the exercise . importantly , to facilitate the development of a training habit , it is better that exercisers do not feel fatigue . muscle strength significantly decreases when training is stopped ; if training is resumed , its muscle strengthening effects are then less than they were previously24 . however , no studies have examined the effects of moderate endurance training ( except for the bench step exercise25 ) on muscle strength . the muscle activity of the vm during the turn phase of swt was twice that seen during regular walking . gazendam and hof26 reported the emg profile during walking and jogging at 4.5 to 8.1 km / h ; the emg amplitude of the vm during the turn phase of swt at 2.5 to 4.5 km / h in the current study was equal to what they reported during jogging at 8.1 km / h and brisk walking at 5.2 km / h . thus , muscle activity that occurs while turning during swt is equal to or greater than that seen during brisk walking or jogging . because brisk walking and jogging have been confirmed to increase the muscle mass and strength of the thigh , our results suggest that swt may also increase muscle mass and strength . additionally , in a study comparing running and soccer in young and middle - aged subjects , the vl cross - sectional area and knee extensor strength were increased only by soccer27 , 28 . training consisting of varying intensities and motions , such as swt , has the potential to result in muscle hypertrophy and strength gains . at present , the research on muscle hypertrophy and strength gains caused by aerobic exercise has been limited to the lower limbs ; further studies clarifying the effects of aerobic exercise on the muscles of other regions of the body , including the trunk , are needed . the purpose of this study was to investigate whether swt is useful for older people ; however , we chose , young people as subjects . the characteristics of walking in older people are a decreased : stride caused by a reduced range of motion of the hip and ankle , a lower walking speed cause by the decreased stride , and extension of the two - leg supporting moment . however , walking adjusted to 180 strides per minute is unlikely to be affected by the characteristics , showing an operation pattern similar to that of young people29 . in future work we will examine the effects of swt on the physical function of the older adults in terms of increased aerobic capacity , prevention of muscle ageing , and improvement in balance . our findings suggest that swt may help to preserve the muscle strength and mass of the trunk and lower limbs that is needed to maintain an independent lifestyle . even slow walking can be performed easily by older people , and the exercise intensity can be adjusted as required for each individual . furthermore , swt requires the participant to have greater control of their center of gravity during lateral , sagittal , and up and down movements than during regular walking . complex exercise training consisting of strength , balance , and walking training for older adults has been expected to effectively prevent falls30 . swt is done over short distances ( maximum of 3.5 m ) , which allows its performance in a small indoor space and eliminates the need for supervision or equipment .

[ purpose ] to maintain an independent lifestyle , older adults should improve muscle strength and mass , or aerobic capacity . a new exercise pattern , called slow walking with turns , which incorporates turning as an extra load additional to walking . the purpose of this study was to measure oxygen consumption during exercise and muscle activity while turning . [ subjects and methods ] recreationally active volunteers participated . the participants performed 20 turns per minute while walking back and forth over distances of 1.5 to 3.5 m. we measured oxygen consumption , heart rate , and rating of perceived exertion and performed electromyography during the exercise . [ results ] the metabolic equivalents of the exercise were 4.0 0.4 to 6.3 4.0 mets . activity was significantly greater in the vastus medialis , vastus lateralis , and erector spinae during the turn phase of slow walking with turns than during the stance phase of treadmill walking . [ conclusion ] these findings suggest that slow walking with turns may help to preserve the muscle strength and mass of the trunk and lower limbs that are needed to maintain an independent lifestyle . slow walking can be performed easily by older people , and in slow walking with turns , the exercise intensity can be adjusted as required for each individual .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2989444

all experimental procedures conform to the uk animals ( scientific procedures ) act 1986 and to ethical standards set out by the university of leeds ethical review committee . 1012 day old wistar rats ( n=89 ) of either sex were deeply anaesthetized with urethane ( 2 g / kg of body weight intraperitoneally ) . animals were transcardially perfused with ice cold sucrose artificial cerebrospinal fluid ( sucrose acsf ) containing ( in mm ) : sucrose ( 215 ) , nahco3 ( 13 ) , glucose ( 5 ) , kcl ( 1.5 ) , nah2po4 ( 1.5 ) , mgso4.7h2o ( 1 ) and cacl2 ( 1 ) , then humanely killed by decapitation . the thoracic spinal cord was dissected out and 500 m transverse slices cut using a vibraslice ( campden instruments ) . slices were stored at room temperature submerged in a holding chamber of acsf containing ( in mm ) : nacl ( 124 ) , nahco3 ( 26 ) , glucose ( 10 ) , kcl ( 3 ) , nah2po4 ( 2.5 ) , mgso4.7h2o ( 2 ) , cacl2 ( 2 ) , equilibrated with 5 % co2/ 95% o2 . slices were allowed to equilibrate for at least 20 min in a custom built interface recording chamber , superfused with gassed acsf at 3035 c . to keep slices moist , humidified 95 % o2/ 5% co2 was circulated in the recording chamber . a glass micropipette ( 24 m tip diameter ) signals were amplified 10 by an axoclamp2b amplifier ( axon instruments , usa ) , and a further 1000 amplification was achieved with two neurolog nl106 ( digitimer , uk ) amplifier modules . signals were low - pass filtered ( < 35 hz ) , notch filtered for 50 hz mains noise ( humbug , digitimer ) , digitized ( ced1401plus , cambridge electronic design , uk ) and captured at 5 khz on a pc running spike2 software ( cambridge electronic design , uk ) . extracellular field potential recordings were made , at depths of approximately 100 m below the surface , from the iml of 157 transverse spinal cord slices ( which retain the mediolateral dendrites that are predominant in spns at this age ( markham and vaughn , 1991 ) ) from 89 neonatal rats . extracellular iml activity had peak amplitudes of 2060 v which did not vary appreciably during the course of a recording . background noise levels were determined by comparing traces captured with the electrode placed in the iml ( fig . the amplitude of the activity was much lower with the electrode in the bath ( < 10 v ) , as expected . 1a , b ) revealed that the frequency components of the two traces were different : the background noise consisted mainly of a small peak at 3 hz , whereas a larger proportion of higher frequencies was present in the neuronal trace . to enable post hoc verification that the recording electrode was correctly positioned , power spectra ( fast fourier transform ( fft ) with hanning window , fft size=8192 and resolution=0.6 hz ) and autocorrelograms were generated from 1 min of activity . the field potentials were considered to be rhythmic if there was a peak in the power spectrum and the first autocorrelogram peak to the right of the central peak indicated a correlation coefficient of greater than 0.1 . the principal frequency of rhythmic activity was assessed from the highest peak in the power spectrum . the degree of rhythmicity ( area power ) was quantified by measuring the area under the power spectrum in a 3 hz band surrounding the highest peak . note that due to this analysis protocol , slices that lacked rhythmic discharges nevertheless had an area power considerably greater than zero because a degree of activity in the frequency band of interest is to be expected at all times . if the frequency of the highest peak changed following drug treatment , the power under this new peak was calculated , not the power at the original frequency . if no power spectrum peak was present ( for example , after blockade of oscillations with a drug ) , then area power was measured from the same 3 hz range as in control conditions . statistical comparisons involving area power were carried out with wilcoxon matched pairs signed rank test because these data do not fit a normal distribution . frequency data are presented as meanstandard error of the mean ( sem ) , whereas area power data are presented as median and interquartile ( iq ) range . stock solutions of drugs ( apart from 5-ht which was made up fresh on the day ) were diluted in acsf on the day of the experiment and superfused over the slice without recirculation . drugs were obtained from tocris bioscience ( uk ) except where stated : 5-hydroxytryptamine hydrochloride ( 5-ht , sigma - aldrich , uk ) , methyl-5-hydroxytryptamine maleate ( me5-ht ) , 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride ( mk212 ) , potassium ( 2s,4as,6ar,6as,6br,8ar,10s,12as,14br)-10-hydroxy-2,4a,6a,6b,9,9,12a-7 heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b - dodecahydro-1h - picene-2-carboxylate ( 18-glycyrrhetinic acid , stock in 50% ethanol ; sigma - aldrich , uk ) , ( as)-rel - a-(2r)-2-peperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol monohydrochloride ( mefloquine ) , n-[2-[[3-(dimethylamino)propyl]thio]phenyl]-3-phenyl-2-propenamide hydrochloride ( cinanserin ) , tetrodotoxin citrate ( ttx ) , bicuculline hydrochloride ( bicuculline ) . power spectral and autocorrelation analysis of iml activity revealed spontaneous rhythmic field potential oscillations in 48/164 ( 29.3% ) slices tested . the rhythmic discharges were manifest as a peak in the power spectrum in the range 7.522 hz ( mean 13.50.7 hz ) and as a sinusoidal autocorrelogram ( e.g. fig . oscillation frequency did not drift appreciably during the recording , even though different slices showed a range of frequencies . this rhythmic activity typically persisted for the duration of a recording , which could be in excess of 3 h. of the slices that did not exhibit spontaneous rhythmic activity , a further 18 previously non - rhythmic slices could be pharmacologically manipulated with 5-ht agonists to induce this activity ( see below ) . the other slices were not able to generate spontaneous or 5-ht - induced oscillations ( see discussion ) and therefore were not considered any further . in six slices where robust rhythmic oscillatory activity was recorded from the iml , moving the electrode to regions outside the iml resulted in a loss of rhythmic activity in all slices tested . these regions included the motor nuclei ( lamina ix ) of the ventral horn ( n=3 ) or superficial dorsal horn ( n=3 ) , ( fig . 1c ) and suggests that this particular rhythm was specific to the iml ( see discussion ) . the positioning of the electrode in the iml was verified by deposition of rhodamine from the electrode at the end of recording ( fig . this was seen as the disappearance of the peak in the power spectrum and the flattening of the sinusoidal morphology of the autocorrelogram ( fig . , ttx significantly reduced the power of iml oscillations , from a median value of 45.8 ( iq range 31.667.1 v ) to 4.6 ( iq range 1.76.2 ; p=0.028 , n=6 ; fig . the frequency of the oscillations ( measured at the latest time the power spectrum peak was still detectable ) was unaffected by ttx ( 14.31.5 hz in control conditions vs. 12.31.4 hz in ttx , p=0.201 , n=6 ; fig . sympathetic preganglionic neurons in the iml express the gap junction channel subunit connexin 36 ( cx36 , marina et al . , 2008 ) and fire synchronous action potentials in slices as a result of electrotonic coupling ( logan et al . , we therefore sought to determine whether direct electrical coupling contributed to the rhythmic population activity in our slice preparation . the gap junction blocker 18-glycyrrhetinic acid ( 100 m , verified as effective at this concentration at blocking spikelets , the hallmark of gap junctions in recordings from single spns ; data not shown ) was continuously bath applied to six spontaneously oscillating slices ( fig . 18-glycyrrhetinic acid reduced the area power of spontaneous oscillations by an average of 68.2% ( median power was 42.9 [ iq range 26.956.1 ] v in control and 5.0 [ iq range 4.810.6 ] v in 18-glycyrrhetinic acid , p=0.028 , n=6 ; fig . in addition , the oscillation frequency ( measured immediately before abolition of the power spectrum peak , where necessary ) was significantly reduced by 18-glycyrrhetinic acid from 14.11.0 hz in control conditions to 11.21.5 hz in the presence of the drug ( p=0.02 , n=6 ; fig . another gap junction blocker , carbenoxolone ( 100 m , a concentration at which spontaneous spikelets were reduced in current clamped spns , not shown ) , had a similar effect ( data not shown ) , reducing the power of iml oscillations by an average of 69.8% . area power was reduced from 5.5 ( iq range 3.6110.5 ) v to 1.4 ( iq range 1.036.6 ) v ( p=0.068 , n=4 ) by this drug ; however , this reduction did not quite reach statistical significance , possibly due to the range of ongoing area powers observed . oscillation frequency , measured just before abolition of the power spectrum peak , was not affected ( 15.22.3 hz in control to 16.74.1 hz in carbenoxolone , p=0.47 ) . finally , we also tested the effects of mefloquine , a gap junction blocker which has been reported to block cx36-containing gap junction channels more effectively than those with alternative subunit compositions ( cruikshank et al . , 2004 ) . bath application of 1 m mefloquine , a concentration also verified to reduce spikelet activity in spns ( not shown ) abolished spontaneous oscillations in 2/3 slices ( fig . j ) , and reduced the power from 33.2 to 5.8 v in the remaining slice . the mean reduction in area power produced by mefloquine was 76.4% ( median 33.2 [ iq range 20.240.0 ] under control conditions , 3.9 [ iq range 3.64.9 ] in mefloquine , p=0.109 , n=3 ; fig . oscillation frequency ( measured just before abolition of the power spectrum peak , where necessary ) was not significantly changed by mefloquine , although a decrease from 16.73.1 hz to 12.51.7 hz ( p=0.153 , n=3 ; fig . 3i ) was observed following application of this drug . to test whether inhibitory gabaergic synaptic transmission is required for rhythm generation in the iml , 10 m bicuculline significantly reduced the power of ongoing oscillations from a median value of 19.1 ( iq range 5.135.1 ) v to 3.6 ( iq range 0.84.4 ) v ( average 65.1% reduction fig . the frequency of the oscillation was not significantly changed by bicuculline ( 11.92.0 hz in bicuculline vs. 10.11.2 hz under control conditions , p=0.149 , n=5 ) . 10m 5-ht was applied to eight slices which were demonstrated to be capable of oscillating , of which five were spontaneously rhythmically active and three were non - rhythmic at the time 5-ht was applied . intermittent application of 5-ht over 30 min using a 5-min - on , 5-min - off protocol resulted in an increase in the area power at the rhythm frequency in all eight slices ( fig . 5 ) . intermittent , rather than continuous , application of 5-ht was employed because this type of protocol was more successful at enhancing inspiratory nerve burst amplitude than continuous application of 5-ht to a brainstem - spinal cord preparation ( lovett - barr et al . , 2006 ) . the increase in area power was from a median value of 4.1 ( iq range 2.110.4 ) v to 14.2 ( iq range 8.734.4 ) v ( average 544.7% increase fig . the 5-ht - induced rhythmic activity lasted at least 10 min and often in excess of 1 h. 5-ht - induced oscillations fell within the normal range for spontaneous oscillations , averaging 9.91.8 hz . in the five slices which exhibited spontaneous oscillations , the oscillation frequency was not changed by 5-ht treatment ( 9.71.1 hz vs. 9.71.0 hz , p=0.655 , fig . selective agonists were also used to determine the receptor subtype which contributed to the effects of 5-ht . 10 m me5-ht , an agonist of 5-ht2 receptors , mimicked the effects of 5-ht . when applied to slices which were demonstrated to be capable of rhythmic activity , methyl5-ht increased area power in all 12 slices , from a median value of 4.9 ( iq range 2.112.9 ) v to 13.3 ( iq range 5.421.6 ) v ( average 574.0% increase ; fig . 6 ; p=0.005 ) . six of these slices were spontaneously active when the drug was applied , whereas the other six were not . similar to the effects of 5-ht , me5-ht did not significantly change spontaneous oscillation frequency ( 11.71.9 hz during control , 11.31.4 hz during methyl5-ht application , p=0.595 ; fig . in addition , the mean frequency of rhythmic activity induced by me5-ht was very similar to that of spontaneous oscillations ( 13.02.2 hz , p=0.617 ) . since me5-ht does not discriminate between the three subtypes of 5-ht2 receptor , and mrna for 5-ht2c is high in the iml ( fonseca et al . , 2001 ) , the action of the selective 5-ht2c agonist mk212 was tested . 10 m mk212 was continuously bath applied to 12 slices deemed capable of generating rhythmic activity , of which four were spontaneously rhythmic . the area power of iml oscillations was significantly enhanced in all 12 slices ( fig . the median control power in these experiments was 12.0 ( iq range 6.016.0 ) v , rising to 18.7 ( iq range 10.750.2 ) v ( average increase of 167.0% , p=0.006 , n=12 ; fig . 7e ) measured after at least 8 min in mk212 . again , the frequency of spontaneous rhythmic discharges was unaffected ( 15.01.9 hz in control , 16.12.5 hz in mk212 , p=0.288 , n=4 ; fig . 7d ) . to determine the contribution , if any , of 5-ht receptors to the spontaneous oscillations , the 5-ht2 receptor antagonist cinanserin was continuously bath applied to three spontaneously oscillating slices . 10 m cinanserin reduced iml oscillations in all three slices , with a mean reduction in power of 33.3% ( from 0.3550.079 to 0.2360.060 , n=3 ; fig . cinanserin did not significantly affect oscillation frequency : the mean frequency in control conditions was 11.11.2 hz , and in cinanserin the frequency was 9.81.8 hz , n=3 . in this study we have recorded field potentials , through electrodes placed in the iml , that have ( often spontaneous ) rhythmical discharge characteristics . we therefore suggest that the neuronal networks present in a spinal cord slice preparation are sufficient to generate and maintain rhythmic sympathetic activity , challenging the current view that brainstem circuits are the key contributors to the patterning of rhythmic sympathetic nerve bursts . rhythmic activity was abolished by application of ttx , indicating that neuronal firing plays a critical role in generating these rhythmic field potentials . we have also observed that iml rhythmic activity is disrupted by gap junction blockers and bicuculline . furthermore , rhythmic activity could be enhanced or induced by agonists that activate 5-ht receptors , including those selective for 5-ht2c receptors , an effect similar to that observed in vivo or in spinalized preparations . it should be noted that these network iml rhythms are a result of ensemble neuronal activity ( probably including spns and interneurons in this region ) and as such are distinct from the membrane potential fluctuations ( similarly termed oscillations ) in single cell recordings such as those reported in hb9-positive interneurons involved in locomotion ( wilson et al . , 2005 ) or indeed spns ( spanswick and logan , 1990 ) . however , this is not to say that these intracellular events reported by logan 's group do not correlate with , or indeed contribute to , our ensemble activity indeed , these events are gap junction - mediated potentials ( spikelets ) ( logan et al . , 1996 ) and thus they are a probable target of our gap junction blockers . nevertheless , it is clear that our network oscillations are not simply trains of synchronized spikelets summating in the extracellular milieu the spontaneous spikelet frequency is much too low in spinal cord slices ( mean value 1 hz ) , and spikelet frequency is increased by application of 5-ht ( pickering et al . , 1994 ) , whereas our network oscillations are increased in power by this drug but their frequency remains unaffected . it is well known that the drugs used here can have non - specific actions ( coker et al . , 2000 ; mcardle et al . , 2006 ) , such as an inhibitory effect of carbenoxolone on ca channels ( vessey et al . , 2004 ) or mefloquine on herg k channels ( traebert et al . , 2004 ) ; although with an ic50 of 2.6 m , higher than the dose used here . mefloquine and 18 -glycyrrhetinic acid can also block volume - regulated anion channels ( maertens et al . , 2000 ; ye et al . , 2009 ) and since other blockers of these channels may also have an effect at glycine receptors ( scain et al . , 2010 ) , there is a chance that these blockers may exert an action at glycine receptors . however , the fact that three different drugs exerted similar effects suggests it is their shared action at gap junctions , rather than other possible non - specific effects , which is responsible for attenuating the oscillations . this is further supported by the observation that all three gap junction blockers used disrupt gap junction - mediated spikelets in spns at these concentrations , without obvious effects on other firing properties of the neurons . although further experiments may prove unequivocally that these effects are mediated solely by an action on gap junctions , this indicates that synchronization of neuronal activity by gap junctions may be an important mechanism underpinning this rhythmic iml activity . at least some of the junctions involved may contain the subunit cx36 because mefloquine , a blocker with selectivity for cx36-containing gap junctions , replicated the effects of general gap junction blockade . a similar role of cx36-containing gap junctions in the inferior olive ( placantonakis et al . , 2006 ) , suprachiasmatic nucleus ( long et al . , 2005 ) , and hippocampus ( buhl et al . , 2003 ) has been confirmed in cx36-knockout mice . for two reasons , it is likely that the gap junctions involved in iml rhythm generation are located between spns . firstly , gap junction coupling tightly synchronizes subthreshold and suprathreshold activity between pairs of spns ( logan et al . , 1996 ) , but gap junction coupling between interneurons or between spn - interneuron pairs has not been observed . secondly , cx36 immunoreactivity has been localized to the dendritic membrane of spns , but not to interneurons , in the iml ( marina et al . , 2008 ) . in common with other cns networks ( e.g. see somogyi and klausberger , 2005 ) , spontaneous iml oscillations were also attenuated by bicuculline , a gabaa receptor antagonist , suggesting that spontaneously active gabaergic interneurons also contribute to the network rhythms . this is relevant to sympathetic control since gabaergic interneurons within the central autonomic area synapse directly onto spns ( deuchars et al . , 2005 ) and provide ongoing phasic inhibitory input in the shape of inhibitory postsynaptic potentials ( ipsps ) recorded in spinal cord slices . therefore , antagonism of the effects of these interneurons is the likely mechanism underlying the effects of bicuculline on oscillation power . these inhibitory interneurons may act to pace or reinforce the activity generated in the coupled networks , as originally observed in the hippocampus ( cobb et al . , iml rhythms were abolished by the voltage - gated na channel blocker ttx in all experiments , indicating that action potential propagation is a vital component of the oscillations . taken together with the results of gap junction and gabaa receptor blockade , these data suggest that iml field oscillations are the emergent property of a network of neurons that is dependent on synaptic properties and patterns of connections between neurons . 5-ht acts as a modulator of iml rhythmic oscillations , able both to enhance the power of existing rhythms on every occasion tested and induce rhythms in otherwise quiescent slices . this effect was mimicked by me5-ht and mk212 , suggesting this is mediated at least in part by 5-ht2 receptors . it is likely that the mechanisms underlying the spontaneous and 5-ht - induced rhythms are similar since the frequencies of spontaneous and 5-ht - induced rhythms are not significantly different and 5-ht does not significantly change the frequency of ongoing rhythms even when it enhances the power . moreover , results from the slices where cinanserin reduced the power of the oscillation provide evidence that , in these cases , at least a proportion of the spontaneous oscillations is mediated by activation of 5-ht2 receptors . where 5-ht or methyl5-ht were unable to elicit rhythmic activity in hitherto quiescent slices , it is likely that these slices were either not viable ( i.e. not healthy enough to generate complex oscillations ) , did not contain enough of the circuitry necessary for generating rhythmic activity , or may oscillate in the presence of drugs other than those of interest to us here . since the spontaneous activity level of many slices was only marginally above background noise levels , it was difficult to be certain of the viability of slices which did not display oscillations . we therefore report the existence of these slices for transparency , and with the admission that we can not say for sure whether they were the result of sub - optimal slice preparation or maintenance , or whether some may have been alive but intrinsically unable to oscillate . possible causes of the latter would include the ladder and rung morphology ( petras and cummings , 1972 ) of the iml , in which small clusters of spn somata are separated by highly variable gaps of 50150 m ( our unpublished estimates in 11-day - old rats ) consisting mainly of dendrites . the number of clusters per 500 m slice is therefore likely to vary , perhaps resulting in some slices containing too few spns to generate oscillatory activity . previous studies support the notion that 5-ht is an important modulator of rhythmic sympathetic activity . for example , the 10 hz rhythm in sympathetic nerve discharge is selectively enhanced by intravenous administration of a 5-ht2 receptor agonist and eliminated by a 5-ht2 antagonist , and these effects are mimicked by exciting or inhibiting serotonergic neurons in the medullary raphe respectively ( orer et al . , 1996 ) . furthermore , microinjections of 5-ht into the upper thoracic iml increased sympathetic outflow to brown adipose tissue ( madden and morrison , 2006 ) . significantly , 5-ht acting at a spinal site induces at least two distinct sympathetic rhythms ( marina et al . , 2006 ; the effects of 5-ht on iml rhythmic activity were reproduced by the selective 5-ht2c receptor agonist mk212 , suggesting that the facilitation is at least partially mediated by this receptor . indeed , 5-ht2c mrna is abundant throughout the spinal cord , including the iml ( fonseca et al . , 2001 ) . the rhythmic activity reported here was confined to the iml region of the spinal cord since , in all slices tested where spontaneous activity was present in this region , moving the electrode to other areas such as the ventral or dorsal horn resulted in loss of this activity . this suggests that , although both dorsal and ventral horn regions are capable of generating rhythmic activity if driven pharmacologically ( asghar et al . , 2005 ; nakayama et al . , 2004 ) , this particular spontaneous or 5-ht - induced rhythm is specific to the iml . it is therefore likely that it is generated locally within the iml , and represents the summed network activity of the spns and/or sympathetic interneurons in this region . the presence of rhythmic activity in the iml of a spinal cord slice strongly supports previous suggestions that rhythmic sympathetic activity can be generated by the spinal cord ( see introduction ) . the frequency of iml oscillations in the current study was in the range 7.522 hz . in vivo , the 10 hz rhythm occurs in a similar , but much more restricted , frequency band ( 812 hz ; cohen and gootman , 1970 ; green and heffron , 1967 ) . thus , it might be speculated that the iml oscillations in the current study represent a stripped - down version of the 10 hz rhythm . in support of this idea , although it is known that at least a component of the 10 hz rhythm is generated in the brainstem , ( barman and gebber , 2000 ) , 10 hz discharges can also be generated within the spinal cord ( kubota et al . mccall and gebber , 1975 ; ootsuka et al . , 1995 ) and the 10 hz rhythm is dependent on the activity of 5-ht2 receptors ( orer et al . , however , since the closest in vivo correlate of these oscillations , the 10 hz rhythm , occurs in both young kittens and swine ( hundley et al . , 2001 ; sica et al . , 1990 ) as well as adult animals the generation of 10 hz discharges by the spinal cord is poorly understood , so this theory remains speculative . further , it will enable the contribution of individual cell types to network oscillations to be determined , since intracellular sharp electrode or patch clamp recordings may be performed in 500 m slices in conjunction with field recordings . our data demonstrate that rhythmic field potential oscillations can be recorded from the iml of neonatal rat spinal cord slices , and this activity is spontaneous in a proportion of slices . these findings suggest that the rhythmogenic capabilities of the sympathetic circuits of the spinal cord have been underestimated , and that spinal oscillators could potentially play a role in generating the physiologically - important sympathetic nerve bursts that have hitherto been credited mainly to supraspinal networks . 5-ht acting at 5-ht2 receptors modulates iml oscillations , inducing rhythmic network activity in at least a proportion of quiescent slices , and significantly enhancing spontaneous rhythmic activity in all slices tested . action potential propagation is required for the oscillations to occur , and both gap junctions ( probably between spns ) and gabaergic interneurons are important components of the spontaneous rhythmogenic network . iml oscillations in slices represent a valuable paradigm for studying the rhythmogenic capabilities of the spinal cord .

neuronal networks generating rhythmic activity as an emergent property are common throughout the nervous system . some are responsible for rhythmic behaviours , as is the case for the spinal cord locomotor networks ; however , for others the function is more subtle and usually involves information processing and/or transfer . an example of the latter is sympathetic nerve activity , which is synchronized into rhythmic bursts in vivo . this arrangement is postulated to offer improved control of target organ responses compared to tonic nerve activity . traditionally , oscillogenic circuits in the brainstem are credited with generating these rhythms , despite evidence for the persistence of some frequencies in spinalized preparations . here , we show that rhythmic population activity can be recorded from the intermediolateral cell column ( iml ) of thoracic spinal cord slices . recorded in slices from 10- to 12-day - old rats , this activity was manifest as 822 hz oscillations in the field potential and was spatially restricted to the iml . oscillations often occurred spontaneously , but could also be induced by application of 5-ht , -methyl 5-ht or mk212 . these agents also significantly increased the strength of spontaneous oscillations . rhythmic activity was abolished by ttx and attenuated by application of gap junction blockers or by antagonists of gabaa receptors . together these data indicate that this rhythm is an emergent feature of a population of spinal neurons coupled by gap junctions . this work questions the assumption that sympathetic rhythms are dependent on supraspinal pacemaker circuits , by highlighting a surprisingly strong rhythmogenic capability of the reduced sympathetic networks of the spinal cord slice .

Experimental procedures Results Discussion Conclusion

PMC4590909

sprains and ruptures of the ligaments supporting the ankle and knee joints as well as muscle strains occur quite often . in some cases , joint injuries are sustained through contact with another player . a classic example occurs when one player collides with another , applying excessive force to the lateral side of the opponent 's knee . this often results in damage to the medial collateral ligament , lateral meniscus , and the anterior cruciate ligament ( acl ) . however , a substantial number of soccer injuries occur through noncontact mechanisms . in these cases , an athlete may plant his or her foot then stop , cut , or turn . as the body changes direction while the foot is stationary , the knee and/or ankle experiences torque . as a result , these include intrinsic factors such as proprioception , muscular strength , ligament properties , and biomechanics as well as extrinsic factors such as the playing surface and other environmental conditions . several studies have focused on this latter factor as important , specifically the use of artificial turf playing surfaces . they suggest that the added friction between the shoe and the surface increases the torque experienced by the ankle and knee [ 1 , 2 ] . in fact , early studies on the first - generation artificial turf ( at ) fields ( astroturf - type surfaces ) did show an increased injury risk compared to natural grass ( ng ) [ 3 , 4 ] . the short - pile carpet laid over a thin pad has been replaced by a surface that contains long grass - like fibers that are embedded with granules of crushed rubber , sand , and/or silica and laid over a thick pad . this results in a more compliant surface and one that results in a considerably lower shoe - surface coefficient of friction . manufacturers of third - generation at surfaces argue that such a design may lower the risk of noncontact joint injury . on the other hand , many coaches , trainers , and athletes remain concerned that playing on at increases injury risk . whether or not third - generation at alters the risk of noncontact injury remains unresolved . several recent studies have focused on comparing injury incidence rates in soccer players who train and/or play matches on both at and ng . unfortunately , the results are somewhat variable , with some studies showing reduced risk of some injuries on at and others showing no difference or slightly increased risk . thus , the purpose of this investigation was to resolve this issue . to accomplish this , we used previous research and a meta - analytic approach to compare injury rates in soccer players performing on at and ng . key search terms were soccer , injury , grass , synthetic turf , and artificial turf . to be included in the meta - analysis , studies met several criteria : ( 1 ) focused on competitive soccer players , ( 2 ) compared acute injuries sustained during soccer matches and/or training on ng and on third - generation at surfaces , ( 3 ) examined players who participated on both surfaces during the study period rather than teams that used one surface exclusively , ( 4 ) used the injury definition as described by fuller et al . , ( 5 ) reported both exposure times and injury occurrences for play on at and ng , and ( 6 ) written in english or an english translation . the studies meeting our criteria were examined and exposure times , injury occurrences , and playing surface were recorded . in addition , the playing condition ( match or training ) , gender , and age ( youth or adult ) were noted . chronic injuries were not considered as these could be attributed to both surfaces or to other causes . acute knee injuries , ankle injuries , foot injuries , sprains , and muscle strains were recorded . these specific locations and types of injuries were selected because they were the most consistently defined across all of the included studies . crude injury rates were computed using the unweighted sum of total injuries and exposure times from all eight studies . statistical analyses were performed on adjusted injury incidence rate ratios ( irrs , at / ng ) using the mantel - haenszel method for fixed effects . the data were then categorized based on condition ( match or training ) , gender , and age ( youth or adult ) . an initial literature search on pubmed returned more than 25 citations . of those , eight studies met our criteria . these are shown in table 1 along with key characteristics of each study . as can be seen , individual studies varied in terms of the number of players and teams examined . in addition , the period of investigation ranged from several months to several years resulting in varying exposure times . three studies focused on either match or training injuries exclusively while five included both conditions . five of the studies focused on adult players , generally college or professional players , whereas three studies focused on youth players that were 12 to 17 years of age . of that , 571,196 hours were played on at ( 34.5% ) and 981,147 on ng ( 65.5% ) . the amount of time spent in training and playing on at and ng varied between studies . in the study by soligard et al . , players competed on at for less than 10% of the total time played . at the other end of the range , ekstrand et al . studied players who trained and competed on at nearly three times as long as on ng . matches accounted for 271,022 hours ( 18.1% ) of total exposure time whereas training accounted for 1,227,321 hours ( 81.9% ) . of the total exposure time , males accounted for 949,568 hours ( 66.1% ) and females for 486,178 hours ( 33.9% ) . did not distinguish between male and female players and was not included in the analysis of gender . a total of 9737 injuries were recorded with 2670 occurring on at ( 27.4% ) and 7067 on ng ( 75.6% ) . this resulted in crude injury incidence rates of 5.16 and 7.20 injuries per 1000 hours for at and ng , respectively . crude incidence rates for matches were 20.26 and 24.45 injuries per 1000 hours for at and ng . for training injures on at and ng , the incidence rates were 2.91 and 2.68 injuries per 1000 hours . figure 1 shows the injury irrs for each of the studies examined along with the overall adjusted irr of 0.86 ( 0.740.93 , p < 0.05 ) . five of the eight studies showed irrs that were significantly lower than 1.0 , indicating a lower incidence rate on at [ 79 , 12 , 13 ] , while the other three showed nonsignificant differences . the overall adjusted irr was statistically significant with a lower incidence rate on at . ankle , and foot injuries as well as sprains , incidence rates were significantly lower on at compared to ng . analyses of male , female , youth , and adult subcategories for all injuries showed irr values significantly lower than 1.0 . for knee injuries , incidence rates on at were lower for training injuries and adults . for muscle strains , incidence rates were lower on at for match injuries , males , and both young and adult players . in no case was injury incidence increased on at . for the entire data set , including all injuries as well as both conditions , gender and age ( figure 1 ) , the i value was 80% . a portion of the heterogeneity was due to different injuries and locations . for specific injuries and sites ( figure 2 ) , i ranged between 43% and 74% . condition , gender , and age also contributed as i values for these categories ranged from 0 to 80% . we found that variations in exposure to at accounted for some of the heterogeneity . figure 3 shows a significant negative correlation between the relative amount of time spent on at ( at exposure / ng exposure ) and the irr for each study . as can be seen , the two studies in which the greater exposure time on at had the lowest irr . this meta - analysis examined eight studies that compared soccer injury rates occurring on at and ng . in total , these studies report nearly 1.5 million hours of training and match play and almost 10,000 injuries . the adjusted irr for all injures was significantly less than 1.0 indicating lower incidence rates for playing and training on at . for specific categories and specific injuries , thus , the overall results of our analyses do not support the idea that playing or training on at increases the risk of injury compared to ng . in fact , our analyses suggest that at surfaces may reduce injury incidence for some types of injuries within specific categorizations . first , despite the guidelines provided by fuller et al . , the eight studies varied in terms of their injury descriptions . for example , ekstrand et al . [ 8 , 9 ] reported the incidence of injuries based on location and type ( e.g. , knee sprains ) , whereas bjrneboe et al . , fuller et al . [ 10 , 11 ] , soligard et al . , and steffen et al . described injuries by location ( e.g. , knee ) or type ( e.g. , sprain ) . only ekstrand et al . noted specific injuries such as anterior cruciate ligament tears . thus , our results are limited to the general classifications of location ( knee , ankle , and foot ) or type ( sprains and muscle strains ) . second , in one study , exposure and incidence values for males and females were pooled . third , the severity of injuries is categorized based on the number of training and match days missed . for example , a minor or mild injury was defined as 17 days [ 7 , 10 , 11 , 13 ] , 47 days [ 8 , 12 ] , and 12 weeks missed . given these discrepancies , we were not confident using severity as a subcategory for analysis . fourth , the studies failed to report environmental conditions such as temperature , wet / dry field conditions , or shoe type / cleat design . finally , only three of the studies differentiated between contact and noncontact injuries [ 10 , 11 , 13 ] . this is an important limitation since contact injuries may or may not be directly attributable to playing surface . our results do not provide an explanation for possible reductions in injury risk on at . artificial turf surfaces provide a more consistent playing surface than ng , free of bare sports , ruts , and divots which could affect injury risk . however , the studies examined focused on teams competing at a high level , so it is likely that the ng surfaces were of high quality . traditionally , physical characteristics of at and frictional resistance at the shoe - surface interface have given rise to suggestions of greater injury risk on at . however , laboratory experiments yield conflicting results regarding this idea . several studies show greater rotational torque between the shoe and at compared to ng [ 2 , 14 ] , whereas cawley et al . further argue that stiffness ( the rate of change in torque ) may play a role in injury risk . yet , greater peak torque on at does not result in increased stiffness . in human studies , ford et al . found different foot loading patterns during cutting maneuvers performed on at and ng . on the other hand , potthast reported no differences in rear foot and ankle movements of the plant foot when goal kicking between ng and rubber - filled at . mcghie and ettema found that impact forces experienced during cutting maneuvers on at were not indicative of a hazardous condition . to further complicate this issue , torque and stiffness can be affected by the type of cleat worn on a particular surface as well as the temperature of the surface [ 2 , 14 ] . thus , it is difficult to conclude with confidence which of the physical characteristics of at accounts for difference in injury incidence rates found in the present study . match analyses show that the amount of time spent running at different intensities is similar between surfaces , but fewer slide tackles and shorter passes are executed on at . the reduction in slide tackles may stem from the fear of skin abrasions but may limit the number of high risk they feel that heat as well as speed of play on at requires greater physical effort compared to grass [ 18 , 19 ] . however , these perceptions have been difficult to confirm as some controlled studies show increased energy expenditure while others show no differences in fatigue [ 2023 ] . note that negative player perceptions of at are stronger in those who have limited exposure to at or are exposed to at later in their careers . figure 3 suggests that more time spent in training and competing on at is associated with lower injury risk . perhaps with increased exposure to at , players develop a less aggressive play with fewer slide tackles that reduces injury risk . it should be pointed out , however , that this idea is speculative at this time . clearly more research is needed regarding player movements , energy expenditure , and fatigue on at and ng surfaces . this study highlights several areas that need to be addressed before firm conclusions can be drawn . first , three of the eight studies focused only on male players [ 6 , 7 , 9 ] . given the concern over the high incidence of acl injuries in women , it seems reasonable to suggest that more information is needed regarding playing surface as a potential risk factor in females . fourth , more studies are needed on energy expenditure and fatigue on at and ng , especially using activities that replicate match play . given that fatigue can increase the risk of joint injury , it is important to understand if player perceptions of increased effort on at have a physiological basis . fifth , while preliminary studies suggest that players adjust their style of play on at , it is not clear if this occurs across levels of play and across genders . identifying specific movement patterns that either increase or decrease injury risk on at could impact player training . as the use of at in club and school settings increases , a thorough understanding of safety and injury risks in youth and adolescent players is essential . in this investigation , we found no evidence that playing matches or training on at raises the risk of soccer players sustaining injury . in fact , the evidence suggests that the risk of some injuries and some subgroups might be lowered . however , until more is known about how issues such as altered playing styles affect injury incidence , it is difficult to make firm conclusions regarding the direct and indirect roles of at in player safety .

the goal of this investigation was to determine if playing or training on third - generation artificial turf ( at ) surfaces increases the incidence rate of injuries compared to natural grass ( ng ) surfaces . this was accomplished by a meta - analysis performed on previously published research . eight studies met the criteria of competitive soccer players , participation on both surfaces , and presentation of both exposure time and injury occurrence . exposure time and injury incidence values were used to generate injury rate ratios ( irrs , at / ng ) for all injuries as well as specific injuries . subgroup analyses were also performed by condition ( match or training ) , gender , and age ( youth or adult ) . the overall irr was 0.86 ( p < 0.05 ) suggesting a lower injury risk on at than ng . however , there was considerable heterogeneity between studies . analyses of individual injuries and subgroups found that in many cases irr values were significantly less than 1.0 . in no case was the irr significantly greater than 1.0 . based on this , it appears that the risk of sustaining an injury on at under some conditions might be lowered compared to ng . however , until more is known about how issues such as altered playing styles affect injury incidence , it is difficult to make firm conclusions regarding the influence of at on player safety .

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

PMC5382572

self - organized tio2 nanotube layers have attracted remarkable attention within the past 15 years due their unique architecture , high surface area , semiconductive properties , and biocompatibility . in addition , they are produced by a low - cost electrochemical anodization of ti substrates in suitable electrolytes containing fluorides . all these features enabled utilization of tio2 nanotube layers in a wide number of applications as photocatalyst , anode of dye - sensitized solar cells ( dssc ) and perovskite solar cells ( psc ) , gas sensors , and biomedical materials , among others . in all these applications , tio2 nanotube layers have shown superior performance compared to other tio2 nanostructures . in parallel , important efforts have been carried out to tune the nanotube aspect ratio and to improve the nanotube ordering and crystallinity . annealing treatment of amorphous as - synthesized tio2 nanotube layers leads to their crystallization into anatase ( > 280 c ) , a combination of anatase and rutile ( > 450 c ) , or rutile ( > 550 c ) . the anatase nanotubular structure has shown to be more favorable than rutile for photoelectrochemically assisted applications , such as photocatalysis and dssc . thus , the stability of anatase nanotubular structure is highly desired , and numerous efforts have been focused on this target , especially at high temperatures . the introduction of alloying elements as nb or c was reported to induce a shift of the anatase to rutile transition ( further noted as art ) to higher temperature and increased thermal resistance against collapse . however , the main disadvantage of alloying is the formation of undesired secondary impurity phases , e.g. , ti nb2o5 . the art threshold depends on whether the nanotube layers are attached or separated from the ti substrate . high temperature stability ( up to 700 c ) of tio2 nanotube arrays , preserving the nanotubular integrity and anatase structure , was reported for free - standing tio2 nanotube arrays . for tio2 nanotube layers attached to ti substrate , anatase structure and no structural collapse is maintained either by a previous solvothermal treatment or previous annealing at lower temperature . the highest published temperature without nanotube collapse ( 1048 c ) was reached during the flame annealing process . however , such flame high temperature processing led to undesired transition to rutile structure and a significant uptake of carbon from the flame . despite numerous efforts focused onto the high temperature stability of tio2 nanotube layers , the temperature working window is still restricted . another constrain for applications of tio2 nanotube layers is their limited chemical stability in harsh acidic environments , where nanotube layers undergo chemical dissolution . the improvement of the thermal , chemical , and eventually also mechanical properties of tio2 nanotube layers would enable their utilization in previously nonimaginable working environments and surely interesting expansion of their application range . in principle , addition of a thin continuous coating of an appropriate secondary material ( with excellent thermal and chemical stability ) within nanotubes should significantly alter also their stabilities . so far , however , no such treatment has been shown . to date , the atomic layer deposition ( ald ) technique is the only method that enables homogeneous , continuous , and conformal coating of secondary materials into tio2 nanotube layers . deposition of al2o3 and zno coating by ald into tio2 nanotube layers has been reported yielding interesting synergic effects . the resulting composite heterostructures revealed significant improvement of their photovoltaic and photocatalytic performance due to enhanced charge separation induced by coatings of secondary materials . therefore , in the present work we investigated the thermal , chemical , and mechanical properties of self - organized tio2 nanotube layers uniformly coated with al2o3 layers of different nominal thicknesses : 1 , 10 , and 42 nm . these coatings were carried out by atomic layer deposition ( ald ) using different number of identical deposition cycles . after the al2o3 coating , the tio2 nanotube layers were annealed at temperature up to 870 c for 1 h to evaluate their thermal stability . the resulting crystal structure and composition were analyzed through x - ray diffraction ( xrd ) and scanning transmission electron microscopy ( stem ) . mechanical properties ( hardness ) were characterized by nanoindentation measurements using an atomic force microscope . the chemical stability was tested by soaking the al2o3-coated tio2 nanotube layers into h3po4 solutions of different concentrations for 48 h at laboratory temperature and for an additional 8 h in solutions with temperature of 60 c . self - organized tio2 nanotube layers with a thickness of 20 m and a nanotube diameter of 110 nm ( aspect ratio 180 ) were fabricated via anodization of ti foils using a previously published approach . prior to anodization , the ti foils ( sigma - aldrich , 0.127 mm thick , 99.7% purity ) were degreased by sonication in isopropanol and acetone , then rinsed with isopropanol , and dried in air . the electrochemical setup consisted of a two - electrode configuration using a platinum foil as the counter electrode , while ti foils ( working electrodes ) were pressed against an o - ring of the electrochemical cell , leaving 1 cm open to the electrolyte . a high - voltage potentiostat ( pgu-200 v , ips elektroniklabor gmbh ) was employed to carry out the electrochemical experiments at room temperature . ethylene glycol containing 1.5 vol % deionized water and 176 mm nh4f was used as electrolyte . electrolytes were aged before the first use for 15 h by anodization of blank ti foils at 60 v under the same conditions for the anodization experiments ti foils were anodized for 4 h after sweeping the potential from 0 to 60 v with a sweeping rate of 1 v / s . the tio2 nanotube layers by were coated with al2o3 by atomic layer deposition tool ( thermal ald , tfs 200 , beneq ) . this technique based on sequential and self - limiting gas surface reactions allows conformal deposition of various coatings within tio2 nanotube layers with a nanometer scale accurate thickness , as shown previously . . grade , 99.999+% ) and deionized water ( 18 m ) were used as aluminum and oxygen precursors , respectively . under these deposition conditions , one growth ald cycle was defined by the following sequence : tma pulse ( 1 s)n2 purge ( 3 s)h2o pulse ( 1 s)n2 purge ( 3 s ) . all processes were carried out at a temperature of 200 c and using n2 ( 99.9999% ) as carrier gas at a flow rate of 400 standard cubic centimeters per minute ( sccm ) . al2o3 deposition was carried out running 8 , 88 , and 366 ald cycles , leading to coatings of different nominal thicknesses : 1 , 10 , and 42 nm , respectively . the number of cycles required for the different al2o3 thicknesses was estimated from the growth per cycle value of the al2o3 process at 200 c ( 1.1 / cycle ) . the thicknesses of al2o3 coatings were confirmed by variable angle spectroscopic ellipsometry ( vase ellipsometer , j.a . woollam , co. , inc . ) of al2o3 coatings on si wafers . upon the al2o3 coating process the annealing process was carried out in a muffle oven in an air atmosphere applying a heating rate of 15 c / min , until the target temperature ( 870 c ) was reached . the annealing process proceeded at such temperature for 1 h. afterward , the layers were allowed to naturally cool down . the morphology of the tio2 nanotube layers was characterized by a field - emission sem ( fe - sem jeol jsm 7500f ) and a scanning transmission electron microscope ( stem , fei tecnai f20 x - twin ) fitted with a high angle annular dark field ( haadf ) detector and operating at 200 kv . the cross - sectional views were obtained from mechanically bent samples . because of the rupture of the nanotube layers via this bending , it was possible to visualize nanotubes within the layers and coatings within nanotubes in various directions and nanotube layer depths . average values and standard deviations were calculated from at least three different locations with a high number of measurements ( n > 100 ) . diffraction analyses of the al2o3-coated tio2 nanotube layers carried out using x - ray diffractometer ( xrd , d8 advance , bruker axe ) using cu k radiation with secondary graphite monochromator and na(tl)i scintillation detector . nanoindentation measurements were performed to analyze the mechanical properties ( hardness ) of the tio2 nanotube layers . they were determined by an atomic force microscope ( afm , solver next , nt mdt ) equipped with a nanoindentation head ns01ntf and a berkovich type of tip ( three - sided pyramid geometry with a parameter of static stiffness , k = 10.2 0.3 kn / m ) . the nanotube layers were measured in longitudinal direction for compressive force of 0.5 mn , loaded for 100 s. fused silica sio2 was used as a calibration sample ( hardness , h = 9.5 0.5 gpa by iso 9450 - 76 ) . the penetration depth of the tip was up to a maximum 10% of the total thickness of the nanotube layer the hardness was evaluated at 30 different areas of each nanotube layer to ensure statistically relevant data set / significant results . the chemical stability of al2o3-coated tio2 nanotube layers was analyzed by soaking in h3po4 solutions of different concentrations : 25 , 50 , 70 , and 85 wt % ( prepared from 85 wt % h3po4 , penta ) . tio2 nanotube layers were soaked in these solutions for 48 h , including a thermal treatment for 8 h by which solutions were heated up at 60 c to further study the nanotube chemical stability under warm acidic conditions . before the subsequent sem analysis highly ordered tio2 nanotube layers , with a thickness of 20 m and an average diameter value of 110 nm ( aspect ratio 180 ) , were prepared by anodization of ti foils as described in detail in the experimental section . as - prepared amorphous tio2 nanotube layers were coated with al2o3 of different nominal thicknesses , namely 1 , 10 , and 42 nm by ald , as verified by sem and ellipsometric measurements ( 1.1 0.2 , 10 0.5 , and 44 2.1 nm ) . freshly coated nanotube layers were annealed at 870 c for 1 h along with reference uncoated tio2 nanotube layers . figure 1 shows sem images of the tio2 nanotube layers with and without al2o3 coating annealed at 870 c . uncoated tio2 nanotube layers ( figure 1a ) collapsed during the annealing process into a pillar nanostructure ( figure 1b ) . when coated , the nanotube layers were preserved after the annealing process , regardless of the thickness of the al2o3 , as apparent for coatings of either 1 nm ( figure 1c , d ) , or 10 nm ( figure 1e , f ) . it is quite fascinating that even 1 nm thin al2o3 coating can built a very thermally robust cage all over tio2 nanotubes with some 2040 nm thick tube walls . sem top - view images of uncoated tio2 nanotube layer ( a ) before and ( b ) after annealing ; al2o3 coated ( 1 nm ) tio2 nanotube layer ( c ) before and ( d ) after annealing ; al2o3 coated ( 10 nm ) tio2 nanotube layer ( e ) before and ( f ) after annealing . the annealing was carried out at 870 c for 1 h. insets : magnification of the corresponding sem images . all the scale bars denote 100 nm . figure 2 shows representative stem - haadf images of the nanotube body ( separated from the annealed al2o3 coated ( 10 nm ) tio2 nanotube layer by mechanical bending of the layers followed by sonication in methanol ) at a low ( a ) and at a high magnification ( b ) . especially from figure 2b , the interface between the tio2 wall and al2o3 coating is well distinguishable . there are actually two interfaces between the tio2 wall and al2o3 coating , as the al2o3 coating is deposited inside ( interior coating ) and outside ( exterior coating ) the tio2 tube walls . this feature is in accordance with our previous ald work , where we showed very good uniformity of al2o3 coatings on the amorphous tubes and absence of any pinholes in the coating . as apparent from figure 2 , al2o3 coatings remained continuous and pinhole - free even after annealing , during which thermally induced crystallization of tio2 tube walls occurred . some delamination of the coating seen at the outer and inner interface between tio2 wall and al2o3 coating ( especially at figure 2b ) stems most likely from the stress that these layers are exposed to during the preparation of specimens for sem and stem observation , which includes mechanical rupture of layers . these roughening and delamination events have no detrimental effect on coated nanotube layers that were not submitted for sem and stem and that completely survived soaking in h3po4 solutions ( described later in text ) . representative stem - haadf images of ( a ) a fragment of al2o3 coated ( 10 nm ) tio2 nanotube and ( b ) the corresponding higher magnification of the nanotube wall . interfaces between individual parts of the tubes are distinguished by solid lines and appropriate description . it is generally accepted that the annealing process influences crystal structure , phase transition , and structural integrity of the tio2 nanotube layers . it has also been accepted that amorphous as - prepared tio2 nanotube layers crystallize into anatase above 280 c in air . the anatase to rutile transition ( art ) has been reported at different temperatures , most usually in the range of temperature between 500 and 600 c , depending on the nanotube dimensions ( diameter , thickness , and composition ) . annealing at temperatures higher 600 c leads to the coexistence of anatase and rutile structures , while total conversion to rutile structure takes place above 800 c . figure 3a shows the xrd pattern obtained for reference uncoated tio2 nanotube layers annealed for 1 h at either 400 or 870 c , respectively . in line with literature , the former exhibits pure anatase crystal phase identified by typical anatase peaks associated with planes ( 101 ) , ( 004 ) , ( 105 ) , and ( 211 ) , with a dominant orientation ( 101 ) . the latter reveals pure rutile crystal phase with well - defined diffraction peaks of planes ( 110 ) , ( 011 ) , ( 111 ) , ( 211 ) , and ( 220 ) . the intensity of the peak at 2 = 27.4 indicates a preferred orientation along the ( 110 ) direction ; no trace of anatase polymorphic phase is detected . xrd patterns of ( a ) uncoated tio2 nanotube layers annealed at 870 and 400 c for 1 h ; ( b ) al2o3-coated tio2 nanotube layers with different coating thicknesses ( 1 , 10 , and 42 nm ) annealed at 870 c for 1 h ; and ( c ) al2o3-coated ( 1 and 10 nm ) tio2 nanotube layers preannealed ( 400 c , 1 h ) and second annealing at 870 c . a = anatase , r = rutile , and t = titanium substrate . the xrd patterns , obtained from al2o3-coated tio2 nanotube layers annealed at 870 c for 1 h , are shown in figure 3b . therein the coexistence of anatase and rutile structures can be clearly seen , in contrast to the uncoated nanotube layers ( figure 3a ) where rutile was exclusively formed at this temperature , in line with the previous work . the incomplete art of coated tio2 nanotube layers annealed at 870 c stems from the hindered surface reconstruction of the tio2 due to al2o3 coating and also the impact of al as the phase transformation inhibitor . in contrast , for uncoated nanotube layers ( annealed at 870 c ) the surface s reconstruction can easily take place ( no space constrains are present ) , allowing for the mass flow and rearrangements that yield complete rutile conversion , expected at this temperature . another evidence for limited reconstruction of coated tio2 nanotube layers annealed at 870 c is the fact that they do not collapse ( sinter ) , which they would otherwise do without coating . in the case of thermally stable coated nanotube layers , the coating acts in similar fashion as it does for nanoparticles and nanorods that can be annealed , when coated , at high temperatures without undergoing sintering events . quantification of the content of each crystal phase and an average of the corresponding crystallite size are given in table 1 . the anatase : rutile ratio was found to be dependent on the al2o3 coating . the nanotube layer with the thickest al2o3 coating ( 42 nm ) exhibited dominant rutile structure ( 62% ) with peaks corresponding to the planes ( 110 ) , ( 001 ) , ( 111 ) , ( 211 ) , and ( 220 ) and only one minor anatase peak corresponding to ( 101 ) plane . in clear contrast , the anatase content for 10 and 1 nm thin al2o3 coating was found to increase up to 73% and 83% , respectively , on account of rutile . in addition , identical rutile peaks were revealed for them as for the nanotube layer with thickest al2o3 coating ( 42 nm ) . it is noteworthy that the 1 nm thin al2o3 coated nanotube layer revealed anatase peaks with preferential orientation along the ( 004 ) plane , instead of usual ( 101 ) plane . who associated a particular thermal stability of the nanotube layers to such anatase ( 004 ) plane . based on the xrd results in figure 3b , there is obvious retardation of the anatase to rutile transition ( further noted as art ) with decreasing thickness of al2o3 coatings ( 1 and 10 nm ) . in order to give a physical description of the results , factors affecting the art first , we should consider the influence of the number of oxygen vacancies within the tio2 on the art temperature . reported that the larger is the number of oxygen vacancies within tio2 , the lower is its art temperature , or the art does not proceed at all and tio2 remains in anatase form . the defects ( in this case oxygen vacancies ) provide a low energy mass transport route and lower such art temperature . the number of oxygen vacancies within tio2 is also strongly influenced by the annealing atmosphere . previous works clearly indicated that dry annealing atmospheres such as ar or co led to more oxygen vacancies in tio2 than those performed in o2 or air . thus , the art was enhanced in oxygen - free atmospheres and resulted into larger rutile crystallites . second , it is important to define the fundamental reasoning behind the art origin , which has been subject of controversy . tio2 nanotube interface , where ti metal would be directly thermally oxidized into rutile structure . the presence of oxygen vacancies in between the ti substrate and tio2 nanotube layer was believed to induce the art at such interface , spreading toward the whole nanotube walls in the course of time , as reported by zhu et al . in contrast , yu et al . proposed that art does not stem from metal ti , but from the anatase ( created at lower temperatures , while ramping up the temperature ) at the interface between tio2 nanotubes and ti substrate , which converts to rutile at temperatures 600 c . in addition , works on annealing and crystallization of free - standing nanotube layers ( i.e. , nanotubes were detached from the ti substrate before annealing ) reported both the preservation of the anatase structure in the nanotube walls at temperatures higher than 600 c and much higher triggering art temperature . those results would point on a significant role of metal ti substrate tio2 nanotube interface on the art . there is a clear link between the experimental results obtained in this work and the literature about factors affecting the art . according to figure 3 , the anatase : rutile ratio is clearly dependent on the al2o3 coating thickness . that larger number of oxygen vacancies promotes the art , it is clear that the al2o3 coating within our tio2 nanotube layers influences the number of oxygen vacancies as it possesses a barrier against the oxygen diffusion . for example , the thickest al2o3 coating ( 42 nm ) hinders the oxygen diffusion most significantly from all used coatings in this work , leads to highest number of oxygen vacancies within tio2 nanotubes , and boosts the art process that ends up with the highest rutile content . in contrast , the oxygen diffusion into tio2 takes place more easily through thinner al2o3 coatings ( 1 and 10 nm ) , resulting in a lower number of oxygen vacancies , retarding the art within tio2 nanotubes . in addition , the largest rutile crystal size , calculated by the scherrer equation ( table 1 ) , corresponds to the thickest al2o3 coating , which also corroborates previously published findings on art and size of rutile crystals . to get a complete picture about the art , we also fully explored anatase tio2 nanotube layers ( annealed at 400 c for 1 h ) , shown in figure 3a , for al2o3 coating . we coated these nanotube layers with 1 and 10 nm of al2o3 by ald , before undergoing a second thermal treatment at 870 c for 1 h. first , the figure 3c shows that the al2o3-coated ( 10 nm ) tio2 nanotube layer consisted of 100% rutile , while in the al2o3-coated ( 1 nm ) tio2 nanotube layer rutile content was only 26% . these results confirm the active role of the al2o3 coating for tio2 crystal structure and are in line with the results and theories discussed in figure 3b . second , the significantly different crystal structure of the both investigated types of al2o3 ( 10 nm ) tio2 nanotube layer were revealed . the formerly annealed al2o3 coated ( 10 nm ) tio2 nanotube layer ( fully anatase comprised ) underwent a complete art and was 100% rutile comprised ( see figure 3c ) . in contrast , the initially amorphous tio2 nanotube layer revealed a predominant anatase content of 74% ( see figure 3b ) . this comparison clearly confirms that the tio2 structure influences the art and that it is clearly promoted for the tio2 nanotube layers annealed to anatase before art and ald coating . in other words , the lack of coating induces during the thermal annealing to 400 c more oxygen vacancies in the tio2 nanotube layers than it does when coatings are present during this annealing step . the mechanical integrity of the tio2 nanotube layers is of significant importance , especially for synthesis of devices based on flow - through membranes utilizing nanotube layers opened on both sides . even though some nanoindentation analyses of the tio2 nanotube arrays were already carried out , nanotube layers modified with additional coatings , as in the present case , have not yet been analyzed . figure 4 shows hardness of tio2 nanotube layers with al2o3 coatings of different thicknesses as well as two reference nanotube layers . if not denoted otherwise , all nanotube layers were annealed at 870 c for 1 h as the last processing step . first , the uncoated amorphous ( i.e. , did not undergo annealing ) tio2 nanotube layer displayed lower hardness value than the annealed uncoated counterpart , fully rutile structure comprised . that was expected as the crystal structure has ( as a rule of thumb ) higher hardness than amorphous mass of the same compound . second , the annealed al2o3-coated tio2 nanotube layers exhibited larger hardness with the increasing al2o3 coating thickness . this can be ascribed to increasing content of rutile ( table 1 ) and to an increasing al2o3 mass within the nanotubes . , all nanotube layers were annealed at 870 c for 1 h. even though rutile and anatase are similar in structure , the reason to rutile to be more mechanically robust than anatase is that its octahedra shares four edges instead four corners ( anatase case ) , which leads to the formation of chains arranged subsequently in a 4-fold arrangement . this also explains why the al2o3-coated ( 1 nm ) tio2 coated layer ( which has mainly anatase structure as shown in figure 3b ) has lower hardness than uncoated annealed layers ( completely rutile based , also shown in figure 3a ) . the hardness values presented here for high aspect ratio ( 180 ) nanotube layers were larger than those found in the literature that reports typical hardness in the range from 94 mpa to 3.5 gpa . however , it is difficult to establish a comparison , principally because the published reports show results for exclusively uncoated and lower aspect ratio tio2 nanotubes layers with thicknesses from 625 nm to 8.5 m ( compared to 20 m in the present case ) . moreover , the use of different indenter tips ( vickers tips for microhardness , berkovitch tips for nanohardness , cube corner tips for nanohardness , etc . ) entails different hardness values . nevertheless , it can be concluded that both the annealing treatment and the al2o3 ald coating resulted in a substantial enhancement of the mechanical properties of tio2 nanotube layer . finally , the chemical stability of al2o3-coated tio2 nanotube layers was investigated in strongly acidic environment , namely in h3po4 solutions with different concentration . such stability is significant for the nanotube layers to sustain in environments , where up to now they could not preserve their morphological integrity . for example , in various biological environments with low ph , the knowledge about the stability threshold is important . as it can be seen in figure 5 , even the thinnest coating ( 1 nm al2o3 ) completely preserved the tio2 nanotube layers from degradation in concentrated h3po4 . in line with that no degradation was observed for any of the thicker al2o3 coatings : 10 and 42 nm ( data not shown here ) . the soaking tests were performed on the 40 h time scale for all nanotube layers . this expands the already wide range of environments , where ald al2o3 coatings are stable , in addition to published stability results of these coatings in various acidic ( h2so4 , hno3 , hcl ) and alkaline ( koh ) environments and water . to make the h3po4 environment even more harsh , the h3po4 solutions were heated up to 60 c , and soaking was carried out for an additional 8 h ( in total 48 h ) . since again no visible changes were observed , soaking experiments were terminated afterward . reference uncoated layers ( namely as - anodized amorphous and annealed ( 400 c for 1 h ) nanotube layers ) did not survive these conditions . in order to determine the chemical threshold conditions for these reference uncoated layers , lower h3po4 concentrations had to be used . the stability threshold was revealed to be 10 wt % ( figure 5e ) and 40 wt % ( figure 5f ) on the scale of 24 h for the amorphous and annealed case , respectively , without any heating . all in all , the results presented in figure 5 for al2o3-coated nanotube layers confirm the outstanding enhancement of the chemical stability of tio2 nanotube layers provided by uniform al2o3 coatings . sem top - view images of annealed al2o3-coated ( 1 nm ) tio2 nanotube layers before ( a ) and after soaking in h3po4 solutions with different concentrations : ( b ) 50 wt % , ( c ) 70 wt % , and ( d ) 85 wt % in total for 48 h ( last 8 h at 60 c ) . sem top - view images of reference uncoated amorphous ( e ) and anatase ( f ) tio2 nanotube layers after soaking in h3po4 solutions of 10 and 40 wt % , respectively , for 24 h. all the scale bars denote 100 nm . thus , the present results , especially for the thinnest al2o3 coating ( 1 nm ) , are very useful and promising for practical applications of the nanotube layers . as - treated tio2 nanotube layers : ( i ) maintain anatase structure ( more favorable for photovoltaics and photocatalysis than rutile ) in the tubes over a very broad temperature range , ( ii ) possess significantly improved charge separation on the interface with various electrolytes ( especially because electrons can tunnel to tio2 via al2o3 coatings thinner than 2 nm ) , ( iii ) possess strong mechanical integrity , and ( iv ) provide extremely good stability in strongly acidic environments . all these features pave favorable way for the functionalization of tio2 nanotube layers by secondary materials . it is foreseen that additional materials , such us various oxides , nitrides , sulfides , etc . thermal and chemical stability of al2o3-coated tio2 nanotube layers can extend the utilization of nanotube layers for catalytic applications and sensing of gases ( such as co , nox , ch3ch2oh , h2 , and o2 ) at high temperatures and/or in harsh acidic environment , so far unfeasible for uncoated tio2 nanotube layers counterparts . in parallel , membranes composed of ultrahigh aspect ratio tio2 nanotube layers that have been used for photocatalytic or flow - through experiments may be prone to mechanical instabilities . thus , they could greatly benefit from a thin al2o3 coating to become mechanically more robust . in this work , effects of al2o3 coating produced by ald on the crystal structure , mechanical , and chemical properties of tio2 nanotube layers were explored . noteworthy improvement of the thermal stability upon annealing in air was revealed up to temperatures of 870 c , even with an extremely thin al2o3 coating ( 1 nm ) . in contrast to uncoated tio2 nanotube layers ( 100 vol % rutile ) , a high fraction of anatase structure ( 83 vol % ) was determined for al2o3-coated ( 1 nm ) tio2 nanotube layers upon annealing at 870 c , which is highly desired due to its optical and electronic properties for photovoltaic and photocatalytic applications . an enhanced hardness was revealed for al2o3-coated tio2 nanotube layers with a positive impact on the mechanical properties of nanotube layers . in addition , al2o3 coatings provided to the tio2 nanotube layers extremely good stability in extremely acidic environments of h3po4 solutions with different concentrations . all in all , self - organized tio2 nanotube layers coated with thin al2o3 coatings yield superior thermal , chemical , and mechanical stabilities that will extend their application range to previously nonimaginable working environments .

we report on a very significant enhancement of the thermal , chemical , and mechanical stability of self - organized tio2 nanotubes layers , provided by thin al2o3 coatings of different thicknesses prepared by atomic layer deposition ( ald ) . tio2 nanotube layers coated with al2o3 coatings exhibit significantly improved thermal stability as illustrated by the preservation of the nanotubular structure upon annealing treatment at high temperatures ( 870 c ) . in addition , a high anatase content is preserved in the nanotube layers against expectation of the total rutile conversion at such a high temperature . hardness of the resulting nanotube layers is investigated by nanoindentation measurements and shows strongly improved values compared to uncoated counterparts . finally , it is demonstrated that al2o3 coatings guarantee unprecedented chemical stability of tio2 nanotube layers in harsh environments of concentrated h3po4 solutions .

Introduction Experimental Section Results and Discussion Conclusions

PMC2759361

the last several years have seen substantial theoretical developments related to the hypothesized behavioral functions of nucleus accumbens dopamine ( da ) . it has become evident to many investigators that there are conceptual limitations and empirical problems with the traditional da hypothesis of reward ( baldo and kelley , 2007 ; barbano and cador , 2007 ; salamone et al . , 1997 , 2005 , 2007 ; even the use of the term reward itself often is problematic ( cannon and bseikri , 2004 ; salamone et al . , 2005 ; salamone , 2006 ; sanchis - segura and spanagel , 2006 ; yin et al . , 2008 ) . researchers rarely define what they mean by reward when they are using it to describe a psychological process ; some use it as though it were a synonym for reinforcement , or in reference to appetite or primary motivation , while still others employ it as a code word to mean pleasure . in some papers , the word reward seems to be used as a rather monolithic , all - encompassing term that refers to any and all aspects of appetitive learning , motivation and emotion , whether conditioned or unconditioned . used in this way , the term reward is a rather blunt instrument . these problems are not merely semantic , as it is difficult to test a hypothesis which maintains that a neurotransmitter mediates such an ill - defined set of functions . it has been suggested that it is advantageous to maintain the distinction between the terms reward and reinforcement ; with this usage , reinforcement refers more directly to instrumental learning mechanisms ( wise , 2004 ; sanchis - segura and spanagel , 2006 ) , while reward connotes the primary motivational and emotional effects of reinforcing stimuli ( everitt and robbins , 2005 ; salamone et al . , 2005 , 2007 ) . against the backdrop of these conceptual and terminological issues , there is a tremendous weight of empirical evidence that has built up against the various iterations of the da hypothesis of reward . it is somewhat ironic that the processes most directly linked to the use of the term reward ( i.e. , primary motivation , subjective pleasure ) are the ones that have proven to be most problematic in terms of demonstrating the involvement of mesolimbic da ( salamone et al . , 2007 ) . for example , low doses of da antagonists and depletions of nucleus accumbens da have been shown to produce effects that do not closely resemble extinction ( salamone , 1986 ; salamone et al . , 1995 , 1997 ; rick et al . , 2006 ) , pre - feeding ( salamone et al . , 1991 ; aberman and salamone , 1999 ) , or appetite suppressant drugs ( cousins et al . , 1994 ; salamone et al . , 2002 ; although it is well known that whole forebrain da depletions can produce aphagia ( i.e. , lack of eating ) , it is da depletions in the lateral or ventrolateral caudate / putamen , rather than the nucleus accumbens , which have most conclusively been linked to this effect ( ungerstedt , 1971 ; dunnett and iversen , 1982 ; salamone et al . , 1993a ) . it has been shown repeatedly that nucleus accumbens da depletions or antagonism do not substantially impair appetite for food , or produce a general disruption of primary food motivation ( ungerstedt , 1971 ; koob et al . , 1978 ; bakshi and kelley , 1991 ; salamone et al . da deficient mice , restoration of da production in caudate putamen , but not nucleus accumbens , was able to rescue feeding behavior ( szczypka et al . , 2001 ) . in summarizing their findings that injections of da d1 or d2 family antagonists into either the core or the shell subregions of nucleus accumbens impaired locomotion and rearing , but did not suppress food intake , baldo et al . ( 2002 ) stated that da receptor blockade did not abolish the primary motivation to eat furthermore , the idea that nucleus accumbens da mediates the pleasure associated with positive reinforcers has been strongly challenged ( berridge , 2007 ; salamone et al . , 2007 ; berridge and kringlebach , 2008 ) . interference with accumbens da transmission does not impair appetitive taste reactivity for sucrose ( berridge , 2007 ; berridge and kringlebach , 2008 ) . several studies in humans have reported that da antagonists did not blunt the subjective euphoria produced by drugs of abuse ( gawin , 1986 ; brauer and de wit , 1997 ; haney et al . , 2001 ; nann - vernotica et al . , 2001 ; wachtel et al . , moreover , the potential role of da systems in instrumental behavior or learning is not limited to situations involving appetitive motivation . there is considerable evidence that striatal mechanisms in general , and mesolimbic da in particular , also participate in aspects of aversive learning and aversive motivation ( salamone , 1994 ; munro and kokkinidis , 1997 ; blazquez et al . , 2002 ; pezze and feldon , 2004 ; delgado et al . , 2008 ; faure et al . , 2008 although imaging studies often are used to support the idea that nucleus accumbens mediates pleasure ( e.g. , sarchiapone et al . , 2006 ; wacker et al . , 2009 ) , this appears to be oversimplified ; indeed , research employing various imaging methods has demonstrated that the human nucleus accumbens also responds to stress , aversion and hyperarousal / irritability ( liberzon et al . , 1999 ; jensen et al . , 2003 ; pavic , 2003 ; phan et al . , 2004 ; pruessner et al . , 2004 ; levita et al . , physiological and neurochemical studies in animals clearly indicate that da neuron activity is not simply tied to the delivery of primary reinforcers or pleasurable stimuli . rather , vta neuron activity and da release can be activated by a number of different appetitive and aversive conditions ( mccullough and salamone , 1992 ; mccullough et al . , 1993 ; , 2004 ; young , 2004 ; anstrom and woodward , 2005 ; broom and yamamoto , 2005 ; marinelli et al . , 2005 ; schultz , 2007a , b ; brischoux et al . , 2009 ) , with changes seen across varying time scales , including tonic , slow phasic and fast phasic signals ( salamone , 1996 ; salamone et al . , 2007 ; schultz , 2007a , b ; salamone , in press ; see also lapish et al . , 2007 for a discussion of various time scales associated with the postsynaptic effects of da release and da receptor stimulation ) . of course , one would not want to throw the baby out with the bathwater . it is apparent that mesolimbic da participates in several complex functions related to aspects of instrumental behavior , learning and incentive motivation , and pavlovian / instrumental interactions ( wise , 2004 ; everitt and robbins , 2005 ; kelley et al . berridge , 2007 ; robbins and everitt , 2007 ; redgrave et al . , 2008 ; yin et al . , the more difficult aspect of research and theory in this area is to ask which specific aspects ? exploration of these diverse areas of dopaminergic function has become a rich and fruitful area of inquiry . indeed , this literature is so extensive that a thorough review of the behavioral functions of nucleus accumbens da is beyond the scope of the present article ( see salamone et al . , 2007 ) . for the purposes of this special issue , the present review will focus upon the role of nucleus accumbens da in effort - related processes , with a special emphasis on effort - based choice behavior that depends upon cost / benefit analyses . even as the popularity of the da hypothesis of reward was growing during the 1980s , it was becoming apparent that there were alternative conceptual frameworks available for organizing what was known about the behavioral functions of da systems , particularly mesolimbic da . ( 1980 ) suggested that nucleus accumbens acted as a functional interface between the limbic system and the motor system , facilitating the ability of information related to emotion and motivation to impinge upon the neural systems involved in the instigation of action . it had been emphasized for several decades that behavioral activation , i.e. , the vigor , persistence and effort seen in the pursuit of motivational stimuli , and the heightened activity induced by conditioned stimuli that predict reinforcers , was a fundamental aspect of motivation ( e.g. , cofer and appley , 1964 ) . da antagonists or accumbens da depletions were shown to suppress the activities such as excessive drinking , wheel running , and locomotion that are induced by scheduled presentation of food ( robbins and koob , 1980 ; wallace et al . , 1983 ; salamone , 1986 , 1988 ) . it also was reported that the effects of da antagonists on reinforced behavior interacted powerfully with the kinetic requirements of the instrumental response . for example , doses of da antagonists that suppressed reinforced lever pressing had minimal effects on reinforced nose poking behavior ( ettenberg et al . , 1981 ; mekarski , 1988 ) . although 0.1 mg / kg haloperidol severely reduced responding on a fixed ratio ( fr ) 20 schedule of lever pressing , a dose four times that size had no effect on the reinforced response of simply being in proximity to the food dish on a fixed interval 30 s schedule ( salamone , 1986 ) . as this research was being reported , investigators began to employ economic concepts , such as exertion of effort and cost - benefit analyses , to describe the behavioral functions of accumbens da . neill and justice ( 1981 ) hypothesized that injection of amphetamine into nucleus accumbens could be increasing the willingness of rats to exert effort to obtain a given level of reinforcement . in a contemporary review of the behavioral functions of da systems ( salamone , 1987 ) , it was noted that da in nucleus accumbens could be involved in the exertion of effort , and it was suggested that future experiments could offer animals choices between various reinforcers that are associated with operants of varying difficulty ( p. 602 ) so that researchers could determine if the allocation of behavioral resources could be biased toward or away from more or less effortful responses by administration of dopaminergic drugs . this recognition of dopaminergic involvement in the exertion of effort , and effort - based choices related to cost benefit analyses , fit nicely with an emerging emphasis in the behavioral literature on work , response costs or constraints , and economic models of operant behavior . several behavioral investigators have emphasized how response costs or constraints affect operant response output ( staddon , 1979 ; kaufman , 1980 ; kaufman et al . , 1980 ; collier and colleagues studied how work requirements , such as the number of lever presses necessary for obtaining food , could serve as determinants of response output and affect consumption parameters ( collier and jennings , 1969 ; johnson and collier , 1987 ) . economic models of operant behavior have emphasized how a number of factors , including not only reinforcement value , but also conditions related to the characteristics of the instrumental response , can determine behavioral output ( lea , 1978 ; allison , 1981 , 1993 ; bickel et al . , 2000 ) . ( 1988 ) suggested that , in terms of behavioral economics , the price of food reinforcement as a commodity is a cost / benefit ratio expressed as the effort expended per unit of food value consumed . optimal foraging theory was proposed to account for the observation that the amount of effort or time expended to obtain motivational stimuli was an important determinant of foraging choice ( krebs , 1977 ) , an idea that is still very influential in the ethology research today ( e.g. , hengeveld et al . , , several lines of evidence have converged to strengthen the original observation that the effects of interference with da transmission interact powerfully with the work requirements of an instrumental task . one of the ways of controlling work requirements in an operant schedule is to vary the ratio requirement ( i.e. , the number of times the animal must press the lever to receive a unit of reinforcement ) . the effects of the da antagonist haloperidol on food - reinforced behavior were shown to be dependent upon the particular ratio schedule that was used [ i.e. , fr1 vs. progressive ratio ; caul and brindle ( 2001 ) ] . accumbens da depletions also produce effects that interact powerfully with the ratio requirement of the schedule employed . 2004 ) found that accumbens da depletions that substantially impaired fr5 lever pressing had no significant effect on fr1 performance . aberman and salamone ( 1999 ) systematically studied a wide range of ratio schedules ( fr1 , 4 , 16 , and 64 ) to assess the effects of accumbens da depletions . while fr1 performance was not affected by da depletion , and fr4 responding was only transiently and mildly suppressed , the schedules with large ratio requirements ( i.e. , fr16 and fr64 ) were severely impaired ( figure 1a ) . in fact , da depleted animals responding on the fr64 schedule showed significantly fewer responses than those responding on the fr16 schedule ( aberman and salamone , 1999 ) . this pattern indicates that accumbens da depletions exacerbate an effect known as ratio strain . in untreated animals , the overall relation between ratio size and response output is inverted - u shaped . up to a point , as ratio requirements get larger , animals adjust to this challenge by increasing response output . however , if the ratio requirement is high enough ( i.e. , if the cost is too high ) , the animal reaches the point at which additional responses being required actually tend to suppress responding . for normal rats , responding at levels of fr64 , fr100 or higher , even if there is only one 45 mg food pellet being delivered , does not seem to be problematic . a completely different function is shown by rats with accumbens da depletions , which are much more sensitive to the size of the ratio requirement . in behavioral economic terms , this pattern can be described as reflecting an increase in the elasticity of demand for food reinforcement ( salamone et al . , 1997 ; aberman and salamone , 1999 ; see figure 1b ) . the term elasticity is widely used in economics , but price elasticity of demand refers to the sensitivity of consumption to changes in price ( vuchinich and heather , 2003 ) . thus , if the ratio requirement is analogous to the price of the commodity ( in this case , reinforcement pellets ) , it appears that rats with accumbens da depletions are more sensitive than control animals to the price of the food reinforcers . of course , rats do not use currency to purchase operant pellets ; rather , it has been argued that an operant procedure is more of a barter system , in which the rat trades its work ( or reductions in leisure ) for a commodity ( rachlin , 2003 ) . thus , another way of describing this effect of impaired da transmission is to say that rats with accumbens da depletions are more sensitive than control animals to work - related response costs , or that they are less likely to trade high levels of work for food . in another study ( salamone et al . , 2001 ) , the increased effects of accumbens da depletions with increasing ratio requirements were observed when rats were tested across a broader range of ratio schedules as high as fr300 , even when the overall relation between lever pressing and food delivered per lever press was kept constant ( i.e. , fr50 , one pellet every 50 responses ; fr100 , two pellets every 100 responses ; fr200 , four pellets every 200 responses ; fr300 , six pellets every 300 responses ; salamone et al . thus , both the magnitude and the organization of the ratio requirement appear to be critical determinants of the sensitivity of an operant schedule to the effects of accumbens da depletions . this figure shows the effect of ratio requirement on the number of lever presses emitted and operant pellets consumed in rats with accumbens da depletions compared to rats in the vehicle control group . figure ( a ) is re - drawn based upon aberman and salamone ( 1999 ) ; these data are depicted in terms of number of responses , as in the original article . for ( b ) , the data are represented as number of operant pellets consumed . each data point shown is the mean value from each group at each ratio level . although comparable levels of consumption in da depleted and control groups were seen with the fr1 schedule , da - depleted rats showed markedly reduced consumption relative to the control group at higher ratio levels . in order to be sure that these results reflected the influence of ratio size , as opposed to other variables such as time , additional studies examined the effects of accumbens da depletions on tandem schedules , in which a ratio requirement was attached to an interval requirement . in a conventional variable interval ( vi ) schedule , a time interval must elapse before the first response is reinforced , and the particular time interval varies around an average value . a tandem vi / fr schedule has an additional ratio requirement attached to the interval . for example , with a tandem vi 30 s / fr5 schedule , the animal is reinforced for the fifth response after the interval elapses , rather than the first . in this way , one can vary the ratio requirement of a schedule while keeping the programmed time intervals the same . research employing tandem vi / fr schedules with varying combinations ( e.g. , vi 30 s / fr5 , vi 60 s / fr10 , vi 120 s / fr10 ) has yielded a consistent pattern ; accumbens da depletions do not impair overall response output in rats responding on the conventional vi schedules ( i.e. , those requiring only one response after the interval ) , but do substantially reduce responding on the corresponding vi schedule with the higher ratio requirement attached ( correa et al . these results are consistent with research showing that accumbens da antagonism did not impair performance on a progressive interval task ( wakabayashi et al . , 2004 ) , and suggest that interval requirements per se do not pose a severe constraint to rats with compromised da transmission in nucleus accumbens . this serves to underscore the critical importance of ratio requirements as providing a work - related challenge to rats with accumbens da depletions or antagonism . in summarizing these results , salamone and correa ( 2002 ) stated that nucleus accumbens da depletions appear to have two major effects : ( 1 ) they reduce the response - enhancing effects that moderate - size ratio requirements have on operant responding ( i.e. , the ascending limb of the function relating ratio requirement to response output ) , and ( 2 ) they enhance the response - suppressing effects that very large ratios have on operant responding ( i.e. , the descending limb of the function , enhancing ratio strain ) . furthermore , finer grained analyses of detailed patterns of responding reveal more insights into the behavioral manifestations of accumbens da depletions . accumbens da depletions produce a slight reduction in the local rate of responding , as indicated by the distribution of inter - response times ( salamone et al . the latter may indicate a fragmentation in the pattern of responding ( mingote et al . , 2005 ) , a reduction in the ability to sustain uninterrupted response output , or a lack of engagement in the task ( nicola , 2007 ) . recently , computational approaches have been used to analyze these effects of accumbens da depletions on response rate ( e.g. , niv et al . this relation between response output and da function has been interpreted to mean that da release in nucleus accumbens could provide a window of opportunistic drive during which the threshold cost expenditure to obtain the reward is decreased ( phillips et al . , 2007 ) . in discussing the effects of dopaminergic manipulations on ratio performance , it is useful to mention the term reinforcement efficacy , which is sometimes used to describe the effects of drug manipulations on progressive ratio performance . with progressive ratio schedules , the ratio requirement increases as successive ratios are completed , and the break point is said to occur at the point at which the animal essentially ceases to respond . one can operationally define reinforcement efficacy in terms of the break point in a progressive ratio schedule ( and also by measuring ratio strain in rats responding across different fr schedules ) . the determination of reinforcement efficacy can be a very useful tool for characterizing some of the fundamental reinforcing actions of drugs that are self - administered , and for comparing self - administration behavior across different substances or classes of substances ( e.g. , richardson and roberts , 1996 ; marinelli et al . reinforcement efficacy is essentially being employed as an empirical descriptor of a particular behavioral outcome . nevertheless , given the terminological problems mentioned above , it is worth emphasizing that the term reinforcement efficacy should not be used simply as a replacement for reward , nor should progressive ratio breakpoints be viewed as necessarily providing some direct and unambiguous measure related to the subjective pleasure produced by the stimulus ( salamone , 2006 ) . changes in progressive ratio break points can reflect more than just changes in the appetitive motivational properties of a reinforcing stimulus ( richardson and roberts , 1996 ; hamill et al . , 1999 ) . for example , changing the kinetic requirements of the instrumental response ( e.g. , increasing the height of the lever ) was shown to decrease progressive ratio break points ( skjoldager et al . although some researchers have maintained that the break point provides a direct measure of the appetitive motivational characteristics of a stimulus , it is , as explicitly stated in a classic review by stewart ( 1974 ) , more directly a measure of how much work the organism will do in order to obtain that stimulus . progressive ratio break points and measures of ratio strain are essentially outcomes that result from effort - related decision making processes . the animal is making a cost / benefit choice about whether or not to continue to respond , based partly on factors related to the reinforce itself , but also upon the work - related response costs and time constraints imposed by the ratio schedule . for these reasons , interpretations of the actions of drugs or lesions on progressive ratio break points should be done with caution , as should be the case for any individual task . a drug that alters the break point could do so for many different reasons ; it may be affecting functions related to the processing of reward value , or alternatively it could be affecting exertion of effort , or decision making processes . the ability to exert effort , sustain work , overcome obstacles , and attain access to motivationally relevant stimuli is necessary for survival . but it is only part of the story . in a complex environment , which affords many opportunities for obtaining significant stimuli , and multiple paths for accessing them the variables that need to be evaluated to make these decisions are complex and multidimensional , but among the most important are those involving cost / benefit assessments based upon effort and reinforcement value ( salamone and correa , 2002 ; salamone et al . , 2003 , 2005 , 2007 ; van den bos et al . , 2006 ; walton et al . , 2006 ) . considerable evidence indicates that nucleus accumbens da , along with other transmitters and structures , participates in the overall circuitry that regulates effort - based choice behavior ( salamone et al . , 2003 , 2005 , 2007 ; floresco et al . , 2008a ; hauber and sommer , 2009 ) . one of the procedures that has been used to assess the contribution of accumbens da to response allocation and effort - related choice behavior is a task that offers rats the option of either lever pressing to obtain a relatively preferred food ( e.g. , bioserve pellets ; usually obtained on a fr5 schedule ) , or approaching and consuming a less preferred food ( lab chow ) that is concurrently available in the chamber . well trained rats under baseline conditions typically get most of their food by lever pressing , and consume only small quantities of chow ( salamone et al . , 1991 ) . low - to - moderate doses of da antagonists , which block either d1 or d2 family receptor subtypes , produce a substantial alteration of response allocation in rats performing on this task . the da antagonists cis - flupenthixol , haloperidol , raclopride , eticlopride , sch 23390 , skf83566 , and ecopipam all decreased lever pressing for food but substantially increased intake of the concurrently available chow ( salamone et al . , 1991 , 1996 , 2002 ; cousins et al . , 1994 ; sink et al . , 2008 ; worden et al . , the use of this task for assessing effort - related choice behavior has been validated in many ways . for example , the low dose of haloperidol that produced the shift from lever pressing to chow intake ( 0.1 mg / kg ) did not affect total food intake or alter preference between these two specific foods in free - feeding choice tests ( salamone et al . , 1991 ) . although da antagonists have been shown to reduce fr5 lever pressing and increase chow intake , appetite suppressants from different classes , including amphetamine ( cousins et al . , 1994 ) , fenfluramine ( salamone et al . , 2002 ) , and cannabinoid cb1 antagonists ( sink et al . , 2008 ) , failed to increase chow intake at doses that suppressed lever pressing . similarly , pre - feeding to reduce food motivation was shown to suppress both lever pressing and chow intake ( salamone et al . , 1991 ) . furthermore , attachment of higher ratio requirements ( up to fr20 ) caused animals that were not drug treated to shift from lever pressing to chow intake ( salamone et al . together with other results , these findings demonstrate that interference with da transmission does not simply reduce appetite , but does act to alter response allocation between alternative sources of food that can be obtained through different instrumental responses . the shift from lever pressing to chow intake in rats performing on this task is associated with da depletions in nucleus accumbens , but not the neostriatum . although it has been suggested that caudate / putamen da may have some types of motivational functions related to feeding ( palmiter , 2007 ) , da depletions in anteroventromedial neostriatum , which is dorsal to nucleus accumbens , had no behavioral effect , while ventrolateral neostriatal da depletions produced severe motor impairments that merely decreased both lever pressing and feeding ( cousins et al . , 1993 ) . in contrast , decreases in lever pressing and increases in chow intake occur as a result of accumbens da depletions , as well as intra - accumbens injections of d1 or d2 antagonists ( salamone et al . , 1991 ; cousins et al . , 1993 ; cousins and salamone , 1994 ; sokolowski and salamone , 1998 ; koch et al . , 2000 the shift from lever pressing to chow intake on this task has been shown to occur in rats if d1 or d2 family antagonist are injected into the medial core , lateral core , or dorsal shell subregions of the accumbens ( salamone et al . , 1991 ; nowend et al . , thus , although lever pressing is decreased by accumbens da antagonism or depletions , the rats show a compensatory reallocation of behavior and select a new path to an alternative food source . consistent with these effects observed in rats that have impaired da transmission , da transporter knockdown mice , which have enhanced da transmission , show increased selection of lever pressing relative to chow intake when tested with this task ( cagniard et al . , 2006 ) . ( 1994 ) also developed a t - maze procedure in order to assess the effects of da antagonists and accumbens da depletions on effort - related decision making . with this procedure , the two choice arms of the maze can have different reinforcement densities ( e.g. , four vs. two food pellets , or four vs. zero food pellets ) , and under some conditions a 44-cm barrier can be placed in the arm with the higher density of food reinforcement to present an effort - related challenge to the rat . when no barrier is placed in the arm with the high reinforcement density , rats mostly choose that arm , and neither haloperidol nor accumbens da depletion alters their response choice ( salamone , 1994 ) . when the arm with the barrier contained four pellets , but the other arm contained no pellets , rats with accumbens da depletions were very slow , but still managed to choose the high density arm , climb the barrier , and consume the pellets ( cousins et al . , 1996 ) . yet accumbens da depletions dramatically altered choice behavior when the high density arm ( four pellets ) had the barrier in position , and the arm without the barrier contained an alternative food source ( two pellets ) . in this case , da depletions or antagonism decreased choice for the high density arm , and increased choice for the low density arm ( salamone , 1994 ; cousins et al . , 1996 ; denk et al . , 2005 ; mott et al . , , the t - maze task for measuring effort - based choice behavior also has undergone considerable behavioral validation and evaluation ( salamone , 1994 ; cousins et al . , 1996 ; for example , in a recent t - maze choice study with mice , it was confirmed that haloperidol reduced choice of the arm with the barrier , and it also was demonstrated that haloperidol had no effect on choice when both arms had a barrier in place ( correa et al . , 2009 ) . thus , dopaminergic manipulations did not alter the preference for the high density of food reward over the low density , and did not affect discrimination or memory processes related to arm preference . over the last several years , variants of this task have been used by several laboratories to characterize the effects of brain lesions or drug manipulations ( salamone , 1994 ; walton et al . , 2003 ; denk et al . , 2005 ; schweimer and hauber , 2005 ; van den bos et al . , 2006 ; bardgett et al . , 2009 ; the results of the t - maze studies in rodents , together with the findings from the operant concurrent choice studies reviewed above , indicate that low doses of da antagonists and accumbens da depletions cause animals to reallocate their instrumental response selection based upon the response requirements of the task , and select lower effort alternatives for obtaining rewards ( see reviews by salamone et al . ( 2008b ) investigated the effects of dopaminergic and glutamatergic drugs on both effort and delay discounting . the da antagonist haloperidol altered effort discounting even when the effects of time delay were controlled for ( floresco et al . a t - maze effort discounting task was recently developed ( bardgett et al . , 2009 ) , in which the amount of food in the high density arm of the maze was diminished each trial on which the rats selected that arm ( i.e. , an adjusting - amount discounting variant of the t - maze procedures , which allows for the determination an indifference point for each rat ) . administration of both the d1 family antagonist sch23390 and the d2 family antagonist haloperidol altered effort discounting , making it more likely that rats would choose the arm with the smaller reward . increasing da transmission by administration of amphetamine blocked the effects of sch23390 and haloperidol , and also biased rats toward choosing the high reward / high cost arm , which is consistent with operant choice studies using da transporter knockdown mice ( cagniard et al . , 2006 ) . together with other results , the findings reported by bardgett et al . ( 2008b ) support the suggestion that , across a variety of conditions , da transmission exerts a bidirectional influence over effort - related decision making . as reviewed above , considerable research has demonstrated that da antagonists and accumbens da depletions affect behavioral activation , instrumental response output , response allocation , and effort - related processes ( salamone et al . , 1991 , 2007 ; salamone and correa , 2002 ; phillips et al . , 2007 ; robbins and everitt , 2007 ; clearly , da does not participate in effort - related processes in isolation , and for that reason it is important to review how other brain areas and neurotransmitters interact with dopaminergic mechanisms . within the last few years , considerable emphasis has been placed upon interactions between da and adenosine . caffeine and other methylxanthines , which act as minor stimulants , are non - selective adenosine antagonists ( ferr et al . , 2008 ) . recently , there has been a rapid growth of research on adenosine receptor neurochemistry and pharmacology , particularly concerning the a2a subtype of adenosine receptor . da - rich striatal areas , including both the caudate / putamen ( neostriatum ) and the nucleus accumbens , have a very high degree of adenosine a2a receptor expression ( schiffmann et al . , 1991 ; demet and chicz - demet , 2002 ; there is considerable evidence of a functional interaction between striatal da d2 and adenosine a2a receptors ( fink et al . , 1992 ; ferr , 1997 ; hillion et al . , 2002 ; fuxe et al . , this interaction frequently has been studied in regard to neostriatal motor functions that are related to parkinsonism ( ferr et al . , 1997 , 2001 ; hauber and munkel , 1997 ; svenningsson et al . , 1999 ; , 2001 ; wardas et al . , 2001 ; morelli and pinna , 2002 ; correa et al . , 2004 ; jenner , 2005 ; pinna et al . , 2005 ; ishiwari et al . , 2007 ; several reports also have characterized aspects of adenosine a2a receptor function related to cognitive processes ( takahashi et al . , 2008 ) , anxiety ( correa and font , 2008 ) , and motivation ( salamone et al . adenosine a2a receptors also are involved in aspects of behavioral activation and effort - related processes ( farrar et al . , 2007 ; font et al . , 2008 ; mingote et al . , 2008 ; mott et al . , injections of the adenosine a2a agonist cgs 21680 directly into the accumbens can produce effects that resemble those of accumbens da depletions or antagonism . intra - accumbens injections of cgs 21680 were shown to reduce locomotor activity ( barraco et al . , 1993 ) . local infusions of cgs 21680 into the accumbens reduced responding on a vi 60 s schedule with a fr10 requirement attached , but did not impair performance on a conventional vi 60 s schedule ( mingote et al . , 2008 ) ; this pattern is similar to that previously shown with accumbens da depletions ( mingote et al . , 2005 ) . in rats responding on the operant fr5/chow feeding concurrent choice procedure , injections of cgs 21680 into the accumbens decreased lever pressing and increased chow intake ( font et al . , 2008 ) , a pattern of effects similar to that produced by accumbens da depletions and antagonism . consistent with the observation that an adenosine a2a agonist could produce actions similar to those resulting from da depletion or blockade , it also has been reported the locomotor suppression induced by the da antagonist haloperidol was reduced by injections of the adenosine a2a antagonist msx-3 into nucleus accumbens core , but not into the shell or the ventrolateral neostriatum ( ishiwari et al . , 2007 ) . furthermore , it has been demonstrated that adenosine a2a receptor antagonists can reverse the effects of da d2 antagonists on both the operant concurrent choice task ( farrar et al . 2009 ) and the t - maze choice procedure ( correa et al . , 2009 ; mott et al . , 2009 ) . recently , studies with intracranial injections revealed that systemic or intra - accumbens injections of the adenosine a2a antagonist msx-3 were able to block the effects of intra - accumbens injections of the d2 antagonist eticlopride in rats responding on the operant concurrent choice task ( farrar , 2009 , unpublished doctoral dissertation , university of connecticut ) . these studies afford an interesting opportunity to assess the overall interaction between da and adenosine receptor subtypes . adenosine a2a receptor antagonists msx-3 and kw 6002 reliably attenuate the effects of d2 antagonists such as haloperidol and eticlopride in rats responding on the operant concurrent choice procedure ( farrar et al . , 2007 ; in contrast , msx-3 was relatively ineffective at reducing the effects of the d1 antagonist ecopipam ( sch 39166 ; worden et al . , 2009 ) on this task . although the non - selective adenosine antagonist caffeine was able to partially reverse the effects of haloperidol on the concurrent choice task , dpcpx , which is highly selective for the adenosine a1 receptor subtype , was ineffective ( salamone et al . , similar results were obtained with rats and mice responding on the t - maze barrier choice task . although msx-3 was able to reverse the effect of haloperidol on selection of the arm with the barrier , the a1 antagonists dpcpx and cpt were not ( correa et al . , 2009 ; mott et al . , 2009 ) . the results described above demonstrate that there is a relatively selective interaction between da d2 and adenosine a2a receptor subtypes ( table 1 ) . based upon anatomical studies , it appears that this is likely to be due to the pattern of cellular localization of adenosine a1 and a2a receptors in striatal areas , including the nucleus accumbens ( ferr , 1997 ) . adenosine a2a receptors are typically co - localized on striatal and accumbens enkephalin - positive medium spiny neurons with da d2 family receptors , and these receptors converge onto the same signal transduction pathways and show the capacity for forming heteromeric complexes ( fink et al . , 1992 ; ferr , 1997 ; svenningsson et al . , 1999 ; hillion et al . , thus , adenosine a2a receptor antagonists appear to be so effective in reversing the effort - related actions of d2 antagonists because of direct interactions between da d2 and adenosine a2a receptors located on the same neurons ( figure 2 ) . on the other hand , da d1 receptors are more likely to be co - localized with adenosine a1 receptors ( ferr , 1997 ) , which could help to explain why it is more difficult for adenosine a1 receptor antagonists to reverse the effects of d2 receptor blockade . interestingly , despite the fact that d1 and a1 receptors tend to be co - localized on the same neurons , the a1 antagonists dpcpx and cpt were unable to reverse the effects of the d1 antagonist ecopipam in rats responding on the concurrent choice operant procedure ( nunes et al . , 2009 ) . this suggests that a2a antagonists exert an overall greater effect than a1 antagonists on effort - related functions of nucleus accumbens . there was a mild increase in lever pressing in ecopipam - treated rats that received the a2a antagonist msx-3 , but no reversal of the chow intake effect of the d1 antagonist . data from pardo ( 2009 ) , unpublished masters thesis , university of jaume i. anatomical diagram depicting the pattern of da and adenosine receptor localization in nucleus accumbens . mgp , medial globus pallidus ; epn , entopeduncular nucleus ; s. nigra , substantia nigra ; vta , ventral tegmental area . research on the brain mechanisms involved in effort - related processes may lead to new ways of thinking about behavioral analysis and theory in behavioral economics . one of the contributions that behavioral neuroscience can make to behavioral theory is to use manipulations ( e.g. , drugs , lesions ) that dissociate complex behavioral processes into component parts ( salamone et al . , 2007 ) . in this regard , it is useful to consider that a given parameter that is generated from curve - fitting analyses , when viewed in terms of its biological characteristics , has many factors that contribute to it . a good example of this is the ed50 , which is used in pharmacology to provide a measure of the potency of a drug based upon dose - response analysis . empirically , the ed50 is the dose that produces an effect that is 50% of the maximal effect . although the ed50 is expressed as one number , that simplicity is deceptive because many biochemical factors contribute to it , including the affinity of a drug for a receptor , duration of action , drug metabolism , and penetration into the target tissue . a useful example of this principle from the behavioral neuroscience literature is the progressive ratio break point ; as discussed above , this measure also has many factors that can contribute to it . another case in which this point is important to consider is threshold measures used in intracranial self - stimulation studies . such measures often are viewed as providing rate - free indices of reinforcement value , nevertheless , they are influenced by lever pressing ratio requirements as well as the electrical current level ( fouriezos et al . , some research related to behavioral economics , reinforcer value , and the functions of da systems has used response - reinforcement matching methods ( e.g. , heyman and monaghan , 1987 ; aparicio , 2007 ) . matching equations have been employed to describe the results of studies with both conventional and concurrent vi schedules , and one of the parameters ( re ) can be used to represent reinforcement value ( e.g. , herrnstein , 1974 ; see equation below for single - lever conventional vi schedules , in which b represents response rate , r represents reinforcement density , k is the constant for maximal responding , and re represents the reinforcement level that generates 50% of maximum responding ) . however , used in this way , re does not selectively represent only the reinforcement value of food per se ; actually , it reflects the relative value of lever pressing for and consuming the food reinforcer compared to the reinforcing value of all other stimuli and responses available ( salamone et al . , 1997 ) . several factors can contribute to this composite measure , which is one of the reasons why other matching equations have been developed that account for deviations from matching by allowing for estimates of reinforcer sensitivity , as well as response preference or bias ( baum , 1974 ; williams , 1988 ; aparicio , 2001 ) . clearly , a drug or lesion manipulation could yield apparent effects on reinforcement value that actually reflect changes in response bias ( salamone , 1987 ; salamone et al . , 1997 ) . for these reasons , it may be useful to think more deeply about how terms such as value are used in neuroeconomics research . the aggregate reinforcement value of an instrumental activity ( e.g. , lever pressing for and consuming food ) should perhaps be viewed as a composite measure that includes both the reinforcing value of the reinforcer itself , plus any net value or costs associated with the instrumental response that is required to obtain the reinforcer . viewed in this way , the effects of dopaminergic manipulations on effort - related choice behavior could be described in terms of actions upon the response costs associated with the particular reinforcer , rather than the reinforcing value of the food stimulus itself . although the effects of haloperidol on bias may be minimal when two levers that are relatively similar are used ( e.g. , aparicio , 2007 ) , they may be much larger when very different responses are compared ( e.g. , lever pressing vs. sniffing ; lever pressing vs. unrestricted access to food ; barrier climbing vs. locomotion ) . future research will determine if measures of bias based upon the matching equations , or some other type of mathematical formulation , would be the best way to capture these drug effects quantitatively . in summary , da and adenosine in the nucleus accumbens interact to regulate effort - related functions . additional research has shown that a number of components of the cortico - striato - pallidal loop system also are involved ( walton et al . , 2006 ; floresco and ghods - sharifi , 2007 ; farrar et al . , 2008 ; mingote et al . , 2008 ; hauber and sommer , 2009 ) . disconnection studies have revealed that serial connections between basolateral amygdala , anterior cingulate cortex , nucleus accumbens , and ventral pallidum are involved in the exertion of effort and effort - related choice behavior ( floresco and ghods - sharifi , 2007 ; farrar et al . , 2008 ; mingote et al . , 2008 ; hauber and sommer , 2009 ) . within the last few years , there has been considerable progress in characterizing the functional anatomy underlying this important aspect of motivation and decision making . several transmitters across multiple brain regions are involved in effort - related functions , and researchers are only beginning to piece together the complex puzzle of all the potential brain systems that are involved . presently , the specific way in which each structure contributes to the overall function of the system is unclear . it is uncertain which brain areas are involved in the exertion of effort , or the perception of effort , vs. the actual decision making process itself . for example , it is possible that nucleus accumbens is involved in the actual decision making processes , but it also is possible that it is mainly involved in regulating energy output , or setting effort - related constraints or feedback that in turn influences decisions made at other levels in the system . if the latter is true , then it is possible that the decision making effects of drug or lesion manipulations of nucleus accumbens are an outcome reflecting the constraints that are set after compromised da function in accumbens , rather than a direct effect upon decision making processes per se . future research will be necessary to tease apart these distinct aspects of effort - related function . in addition to providing insights into aspects of animal behavior and natural motivation , research on effort - related processes also has clinical implications . within the last few years , there has been a greater emphasis upon effort - related functions involved in drug self - administration ( e.g. , vezina et al . , addicts will go to great lengths to obtain their preferred drug , overcoming numerous obstacles and constraints , both behavioral and economic . furthermore , addiction is characterized not only by a re - organization of the preference structure of the person , but also by a dramatic change in the allocation of behavioral resources toward the addictive substance ; there is a heightened emphasis upon drug seeking and drug taking , typically at the expense of other motivational activities . as well as being related to aspects of drug taking and addiction , research on behavioral activation and effort has implications for understanding the neural basis of psychiatric symptoms such as psychomotor slowing , anergia , fatigue and apathy , which are seen in depression as well as other psychiatric or neurological conditions ( salamone et al . , 2006 , 2007 ) . these motivational symptoms , which can have devastating behavioral manifestations ( stahl , 2002 ; . , 2005 ) , represent impairments in aspects of behavioral activation and effort that can lead to problems in the workplace , as well as limitations in terms of life function , interaction with the environment , and responsiveness to treatment . there is considerable overlap between the neural circuitry involved in effort - related functions in animals and the brain systems that have been implicated in psychomotor slowing and anergia in depression ( salamone et al . , 2006 ) . thus , research on effort - related behavioral processes , and their neural regulation , could have substantial impact on clinical research related to addiction , depression , and other disorders . 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 .

there are numerous problems with the hypothesis that brain dopamine ( da ) systems , particularly in the nucleus accumbens , directly mediate the rewarding or primary motivational characteristics of natural stimuli such as food . research and theory related to the functions of mesolimbic da are undergoing a substantial conceptual restructuring , with the traditional emphasis on hedonia and primary reward yielding to other concepts and lines of inquiry . the present review is focused upon the involvement of nucleus accumbens da in behavioral activation and effort - related processes . viewed from the framework of behavioral economics , the effects of accumbens da depletions and antagonism on food - reinforced behavior are highly dependent upon the work requirements of the instrumental task , and da depleted rats are more sensitive to increases in response costs ( i.e. , ratio requirements ) . moreover , interference with accumbens da transmission exerts a powerful influence over effort - related choice behavior . rats with accumbens da depletions or antagonism reallocate their instrumental behavior away from food - reinforced tasks that have high response requirements , and instead these rats select a less - effortful type of food - seeking behavior . nucleus accumbens da and adenosine interact in the regulation of effort - related functions , and other brain structures ( anterior cingulate cortex , amygdala , ventral pallidum ) also are involved . studies of the brain systems regulating effort - based processes may have implications for understanding drug abuse , as well as energy - related disorders such as psychomotor slowing , fatigue or anergia in depression and other neurological disorders .

Limitations of the Reward Hypothesis of Dopaminergic Function Behavioral Activation, Exertion of Effort, and Nucleus Accumbens DA Response Allocation, Effort-Related Choice Behavior, and Nucleus Accumbens DA Interactions between DA and Adenosine Behavioral Theory and Analyses: Further Evaluation of Effort-Related Processes Summary and Conclusions Conflict of Interest Statement

PMC4736226

osteoporosis is among the ten most important and prevalent chronic diseases [ 1 , 2 ] affecting approximately 200 million people worldwide . osteoporosis is characterized by a progressive decrease in bone mass and microarchitectural deterioration , resulting in increased bone fragility and ultimately an increased risk of fracture [ 46 ] . osteoporotic fractures , especially hip and vertebral fractures , are associated with significant morbidity and mortality in the elderly and also pose a significant burden on the health - care system . as the population worldwide continues to age , the prevalence of osteoporosis will continue to climb , with a growing need for effective therapies to prevent fractures [ 1 , 2 ] . bisphosphonates have been shown to effectively reduce the risk of vertebral and hip fractures in osteoporotic patients . unfortunately , several large studies have found that the majority of postmenopausal women stop bisphosphonate therapy within 1 year of beginning treatment [ 79 ] . reasons for poor persistence include conflicting patient beliefs , patient preferences , financial limitations , and previous adverse effects including rash , myalgia , nausea , and diarrhea [ 7 , 10 , 11 ] . impaired compliance and persistence to long - term bisphosphonate treatment can ultimately lead to an increased risk of fracture . denosumab is a fully human monoclonal antibody against rank ligand used in the treatment of osteoporosis . in osteoporosis , the thinning and increased porosity of cortical bone and disruption of cortical and trabecular architecture is the result of an imbalance in bone remodeling where the rate of bone resorption exceeds bone formation via changes in the rank / rank ligand pathway , an important regulator of osteoclast activity [ 1217 ] . in postmenopausal women , increases in rank ligand production have been associated with an increase in osteoclast activity and overall net bone resorption [ 18 , 19 ] . denosumab has an affinity and specificity for rank ligand and is responsible for inhibiting the proliferation and maturation of osteoclast precursors and functioning of mature osteoclasts . denosumab has been found to increase bone mineral density ( bmd ) annually and decrease bone turnover markers for at least six months after injection . clinical trials have demonstrated that denosumab treatment given subcutaneously once every six months is well tolerated and results in significant decreases in hip , nonvertebral , and vertebral fracture risk [ 21 , 22 ] . the objective of this study is to assess the effectiveness of denosumab therapy in the real world setting and the impact that noncompliance with the regular dosing regimen has on bmd ( measured at the lumbar spine [ ls ] and femoral neck [ fn ] ) compared to patients who receive their scheduled dosing regimen . all patients from the charlton centre for specialized treatments in hamilton , ontario , canada , on denosumab were screened for eligibility in the review . a retrospective cohort study was performed from august 2012 to december 2013 for all osteoporotic patients who received a minimum of two subcutaneous injections of denosumab since may 2010 . patients were eligible for this inclusion if they were above 50 years of age with or without prevalent fractures , received at least two subcutaneous injections of denosumab , and had at least two sequential bmd measurements performed one year apart . patients were excluded if they had a subsequent bmd measurement performed more than one year before or after the initial injection of denosumab , a change in bmd dxa - measurement machine on subsequent bmd measurements , or if they had a prevalent fracture in the femoral neck and/or lumbar spine during the review period , thus making the results incomparable . included patients were treatment nave ( 46 ) or have previously been treated with oral bisphosphonates [ risedronate ( 210 ) , etidronate ( 4 ) , and alendronate ( 95 ) ] , the intravenous bisphosphonate [ zoledronic acid ( 29 ) ] , raloxifene ( 29 ) , or teriparatide ( 23 ) . patients were allocated to one of three groups based on their compliance to the product monograph of injections given every 6 months : subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection . compliance was determined by calculating time elapsed between the initial and subsequent injection of denosumab . patients were considered to be compliant if the two injections of denosumab were 6 months + / 4 weeks apart . fractures were considered to be incident if they occurred during the time frame of this retrospective review . multivariable regression analyses were performed to evaluate the differences among the three prespecified groups in bmd change ( g / cm ) after one year of denosumab therapy at both ls and fn . the group with subsequent injection between five and seven months was considered the reference group . all multivariable regression analyses were adjusted for baseline bmd values , age of the patient , proton pump inhibitors ( yes / no ) , selective serotonin reuptake inhibitors ( yes / no ) , serotonin - norepinephrine reuptake inhibitors ( yes / no ) , tricyclic antidepressants ( yes / no ) , and the number of months between the first and second bmd measurements . all statistical analyses were performed using the sas / stat ( version 9.2 ; sas institute , cary , nc , usa ) software package running on windows xp professional . out of the 924 charts reviewed , 403 female ( 92.4% ) and 33 male ( 7.6% ) with osteoporosis and on denosumab therapy were eligible . the mean age and standard deviation ( sd ) of patients were 67.8 ( 10.7 ) years . the baseline bmd at the lumbar spine and femoral neck was 0.803 ( 0.128 ) and 0.622 ( 0.099 ) g / cm . a total of 54.13% of patients had a history of at least one nonvertebral fracture while 23.17% had a history of a vertebral fracture prior to denosumab therapy . upon completion of the analysis , patients were allocated to one of three groups based on their compliance to denosumab therapy ( subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection ) . twelve patients received a subsequent injection of denosumab less than 5 months , 365 patients between five and seven months , and 59 patients greater than seven months . baseline bmd results are presented for each of the defined groups along with the history of nonvertebral and vertebral fractures . moreover , the frequency of proton pump inhibitors , selective serotonin reuptake inhibitors , serotonin - norepinephrine reuptake inhibitors , and tricyclic antidepressants for each group are reported . descriptive statistics are provided along with the bmd values after one year of denosumab treatment . the mean duration and standard deviation ( sd ) of treatment from baseline injection were 3.03 months ( 1.18 ) , 6.05 months ( 0.38 ) , and 9.43 months ( 3.06 ) for patients who received a subsequent injection of denosumab less than 5 months , between 5 and 7 months , and greater than 7 months , respectively . to compare the change in bmd after one year of denosumab therapy , multivariable regressions were performed for the lumbar spine and femoral neck . using the group receiving a subsequent injection between 5 and 7 months as a reference , the groups receiving a subsequent injection results are provided in table 3 . the difference in lumbar spine bmd change ( 95% confidence interval ) after one year was 0.00035 g / cm ( 0.0124 , 0.0131 ) for patients receiving a subsequent injection greater than 7 months and 0.020 g / cm ( 0.0058 , 0.0479 ) for patients receiving a subsequent injection less than 5 months , as compared with the reference group receiving an injection between 5 and 7 months after their initial injection , respectively . the difference in femoral neck bmd change ( 95% confidence interval ) after one year was 0.0054 g / cm ( 0.0171 , 0.0063 ) for patients receiving a subsequent injection greater than 7 months and 0.0079 g / cm ( 0.0165 , 0.0324 ) for patients receiving a subsequent injection less than 5 months , as compared with the reference group receiving an injection between 5 and 7 months after their initial injection , respectively . the relationship between drug administration and change in bmd was not clinically or statistically significant ( p > 0.05 ) . the efficacy of a treatment depends on both the effectiveness of the therapy and the adherence to recommended dosage regimens . osteoporosis , like other chronic medical conditions , must have strategies to maintain patient compliance to therapy . noncompliance to long - term bisphosphonate or denosumab treatment can result in the decline of bmd values and ultimately result in an increased risk of fracture . it is a fully human monoclonal antibody that binds to the receptor activator for the rank ligand , leading to increased bone mineral density and decreased risk of fractures . while the pharmacology of denosumab is ideal , the success of the drug as therapy for osteoporosis hinges on patient adherence to established clinical guidelines this highlights the importance of identifying reasons for poor adherence and adapting clinical guidelines accordingly . this study explores the extent to which noncompliance to denosumab can affect the changes in bmd . in this retrospective cohort study , the multivariable regressions revealed that there were no statistically significant differences in the bmd values between patients who received a subsequent subcutaneous injection of denosumab less than 5 months , between 5 and 7 months , and greater than 7 months . however , our study does not suggest that patients and clinicians can extend the duration between subsequent injections of denosumab , since there is a steady decline in drug levels in circulation 6 months following administration , with complete elimination of the drug at the 9-month mark . this study does suggest that , only under unforeseen circumstances , when a patient is otherwise unable to receive a subsequent injection of denosumab in a timely manner , then a delay may be acceptable . otherwise , all patients should continue to receive their subsequent doses every 6 months , as per the product monograph . there are factors inherent to the nature and administration of prolia , as well as factors unique to this patient population , that may explain improved compliance rates . firstly , it must be acknowledged that denosumab can only be administered through subcutaneous injection by a healthcare professional . therefore , physicians have significant control over the adherence to denosumab , being able to establish a dosing course , directly monitor adherence to the treatment , and communicate the importance of adherence to patients when doses are missed . furthermore , there are factors that may explain the higher adherence rates in this study compared to other observational studies . one such reason is that the subjects of the study are recommended to use the provital program , a support program aimed at helping patients stay on their dosing schedule . patients receive a call from a nurse a week before their next scheduled injection as a reminder . additionally , at our academic center , a specialty pharmacy exists , which also reminds patients about their appointment one week in advance . together , these two factors have contributed to the high compliance rate for patients in this study and may be significantly higher than many community practices . the limited number of patients in the less than 5 months and greater than 7 month groups makes it difficult to draw definitive conclusions . we are also unable to comment on differences in fracture rates between the groups given the small number of events owing to the small size of the cohort and short follow - up duration . a longer follow - up duration in a larger cohort may in fact show a difference in bmd or even fracture rates in patients not compliant to therapy . furthermore , there is uncontrolled variability among patients in each group as additional medications can influence bmd , and this was not accounted for in the multivariable analysis . finally , the patients excluded due to a fracture in the femoral neck and lumbar spine during the review period along with a change in dxa machine could affect the results of the investigation . patient compliance is a simple and economic way for patients to benefit from drug efficacy . no matter how effective a drug is , without the proper adherence to the recommended dosing regimens , the patient may not fully benefit from the course of therapy . in summary , our study found that there were no statistically significant differences in bmd values between patients who received a subsequent injection of denosumab less than five months , between five and seven months , and greater than seven months . these findings indicate that clinicians may have some flexibility for administering denosumab if there are extenuating circumstances for which patients must delay their subcutaneous injection . however , the short follow - up duration and small sample size do not allow us to make such a conclusion . patients should continue to receive their denosumab therapy every six months if this is possible . a follow - up study with a larger sample size and longer follow - up duration will allow for a more in - depth characterization of this relationship . furthermore , our study suggests that utilizing different patient adherence programs either at a local or at a national level is an effective method to maintain a high level of patient compliance , thus ultimately improving the real - world effectiveness of this therapy .

although denosumab ( prolia ) has been shown to be a safe and efficacious therapy for osteoporotic patients in numerous clinical trials , few studies have determined its effectiveness in real world clinical practice . a retrospective review of patients prescribed prolia assessing the impact that noncompliance from the regular dosing regimen of six months for denosumab has on bone mineral density ( bmd ) was performed . 924 patient records were reviewed between august 2012 and september 2013 with 436 patients meeting the eligibility criteria . patients were divided into three groups : subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection . a multivariable regression analysis was conducted comparing the differences among the three prespecified groups in bmd change ( g / cm2 ) after one year of denosumab therapy at both the lumbar spine ( ls ) and femoral neck ( fn ) . the differences in ls and fn bmd have shown that the relationship between the timing of drug administration in these three groups and change in bmd over 1 year was not clinically or statistically significant ( p > 0.05 ) . a follow - up study with a larger sample size and longer follow - up duration is required to further characterize this relationship .

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

PMC4006140

reflecting in 2008 on aids in south africa during the final phase of the anti - apartheid struggle and the transition to democracy , edwin cameron , aids activist and justice of the constitutional court , observed that the transition period brought to the fore the intricate relationships that existed between illnesses , ideologies and ( intimate ) identities , resulting , in the unavoidable politicisation of homosexuality. indeed , as the oral testimonies in this article reveal , this politicisation of homosexuality often affected both public and private spheres , and , for two health care professionals on whose narratives this article focuses , their own sexual identity and personal and professional relationships with ( gay ) men dying of aids mediated the urgency of responding to aids . in july 1983 an article by five medical professionals reporting on the first officially documented aids cases in south africa appeared in the south african medical journal . while the two cases were not named in the journal article the initial responses by the general public to the first documented cases were primarily characterised by fear and stigma and reflected prejudice , homophobia and moral panic that were deeply rooted in the dominant conservative morality that pervaded south africa in the 1980s . white , flight stewards , not as two people who had died and would be mourned . ralph kretzen died on 26 august 1982 and pieter danil ( charles ) steyn died on 1 january 1983 , and we know very little about their personal histories . we do however know that the association of the new syndrome with an undefined idea of homosexuality replicated the initial link between homosexuality and aids that had been made by the centers for disease control in atlanta , usa ( cdc ) in june 1981 . white homosexual men by elites both medical professionals guiding and directing early aids research and advising government departments and ministers ; and people or organisations in positions of political power including government health departments and ministers , mps and political parties shaped and influenced responses , research , and narratives about aids in these and other spheres . the resultant moral outrage expressed in parliament , from pulpits and in conservative media provoked by this initial association did not allow for reflective discussion about sex and sexuality , but rather positioned homosexuality and homosexuals negatively in broader public spheres and , in so doing , politicised homosexuality . moreover , authors of the hegemonic scientific narratives whether in medical journals , health care organisations or parliament often used the term homosexuality only to refer to undefined sexual contact between white men in a narrative which was preoccupied with an imagined middle - class , white , male , sexuality that was the primary referent against which normative and non - normative behaviours were judged . drawing on narratives about aids primarily from the uk and the usa , influential medical practitioners and scientists , and politicians responsible for addressing aids in south africa focused attention on this understanding of homosexuality and in doing so rendered invisible heterosexuals , lesbians , bisexuals , transgendered and intersexed people , and linked male homosexuality only to whiteness . in the 1980s , being gay in south africa was a process mediated by apartheid legislation and questions of morality , race , sex , gender and class amplified by the socio - political and economic turmoil of the time . the oppressive , conservative , moral climate of south africa did not allow sexuality organisations or lgbti communities to flourish , especially not in a context where ( male ) homosexuality or more specifically , sodomy was illegal . while gay organisations and communities in the usa and uk had fought for , and built on , social changes in the 1960s and 1970s , in south africa apartheid s effect on sexuality politics meant that corresponding socio - political gains had not been made . despite this , some gay activists maintained international contacts with gay organisations and aids education organisations , resulting in a flow of information and exchange of ideas between south africa and countries with more established and politically active gay communities . gay groups in south africa were not necessarily concerned with addressing sexism , racism , homophobia or sexuality politics . agendas ranged from providing safe social spaces to gather and talk , directing legal challenges to homophobic legislation and laws that discriminated against people because of their hiv status , to aligning the struggle for sexuality rights within broader anti - apartheid and international human rights campaigns . as mark gevisser and edwin cameron noted in one of the earliest and most influential works on the histories of sexualities in south africa the two factors that exemplify homosexual experiences in south africa are the history of division and resistance and the demographic divergence our country reflects. they note that from the developed world , we inherit notions of sexual freedom and gay subculture ; from the developing world we gain the imperatives of struggle , resistance , and social transformation. importantly , they add , there is no single , essential what has passed for the gay experience has often been that of white , middle - class urban men. at the outset it was predominantly communities of urban , middle - class , white men with multiple gay identities who came together to organise around aids and it is this this article is drawn from a larger thesis project which explored how narratives about aids were constructed by elites in medico - scientific and political communities , and examined practical responses to aids by governments , the african national congress ( anc ) liberation movement in exile and progressive health movements . the thesis revealed the complex relationships between these communities , tracked the changing hegemonic aids narratives , and revealed the constructions of morality , it also showed how the relationships and narratives shaped and influenced practical responses to aids . most of the sources used for this project were written sources which occasionally provided glimpses of the effects of narratives and practice on personal experiences , but none provided insights into the specific experiences of gay health care professionals . the relationships ( nationally and internationally ) between medico - scientific and political elites could be drawn out by combining written and oral sources , but the personal accounts of people at the nexus of the polemic and politics that combined to influence practical responses were hidden . while accessible published work and archived sources provide insights into the experiences of progressive figures and sexuality activists , the stories of gay health care professionals living and working in a predominantly homophobic society and medical community at the start of an epidemic linked explicitly to white gay men while important , were not readily available . of the twenty oral interviews undertaken with medical professionals , political figures , activists and academics for the thesis , only two were with health care professionals who had openly declared their sexual orientation during the time period under discussion . their stories offered unique perspectives and a way to record the effects of politics and polemics on people s lived experiences . the article uses oral interviews to examine the experiences of two gay health care professionals addressing aids during the early years of the epidemic . it follows the example set by gerald oppenheimer and ronald bayer of recording the experiences of physicians and nurses in their book in shattered dreams : an oral history of the south african aids epidemic . their book incorporates interviews with gay doctors , including dennis sifris and steve miller , and , in recording their oral histories , begins to address how some gay health care professionals experienced the epidemic . this article expands on the oral testimony of sifris that appears in shattered dreams and contributes the oral testimony of pierre brouard . the face - to - face interviews with sifris and brouard were carried out in single sessions lasting from one and a half to two hours . they were recorded in 2008 using a digital voice recorder and verbal permission was sought and obtained from interviewees to record the interviews . while specific questions relevant to the professional experiences of each interviewee were devised , all interviews were predominantly open - ended and unstructured . during the interview notes were taken by the author , and the interviews were later transcribed . the purpose of the interviews and the nature of the research were also explained to each interviewee and they confirmed the accuracy and representation of their statements in the original chapter from which this article is drawn . sifris is a medical doctor who worked with one of the first south african medical researchers investigating aids , ruben sher , and helped to establish the aids clinic at the large public and academic johannesburg general hospital ( also widely known as joburg gen ) and has been involved in aids education campaigns since the early 1980s . a clinical psychologist , brouard was involved in some of the earliest counselling and support initiatives for people who were hiv - positive at a variety of venues , including the clinic at joburg gen , at atic ( the first aids training and information centre ) that opened at the south african institute for medical research ( saimr ) in 1987 , and at the city of johannesburg s esselen street sexually transmitted infection ( sti ) clinic in hillbrow . both sifris and brouard moved between and through the spheres of medical science , activism and sexual identity while attempting to address aids . while sifris and brouard are both professional , white , middle - class , urban , gay men , whose paths overlapped and who shared some similarities in their experiences of the early days of the aids epidemic , their professions , personalities and individual contexts resulted in different experiences . these early experiences laid the foundations for two men who , now both in their fifties , have spent almost three decades of their professional lives involved in aids health care and education . their personal reflections provide vignettes that contribute to understanding how narratives about , and responses to aids ( and sexual orientation ) , affected individuals . their stories provide insight into personal experiences of addressing aids in the early years of the epidemic when researchers and decision - makers were constructing a hegemonic aids narrative that was fundamentally conservative and homophobic . their stories are micro - narratives that act as a counterpoint to the meta - narratives about aids featured elsewhere and act as a reminder of the emotional and psychological realities of an epidemic that continues to take lives , thus contrasting the unemotional writing about aids in medical journals , the politically charged language in parliament or the official correspondence of political organisations . the article presents first sifris and then brouard s recollections of the early years of the epidemic and some of their reflections on sexuality , health care provision and aids after almost three decades as health care practitioners . sifris and sher began working together in the late 1970s after sher had heard that sifris had a large gay practice and asked if he would collect serum samples for a hepatitis b study . after the first official aids death in south africa in 1982 they met to discuss further collaboration . sher was based at the saimr and sifris had his own practice in the city centre . while these collaborations would result in a long - term professional relationship , sifris s experience as a physician involved in aids research was mediated by how colleagues and government officials responded to his sexual orientation as a gay man . soon after the first recognised aids deaths sifris sought to address aids among the gay community and contacted other gay doctors ( an anaesthetist , a psychiatrist , and a neurosurgeon ) to discuss what could be done . he recalled that , as a starting point in 1983 , they put the word out in the gay community that there was this disease which nobody really knew about , while calling for volunteers to give blood samples to be sent to sher at the saimr . sher and sifris compiled a questionnaire that followed research in the usa and uk and included questions about the number of sexual partners volunteers had , their medical history and current state of health , and their use of recreational drugs . to facilitate the work sifris extended his practice hours to include saturday mornings : word got out and we actually got about 700800 people coming to my office over a period of about four or five months and we pulled the bloods and we did nt quite know what we were looking for . we did a full blood count , we did an std [ sexually transmitted disease ] profile and we stored bloods to see if there was anything we could find . sifris kept a list of names that linked volunteers the majority of whom came from south africa but also from countries such as lesotho , swaziland , malawi , zambia and zaire to their samples , but there was little that could be done medically to assist the increasing number of people who in the mid-1980s started to become sick and die . the lack of an effective medical response did not necessarily make the medical community in johannesburg more interested in learning about other , non - medical , engagements and responses , however , as is evident from sifris s account of a visit to johannesburg by glen margo in 1985 . margo , who was originally from south africa , was involved in aids education in san francisco . he contacted the gay association of south africa ( gasa ) as he wanted to deliver talks about aids in johannesburg . sifris , a gasa member , agreed to arrange the lectures for margo and organised one at the department of medicine at joburg gen . sifris recalls : there were all these cardiologists and respiratory physicians and this hippy san francisco guy came along with handmade sandals and ringlets in his hair and told them about this funny [ sic ] disease that was affecting gay men that they should all be aware of and he was almost laughed off as a joke . it was a wee bit embarrassing but that was the context of the medical community . it is unsurprising that medical elites in johannesburg did not pay serious attention to a hippy social worker talking about something that apparently only affected gays. however , as sifris was to discover , even being a medical doctor defined as white and male , did not guarantee an attentive audience if one did not also despite the apparent lack of interest , sifris and sher persuaded hospital authorities to allow them to establish an aids clinic at joburg gen in 1983 . by 1985 sifris was managing the daily running of the clinic and sher moved between the clinic and the saimr . they decided to bring specialists from various fields , including respiratory illnesses and dermatology , into the clinic . despite the dominant medical and popular association of aids with gay men , sifris recalls that the first person they saw as an outpatient was an hiv - positive ( then htlv - iii positive ) womyn : she came in and we sat there , eight of us doctors this poor woman came in , i still remember her sitting there and i think she was intimidated by all these doctors sitting there in their white coats . during the mid-1980s there was an increase in the number of patients attending the clinic and being admitted to the hospital with aids - related complex . the guys we had seen two or three years ago , who were friends of mine . a lot of them were saa ( south african airways ) people who were now getting sick and dying with this disease , and this was the time in the hospital where they were doing barrier nursing. in 1985 the advisory group on aids ( aga ) was established by the department of health to advise the conservative and ostensibly christian government led by the nationalist party on aids policy and sher was invited to become a member . however , a medical background , an interest in aids , and experience researching and monitoring the epidemic did not guarantee membership of the group , as sifris discovered . high - risk group and if we have a gay man on the ( aga ) we have to have a prostitute , we have to have a haitian , and we have to have a black . this decision made sifris determined to just go ahead and do my own thing so he called on medical professionals such as steve miller , des martin and clive evian ( all of whom would become key aids researchers ) to work with him at joburg gen ; and , in conjunction with gasa , established an alternative aids group . sifris and other concerned professionals and volunteers formed the aids action group in 1985 , with the intention of providing psychological , social and concrete support services for people with aids and the aids - related complex. the group shared information about aids with interested parties and undertook education campaigns amongst sections of johannesburg s gay community . on trips to the usa and uk , sifris gathered literature and came home with bundles of it. the aids action group decided to distribute information leaflets and safe - sex cards in gay clubs in johannesburg and approached club owners for support . in one club the group set up a little ( information ) thing outside and people walked past and we said , hey , we are giving out safe sex cards , and everyone kept away from us!. on one occasion sifris asked if the music in the club could be stopped at midnight so that he could make an announcement : so they stopped the music and i came out onto the dance floor with a microphone and i said , listen this is not a police raid or anything , just relax , nothing is happening here , i just want to make everybody aware that there is a problem called htlv - iii which is affecting gays in america and all over the world and everyone just moved away from me . i stood there in the middle of the dance floor trying to say , now listen people it is up to you to volunteer , to create awareness , to get the people aware , to give out pamphlets and please we have a desk in front , please come and sign . there was no interest , just apathy like we were ruining the party . the pre - emptive declaration in sifris s statement that his announcement was not part of a police raid speaks to the constant stress , suspicion and fear of arrest that was associated with being gay in south africa . the majority of people in south africa faced the constant threat of police and military violence by virtue of being declared black in the apartheid state , and whiteness usually afforded privilege and protection to people declared white gay men maintained significant privilege in most socio - economic and political spheres , they did not maintain sufficient privilege to remain exempt from raids because of their sexual orientation . black gay men maintained some privileges by virtue of being male in a patriarchal system , but faced persecution for being both black and gay . the unwillingness of people to listen or respond to a potential health concern mirrors responses in most communities when faced with frightening news . it is not possible to determine how effective these interventions ultimately were , nor how much impact the distributed literature may have had on awareness raising . nonetheless , it is significant that at a time when official responses to aids failed to cater for a group of people identified and constructed as high - risk , alternative , non - official efforts were made to raise awareness and provide support . the group decided to focus their energy on talks , engaging with the press and fundraising for aids - related work . sifris and sher were already giving talks for audiences ranging from medical professionals to staff and students at schools , and to a group of jewish single people over the age of 40. sifris gave talks to gay groups around the country and spoke at the official opening of anti - apartheid activist simon nkoli s township aids project ( tap ) in soweto in 1990 . sifris both received calls from journalists seeking his expert medical opinion , and challenged journalists and editors about homophobic or inaccurate content in articles . he felt frustrated that many journalists were more concerned with writing sensationalist articles than with conveying educational information . these challenges extended to his engagements with the editor of exit the longest running gay magazine in south africa . exit ( originally known as link / skakel ) started in the early 1980s as gasa s newsletter . it was renamed exit in 1985 and published its 200th edition in 2006 when it cast a reflective eye on some aspects of its reporting on aids . sifris criticised moolman for running a sensationalist article about a person apparently getting aids from their dentist , rather than featuring comprehensive aids education information . botha reflected on the early years of exit : during the 1980s a new gay disease was discovered in america and eventually found its way here . although we started publishing safe sex guides and lists of symptoms , very little was known about this mysterious killer affecting at that stage mostly gay men . i am still haunted by the front - page headline that said something to the effect aids [ sic ] scare overrated . an exclusive black - tie event and art auction launched the aids action group s fundraising initiative . the event raised close to r10,000 for what was referred to as the aids action fund. another shaft 8 included information and education stalls and provided an opportunity to remember those who had died . i bought those jewish yahrzeit candles , and i laid out a table with one candle for each person who had died of aids . the shaft 8 event raised a significant amount of money and brought all the gay communities together a gay christian community , a support group called outreach , a gay motorcycle group , and gay lesbians. while aids may have connected these communities , it hardly united them . sifris noted that there was all sorts of internal politics and stress in the organisation . black gays or lesbians and often a lack of gender sensitivity or awareness of racial prejudice . aids may have created spaces for collaboration and co - operation , but these spaces were not necessarily all - inclusive or self - reflexive ; and addressing aids amongst a certain group of gay men who experienced discrimination did not amount to challenging the economic , social or racial position of that group of men , nor other forms of discrimination . in order to increase fundraising opportunities and more effectively manage funds , the group needed an official fundraising number . it prepared a proposal outlining its plans to print pamphlets and undertake a countrywide outreach programme and had it endorsed by medical professionals , a professional in the finance sector , and people in the marketing and advertising sectors . the group presented the proposal to a representative from the department of health and asked for a grant of r20,000 . however , because the organisations associated with the project were sexuality organisations , the fundraising number and the grant request were denied . lombard apparently listened carefully to the presentation and then said , you know , aids is not a problem in this country , tb is a problem in the country . besides which homosexuality is illegal , and we do nt have homosexuals , so we do nt have aids. the department of health s rejection of the funding request meant the group was unable to initiate extensive aids education programmes based on those of the san francisco aids foundation , the gay men s health crisis , london lighthouse , and the terence higgins trust , whose pamphlets and programmes were the template for their own local campaigns . thus , the content , politics and practice of aids education programmes developed by , and for , male gay communities in the usa and uk , shaped and influenced responses amongst some sectors of the male gay community in south africa . for sifris the frustration at not being able to implement ready - made education programmes that could save lives was compounded by the denial of the very existence of homosexual men . invisible in official apartheid discourse . had the grant had been successful , there is no guarantee that imported education campaigns would have been appropriate or effective in the south african context . however , in the absence of official responses or help for gay communities , they would at least have bolstered existing education efforts by gay organisations to prevent new infections . access to medical scientists and research organisations in the uk and usa shaped both sifris s and sher s professional engagement with aids , and contact with gay activists in these countries deeply influenced sifris s personal and political outlook . sifris remembered going with sher to a research laboratory in bethesda , maryland , and being drag[ged ] into the halls of the walter reed hospital in washington or to some top - level research conference. in 1985 sifris went on ward rounds at the san francisco general hospital and at the aids unit in london , experiences that really opened ( his ) eyes to the realities of the epidemic , the strategies being employed to address aids , and the remarkable solidarity and politicisation occurring around aids . sifris had his most profound experiences at the points of intersection between personal , professional and political spheres . in the mid-1980s he attended an aids conference in washington where members of the aids coalition to unleash power ( act - up ) protested over the lack of action in addressing aids . the conference changed sifris s understanding of how people could organise politically to challenge medical and discursive responses to aids : larry kramer was there from act - up and he was wearing a concentration camp uniform . at the time , and i thought this was very strange , he would shout and scream and carry on . they had a scientific session , and they had one or two sessions of people with aids and it was very funny because people stood up and said , i am so and so , and i have got hiv and everybody applauded , and everybody clapped for support i remember that to this day and i really learnt a hell of a lot [ particularly ] that there was a lot of mobilisation . during trips to london , new york and san francisco , sifris took part in aids memorials that made a lasting impression on him : i was in castro street and they had it at castro s station there , a candlelight memorial for rock hudson [ it was ] very moving . they unfurled the quilt and there was nt a dry eye in the house , everybody was crying . contacts with gay organisations and ( gay ) doctors working on aids in the usa and uk were important in providing models of engagement , education , commemoration and grieving . for sifris and others like miller , who participated in conferences and marches overseas , these were spaces that demonstrated the potential for public protest , and subsequently influenced education and mobilisation efforts among sectors of the gay community in south africa . on a trip to san francisco a group called inspired by the experience of seeing doctors organise around political issues relating to the realisation of rights , sifris decided to start a gay doctors group in johannesburg that numbered about twenty clinicians and met monthly at the hiv clinic . brought together by an interest in hiv , the primary aim was just to keep everybody up to date with hiv news , but eventually it just sort of fell to pieces because nobody was interested in coming anymore because there was not much more we could do. it was only in the late 1990s that sifris , des martin and others decided to establish a new association , the southern african hiv clinicians association . this was open to all clinicians involved in hiv work and included a number of gay doctors . martin was appointed the first chair of the organisation and the first meetings were held at the national institute of virology . from small beginnings the organisation continued to grow and recently claimed to be the largest professional hiv interest group in the world , with over 12,500 members. the mid-1980s were particularly turbulent times in south africa , both for anti - apartheid and sexuality activists . in 1986 gasa collapsed , but its demise , gevisser notes , resulted in two new forms of gay political activity. one found the notion of single - issue gay politics to be untenable in south africa and sought to align sexuality struggles with broader human rights and anti - apartheid struggles ; the second insisted on fighting specifically for the reform of laws that discriminated against homosexuals. in 1985 president p.w . interracial ( hetero)sex which had been made illegal by the immorality act , 1927 ( amended in 1950 ) . the immorality act also covered other sexual offences such as prostitution and sodomy between men and botha tasked an ad hoc committee with investigating these sexual offences , including homosexuality . this committee proposed : a strengthening of legal prohibitions against homosexuals ; that greater effort should be made to determine how to appropriately rehabilitate or punish homosexuals ; and that a strategy should be formulated to express society s the collapse of gasa and the suggested law reforms had a direct impact on the aids action group and on organisations that had come together to raise money for aids programmes . key gay activists established a national law reform fund ( nlrf ) to fund challenges to the proposed legislation . the organisers of shaft 8 were asked to consider reallocating the money they had raised to support the nlrf . numerous meetings ensued and sifris recalls that it was decided among the committee that the law reform fund was now more of an emergency than aids , so all the money was given over to the law reform committee . we thought we could always just raise more money for aids. determining which threat aids or the further criminalisation of homosexuality was the more pressing could not have been an easy task , but the historical coincidence of the two issues coming into the spotlight at the same time detracted from plans to address aids . as it turned out , the investigations into homosexuality and law reform never got underway and the nlrf was transformed into a charitable trust . the late 1980s were also difficult times for sifris and others working at the hiv clinic at joburg gen , primarily because of the inherent homophobia and stigma associated with aids . although sifris and sher had convinced hospital authorities to provide space for the hiv clinic it was not easily accessible to clients or visible , and both the name of the clinic , and who attended it , became contentious points . immune disorders clinic but sifris was adamant that after the identification and naming of the hi virus it was important for the clinic to be known as the hiv clinic to reduce the secrecy and fear associated with hiv . after several years of running the clinic on an honorary basis , sifris was formally employed as its director in 1985 . despite vocal opposition , one of the first things sifris did as director was to rename the clinic the hiv clinic. it retained that name until sifris s resignation in 1990 , when it was renamed the immunity clinic. the hegemonic aids narrative of the time constructed gay men with aids as guilty offenders who got aids because of their sexual orientation and deviant lifestyles. conversely , haemophiliacs infected through contaminated blood products were constructed as innocent victims. so strong was the stigma and homophobia that a separate clinic was established for haemophiliacs and others . sifris recalled the haemophiliacs had their own clinic because they did nt want to come to the gay clinic they did nt want to come to the hiv clinic , so they had their own clinic . we never had anything to do with them they did nt want anything to do with us because we were gays . things came to a head for sifris and miller because of a devastating interplay between discrimination , drug pricing and public health care provision . in 1986 one of the first antiretroviral ( arv ) drugs , azido - thymidine ( azt ) also known as zidovudine , came onto the market in the usa . as the first significant medical intervention for aids it was a breakthrough , but it was staggeringly expensive , costing approximately $ 10,000 a year in the us at the time . the number of south africans who could afford it when it was first distributed to the country in 1987 was minimal . while paying for the drug was completely impossible for most patients attending the hiv clinic , it did at least provide hope because it prolonged the life of aids patients . the hospital eventually purchased azt but only wanted to prescribe it to people with haemophilia . the hospital said we will give azt to the haemophiliacs but we wo nt give it to the gay people because it is their own fault and haemophiliacs are innocent victims. livid at this blatant discrimination , sifris contacted reporters and informed them of the hospital s policy , resulting in the story being featured in newspapers . shortly afterwards the superintendent of the hospital informed sifris that they had been called to a meeting with the secretary of health in pretoria . at this meeting sifris was reminded that as a member of staff of a public hospital he was not allowed to make statements to the media about hospital policy , and was duly admonished by the health secretary . i do nt want anything more to do with the hospital. after sifris s resignation miller took over as head of the clinic , but was not in that position for long as he was told to leave by hospital authorities after supporting a patient s legal claim against the hospital . a final example of the ongoing frustrations experienced by miller and sifris related again to the aga . miller , like sifris , had been excluded from joining the group because of his sexual orientation and his designation as a member of a high - risk group. nonetheless sifris made a second attempt to join the group when it decided to expand its membership . frustrated that the government had sewed together a bunch of old fogies who did nt do anything , sifris thought that membership of the group might afford him the chance to facilitate change or action . in his application sifris argued that as head of the largest hiv clinic in the country he had considerable experience in addressing aids , which was augmented by the lectures he had given , the travelling he had done both regionally and internationally in connection with aids work , and the conferences he had attended . own coffee cup in case you are worried that a gay man is going to infect you. sifris s application was rejected , and the group appointed someone who had never seen an hiv patient in his life. while sifris has never explicitly commented in interviews on the racial dynamics within gasa or any of the aids - related organisations that he was involved in , he did discuss the problems he and sher faced at the clinic in the late 1980s getting black doctors involved. they organised a world aids day event and wanted to invite black doctors to attend but were unsuccessful : we searched and searched and searched and searched and could nt find any black doctors to come out. they approached a prominent black doctor in soweto with known links to the anc but were turned down . black doctors were nt interested because it was not a black problem , it was a gay disease . it was a gay disease and nobody worried about the gays. because aids was not being identified among black patients , and places like the hiv clinic were either not legally integrated or welcoming of black patients , it was not seen as important amongst black doctors may not have wanted to participate in the events , not least of which were the other health concerns exacerbated by apartheid which at the time were regarded as being more pressing . in the early days of the epidemic , sifris recounts , it was difficult coping with the deaths , government and community inaction , and general homophobia . burnout. added to the stresses and strains of battling government and hospital officials , he experienced a personal loss that caused him to withdraw from professional and public spheres . sifris met an hiv - positive man at an aids conference in stockholm and the two fell in love and lived together for five years . as his partner s health began deteriorating , the couple travelled to sweden for treatment twice a year . after his partner suffered a mini - stroke that left him paralysed , the couple decided that he needed to return to sweden to see his doctors and be with his family . as he recalled some twenty years later , while they were in transit at brussels airport , sifris s partner suffered a major stroke and a massive brain haemorrhage : as we were getting onto the plane he collapsed and died so , i sort of withdrew at that time a little bit , but ( since then ) i ve slowly gotten back. in 2013 , sifris has semi - retired but continues to deliver lectures on aids , acts as a medical consultant to a disease management company , and contributes health information to exit and other media fora . having seen so many people die of aids over the course of three decades , sifris expressed disappointment in the interview that successive post-1994 democratically - elected south african governments have put up so many barriers to procuring and distributing life - saving drugs . his recollections reveal the inherent and blatant homophobia that gay men experienced , regardless of their race or class . frustration at official inaction around aids and the reality of friends , colleagues and lovers dying , provided the impetus for sifris and others to mobilise in the early 1980s , soon after the seriousness of the epidemic was becoming apparent to them . the importance of international connections and experiences is evident in sifris s personal politicisation around aids . as a doctor involved in the first hiv clinic in south africa , his professional status and resources allowed him to travel to international conferences and connect with other doctors and with gay organisations addressing aids , and to bring important information and ideas back to sectors of the gay community in south africa . while sifris and brouard were both involved in addressing aids as health care practitioners , they occupied different positions in the health care facilities they worked in , and their experiences correspond and contrast each other , as will be shown below . pierre brouard is a clinical psychologist and currently the deputy director of the centre for the study of aids at the university of pretoria . brouard s recollections on the early days of the aids epidemic are noticeable for their reflexive and contemplative nature . his discussions about the epidemic , and his involvement in various counselling and support positions , are imbued with a critical awareness of the broader socio - political landscape of south africa during the 1980s and early 1990s . brouard came out in the late 1970s and believes that coming out before aids was on the scene shaped his sexual identity in a different way to people who were coming out during the epidemic and helped form his perspective on the fears and considerations that framed peoples sexual lives . reflecting on the late 1970s , brouard view was that one of the key issues that concerned many ( white ) gay men at the time was that homosexuality was illegal . under the immorality act and subsequent amendments , there was the continued threat of being arrested , harassed , hounded , shamed or imprisoned . legal concerns aside , from a personal physical health perspective the most immediate health concerns were stis such as herpes , syphilis or gonorrhoea , all of which were treatable . while fear of the law remained , in the early 1980s physical health priorities were to be radically altered as the first murmurings that something was amiss started to surface with news often coming from middle class , white , gay men who travelled internationally , and from people working in the airline industry . new disease that was framed entirely as a gay disease , complete with the acronym grid ( gay - related immune deficiency ) . central to both brouard s and sifris s narratives , is the importance of international travel and networks for transmitting information about aids . brouard remembers that people who travelled internationally brought back newspapers , pamphlets and magazines or knew or corresponded with people overseas. in the early 1980s brouard and his partner at the time noticed that one of their friends , who had moved to the uk but who regularly visited south africa , was getting increasingly ill . they speculated as to whether their friend had aids , but he never told them and they never asked . the friend subsequently died , ensuring that from early on in the epidemic , brouard was acquainted with the reality of aids - related deaths . brouard saw that aids - related illness and death mobilised people both as individuals and as members of organisations : those early responses were really located in the gay community . people mobilised around friends , tried to get support groups going tried to understand the treatment options available , and set up informal networks . the latter was the director of gasa-6010 , was hiv - positive and died of aids - related illnesses . brouard commented on different political outlooks between organisations : gasa was the main organisation for more middle - class , mainstream , men and women and was apolitical. he recalls that gay organisations that were more left - orientated appeared to be more active in cape town , than in johannesburg . regardless of geographic location however , brouard s retrospective sense of the situation was that middle of the road , white , gay men were really just trying to hold it all together in the absence of really much available [ sic]. reflecting on the arrival of a serious new health concern brouard suggested that aside from the emotional impact of the deaths of friends , family members , lovers and partners , the association of aids with homosexuality required people to address their sexuality without internalising aids as something that was inherently related to being gay . at the same time as challenging ideas that being gay was wrong , evil or sick , gay men were faced with a health crisis that was portrayed as being intrinsically linked to what was apparently gay sexuality. gay men thus had to address both internalised homophobia and societal perceptions of homosexuality being evil or sick , in the context of a new fatal disease that many medical professionals , politicians , media and religious groups directly linked to sexuality . the ramifications of aids affected both individual and community identity formation , as brouard recalls : just to try and retain a sense of healthy sexuality and healthy identities when aids spoils your identity and spoils your sexuality that was a real challenge of those times . i remember having feelings like the community is self - destructing , there s just so much stuff going on , and people just are nt caring for themselves or each other . it was an incredibly distressing and painful time to live through and people were just getting sick and aids struck the old and the young , and the beautiful and the ugly , the fat and the thin it was a great equaliser . my sense of the time , perhaps it was a psychological sense , was that some people who were the beautiful glittering stars of the gay firmament , were deeply shamed and humiliated and distressed about being hiv positive because it reduced them to their physicality and was ultimately a reminder that we are physical creatures and we get ill and we die and nobody is immune from that no matter how beautiful or glamorous you or your lifestyle are . it was really an incredibly fraught time to be gay and i do nt know that , historically , if we look back , how that whole time affected the psyche of the gay community . to what extent it damaged it , or created a particular response or a feeling of what it means to be gay in the twentieth century . research into how aids , the illegality of homosexuality , socially - sanctioned homophobia , apartheid ideologies and race shaped gay male identity in south africa historically is another area of research that requires further exploration . the hegemonic narratives around men and aids created by medico - scientific and political elites in south africa and elsewhere in the early 1980s and not challenged significantly until the mid-1990s strongly suggested that it was not types of sexual practices such as unprotected anal or oral sex , or higher - risk behaviours that needed to be examined , but rather something the focus in medical journals and parliamentary discussions , throughout the 1980s and mid - way into the 1990s , was thus not on sexual acts and practices that could be performed by sexually active people regardless of their sexual orientation , but rather on constructed aids avatars of deviant gays , dirty prostitutes , infected foreigners , and sexually rapacious this prevented serious attention from political and medico - scientific elites falling on male sexuality more broadly and assumed a normative , healthy , while these avatars were used to construct a hegemonic aids narrative , this does not mean that the narratives were not contested , challenged and reconfigured or rejected by some health care professionals such as brouard . conflated identity with risk and assumed that something about the gay condition ( or ) the gay being presupposed vulnerability to aids to hiv. this fitted in with broader public health paradigms of the time that were modelled on popular understandings of sexuality , that did not sufficiently interrogate the relationships between sexual practice , sexual identity and basic biology . in this paradigm it was not the fluid that was the problem , it was who you were that posed the risk. this understanding of sexuality and confusion over sexual practice and sexual identity resulted in a lot of mythologizing even in the medical sphere. brouard recalled counselling a heterosexual couple in the esselen street clinic where the man was concerned about their risk of hiv infection because they practised anal sex . further questioning revealed that they were both hiv - negative and in a committed monogamous relationship . brouard explained that , in light of this , they were not likely to be at risk of hiv as it was not the practice of anal sex per se , that was the primary risk , but rather bodily fluids containing the hi virus . these and other experiences left brouard with a sense that , for many people , understandings of aids and hiv transmission , and sexuality more broadly , were very mystifying and mystified. brouard started counselling people in the early to mid-1980s through his involvement with gab ( gay advice bureau ) a counselling service providing support for gay people or people exploring their sexuality . at that time gab was not dealing with many hiv - positive people but one of brouard s colleagues was diagnosed with aids so gab established an informal support system for him and his partner . after his death , gab launched an unsuccessful support group for people living with hiv . while completing his master s degree brouard volunteered at the aids centre at the saimr , which opened to the public in january 1988 . it offered an evening counselling and testing clinic where brouard did pre- and post - test counselling with the predominantly white , gay clients . he and a friend also established an hiv support group attended by a small number of gay men , which allowed brouard and his friend to explore what it means to be positive , and what support one needed , and what was out there. during his internship year as a master s student in 1989 , brouard volunteered at the hiv clinic at joburg gen doing informal counselling and support work , and acting as a resource person. he recalls that people like ruben sher , dennis sifris and steve miller ran the clinic and it was a fascinating period because it was very other. while the hospital was still racially segregated and primarily served the needs of other because of their sexual orientation or because of the demands that they made on the hospital on behalf of gay people : suddenly this group of gay men who were feeling more entitled in some ways , or more informed , or more empowered , were coming in and it created some envy and resentment among the medical fraternity because why was there a need for a special clinic ? aids was still very much a disease of gay men , it was still stigmatised , and dennis , ruben and steve had to really fight to get staff , to get funds . they had constant battles with hospital management . in the context of apartheid ideology that sought to categorise people in unambiguous ways and thereby create specific , often binary , identities , white gay men were problems as they not only transgressed accepted heteronormative identities , but were also criminalised . they were men and so were expected to occupy a particular place in patriarchal structures , and they were race. they were , however , gay men and therefore their privileged status in terms of masculinities could be called into question . instead of being shamed into silence by their sexual orientation , these men took advantage of the social capital they had as white elite medical professionals , and challenged hospital authorities . unsurprisingly they antagonised others in positions of privilege both within , and beyond , the health sector . brouard recalled how the accessibility of arvs at joburg gen highlighted the class dynamics inherent in a health care system divided into public and private sectors . he felt ineffectual at the clinic because it seemed to be an overwhelming problem and you just saw so much death and dying , and just sadness and grief. sifris and miller saw patients who could afford arvs at their private practices , but there was no hope of this at the joburg gen . by the early 1990s both sifris and miller had left the clinic and worked predominantly in their private practices . brouard in the meantime , continued to volunteer at joburg gen and got a job in hillbrow at the esselen street clinic with mary crewe and clive evian , who were starting up the johannesburg city council aids programme . despite being state - funded , and having to negotiate the complexities of national and local budgetary funding processes , brouard maintains that he , evian , crewe and others pioneered several innovative programmes relating to sexual health and stis at the esselen street clinic . brouard , for instance , started a closed support group along strict psychotherapeutic lines for hiv - positive gay men which ran for three years . with so little access to azt the primary focus of the group was on how to adapt to living with hiv and preparing to die of aids . brouard remembers those years as his most significant immersion into counselling people who were dying or confronting mortality. brouard recounts that the clinic was not a specific aids or hiv clinic but rather provided comprehensive sexual health services with an aids training and support component . it had family planning services , a tb clinic , an sti clinic that incorporated hiv antibody testing , and an outreach programme focused on sex workers and gay men . brouard remembers the clinic being used by all sectors of the hillbrow community and being told by clients that it developed a reputation for meeting the needs of all people in the community and for being gay friendly. brouard knew many of the people who came to the clinic for help , and , despite minimal resources , recalls that they never turned people away . the clinic became a space where community members or groups could collaborate and develop new aids intervention strategies such as drama productions , comic strips and other innovative education materials . aids weeks , and in 1991 piloted aids education adverts on public buses . brouard maintains that they also developed interesting training models and counselling courses hailed as innovative by other health care centres , and provided non - judgemental testing services and sti treatments . in addition , brouard suggested the background and politics of the staff and a guiding assumption that responses to stis required a multi - disciplinary rather than a solely medico - scientific approach influenced the integrative outlook of the clinic . brouard referred to the interesting mix of people and the variety of skills that coalesced at the clinic , with clive evian bringing a community medicine perspective while mary crewe applied her academic knowledge to understanding the sociological roots of aids to provide a foundation for implementing programmes at the clinic . while sexuality organisations or gay professionals could exert pressure on policy makers in the uk and usa , or influence the aids policy - making process itself , there was less opportunity for this in south africa . in the 1980s the criminalisation of homosexuality and the government s homophobic stance made engagements between sexuality activists and government officials complicated . the political transition in the early 1990s created more opportunities for sexuality activists to engage with decision - makers but the focus for sexuality activists was no longer only on fighting legislation , or even raising awareness about aids , but rather on securing citizenship rights and equality for people of all sexual orientations . in the late 1980s and early 1990s the clinic built more alliances , both nationally and locally with progressive organisations in the gay communities like the gay and lesbian organisation of the witwatersrand ( glow ) and the tap . alliances were also built between progressive organisations involved in aids work ; and between gay organisations , activists , and the clinic . simon nkoli was involved in glow and tap , and with brouard ran hiv prevention workshops in hillbrow for black , gay men based on sex - positive , risk reduction approaches . in addition to providing information and distributing condoms , they used the workshops to explore relationships , sexuality and identities . brouard and nkoli criticised the abstain , be faithful , use condoms ( abc ) programmes and messages that were most prominent at the time , concluding that gay communities would not be receptive to the moralistic , conservative messages . nkoli had an office in esselen street and started an organisation for hiv - positive black , gay men . brouard recalls how you would walk into his office and there were all these explicit posters of naked black men so he was fantastically subversive. nkoli , brouard and sifris , as well as organisations like the gay peoples health forum , and gasa-6010/aset , were responsible for subverting the conservative , homophobic , moralistic narratives about sex and sexuality by virtue not only of their sexual orientation and politics , but also because of the safer sex material that they distributed or produced . in stark contrast to the government s abc messages , the aids education material developed by gay organisations or aids support groups in the mid-1980s , was radically different in both messaging and in assumptions about the intended audience . as the examples pictured below reveal , images and messages from gay organisations treated the audience as sexually active and did not assume that abstinence was realistic . instead , the images and words encouraged safer sex practices as a normalised part of enjoyable , erotic , healthy , sexual activity , and depicted sex across colour lines. brouard was on an editorial board that produced a comic pamphlet called keep it hot , safe and gay which featured two characters , hot shot and safe sex a lycra - clad super hero and a condom who delivered unabashed information about anal sex , oral sex , thigh - sex and mutual masturbation ( figure 1 ) . information about hiv infection and transmission was underscored by a central message that playing safe with the one you love puts the fun back in and takes the danger out. the comic provided contact information for the esselen street clinic and gay organisations . figure 1:panels from keep it hot safe and gay. panels from keep it hot safe and gay. safer sex cards from the johannesburg - based gay peoples health forum ( gphf ) were designed as actual postcards with writing space , and included the organisation s contact information . some of the cards showed a white man and a black man in intimate poses and reminded recipients to protect their partners and declared that healthy sex was not boring ( figure 2 ) . the gay peoples health forum s safer sex postcards . the significance of these media needs to be considered in light of homosexuality being illegal , and interracial hetero(sex ) only having been decriminalised in 1985 . producing and distributing these education materials was socially ( and legally ) subversive and the content of their sexual health messages was radical by the standards of the day . much like the keep it hot pamphlet and the gphf postcards , safer sex cards distributed by aset in cape town combined basic facts about hiv transmission with contact information for the organisation . the cards also used frank language and ( for the time ) explicit images , such as a the photographic credits indicate that some images were reproduced from the terence higgins trust in london , while others were created for the organisation . in 1991 john pegge , the director of the organisation , confirmed the importance of information exchanges between international organisations : we run a large safer sex campaign amongst the gay [ sic ] minority and for this purpose we use safer sex posters donated to us by gay aids service organisations in other countries . we have a wide selection from australia , new zealand , the united kingdom , the netherlands , canada and the united states of america . these images depicting a sexuality that was still criminalised flew in the face of south africa s conservative pornography laws which organisations managed to circumvent . the state of transition in the country was such that aset was able to receive such material , despite it running foul of pornography legislation , because authorities no longer appeared certain of quite which legislation was applicable . when apartheid laws were being more rigorously implemented , pornography received by mail did not result in criminal prosecution if the recipient could prove that they had not solicited the material . nonetheless , pegge urged caution : i would suggest that you send any materials that might be deemed pornographic in a separate envelope without a covering letter. despite the caution urged by pegge , the socio - political milieu of the country seemed to be changing . in the early to mid-1990s , brouard witnessed a change in clinic clients . rather than the initial client base of predominantly black south africans returnees who had either lived in exile communities or travelled to other countries . one womyn that he counselled frequently had been involved with a senior operative of the armed wing of the anc umkhonto wesizwe ( mk ) . the mk soldier attended counselling only once and later died of aids . yet , this time also saw a renewed sense of hope derived both from the country s political transition to a constitutional democracy and from developments in arv treatments . at the same time , progressive health organisations became increasingly involved in aids work and approached the clinic to obtain training materials and discuss mobilising health workers . othered in south africa it was the build up to 94 and the whole nacosa ( national aids convention of south africa ) process had generated an incredible amount of coming together and talking and debating a first national ( aids ) plan . people were pulling together more it was a fascinating time to be living through . the space for gay health care professionals to influence national aids programmes and policy throughout the 1980s and into the early 1990s was , brouard suggests , hampered by the level of homophobia in public health structures . while gay men , like brouard himself , were involved in the government - funded aids training and information centres ( atics ) , they worked within a conventional public health paradigm ( doing ) testing , counselling training , community prevention , ( and ) fairly dull aids in the workplace stuff. within this conventional paradigm , it was difficult for gay activists to bring about radical changes in aids education messaging or campaigns , so their influence would have been primarily at the individual level , with people they counselled , or , possibly , within the atic structures . brouard maintains that even people such as ruben sher , as an aga member , would have had limited opportunities to make substantive changes to national aids policies or programmes . with aids ring - fenced as something other and outside the public health care system , it was seen as a challenge to existing health care systems and structures . brouard recalls that even at the clinic , which championed an integrated approach to sexual health , nursing and other staff who were less involved in aids work saw the aids people as different. brouard noted that the perception of aids as when interviewed for this project , brouard remarked that he was struck by the irony that state resources for aids programmes amongst lgbti communities were not available either during or after apartheid . brouard had met rina venter ( the last np national minister of health ) in the early 1990s and he recalled that she had made it clear that because aids was a minority issue the gay community needed to look after itself. this attitude , combined with the criminalisation of homosexuality , meant that state resources were not made available to gay people . similarly , in the post-1994 period , when the focus of government interventions shifted to heterosexual people and pregnant womyn and people started claiming their constitutionally - guaranteed rights to health care and treatment ( in this instance arv therapy ) and demanding responses from the anc - led government , gay communities were again sidelined as minority groups . this continued marginalisation has resulted in a lack of basic information about hiv transmission and infection rates amongst gay communities in post - apartheid south africa . of the early years of the epidemic brouard primarily recollects death : there was a lot of death that s what i remember of those years the number of funerals one went to , the number of people who disappeared , it was just horrible. while in 2013 the funerals continue , the option for a longer life is now available to people if regular and sustained access to arvs can be maintained by the state , and in 2013 south africa has what is possibly one of the largest arv rollout programmes in the world . brouard continued working at the clinic until 1997 and then joined the centre for the study of aids ( csa ) in 1999 . as the deputy director of the csa , brouard continues to conceptualise and develop training methods and materials , and contributes to academic publications and other fora on topics such as psychosocial issues , gender , sexuality , human rights , testing and tertiary responses to hiv and aids . brouard s work still involves the realities of aids - related deaths , but now also includes components that address prolonging and living life fully , regardless of hiv status . personal and professional relationships and networks organised around sexual identity were important to the first responses to aids in south africa in both medical and social spheres . sexuality organisations had to take responsibility for addressing the aids epidemic in the face of government inaction , but much of this response was limited to specific sections of the gay community , individuals , and to specific geographic areas . while aids provided a point of mobilisation and support in south africa , as this article has explored , sifris and brouard were active in both sexuality organisations and in two key public health sites involved in addressing aids . their accounts of the time provide insights into the experiences of two gay men at the start of an epidemic written on and around gay men s bodies . their stories , as reflected here show how even within hostile government - aligned health care spaces , they were able to provide support and treatment to some people , and to engage in , what were for the time , socially subversive activities . the different contexts and professional fields in which they found themselves mediated their individual experiences , but in both men s case , the hegemonic aids narratives about homosexuality influenced and shaped their responses even as they challenged them . homophobic beliefs , conservatism , or any other invisible or unquestioned ideologies that influence understandings of epidemics and practical responses to them , have tangible repercussions . these repercussions may be evident in the lack of legal protection or in the emotional costs of prejudice and intolerance . brouard , like miller and sifris , challenged hospitals to provide care for people with aids . joburg gen was often reluctant to admit people with aids , as providing palliative care was seen as a waste of resources and of limited value for instructional purposes . even when places could be found for people it was difficult to get staff members to treat them with dignity ; and many staff members did not deal well with gay men , young men , coming in infected and dying of this strange disease ( where ) dementia complicated the death process further. for the partners of the men who died the trauma of loss was aggravated by their lack of legal status or recognition as partners of the deceased . brouard , for instance , recalls counselling loving partners who were not able to claim the bodies of the deceased men . he witnessed both the deaths and the grief of survivors during this time . in the early phases of the epidemic sifris challenged homophobic responses to gay men with aids , but did not necessarily challenge the then dominant narrative that initially linked aids to being gay . given that the professional and personal spaces he occupied bore out the narrative that gay men were the ones dying of aids , this is perhaps not surprising . brouard worked within gay communities he most identified with , but his professional and personal spaces seemed to have presented a more complex narrative about aids and its association with sexual identity and broader identity politics . the socio - political evocation of stereotypes , or constructions of gender , race , and sexual orientation have long shaped experiences of ( ill)health and epidemics . this article has focused on the micro - narratives of individuals and their responses to the epidemic as individuals and as part of communities . it serves as a reminder of the intricate and intimate relationships between ideologies , illness and identities , but also that behind each aids avatar evoked in hegemonic narratives are complex , embodied people .

this article focuses on the micro - narratives of two individuals whose responses to aids were mediated by their sexual identity , aids activism and the political context of south africa during a time of transition . their experiences were also mediated by well - established metanarratives about aids and homosexuality created in the usa and the uk which were transplanted and reinforced ( with local variations ) into south africa by medico - scientific and political leaders.the nascent process of writing south african aids histories provides the opportunity to record responses to aids at institutional level , reveal the connections between narratives about aids and those responses , and draw on the personal stories of those who were at the nexus of impersonal official responses and the personal politics of aids . this article records the experiences of dennis sifris , a physician who helped establish one of the first aids clinics in south africa and emptied the dance floors , and pierre brouard , a clinical psychologist who was involved in early counselling , support and education initiatives for hiv - positive people , and counselled people about dying , and then about living . their stories show how , even within government - aligned health care spaces hostile to gay men , they were able to provide support and treatment to people ; benefited from international connections with other gay communities ; and engaged in socially subversive activities . these oral histories thus provide otherwise hidden insights into the experiences of some gay men at the start of an epidemic that was initially almost exclusively constructed on , and about , gay men s bodies .

Introduction: Apartheid, AIDS, and the Politicisation of Homosexuality Politics, Polemics, Practice, and the Personal: A Complex Nexus Dennis Sifris: The Physician Who Emptied the Dance Floor The Personal as Political: International Influences Pierre Brouard: Counselling for Death, Counselling for life Changes and Transitions Conclusion

PMC3299971

stability of the trunk , often referred to as core stability , has gained considerable attention in the recent years . it can be operationally defined as : the body s ability to control the trunk in response to internal and external disturbances , including the forces generated from distal body segments as well as from expected or unexpected perturbations core stability training has become a major element of training programs in sports as well as rehabilitation ( borghuis et al . it seems plausible that core stability is important for injury prevention and athletic performance , especially in sports with large demands for balance control , such as gymnastics . the importance of core stability for injury prevention in athletes has received some support from epidemiological studies relating low core stability to incidence of injuries of the back ( cholewicki et al . 2005 ) and lower extremities ( zazulak et al . in addition , there is limited evidence to support its role as a determinant of performance . nesser et al . found a moderate correlation between core stability and performance in football players ( nesser et al . it should be noted that , in several of the studies mentioned ( nesser et al . 2004 ) , the measurements used to determine core stability reflect muscle strength and endurance , possible determinants of stability , rather than stability itself . the stability of the spine and trunk depends on contributions of the passive , active and control sub - systems ( panjabi 1992 ) . the passive joint structures such as ligaments and intervertebral discs contribute to stability by providing joint stiffness ( cholewicki et al . the active sub - system , the trunk musculature contributes by further increasing stiffness through cocontraction ( cholewicki et al . 1997 ; gardner - morse and stokes 1998 ; dien et al . 2003 ; stokes and gardner - morse 2003 ) . in addition , the control sub - system , i.e. , the nervous system , controls reflexive and triggered muscle activity contributing to stability ( moorhouse and granata 2007 ) , based on sensory feedback from muscle and joint receptors , and feedback from the visual and vestibular systems ( goodworth and peterka 2009 ) . a model study predicted that the decrease in muscle stiffness that is associated with fatigue ( kirsch and rymer 1987 ; zhang and rymer 2001 ) , may impair trunk stability ( granata et al . 2004 ) . given the importance of reflexive muscle activity for core stability , the reductions in maximum force and rate of force rise that develop with muscle fatigue can also be expected to impair core stability , which may be aggravated by the adverse effects of fatigue on proprioception ( taimela et al . 2004 , 2009 ) , which was attributed to a reduced control over the trunk . granata and gottipati ( 2008 ) showed that fatigue of the extensor muscles had a negative effect on the trunk s local dynamic stability , i.e. , the responses to the small perturbations that are always present due to neuromuscular noise ( dien et al . 2008 ) . herrmann et al . found decreased contact forces during an external perturbation of the trunk induced with a swinging pendulum , which indicates reduced trunk stiffness ( herrmann et al . ( 2010 ) , however , found no change in contact forces . in both of these studies , increased electromyographic amplitudes of the reflex responses indicated compensatory mechanisms to counteract the fatigue effects . moreover , several studies found increased cocontraction in unperturbed standing after inducing trunk muscle fatigue , as a potential compensatory mechanism ( grondin and potvin 2009 ; granata et al . two of these studies also investigated the response to an external perturbation and found no effects of fatigue ( grondin and potvin 2009 ; granata et al . the evidence for the effects of fatigue on trunk stability from the studies reviewed above is not consistent . furthermore , all of these studies involved high fatigue levels ( typically a reduction in force producing capacity by 40% ) induced using isolated , non - functional activity of trunk muscles . it thus remains unclear whether fatigue of the trunk muscles that even well - trained athletes may develop during training or competitive events can induce impairments of trunk stability . the aim of the present study therefore was to investigate the effects of fatigue induced by a set of exercises as performed regularly in training on trunk stability in elite gymnasts . we hypothesized that gymnasts show increased sway amplitudes in a seated balancing task and that they are less able to correct an external perturbation of seated balance after a set of gymnastic exercises . nine gymnasts , all girls , with a mean age of 12.4 ( 2.3 ) years old participated in the study . their mean ( standard deviation ) height and body mass were 1.47 0.12 m and 39.0 12.92 kg , respectively . none of the participants reported any recent history of injuries that did not allow training participation . the experimental protocol , which had been approved by the ethical committee of the faculty of human movement sciences of the vu university amsterdam ( number 2009 - 039 ) , comprised a 10-min fatigue protocol in between the pre- and posttests of trunk stability . the measurements were performed at the beginning of regular training sessions in the afternoon , after school hours . prior activities , either during the day before the training or during warming - up were not controlled . the fatigue protocol contained four series of five dump handstand exercises on the uneven bar . based on subjective report of trainers and gymnasts , this bout of exercises series of dump handstands are a regular part of normal training and the intensity of this bout of exercises was comparable to the more intensive elements of the participant s normal training activities for the measurements of trunk stability , subjects were seated with arms in their lap on an unstable seat , which required them to dynamically balance by trunk movement only ( fig . 1 ) . the seat was mounted over a hemisphere ( radius of hemisphere : 25 cm , height of the seat relative to the lowest point on the hemisphere : 17 cm ) , creating instability in all directions . to trace the center of pressure ( cop ) , the seat was placed on a custom - made strain gauge force plate that was sampled at 200 samples / s . the force plate was calibrated prior to each measurement session , by placing known weights on the plate . the footplate was adjusted to support the feet with the knees and hips at 90 angles . the first task required the subject to sit as still as possible and lasted 30 s. three repetitions of this task were performed before and after the fatiguing exercise , as reliability of single measurements was previously shown to be poor ( dien et al . 2010b ) . in the second task , the subject leaned back on the seat , supported by a strap around the thorax that was attached to two electromagnets on the safety rail in front of the subject . the length of the strap was adjusted to obtain a constant inclination angle for each subject . after a random interval of 37 s after the start of the measurement , the electromagnets were released and the subject had to regain balance as quickly as possible . recording of data was continued for a total of 20 s. nine repetitions were performed within a few minutes before and after the fatigue exercise.fig . 1schematic of the unperturbed seated balancing task ( a ) and the sudden release task ( b ) schematic of the unperturbed seated balancing task ( a ) and the sudden release task ( b ) force plate data were low - pass filtered at 10 hz ( fourth order butterworth ) , as the signals contained only very limited power above 3 hz , while sensitivity analysis showed only minor effects of filtering at either a higher ( 20 hz ) or lower ( 2.5 hz ) cutoff . four independent parameters were derived from the cop time series of the 5th till 30th second of the trial : the rms of the cop in x and y direction ( rmsx , left right ; rmsy , antero - posterior ) and the mean power frequency of the cop time series in both directions ( mpfx and mpfy ) . for the unperturbed trials ( see fig . 2 for an example ) , the mean cop position determined over the first 2 s was first subtracted from the time series . next , the maximum cop position in the y direction was determined , which reflects the maximum backward cop displacement after the sudden release ( maxy ) . subsequently , an exponential decay function was fitted to the cop time series in the y direction from the instant of maxy and the subsequent 7 s ( fig . 2typical examples of : the cop time series in the unperturbed trials with anterior posterior displacement in black and left right displacement in gray ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the rmsx ( black ) and rmsy ( gray ) values for all trials of the same subject ( c ) , the mpfx ( black ) and mpfy ( gray ) values for all trials of the same subject ( d ) . 3typical examples of the anterior posterior cop times series in a sudden release trial with the fitted exponential curve as a thick gray line ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the maxy values for all trials of the same subject ( c ) , the values for all trials of the same subject ( d ) . the fatiguing exercise was performed between trials nine and ten1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y(t ) = y^{\prime } + ( { \text{max}}_{y } - y^{\prime } ) \times e^ { - \lambda t } $ $ \end{document}with time t defined as zero at the instant of maxy , y referring to the y coordinate of cop , y referring to the steady state y position of the cop to which the subject converged , estimated as the median position over the fifth till seventh second after the maximum backward displacement , and maxy referring to the maximum displacement . the parameter was used as an indicator of the rate of recovery after the perturbation , with higher values indicating faster recovery . typical examples of : the cop time series in the unperturbed trials with anterior posterior displacement in black and left right displacement in gray ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the rmsx ( black ) and rmsy ( gray ) values for all trials of the same subject ( c ) , the mpfx ( black ) and mpfy ( gray ) values for all trials of the same subject ( d ) . the fatiguing exercise was performed between trials three and four typical examples of the anterior posterior cop times series in a sudden release trial with the fitted exponential curve as a thick gray line ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the maxy values for all trials of the same subject ( c ) , the values for all trials of the same subject ( d ) . the fatiguing exercise was performed between trials nine and ten for all six dependent variables , median values over the repeated trials per subject were used for statistical analysis to avoid effects of trials with outlying results . comparisons between the fatigued and unfatigued conditions were made based on averages over subjects , which were tested with paired t tests , with = 0.05 . the effect of trial order was tested over the trials performed before the fatiguing exercise using repeated measures anova . figure 2 illustrates a typical example of the data obtained in the unperturbed trials . however , the rmsy values were on average higher and mpfy values were on average lower after than before the fatiguing exercise , whereas no difference was discernible in the x direction . the effects in the y direction ( anterior posterior ) were in line with our hypothesis and were found to be statistically significant for the group ( table 1 ) . no effects were found in the x direction.table 1means and standard deviations of all dependent variables before and after the bout of fatiguing exercise and p values for differences between the fatigued and unfatigued conditions based on paired t testsindependent variablemean sdprmsx ( cm ) unfatigued0.19 0.080.549 fatigued0.21 0.09rmsy ( cm ) unfatigued0.18 0.080.030 fatigued0.31 0.17mpfx ( hz ) unfatigued0.40 0.150.810 fatigued0.41 0.24mpfy ( hz ) unfatigued0.52 0.130.005 fatigued0.35 0.17maxy ( cm ) unfatigued3.81 0.900.003 fatigued5.10 1.00 ( s ) unfatigued1.73 0.580.012 fatigued0.80 0.27 means and standard deviations of all dependent variables before and after the bout of fatiguing exercise and p values for differences between the fatigued and unfatigued conditions based on paired t tests figure 3 illustrates a typical example of the cop data in the perturbed trials . after the sudden release approximately 4 s after the start of the trial , the subject moved further backward to recover and attain a fairly steady position within 3 s after the release . however , the maxy values were on average higher and values were on average lower after than before the fatiguing exercise . these effects were in line with our hypothesis and were found to be statistically significant for the group ( table 1 ) . to test whether the differences between the trials before and after the fatiguing exercise could be explained by order effects , we tested for the effect of measurement order over the trials collected before the fatiguing exercise . none of the variables showed a significant order effect ( p > 0.383 ) . this study was designed to test the hypotheses that gymnasts show increased sway amplitudes in a seated balancing task and that they are less able to correct an external perturbation of seated balance after a set of fatiguing gymnastic exercises . as hypothesized , unperturbed seated balancing performance was decreased in the anterior posterior direction as evidenced by a larger amplitude and lower frequency . perturbations were performed in the posterior direction only . in line with the hypothesis , the maximum displacement after the perturbation was larger and the recovery of balance was slower after the exercise than before . it is at present unclear why effects were found in the sagittal plane ( anterior posterior direction ) only . an explanation could be that the dump hand stands fatigued the abdominal muscles selectively given the mechanics of these exercises . while abdominal muscles can contribute to control of lateral bending of the trunk ( seroussi and pope 1987 ) , the extensor muscles have a higher moment producing capacity in the frontal plane ( dien and kingma 1999 ) and probably allow more precise control in view of their abundant insertions on the multi - segmented lumbar spine . thus , while control in frontal plane could be done mainly by the extensor musculature , the abdominal muscles are obviously indispensible for control in the sagittal plane . hence , selective fatigue of these muscles might explain why effects were restricted to the sagittal plane . as reported in the introduction , the previous studies on the effects of fatigue on trunk stability showed inconsistent effects . most of these tested trunk stability by applying perturbations to the trunk in either standing or sitting positions . it was suggested that compensatory mechanisms such as increased cocontraction and increased reflex gains could explain the absence of fatigue effects in some of these studies . in the present study , we used a dynamic balancing task to test trunk stability . in this task , the center of mass unavoidably sways beyond the surface of support , which is the point of contact of the hemisphere on the force plate . trunk moments , accelerating the upper body relative to the pelvis and seat , must then be used to bring the center of mass back over the surface of support , while stiffening the trunk would not be effective ( cf otten 1999 ) . it has previously been shown that cocontraction , which would stiffen the trunk is not effective in this task ( reeves et al . . the present task may therefore be more sensitive to trunk muscle fatigue than tasks in which the support surface is larger . activity of the trunk muscles was not measured and it is therefore unknown whether subjects increased cocontraction , but this would be consistent with the increase in rmsy ( reeves et al . 2006 ) and decrease in mpfy ( dien et al . with fatigue of an agonistic muscle , the activation levels need to be increased to maintain force output . 2009 ) , which would cause increased sway and thus could explain the increase in rmsy . variability of muscle force would increase even more when antagonistic cocontraction would be increased ( selen et al . 2005 ) which , given the fact that stiffening the trunk does not limit kinematic variability in the present task , could further increase sway amplitudes . fatigue will likely slow down muscular responses due to increased proprioceptive thresholds ( taimela et al . 1999 ) and due to the slower force development in the fatigued muscles ( de ruiter et al . 1999 ; perrey et al . slower responses to balance perturbation would increase sway amplitude ( radebold et al . 2001 ) and likely decrease sway frequency in the unperturbed task and would also increase the amplitude and reduce the recovery in the perturbed task ( reeves et al . . increased proprioceptive thresholds might be reflected specifically in an increase in the maximum displacement after the perturbation ( increased maxy ) , while a decreased rate of force development might be more obvious in a decreased rate of recovery ( decreased ) . finally , fatigue could be associated with an increased respiratory challenge , which might affect trunk stability ( janssens et al . it is to be expected that the respiratory effect would be most obvious in the sagittal plane , which would be in line with the fact that only effects in the y direction were found . unavoidably , trials in the fatigued condition were performed after those in the unfatigued condition and this could lead to order effects , for example due to learning , which might dilute the fatigue effect . the absence of an order effect over the trials performed before the fatiguing exercise suggests that such effects did not occur , possibly due to the fact that the participants were all highly trained athletes . it is unclear to what extent the loss of trunk stability as found in the present study would affect the performance of gymnastic activities . furthermore , impaired trunk stability may limit performance and increase injury risk . therefore , the fact that trunk stability was negatively affected by a series of fatiguing exercises that reflect intensity of typical training activities and competitive events suggests several practical implications . the results suggest that trainers should take fatigue effects into account when planning the order of training activities , e.g. , avoid balance beam exercises after uneven bar or horizontal bar exercises . furthermore , the results may have implications for the intensity of warming up exercises for the competitive events . finally , the results indicate that endurance training of trunk muscles and perhaps balance training may need to be performed not only in rested , but also in fatigued condition . in conclusion , the present study showed that trunk stability in elite gymnasts was negatively affected by a bout of exercises , which reflected normal training activities . both sagittal plane sway in unperturbed balancing and recovery after a backward balance perturbation were affected . these results suggest that fatigue effects on trunk stability should be taken into account in the planning and design of gymnastics training .

the aim of the present study was to test the hypothesis that fatigue due to exercises performed in training leads to a decrement of trunk stability in elite , female gymnasts . nine female gymnasts participated in the study . to fatigue trunk muscles , four series of five dump handstands on the uneven bar were performed . before and after the fatigue protocol , participants performed three trials of a balancing task while sitting on a seat fixed over a hemisphere to create an unstable surface . a force plate tracked the location of the center of pressure ( cop ) . in addition , nine trials were performed in which the seat was backward inclined over a set angle and suddenly released after which the subject had to regain balance . sway amplitude and frequency in unperturbed sitting were determined from the cop time series and averaged over trials . the maximum displacement and rate of recovery of the cop location after the sudden release were determined and averaged over trials . after the fatigue protocol , sway amplitude in the fore - aft direction was significantly increased ( p = 0.03 ) , while sway frequency was decreased ( p = 0.005 ) . in addition , the maximum displacement after the sudden release was increased ( p = 0.009 ) , while the rate of recovery after the perturbation was decreased ( p = 0.05 ) . fatigue induced by series of exercises representing a realistic training load caused a measurable decrement in dynamic stability of the trunk in elite gymnasts .

Introduction Methods Results Discussion Conclusion

PMC5266426

by definition , health literacy is defined as the degree to which an individual has the capacity to obtain , communicate , process , and understand basic health information and services to make appropriate health decisions.(http://www.cdc.gov / healthliteracy / learn / index.html ) results from prior studies provide evidence that patients health literacy has a significant impact on the extent of a patients medication adherence and related health care utilization . adherence can be broadly defined as a patient s ability to accurately follow a prescribed treatment regimen . in research , this is often operationalized as the patient s ability to successfully take the prescribed number of doses of a medication . a common metric of defining adherence is taking at least 90% of the prescribed doses . the annual excess of health care costs as a result of medication nonadherence is roughly us$290 billion in the united states . by understanding barriers to adherence , clinicians can help patients become more adherent in managing chronic disease that can alleviate these costs . between 30 and 34 million american adults fall into the lowest of the 4 levels of health literacy ( below basic , basic , intermediate , and proficient ) , according to results from the 2003 national assessment of adult literacy . even adults with proficient levels of general literacy may have low health literacy due to the stressful nature of health care visits and unfamiliar medical terminology . in a systematic review of literature examining health literacy prevalence rates , up to 50% of us adults were determined to have low health literacy , while approximately 33% of medicare enrollees have low or inadequate health literacy . however , it is difficult to accurately determine the national prevalence of low health literacy due to different definitions and measurements used across the studies . it has been suggested that patients with low health literacy experience a higher rate of medication errors . standard medication guides approved by the food and drug administration have been estimated to require a 10th- to 11th - grade reading level , leaving many patients with a poor understanding of the content . one study by davis et al found that patients in primary care settings who had low literacy were 3 times less likely to correctly interpret medication label warnings . another study by the same authors found that low literacy was associated with misunderstanding medication labels , and only 35% of patients with low literacy could correctly identify how many pills needed to be taken daily . often , these patients do not fully comprehend the benefits and risks of medications or how adherence affects health outcomes . therefore , medical providers can make use of a patient s health literacy to assist patients in understanding their treatment plans . when only 12% of adults in the united states are considered health literate , clinicians must acknowledge an opportunity to improve patient outcomes . therefore , the objective of this study is to evaluate the health literacy levels and medication adherence of patients treated by pharmacists in both the general medicine and the chronic care clinics at the kansas city free health clinic ( kcfhc ) . the study was conducted by pharmacists at the kcfhc , an urban free health clinic in kansas city , missouri . the study estimated health literacy levels and medication adherence of patients in both the general medicine and the chronic care clinics . rapid estimate of adult literacy in medicine ( realm ) was chosen for its ease of use and short administration and scoring time . in order to be eligible for this study , patients had to be 18 to 65 years old , english speaking , and currently receiving medical care at the kcfhc in 2011 . patients were excluded if their current health condition was so poor that it inhibited normal patient provider communication . level of health literacy was assessed using the realm . the entire realm can be administered and scored in less than 5 minutes , which is convenient for many health care settings . the realm can also help practitioners identify those patients who have compensated for their literacy deficiencies by requesting patients to pronounce 66 words that may be used during a medical visit with a health care provider . as this is a word recognition test , it is intended to screen patients rapidly and provide an estimate of their literacy levels , rather than testing comprehension or diagnosing specific reading deficits or learning disabilities . the results of realm are broken down into the following reading level categories : below third grade , fourth to fifth grade , seventh to eighth grade , and high school . patients were asked by the pharmacists on how many medication doses they had missed in the last 3 , 7 , and 14 days prior to their clinic visit . if any doses were missed , patients were asked to volunteer reasons why this had occurred . medication adherence rate ( mar ) was calculated by dividing the number of doses taken in 14 days by the total number of doses available . this calculation was adjusted for each patient s dosing schedule ; for instance , if a medication was prescribed to be taken twice daily , then the total number of doses available in 14 days was 28 . adherent , and patients with an mar less than 90% were categorized as nonadherent . medication adherence barriers were collected by the pharmacist using open - ended questions and emphasized with the probe what else to gather all possible medication adherence barriers experienced by these underserved patients . patients who came to the general medicine clinic were recruited after the patient had been checked into their room and before their examination with their provider . if the patient agreed , the investigator read the institutional review board - approved consent script describing the nature of the research and the realm process . participation in this study was voluntary and did not affect the quality of medical care that the patient would receive . adherence was entered as a dichotomous variable , whereby 1 = mar 90% and 0 = mar < 90% . multivariate logistic models determined associations between adherence , health literacy level , and patient characteristics . results were reported in estimated proportion ( % ) and odds ratio ( or ) with 95% confidence intervals ( ci ) . the study was conducted by pharmacists at the kcfhc , an urban free health clinic in kansas city , missouri . the study estimated health literacy levels and medication adherence of patients in both the general medicine and the chronic care clinics . rapid estimate of adult literacy in medicine ( realm ) was chosen for its ease of use and short administration and scoring time . in order to be eligible for this study , patients had to be 18 to 65 years old , english speaking , and currently receiving medical care at the kcfhc in 2011 . patients were excluded if their current health condition was so poor that it inhibited normal patient provider communication . the entire realm can be administered and scored in less than 5 minutes , which is convenient for many health care settings . the realm can also help practitioners identify those patients who have compensated for their literacy deficiencies by requesting patients to pronounce 66 words that may be used during a medical visit with a health care provider . as this is a word recognition test , it is intended to screen patients rapidly and provide an estimate of their literacy levels , rather than testing comprehension or diagnosing specific reading deficits or learning disabilities . the results of realm are broken down into the following reading level categories : below third grade , fourth to fifth grade , seventh to eighth grade , and high school . patients were asked by the pharmacists on how many medication doses they had missed in the last 3 , 7 , and 14 days prior to their clinic visit . if any doses were missed , patients were asked to volunteer reasons why this had occurred . medication adherence rate ( mar ) was calculated by dividing the number of doses taken in 14 days by the total number of doses available . this calculation was adjusted for each patient s dosing schedule ; for instance , if a medication was prescribed to be taken twice daily , then the total number of doses available in 14 days was 28 . adherent , and patients with an mar less than 90% were categorized as nonadherent . medication adherence barriers were collected by the pharmacist using open - ended questions and emphasized with the probe what else to gather all possible medication adherence barriers experienced by these underserved patients . patients who came to the general medicine clinic were recruited after the patient had been checked into their room and before their examination with their provider . if the patient agreed , the investigator read the institutional review board - approved consent script describing the nature of the research and the realm process . participation in this study was voluntary and did not affect the quality of medical care that the patient would receive . adherence was entered as a dichotomous variable , whereby 1 = mar 90% and 0 = mar < 90% . multivariate logistic models determined associations between adherence , health literacy level , and patient characteristics . results were reported in estimated proportion ( % ) and odds ratio ( or ) with 95% confidence intervals ( ci ) . the average patients age was 48 ( range 20 - 66 ) , and majority was female ( 56% ) and white ( 55% ) . there was a significant association between ethnicity and reading level ( p = .002 ) . white patients displayed higher literacy levels compared to black patients in the bivariate analysis ( table 1 ) . while 33% of patients reported completion of their high school degree , 64% scored as much according to the realm . conversely , 36% of patients could not read at a high school level . in this study , only 21% of patients read at a seventh- to eighth - grade level , which is currently the average american adult literacy level . it is unknown at this time why this discrepancy exists , but with a larger sample size , results may prove representative of the us population . p value for chi - square tests of associations between literacy skills and patient characteristics and between adherence and patient characteristics , respectively . as shown in table 2 , patients with hiv / aids had the highest levels of adherence ( 88% ) , whereas patients with hypertension reported the lowest level of adherence ( 64% ) . when asked to provide a reason for missing a dose , 26% of the patients reported that they simply forgot . chi - square or fisher exact if < 5 in a cell . as shown in table 1 , some patients reported multiple disease states . as such , there may be some overlap in the analysis of adherence versus nonadherence . significance determined at p < .05 . health literacy level as determined by realm scores . we discovered that health literacy was not an indicator of a patient s ability to comply with prescription therapy in treating chronic disease conditions . general trends for decreased adherence included lower dose frequency ( ie , once - daily dosing ) and lower daily pill burden . when patients are separated by disease state and evaluated for adherence , those patients who were hiv - positive showed the greatest trends toward adherence , followed closely by those with diabetes and then hyperlipidemia . this is consistent with the most common reasons given for nonadherence , as the hypertensive population had the lowest daily pill burden and dose frequency . patients with high health literacy level were 21% more likely to be medication adherent ( or : 1.21 , ci : 0.68 - 2.16 ) ; however , it is not statistically significant . we also adjusted this relationship by patients age , gender , education , and ethnicity , but the or and significance level remained the same . male patients were 2.87 times more likely than females to adhere to their medication regimen ( table 2 ) . moreover , ethnicity , age , and education level were not significantly associated with adherence in the multiple logistic regression model . this study examined the varying levels of health literacy and medication adherence in a group of patients with a broad range of disease states and medication regimens . disease state and adherence were only significantly related in the hiv / aids and hypertension subsamples . similar to our study , a recent trend report showed that diabetes and hypertension medication adherence was below the national average , particularly in the midwest . notably , in this population , white patients displayed higher literacy level compared to black patients ( table 1 : p = .002 ) . additionally , patients with high health literacy levels were 21% more likely to be medication adherent ( or : 1.21 , ci : 0.68 - 2.16 ) ; however , it is not statistically significant ( p > 0.05 ) . therefore , the patients literacy levels according to realm were not significantly associated with self - reported medication adherence in this small study sample . we relied only on self - reported medication adherence in this study , which could be imprecise . more accurate and automated medication adherence measures of tracking adherence , such as electronic drug monitoring ( edm ) or partner / proxy reports could have provided more objective data . moreover , medication self - management approach a new conceptual model including patient s knowledge , skills , and behaviors necessary for patients to correctly take medications may be the accurate way to measure adherence . however , the medication self - management approach does not take into consideration patient s initial decision to acquire the medication as their primary treatment management option . in this sample , 21% of patients read at a seventh- to eighth - grade level , which is the average reading level of american adults . therefore , in this underserved population , 64% of the patients read at above average reading level of american adults . similar to general population , this underserved population reports forgetfulness as the most common reason for medication nonadherence . interestingly , in this underserved population , 9% of the participants reported that they ran out of their medication . it is not clear whether this is happening because these patients did not have enough money to refill their medication or not . it is important to mention that many of these underserved patients are on free supply of medication provided by the free health clinic . although from these study findings we can not truly establish the actual reason for their running out of medication , this finding highlights an issue that warrants further investigation . it is possible that low - literacy patients did not fully understand their dosing regimen and thus could not accurately report their adherence to said regimen . if patients with low levels of health literacy were more likely to underestimate nonadherence , this could introduce systematic bias to our results . other methods of tracking adherence , such as edm or partner / proxy reports would provide more objective data . this may explain the discrepancy between our results and those of murray et al , which utilized edm to measure adherence . however , it should be noted that the relationship between health literacy and self - reported adherence has been successfully demonstrated in other studies , such as the sample of hiv - positive patients using triple - drug antiretroviral therapy . indeed , hiv - positive patients may be more motivated to adhere to their drug regimen , given that nonadherence to antiretroviral therapy can have significant consequences , such as reduced viral suppression , poorer immunologic benefit , and overall treatment failure . in comparison , a patient who is nonadherent to their hypertension medication is not susceptible to such risks and therefore may perceive fewer consequences of missing a dose . in light of this , an additional limitation of the current study is the relatively small sample size involving a variety of disease states , which prevented the analysis of the relationship between literacy levels and adherence within each disease category . it is possible that low - literacy patients did not fully understand their dosing regimen and thus could not accurately report their adherence to said regimen . if patients with low levels of health literacy were more likely to underestimate nonadherence , this could introduce systematic bias to our results . other methods of tracking adherence , such as edm or partner / proxy reports would provide more objective data . this may explain the discrepancy between our results and those of murray et al , which utilized edm to measure adherence . however , it should be noted that the relationship between health literacy and self - reported adherence has been successfully demonstrated in other studies , such as the sample of hiv - positive patients using triple - drug antiretroviral therapy . indeed , hiv - positive patients may be more motivated to adhere to their drug regimen , given that nonadherence to antiretroviral therapy can have significant consequences , such as reduced viral suppression , poorer immunologic benefit , and overall treatment failure . in comparison , a patient who is nonadherent to their hypertension medication is not susceptible to such risks and therefore may perceive fewer consequences of missing a dose . in light of this , an additional limitation of the current study is the relatively small sample size involving a variety of disease states , which prevented the analysis of the relationship between literacy levels and adherence within each disease category . in this underserved population , 64% of the patients read at above average reading level of american adults . overall mar was 73% . forgetting to take medication was the most popular reason provided for nonadherence . disease state and adherence were significantly related in patients with hiv / aids and hypertension . although patients literacy levels were not significantly associated with self - reported adherence in this population , availability of a patient s baseline health literacy level as a part of the medical record may help clinicians to individualize their interaction in order to achieve better health outcomes , including improved medication adherence , especially for underserved populations .

background : a patient s health literacy is not routinely assessed during visits with a health care provider . since low health literacy is a risk factor for poor health outcomes , assessing health literacy should be considered as part of the standard medical workup.objectives:to evaluate the health literacy levels and medication adherence of patients treated by pharmacists in both the general medicine and the chronic care clinics at an urban free health clinic.methods:eligible patients from the free health clinic completed the rapid estimate of adult literacy in medicine ( realm ) , a health literacy measurement tool , during their clinic visit in 2011 . medication adherence was self - reported by the patients.results:a total of 100 patients participated ( mean age = 48 ) . the majority of participants were female ( 56% ) and white ( 55% ) . most ( 64% ) of the patients scored at a high school reading level according to realm . only 21% of participants read at a seventh- to eighth - grade level . overall medication adherence rate was 73% . forgetting to take medication was the most popular reason given for nonadherence.conclusion:disease state and adherence were significantly related in patients with hiv / aids and hypertension . patient s ethnicity was significantly associated with literacy levels ( p < .05 ) . although patients literacy levels were not significantly associated with self - reported adherence in this population , availability of a patient s baseline health literacy level as a part of the medical record may help clinicians to individualize their interaction based on the patient s health literacy level in order to achieve better health outcomes , including improved medication adherence , especially for underserved populations .

Introduction Materials and Methods Study Design Measures Medication Adherence Data Collection Statistical Analysis Results Discussion Limitations Conclusion

PMC3894078

tooth brushing plays an important everyday role in personal oral hygiene and effective plaque removal . toothbrushes may become heavily contaminated with microorganisms and these microorganisms may originate not only from the oral cavity but also from the environment where the toothbrushes are stored . this contamination implicates in the possibility of reinfection of a patient by toothbrushes harboring pathogenic microorganisms . as early as 1920 , cobb et al . found that toothbrushes can be heavily infected by mutans streptococci after 24 h. according to glass et al . , microorganisms not only adhere to and reproduce on used toothbrushes but also have the ability to transmit organisms responsible for both local and systemic diseases . procedures for the decontamination of toothbrushes would prevent the risks of reinfection or infection by other pathogenic microorganisms from the environment . several bactericidal agents have been promoted to reduce the possibility of toothbrush bacterial contamination between uses . these include the use of chlorhexidine , brushtox , and several dentifrices . while all these have shown varying degrees of efficacy , none are widely used as a home - based application . a possible reason for the noncompliance with these methods is that they are time consuming and may result in unwanted product residues . recently , few studies indicated that the use of microwave and ultraviolet ( uv ) light is the most effective household method to sanitize the toothbrushes after contamination . furthermore , due to the ease of use however , the extent of bacterial decontamination using the microwave and uv light has not been determined in a clinical setting . therefore , the present study was designed as an investigator - blinded , controlled , microbiological study to compare the efficacy of microwave and uv light in decreasing toothbrush bacterial contamination . thirty male dental graduates residing in a common hostel ( with a common source of water for daily use ) , with at least 28 teeth and age ranging from 22 to 28 years ( mean age 25 2.6 years ) were enrolled into this study . inclusion criteria included subjects in good general health , who were able to give informed consent and comply with the study protocol , having at least 14 natural teeth per arch , and brushing their teeth twice daily . exclusion criteria included the clinical evidence of gross caries or periodontal disease , the presence of systemic diseases or conditions that would affect the oral cavity such as uncontrolled diabetes mellitus , use of any medications associated with xerostomia or any antibiotic therapy within 90 days prior to the start of the study protocol . subjects were randomly assigned to either control ( n = 10 ; group 3 ) or experimental ( n = 20 ) groups . experimental group comprised the microwave group ( n = 10 ; group 1 ) and the uv group ( n = 10 ; group 2 ) . before the start of the study , each subject was given a new , identical multitufted toothbrush with soft nylon tufts ( ajay quest toothbrush , raghav lifestyle products , new delhi , india ) with a tube of toothpaste ( colgate total toothpaste , colgate - palmolive india pvt . ltd . , two unused toothbrushes ( control ) were cultured to check for any microbial growth in packed toothbrushes before starting the study . furthermore , the toothbrush rinsing water from the common tank intended to be used was also subjected to a microbial check before the start of the study . the study was conducted in three phases that included contamination procedures including bacterial culture , sanitization procedures , and postsanitization evaluation . subjects were trained and/or instructed to use the standardized modified bass technique for brushing their teeth for 3 min , twice daily for 1 week . each subject was instructed to rinse the used toothbrush under running tap water without mechanical manipulation for at least 30 s. subjects were also instructed to keep their toothbrushes in their living room within the provided aerated box at room temperature . after 1 week of use , each toothbrush was collected in a sterile paper bag and sent to the central food technological research institute ( cftri ) laboratory , mysore , within 4 h after collection in the morning . various selective and nonselective media used in the study included trypticase soy agar for total counts , mitis salivarius agar for total streptococci , mitis salivarius agar with 2 iu / ml of bacitracin for mutans streptococci , macconkey agar with 1% lactosec for escherichia coli and other coliforms , and rogosa sl agar for lactobacilli . for bacterial extraction , the toothbrushes were individually placed in prelabeled , sterile , 50-ml centrifuge tubes containing 10 ml of the trypticase soy broth ( tsb ) to immerse the bristles , then vortexed vigorously for 1 min , squeezed against the side of the tube to drain , rinsed with 5 ml tsb , and drained again . a series of undiluted and 10-fold dilutions of each sample were prepared and plated onto the surface of selective and nonselective media . a duplicate series of plates was then incubated aerobically or anaerobically at 37c for 24 days , until colony formation was visible . the number of colonies , measured as colony - forming units ( cfus ) , was counted using a colony counter . a new set of standardized toothbrush was once again given to each subject and subjects were instructed to use it for one more week following the same instructions as given earlier . after 1-week usage , the toothbrushes were once again collected and subjected to sanitization procedures . the experimental group toothbrushes were randomly assigned to either the uv light or microwave group . toothbrushes from the uv light group were sanitized by placing the brush in the uv light toothbrush sanitizer ( violight tooth brush sanitizer , violight inc . , sanitization was carried out by placing the brush in the receptacle and exposing the head for 12 min to uv radiation ( manufacturer 's recommendation , 6 min ) . toothbrushes from the microwave group were sanitized by placing the brush in a microwave oven ( 2450 mhz ; kenstar microwave oven , kenstar kitchen appliances india limited , mumbai ) . the wet brush was placed on the revolving table along with a glass of distilled water to protect the magnetron . once again each toothbrush belonging to different groups was collected in a sterile paper bag and sent to the laboratory for further microbial culture and colony count procedure . data obtained for all the microbial counts were log transformed to normalize their distributions prior to analysis . logs of the total bacterial count ( log cfu ) after toothbrush contamination and decontamination were compared and analyzed using one - way anova , tukey 's post hoc procedure for multiple comparisons , and paired t - test for the effect of the pre and postsanitization procedure on the microbial count in specific groups . subjects were trained and/or instructed to use the standardized modified bass technique for brushing their teeth for 3 min , twice daily for 1 week . each subject was instructed to rinse the used toothbrush under running tap water without mechanical manipulation for at least 30 s. subjects were also instructed to keep their toothbrushes in their living room within the provided aerated box at room temperature . after 1 week of use , each toothbrush was collected in a sterile paper bag and sent to the central food technological research institute ( cftri ) laboratory , mysore , within 4 h after collection in the morning . a standard bacterial culture method was followed in the study . various selective and nonselective media used in the study included trypticase soy agar for total counts , mitis salivarius agar for total streptococci , mitis salivarius agar with 2 iu / ml of bacitracin for mutans streptococci , macconkey agar with 1% lactosec for escherichia coli and other coliforms , and rogosa sl agar for lactobacilli . for bacterial extraction , the toothbrushes were individually placed in prelabeled , sterile , 50-ml centrifuge tubes containing 10 ml of the trypticase soy broth ( tsb ) to immerse the bristles , then vortexed vigorously for 1 min , squeezed against the side of the tube to drain , rinsed with 5 ml tsb , and drained again . a series of undiluted and 10-fold dilutions of each sample were prepared and plated onto the surface of selective and nonselective media . a duplicate series of plates was then incubated aerobically or anaerobically at 37c for 24 days , until colony formation was visible . the number of colonies , measured as colony - forming units ( cfus ) , was counted using a colony counter . a new set of standardized toothbrush was once again given to each subject and subjects were instructed to use it for one more week following the same instructions as given earlier . after 1-week usage , the toothbrushes were once again collected and subjected to sanitization procedures . the experimental group toothbrushes were randomly assigned to either the uv light or microwave group . toothbrushes from the uv light group were sanitized by placing the brush in the uv light toothbrush sanitizer ( violight tooth brush sanitizer , violight inc . , sanitization was carried out by placing the brush in the receptacle and exposing the head for 12 min to uv radiation ( manufacturer 's recommendation , 6 min ) . toothbrushes from the microwave group were sanitized by placing the brush in a microwave oven ( 2450 mhz ; kenstar microwave oven , kenstar kitchen appliances india limited , mumbai ) . the wet brush was placed on the revolving table along with a glass of distilled water to protect the magnetron . once again each toothbrush belonging to different groups was collected in a sterile paper bag and sent to the laboratory for further microbial culture and colony count procedure . data obtained for all the microbial counts were log transformed to normalize their distributions prior to analysis . logs of the total bacterial count ( log cfu ) after toothbrush contamination and decontamination were compared and analyzed using one - way anova , tukey 's post hoc procedure for multiple comparisons , and paired t - test for the effect of the pre and postsanitization procedure on the microbial count in specific groups . thirty subjects with an age ranging from 22 to 28 years ( mean age 25 2.6 years ) were enrolled in this study . all the subjects were male and shared the common living environment ( common hostel ) for more than 4 years . all the subjects were able to return their toothbrushes on days 7 and 14 in sealed labeled bags as instructed . moreover , no bacterial growth was observed on culturing the rinsing water obtained from the common tank . the mean microbial growth on toothbrushes in terms of log cfu is shown in table 1 . descriptive statistics of the measured variables of the various groups one - way anova was used to test for differences in mean microbial cfu counts among three groups of toothbrushes sanitized by their respective methods . the mean microbial cfu count done before the sanitization procedure demonstrated no significant difference , f ( 2 , 27 ) = 0.344 , p = 0.712 , among the groups whereas the mean cfu count done after sanitization procedures differed significantly across the all the three groups , f ( 2 , 27 ) = 267.219 , p < 0.001 . tukey 's post hoc comparisons of the three groups after the sanitization procedure indicated that the microwave group ( m = 1.49 , 95% ci [ 1.2729 , 1.7071 ] ) gave a significantly lower cfu count than the uv radiation group ( m = 3.22 , 95% ci [ 2.93 , 3.50 ] , p < 0.001 ) and the control group ( m = 5.88 , 95% ci [ 5.49 , 6.26 ] , p < 0.001 ) . comparisons between the uv radiation group and the control groups also showed statistically significant reduction in the microbial cfu count at p < 0.001 [ table 2 , figure 1 ] . the mean difference is significant at the 0.05 level the paired t - test was also conducted to analyze the microbial cfu count difference between pre- and postsanitization procedures . the results of the paired t - test of the microwave group revealed that there were significant differences in log cfu counts , between the pre- ( m = 5.82 0.89 ) and postsanitization procedure ( m = 1.49 0.303 ) , with t ( 9 ) = 16.18 and p < 0.001 . similarly , the uv radiation group also showed significant differences with t ( 9 ) = 6.77 and p < 0.001 , between the pre- ( m = 5.53 0.974 ) and postsanitization procedures ( m = 3.22 0.40 ) whereas for the control group , the difference between pre- ( m = 5.79 0.68 ) and postsanitization procedures ( m = 5.8 0.54 ) was not statistically significant , with t ( 9 ) = -0.599 , p = 0.564 . the present study was undertaken to quantitatively analyze the microbial growth after toothbrush contamination and to compare the efficacy of two different sanitization techniques ( uv light and microwave irradiation ) after the contamination procedure . the result outcome revealed that the contaminated toothbrushes harbored an increased number of aerobic and facultative anaerobic bacteria species . this finding is in accordance with the results of previous studies that indicated that an actual risk of recolonization exists after each brushing . in the recent years , there has been an increasing interest in the interrelationship between contaminated toothbrushes and systemic reinfections . several studies have also stressed on the role of contaminated toothbrushes and its causation in systemic infections . in this regard , brook and gober suggested that contaminated toothbrushes contributed to the persistence of group a beta - hemolytic streptococci in the oropharynx and to the failure of penicillin therapy in some cases of pharyngotonsillitis . in another study , significant bacteremia has also been reported after tooth brushing , especially in patients with severe periodontitis . therefore , a concern has been raised that the microbial load on toothbrushes might have a significant impact in periodontal patients under therapy . discussions on the modern toothbrushes have suggested the problem of toothbrush construction as a factor of toothbrush contamination . the nylon , multitufted toothbrush has been cited for its design of tufts set too closely to accommodate easy cleaning . the filaments are collected into bundles , bent in half with a metal anchor in the center , and driven into premolded holes in the toothbrush head at a high speed . in toothbrushes , the bristles can harbor inherent microorganisms , further increasing the bacterial contamination . several studies have suggested the need for toothbrush disinfection to reduce the number of microorganisms on the bristles using different methods , including uv radiation , microwave irradiation , boiling water , and chemical agents , such as listerine , plax , cepacol , and chlorhexidine . in addition , some authors have also attempted to incorporate antimicrobial agents , such as silver , chlorhexidine , and others to the toothbrush bristles as a coating layer during the manufacturing process . despite evidence demonstrating that chemical rinses and dentifrices can reduce the total bacterial load on a toothbrush , these methods are not widely used . therefore , recently , devine et al . raised a need of disinfection methods that are rapidly effective , cost - effective , nontoxic , and can be easily implemented . the present study was mainly sought to compare two sanitization techniques used for the decontamination procedure . our result showed that there was a significant reduction in microbial contamination in both microwave and uv groups compared to the control group . furthermore , our results also showed a significant reduction in the microbial count in the microwave group when compared with the uv group . our results confirm the findings of chibebe et al . where previously contaminated toothbrushes when exposed to microwave irradiation at 2450 mhz were reported to get inactivated by the action of microwave . a possible explanation for the effect of microwave irradiation upon formed microbial assemblage on brushes can be validated by the fact that many bacterial species are responsible for biofilm formation on different surfaces like toothbrushes , and microwave irradiation can disrupt the biofilm structure . data suggest that the destruction of microorganisms by microwave is mainly due to a thermal effect of microwave exposure on the microorganism cellular content resulting in cell lysis . another general indication for heat damage is the cell membrane rupture resulting in a leakage of nucleic acid and protein from cells . in this context , some studies have reported that microwave - injured cells often release ninhydrin - positive material , purines , and pyrimidines into a suspension . the presence of these materials in a suspension , in previous studies , has demonstrated the possibility of damage to cells by microwave at the membrane level . in the present study , the uv radiation group showed a significant differential reduction in the microbial count compared to the control group . however , the microbial count did not significantly reduce as compared to the microwave irradiation group . although we exposed toothbrushes for 12 min to uv radiation ( 6 min , manufacturer 's specification ) , the result was not significant as compared to microwave irradiation . previous studies have revealed that the longer exposure to uv light is necessary to ensure a complete inactivation of all microorganisms . the long exposure of uv light inactivates microorganisms by damaging the dna and disrupting the chemical bonds that hold the atoms of dna together in the microorganism . if the damage is severe enough , the bacteria can not repair the damage and are inactivated . however , despite long exposure , a previous literature review has questioned the potential of low - intensity uv radiation in microbial deactivation , and the authors concluded that low - intensity uv rays are not effective against certain microbes and molds . furthermore , tightly packed bristles and other areas are not in direct exposure and have no chance of disinfection . in the present study , these factors might be the reason for the uv radiation to be less efficient in toothbrush sanitization compared to microwave irradiation . in contrast to our results , boylan et al . have reported that a uv light toothbrush holder can effectively reduce an average of 86% total cultivatable bacteria on a toothbrush compared to controls . our result is not in agreement with this result as our result showed only 42% reduction in the microbial count after the uv sanitization procedure . therefore , further microbial studies are required to verify the efficacy of the uv light toothbrush holder in the reduction of the microbial content from contaminated toothbrushes . in the present study , we instructed all the subjects to store their toothbrushes at room temperature in the provided aerated box when not in use . reported that the number of microorganisms in the toothbrushes kept under aerated conditions was lower than that in toothbrushes stored in plastic bags . several authors have reported that bacterial contamination can be reduced by washing toothbrushes after use , and drying under aerated conditions . therefore , as time increases between one tooth brushing and another , more microorganism development can occur in the toothbrushes stored in a wet / moisture environment . our study demonstrated that significant microbial colonization was present after 1 week of repeated use of toothbrushes . however , data suggest that the time necessary for colonization is contradictory varying from 1 to 30 days . according to cesco et al . , the colonization on toothbrushes by mutans streptococci occurs in a short time period , since after a single tooth brushing , they found the development of the microorganism in 24% of the cases . svanberg reported the presence of mutans streptococci on toothbrushes after 3 days and colonization by mutans streptococci was observed on bristles after five consecutive days of toothbrush use . our study also showed similar findings where cultivatable microorganisms were present on the bristles after a short - term ( 1 week ) usage with aerated storage conditions . our results suggested that microwave irradiation is the better option for the sanitization of tooth brushes as compared to uv radiation . however , further studies are required for determining the optimum temperature and duration for the complete eradication of the organisms and spores , thereby achieving sterilization instead of sanitization . moreover , the duration of uv sanitization also needs to be reassessed to achieve optimum results . our results clearly suggest that there is a definite contamination of the toothbrushes after use ; hence need arises for either improving sanitization measures or frequent changes of toothbrushes especially after any infections . the evidence presented in this study suggests that microwave irradiation is an effective disinfectant agent for the microbiota present on the toothbrushes . it may be an important and efficacious oral health measure not only for the debilitated but also for healthy individuals . further in vivo trials are anticipated on more specific bacteria and biofilm of the oral cavity . however , there seem to be good reasons for the daily use of a toothbrush sanitization even before the results of these further trials are available .

background : toothbrushes are rapidly contaminated with different microorganisms representing a possible cause of infection or reinfection especially in the periodontal patients under therapy . the purpose of this study was to evaluate the sanitization of toothbrushes previously contaminated by various oral microorganisms using a domestic microwave oven and commercial ultraviolet ( uv ) light toothbrush sanitizer.materials and methods : thirty male dental graduates were randomly assigned to control or experimental groups and received standardized toothbrushes for home use . each subject was instructed to use it with the standardized modified bass technique for 1 week and submit it to the investigator after use . collected toothbrushes were cultured and analyzed for the number of colony - forming units ( cfus ) . in the next phase , once again a new set of toothbrush was given to each subject and instructed to use it for one more week and follow the same instructions as given earlier . subsequently , the used toothbrushes were again collected and were sanitized by microwave irradiation , uv radiation , or were not sanitized ( control group ) . after the sanitization procedure , toothbrushes were again cultured for the number of cfus . the collected data of the presanitized and postsanitized cfu count were log transformed to normalize their distributions prior to analysis . furthermore , log cfu data were compared and analyzed by one - way anova , tukey 's post hoc procedure , and paired t - test for the difference in the mean at p<0.05.results : result showed that after the sanitization procedure , there was a significant ( p<0.001 ) reduction in microbial contamination in both microwave and uv group toothbrushes compared to control group toothbrushes whereas the microbial count in the microwave group was significantly less ( p<0.001 ) compared to the uv group.conclusions:the evidence presented in this study suggests that microwave irradiation is an effective disinfectant agent for bacteria and fungi on toothbrushes .

INTRODUCTION MATERIALS AND METHODS Contamination procedure Bacterial culture Sanitization procedure Postsanitization evaluation Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC3015928

superior mesenteric artery ( sma ) syndrome , also called wilkie 's syndrome , is a rare clinical phenomenon believed to be caused by compression of the third portion of the duodenum by the overlying superior mesenteric artery . an abnormally acute aorta - sma angle , or high retroperitoneal attachment of the ligament of treitz , or both , this phenomenon has been recognized as a named clinical entity after its original description in 1861 . the incidence of duodenal compression within the aorto - sma angle has been estimated to be as high as 0.3% from upper gastrointestinal barium swallow studies , but the incidence of clinically significant and appropriately confirmed disease has been estimated to be much lower ( range , 0.010.08% ) . acceptance of this cause as a clinically reversible pathology has been controversial , but recent evolution of the literature seems to be clarifying the issue . medically refractory cases have in the past been treated by open release of the ligament of treitz , allowing mobilization of the duodenum or by enteric bypass . as we move into the minimally invasive era , operative intervention for the disease can and , we describe laparoscopic duodenojejunostomy to successfully treat sma syndrome and review the literature on the subject . a 32-year - old woman presented with a 5-year history of vague epigastric pain that intermittently radiated to the back . she had at that time been found on 2 occasions to have elevated pancreatic enzymes and was treated conservatively for pancreatitis . extensive workup at that time including ct scan , mri , and ercp revealed no identifiable anatomic abnormalities . at the time of presentation to our institution , 3 years had passed , and the patient had persistent chronic pain and progressively worsening nausea . over the previous 6 months her epigastric pain increased with meals , and she subsequently developed profuse vomiting after large meals without antecedent symptoms . in an attempt to alleviate these symptoms , she would eat smaller meals and consume fewer liquids , which led to dehydration , fatigue , and constipation . a ct scan revealed a 4-cm dilated duodenal bulb with abrupt decompression to normal duodenum beyond the sma ( figure 1 ) . the distance between the sma and aorta at the level of the duodenal lumen was 7.2 mm . an upper gastrointestinal swallow study revealed a dilated proximal duodenum with an abrupt vertical cutoff ( figure 2 ) . during fluoroscopy , there was significant to - and - fro peristalsis of the second and third portions of the duodenum against an apparent obstruction , with small jets of contrast squirting through . after contrast went through , a narrow lumen at the point of obstruction with distal decompressed bowel could be seen ( figure 3 ) . the diagnosis of sma syndrome was assumed , and the patient was counseled on laparoscopic duodenojejunostomy . computed tomography of the abdomen revealed dilated proximal duodenum ( large white arrow ) , decompressed distal duodenum ( large black arrow ) , and narrow distance between the superior mesenteric artery and aorta ( small double arrow ) . contrast study before surgery demonstrating dilated proximal duodenum with a sharp vertical cutoff ( arrow ) . delayed images showing narrowed duodenal lumen at the level of obstruction ( large arrow ) with distal decompression ( small arrow ) . at operation , the patient was placed in the supine position with both arms tucked . insufflation was attained by using an insufflation needle ( ethicon pneumoneedle , ethicon endo - surgery inc . , a 10-mm optiview port ( ethicon non - bladed trocar 10/12 , ethicon endo - surgery , cincinnati , oh ) containing a 10-mm , 0-degree scope with the camera focused to the tip of the port was used to enter the abdomen under direct visualization . a 12-mm port was then placed in the left mid subcostal region with a 5-mm port between the two . the camera was then switched for a 10-mm , 30-degree lens , and the handle was attached to the robotic endoscopic positioning system for audio - activated control ( aesop , computer motion co. , sunnyvale , ca ) . omentum and transverse colon were retracted cephalad , revealing the large dilated duodenum bulging through a thin , attenuated mesocolon , which was incised to expose the surface of the duodenum . the ligament of treitz was identified , and the proximal jejunum was run approximately 20 cm to the first loop that could be brought easily to the dilated duodenum without tension . utilizing a 2 0 surgidac suture on the laparoscopic stitching device ( ussc endostitch 173016 , us surgical corp . , norwalk , ct ) , a running suture was placed to secure the 2 limbs of bowel and serve as the back row in preparation for a 2-layer , side - to - side anastomosis . the duodenum and jejunum were entered with the harmonic shears ( ultracision harmonic scalpel , ethicon endo - surgery , inc . , cincinnati , oh ) approximately 3 mm above the posterior row , and the stapling device ( 2.5 mm/45 mm ) was deployed . the defect was closed with running absorbable suture using the suture device to complete the inner layer of the anastomosis . the outer layer was then completed with a running nonabsorbable stitch joining the ends of the originally placed posterior row . one intraperitoneal drain was left at the anastomosis and brought out through the right subcostal port site . a swallow study performed on postoperative day 1 revealed no evidence of leakage or stenosis , and the patient was started on a clear liquid diet and advanced to pureed foods over the next 2 days prior to discharge on day 3 . on follow - up a 32-year - old woman presented with a 5-year history of vague epigastric pain that intermittently radiated to the back . she had at that time been found on 2 occasions to have elevated pancreatic enzymes and was treated conservatively for pancreatitis . extensive workup at that time including ct scan , mri , and ercp revealed no identifiable anatomic abnormalities . at the time of presentation to our institution , 3 years had passed , and the patient had persistent chronic pain and progressively worsening nausea . over the previous 6 months her epigastric pain increased with meals , and she subsequently developed profuse vomiting after large meals without antecedent symptoms . in an attempt to alleviate these symptoms , she would eat smaller meals and consume fewer liquids , which led to dehydration , fatigue , and constipation . a ct scan revealed a 4-cm dilated duodenal bulb with abrupt decompression to normal duodenum beyond the sma ( figure 1 ) . the distance between the sma and aorta at the level of the duodenal lumen was 7.2 mm . an upper gastrointestinal swallow study revealed a dilated proximal duodenum with an abrupt vertical cutoff ( figure 2 ) . during fluoroscopy , there was significant to - and - fro peristalsis of the second and third portions of the duodenum against an apparent obstruction , with small jets of contrast squirting through . after contrast went through , a narrow lumen at the point of obstruction with distal decompressed bowel could be seen ( figure 3 ) . the diagnosis of sma syndrome was assumed , and the patient was counseled on laparoscopic duodenojejunostomy . computed tomography of the abdomen revealed dilated proximal duodenum ( large white arrow ) , decompressed distal duodenum ( large black arrow ) , and narrow distance between the superior mesenteric artery and aorta ( small double arrow ) . contrast study before surgery demonstrating dilated proximal duodenum with a sharp vertical cutoff ( arrow ) . delayed images showing narrowed duodenal lumen at the level of obstruction ( large arrow ) with distal decompression ( small arrow ) . at operation , the patient was placed in the supine position with both arms tucked . insufflation was attained by using an insufflation needle ( ethicon pneumoneedle , ethicon endo - surgery inc . , a 10-mm optiview port ( ethicon non - bladed trocar 10/12 , ethicon endo - surgery , cincinnati , oh ) containing a 10-mm , 0-degree scope with the camera focused to the tip of the port was used to enter the abdomen under direct visualization . a 12-mm port was then placed in the left mid subcostal region with a 5-mm port between the two . the camera was then switched for a 10-mm , 30-degree lens , and the handle was attached to the robotic endoscopic positioning system for audio - activated control ( aesop , computer motion co. , sunnyvale , ca ) . omentum and transverse colon were retracted cephalad , revealing the large dilated duodenum bulging through a thin , attenuated mesocolon , which was incised to expose the surface of the duodenum . the ligament of treitz was identified , and the proximal jejunum was run approximately 20 cm to the first loop that could be brought easily to the dilated duodenum without tension . utilizing a 2 0 surgidac suture on the laparoscopic stitching device ( ussc endostitch 173016 , us surgical corp . , norwalk , ct ) , a running suture was placed to secure the 2 limbs of bowel and serve as the back row in preparation for a 2-layer , side - to - side anastomosis . the duodenum and jejunum were entered with the harmonic shears ( ultracision harmonic scalpel , ethicon endo - surgery , inc . , cincinnati , oh ) approximately 3 mm above the posterior row , and the stapling device ( 2.5 mm/45 mm ) was deployed . the defect was closed with running absorbable suture using the suture device to complete the inner layer of the anastomosis . the outer layer was then completed with a running nonabsorbable stitch joining the ends of the originally placed posterior row . one intraperitoneal drain was left at the anastomosis and brought out through the right subcostal port site . a swallow study performed on postoperative day 1 revealed no evidence of leakage or stenosis , and the patient was started on a clear liquid diet and advanced to pureed foods over the next 2 days prior to discharge on day 3 . on follow - up , the patient denies pain and nausea and enjoys a regular diet without symptoms . the abnormally narrow duodenal lumen as it passes under the sma may be secondary to an acute angle between the sma and aorta or a high - riding ligament of treitz wedging the intersecting segment of duodenum tight into the angle . the angle is between 38 degrees and 65 degrees in normal individuals , but may be well less than 10 degrees in patients with sma syndrome . expressed another way , the distance between the vessels at the level of the duodenum is normally between 10 mm to 28 mm , whereas patients with symptomatic compression have been found to have a mean distance of 6 mm . excessive wasting of retroperitoneal fat has also been attributed to the development of this rare syndrome . reports of abnormally low body fat causing sma syndrome need to be viewed with caution given that the gastrointestinal difficulties of these patients leading to weight loss could blur the cause - effect relationship . if dangerously low body weight were a primary cause , it could be plausibly explained by decreased retroperitoneal fat allowing the small bowel and sma to lie more posterior , narrowing the vascular angle . reported cases of successfully reversing symptoms with aggressive caloric augmentation have been attributed to reversing this variable and simultaneously lend credence to the notion that in fact low body weight may be a primary cause . the vascular angle may be iatrogenically narrowed after an operation that fixes the sma into a more posterior position , and this syndrome is reported after ileo - anal anastomosis . expectedly , because a normal , functional intersection between the duodenum and the vascular angle is dependent upon normal anatomy in 3 dimensions , spinal deformities have been associated with the disease . particularly , surgical correction of congenital spinal deformities alters the natural position of the aorta via iatrogenic hyperlordosis , thereby narrowing the angle . recent epidemiologic evidence analyzing patients who underwent posterior spinal fusion for scoliosis identified those patients whose weight percentile for height is under 5% to be at risk for developing postoperative sma syndrome . paralysis and full - body casts have been associated with the development of acute sma syndrome in previously asymptomatic patients . casted / paralyzed patients may experience duodenal compression by the overlying sma when they suddenly are subjected to prolonged supine positioning . this notion is supported by the development of sma syndrome in other types of patients subjected to prolonged supine periods , such as trauma and burn patients . further , immobilized patients with newly diagnosed sma syndrome have been successfully treated by simply altering positions . patients with idiopathic sma syndrome also experience relief from postural changes , although this will not alleviate the disease . however , as many as 40% of patients have no apparent explainable cause , similar to the case described here . in these patients , it is unclear why onset is delayed until early adulthood without evidence of an anatomy - altering event . the great majority of patients will present before age 50 . perhaps natural changes in the gi tract and body habitus with time produce the syndrome in patients fated to have it . familial cases have been reported including a recent report of monozygotic twins who developed idiopathic sma syndrome at ages 28 and 29 . diagnosis currently rests mostly on upper gastrointestinal series and computed tomographic ( ct ) scans . features of sma syndrome on upper gastrointestinal series are a dilated proximal duodenum and vertical or oblique compression of the third portion of the duodenum . flouroscopy during the swallow study was highly suggestive in this case with a to - and - fro pattern of peristalsis proximal to the obstruction and only small jets of contrast squirting beyond the obstruction . this specific fluoroscopic pattern has been previously described with sma syndrome , and it appears to offer a strong indication for the presence of the disease . historically , angiography was recommended . however , it currently offers little information that can not be obtained by ct , it poses significantly greater risks , and it was unnecessary in this case . with appropriate contrast , the ct angiography can be generated in difficult cases with coronal and sagittal reconstruction showing the precise anatomic associations with the vascular angle , the measurement of this angle , and the distance between the vessels . in our case , abdominal ct with intravenous contrast and upper gastrointestinal series were the only studies required for diagnosis . although endoscopy is of minor positive diagnostic value , we feel it is mandatory in all patients to rule out intraluminal pathology before either making the diagnosis or initiating treatment . management of sma syndrome can be initially conservative with nasogastric decompression , intravenous rehydration , and aggressive nutritional support . symptoms in immobilized patients first should be considered related to position and treated accordingly . in idiopathic cases similar to ours of patients who are functioning outside of the hospital but are symptomatic , nonoperative options for definitive treatment are not very realistic , and upon confirming the diagnosis , we feel it is appropriate to discuss surgical options . current surgical options for the disease involve 2 separate concepts : bypass of the obstruction or lysis of the ligament of treitz . liberation of the ligament of treitz has been historically used with success in small numbers of patients . while this operation is advantageous because enterotomy is avoided , the largest comparative study between the 2 operations demonstrated that 21% of patients failed to respond to lysis of the ligament , whereas all patients treated with bypass experienced resolution of symptoms . recurrence after ligament liberation occurs simply as a result of postoperative adhesions tethering the bowel into a similar position as the ligament of treitz had done previously . recurrence has been described after duodenojejunostomy when the entire anastomosis migrated under the sma recreating the obstruction , obviously an unusual event . since the introduction of minimally invasive surgery , the results of the open era have thus far been replicated with laparoscopy , albeit in smaller numbers . laparoscopic lysis of the ligament has been successful in 3 of 4 cases ( 75% ) . there have now been multiple published cases of laparoscopic bypass that have been performed with sucess . our experience reinforces the idea that laparoscopic duodenojejunostomy should be offered to patients with a diagnosis of sma syndrome early in the course , because conservative management is unlikely to provide long - term satisfaction . sma syndrome is a real anatomic clinical pathology resulting in chronic , consistent obstructive symptoms . laparoscopic duodenojejunostomy should be considered the treatment of choice for these patients because it offers a high likelihood of an excellent outcome based on the current literature .

background : superior mesenteric artery ( sma ) syndrome , also called wilkie 's syndrome , is a rare clinical phenomenon believed to be caused by compression of the third portion of the duodenum by the overlying superior mesenteric artery . we present the case of a 32-year - old female who presented with epigastric pain , weight loss , and vomiting.methods:her workup included a normal upper endoscopy as well as an abdominal ct scan and upper gi contrast study that confirmed the diagnosis of superior mesenteric artery syndrome . the patient was taken to the operating room and underwent successful treatment with laparoscopic duodenojejunostomy.results:the patient achieved complete relief of her symptoms and is able to eat a regular diet without difficulty . sma syndrome is a real anatomic clinical pathology resulting in chronic , consistent obstructive symptoms . an upper gi series and ct scan with contrast can confirm the diagnosis.conclusion:laparoscopic duodenojejunostomy should be considered the treatment of choice for these patients , because it offers a high likelihood of excellent outcome based on the current literature .

INTRODUCTION CASE REPORT Presentation Operation Outcome DISCUSSION CONCLUSION

PMC4376467

the accuracy of the measurement of the higgs - boson mass by the atlas and cms collaborations at the large hadron collider ( lhc ) has already reached the level of 300400 mev [ 1 , 2 ] and , being still dominated by statistics , is bound to improve further when the lhc restarts operations in 2015 . this puts new emphasis on the need for high - precision calculations in those extensions of the standard model ( sm ) , such as the minimal supersymmetric standard model ( mssm ) , in which the higgs - boson mass can be predicted as a function of other physical observables . the higgs sector of the mssm consists of two \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$su(2)$$\end{document}su(2 ) doublets , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_1$$\end{document}h1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_2$$\end{document}h2 , whose relative contribution to electroweak ( ew ) symmetry breaking is determined by the ratio of vacuum expectation values ( vevs ) of their neutral components , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta \equiv v_2/v_1$$\end{document}tanv2/v1 . the spectrum of physical higgs bosons is richer than in the sm , consisting of two neutral scalars , \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 \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 , one neutral pseudoscalar , \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 , and two charged scalars , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h^\pm $ $ \end{document}h. at the tree level , the neutral scalar masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh and the scalar mixing angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} can be computed in terms 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 - 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like couplings to fermions and gauge bosons , in which case the tree - level bound on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh has long been disproved by the lep [ 3 , 4 ] . however , radiative corrections can raise the prediction for the lightest - scalar mass up to the value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}\approx 125$$\end{document}mh125 gev observed at the lhc , and they bring along a dependence on all mssm parameters . among the latter , particularly relevant are the masses and mixing of the scalar partners of the third - generation quarks , the stop and sbottom squarks . due to the crucial role of radiative corrections in pushing the prediction for the lightest - scalar mass above the tree - level bound , an impressive theoretical effort has been devoted over more than 20 years to the precise determination of the higgs sector of the mssm.1 after the early realization [ 59 ] of the importance of the one - loop \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t})$$\end{document}o(t ) corrections2 involving top and stop , full one - loop computations of the mssm higgs masses have been provided [ 1013 ] , leading logarithmic effects at two loops have been included via renormalization - group methods [ 1417 ] , and genuine two - loop corrections of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) [ 1825 ] , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}^2)$$\end{document}o(t2 ) [ 18 , 24 , 26 ] , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { b}\alpha _ { s})$$\end{document}o(bs ) [ 27 , 28 ] and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { b}+ \alpha _ { b}^2)$$\end{document}o(tb+b2 ) have been evaluated in the limit of vanishing external momentum in the higgs self - energies . all of these corrections have been implemented in widely used computer codes for the calculation of the mssm mass spectrum , such as feynhiggs , softsusy [ 31 , 32 ] , suspect and spheno [ 34 , 35 ] . furthermore , a complete two - loop calculation of the mssm higgs masses in the effective potential approach ( i.e. , at zero external momentum ) , including also two - loop corrections controlled by the ew gauge couplings , has been presented in refs . [ 36 , 37 ] . some of the dominant three - loop corrections to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh have also been obtained , both via renormalization - group methods [ 3840 ] and by explicit calculation of the higgs self - energy at zero external momentum [ 41 , 42 ] . already at the two - loop level , going beyond the approximation of zero external momentum brings significant complications to the calculation of the higgs self - energies . different algorithms for expressing all two - loop self - energy integrals with arbitrary external momentum in terms of a minimal set of master integrals ( mis ) were developed in refs . however , explicit analytical formulas for the mis can be derived only for special values of the masses of the particles circulating in the loops , whereas in the general case a numerical calculation becomes unavoidable . a method to compute all the mis of ref . by numerically solving a system of differential equations in the external momentum [ 4851 ] . a library of routines for the computation of the mis with the method of ref . a calculation of the two - loop contributions to the higgs self - energies involving the strong gauge coupling or the third - family yukawa couplings , based on the methods of refs . that calculation goes beyond the two - loop results implemented in public codes [ 1921 , 2529 ] in that it includes external - momentum effects , as well as contributions involving the d - term - induced ew interactions between higgs bosons and sfermions . when combined with the effective - potential results of refs . provide an almost - complete two - loop calculation of the higgs masses in the mssm the only missing contributions being external - momentum effects that involve only the ew gauge couplings . however , no code for the calculation of the mssm mass spectrum implementing the results of refs . [ 36 , 37 , 53 ] was ever made available , and the way those results are organized does not lend itself to a straightforward implementation in the existing public codes . on one hand , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization scheme adopted in refs . [ 36 , 37 , 53 ] for the parameters of the mssm lagrangian does not match the mixed on - shell ( os)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme adopted in feynhiggs . on the other hand , implementation of the results of refs . [ 36 , 37 , 53 ] in softsusy , suspect and spheno , which also adopt the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , is complicated by the fact that in refs . [ 36 , 37 , 53 ] the running masses of the higgs bosons entering the loop corrections are defined by the second derivatives of the tree - level potential . while this choice amounts to a legitimate reshuffling of terms between different perturbative orders , it restricts the applicability of the calculation to rather specific ranges of renormalization scale where none of the running higgs masses as defined in refs . [ 36 , 37 , 53 ] is tachyonic . perhaps as a consequence of these complications , a full decade after the publication of ref . its results have yet to be included in phenomenological analyses of the mssm higgs sector . in this paper we present a new calculation of the momentum - dependent part of the two - loop corrections to the neutral higgs masses of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) , i.e. those involving both the top yukawa coupling and the strong gauge coupling . we also compute mixed two - loop corrections that we denote by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) : they involve both the strong gauge coupling and the ew gauge couplings , under the approximation that the only non - vanishing yukawa coupling is the top one . it is natural to consider these two classes of corrections together , because in both of them the dominant terms affecting the lightest - scalar mass are expected to be suppressed by 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}$$\mathcal{o}(m_z^2/{{m}_{t}}^2)$$\end{document}o(mz2/mt2 ) with respect to the zero - momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections ( in practice , we find that both classes of corrections are considerably more suppressed than that , but still comparable to each other in size ) . in our calculation we rely on the integration - by - parts ( ibp ) technique of refs . [ 54 , 55 ] to express the momentum - dependent loop integrals in terms of the mis of ref . , which we evaluate by means of the package tsil . we obtain results for both the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr and the os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization schemes , organized in such a way that they can be directly implemented in the existing codes for the computation of the mssm mass spectrum . we verify that our results are in full agreement with the ones of ref . where they overlap . after describing our calculation in some detail , we briefly discuss the numerical impact of the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to the higgs masses in a set of representative points in the mssm parameter space . while our paper was in preparation , an independent calculation of the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the neutral higgs masses in the mssm appeared , relying on the results of ref . for the decomposition of two - loop integrals into mis and on the package secdec [ 57 , 58 ] for the numerical evaluation of the latter . the results of that calculation are expressed in the os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , and they have been implemented in the latest version of feynhiggs . although we have verified that our results for the contributions of genuine two - loop diagrams involving the strong - gauge and top - yukawa couplings agree numerically with those of ref . , we do not reproduce the overall values of the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the higgs masses . we trace the reason for the discrepancy to an inconsistency in ref . concerning the definitions of the wave - function - renormalization ( wfr ) constants for the higgs fields and of the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. we outline here our calculation of the two - loop corrections to the masses of the neutral higgs bosons in the mssm with real parameters ( we do not consider the possibility of cp violation in the higgs sector ) . we decompose the neutral components of the two higgs doublets into their vevs plus their cp - even and cp - odd fluctuations as follows:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&h_1 ^ 0 \equiv \frac{1}{\sqrt{2}}\,(v_1 + s_1 + i \ , p_1 ) , \nonumber \\&h_2 ^ 0 \equiv \frac{1}{\sqrt{2}}\,(v_2 + s_2 + i \ , p_2 ) . \end{aligned}$$\end{document}h1012(v1+s1+ip1),h2012(v2+s2+ip2).the cp - odd components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_1$$\end{document}p1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_2$$\end{document}p2 combine into the pseudoscalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a and the neutral would - be goldstone boson \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^0$$\end{document}g0 . the cp - even components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_1$$\end{document}s1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_2$$\end{document}s2 combine into the scalars \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h . the squared physical masses of the latter are the two solutions for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2$$\end{document}p2 of the equation2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathrm{det } \left [ \gamma _ s(p^2 ) \right ] = 0 , \end{aligned}$$\end{document}dets(p2)=0,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ s(p^2)$$\end{document}s(p2 ) denotes the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\times 2$$\end{document}22 inverse - propagator matrix in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(s_1,s_2)$$\end{document}(s1,s2 ) basis , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p$$\end{document}p being the external momentum flowing into the scalar self - energies . we can decompose \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ s(p^2)$$\end{document}s(p2 ) as3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \gamma _ s(p^2 ) = p^2 - \mathcal{m}_0 ^ 2 - \delta \mathcal{m}^2(p^2 ) , \end{aligned}$$\end{document}s(p2)=p2-m02-m2(p2),where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 denotes the tree - level mass matrix written in terms of renormalized parameters , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2 ) collectively denotes the radiative corrections . at each order in the perturbative expansion , the latter include both the contributions of one - particle - irreducible ( 1pi ) diagrams and non-1pi counterterm contributions arising from the renormalization of parameters that enter the lower - order parts of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ s(p^2)$$\end{document}s(p2 ) . we express \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 in terms of the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan renormalized in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , and of the physical masses of the pseudoscalar and 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 boson4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathcal{m}_0 ^ 2 = \left ( \begin{array}{cc } c^2_\beta \,m_z^2 + s^2_\beta \,m_a^2 & { } \quad - { { s}_{\beta } } \ , { { c}_{\beta } } \left ( m_z^2 + m_a^2 \right ) \\ - { { s}_{\beta } } \ , { { c}_{\beta } } \left ( m_z^2 + m_a^2\right ) & { } \quad s^2_\beta \ , m_z^2 + c^2_\beta \ , m_a^2 \end{array}\right ) , \end{aligned}$$\end{document}m02=c2mz2+s2ma2-scmz2+ma2-scmz2+ma2s2mz2+c2ma2,using ( here and thereafter ) the shortcuts \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_\phi \equiv \cos \phi $ $ \end{document}ccos and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_\phi \equiv \sin \phi $ $ \end{document}ssin for a generic angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi $ $ \end{document}. neglecting terms that do not contribute at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) , our choices for the parameters entering \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 lead to the following expressions for the two - loop parts of the individual entries of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2):5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{11}&= s_\beta ^2\ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + c_\beta ^2\,\mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\nonumber \\&-\pi _ { 11}^{(2)}(p^2 ) -\delta \mathcal { z}_1^{(2 ) } \left ( p^2- c^2_\beta \ , m_z^2 - s^2_\beta \,m_a^2\right ) \nonumber \\&+ \left ( 1- { { s}_{\beta } } ^4\right ) \frac{~\,t_1^{(2)}}{v_1 } - s_\beta ^2 c_\beta ^2 \frac{~\,t_2^{(2)}}{v_2}\nonumber \\&-2\,s_\beta ^2 c_\beta ^2\ , \left ( m_z^2-m_a^2\right ) \frac{~\delta \tan \beta ^{(2)}}{\tan \beta } , \end{aligned}$$\end{document}m2(p2)11(2)=s2reaa(2)(ma2)+c2rezz(2)(mz2)-11(2)(p2)-z1(2)p2-c2mz2-s2ma2 + 1-s4t1(2)v1-s2c2t2(2)v2 - 2s2c2mz2-ma2tan(2)tan,6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{12}&= - { { s}_{\beta } } { { c}_{\beta } } \left [ \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + \mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\right . s_\beta ^2 \frac{~\,t_1^{(2)}}{v_1 } - c_\beta ^2 \frac{~\,t_2^{(2)}}{v_2 } \right ] -\pi _ { 12}^{(2)}(p^2)\nonumber \\&-\frac{1}{2}{{s}_{\beta } } { { c}_{\beta } } \left ( m_z^2 + m_a^2\right ) \nonumber \\&\times \left [ 2\ , ( c_\beta ^2-s_\beta ^2)\frac{~\delta \tan \beta ^{(2)}}{\tan \beta } \!+\ ! \delta \mathcal { z}_1^{(2)}\!+\!\delta \mathcal { z}_2^{(2)}\!\right ] , \nonumber \\ \end{aligned}$$\end{document}m2(p2)12(2)=-screaa(2)(ma2)+rezz(2)(mz2)-s2t1(2)v1-c2t2(2)v2-12(2)(p2)-12scmz2+ma22(c2-s2)tan(2)tan+z1(2)+z2(2),7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{22}&= c_\beta ^2\ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + s_\beta ^2 \ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\nonumber \\&-\pi _ { 22}^{(2)}(p^2 ) -\delta \mathcal { z}_2^{(2 ) } \left ( p^2- s^2_\beta \,m_z^2 - c^2_\beta \ , s_\beta ^2 c_\beta ^2 \frac{~\,t_1^{(2)}}{v_1 } + \left ( 1- { { c}_{\beta } } ^4\right ) \frac{~\,t_2^{(2)}}{v_2}\nonumber \\&+2\,s_\beta ^2c_\beta ^2\ , \left ( m_z^2-m_a^2\right ) \frac{~\delta \tan \beta ^{(2)}}{\tan \beta } . \end{aligned}$$\end{document}m2(p2)22(2)=c2reaa(2)(ma2)+s2rezz(2)(mz2)-22(2)(p2)-z2(2)p2-s2mz2-c2ma2-s2c2t1(2)v1 + 1-c4t2(2)v2 + 2s2c2mz2-ma2tan(2)tan.in the equations above , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t^{(2)}_i$$\end{document}ti(2 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi ^{(2)}_{ij}$$\end{document}ij(2 ) ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i , j=1,2$$\end{document}i , j=1,2 ) denote the two - loop parts of tadpoles and self - energies , respectively , for the scalars \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_i$$\end{document}si , while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi ^{(2)}_{{\scriptscriptstyle a}{\scriptscriptstyle a}}$$\end{document}aa(2 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi ^{(2)}_{{\scriptscriptstyle z}{\scriptscriptstyle z}}$$\end{document}zz(2 ) denote the two - loop parts of the pseudoscalar and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - boson self - energies . in addition , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i=1,2$$\end{document}i=1,2 ) in eqs . ( 5)(7 ) denote the two - loop parts of the wfr counterterms for the higgs fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h^0_i$$\end{document}hi0 , which we renormalize as follows:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } h^0_i ~\longrightarrow ~\sqrt{\mathcal{z}_i}\,h^0_i ~\simeq ~ \left ( 1 + \frac{1}{2}\,\delta \mathcal{z}^{(1)}_i + \frac{1}{2}\,\delta \mathcal{z}^{(2)}_i \right ) \,h^0_i , \end{aligned}$$\end{document}hi0zihi01 + 12zi(1)+12zi(2)hi0,where in the expansion of the square root we have again neglected terms that do not contribute at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) . we adopt a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{z}_i$$\end{document}zi , which can then be determined from the anomalous dimensions of the higgs fields and from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} functions of the couplings entering the anomalous dimensions . taking from the general formulas of refs . [ 59 , 60 ] only the terms relevant to our approximation , we get9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta \mathcal{z}^{(1)}_1&= \delta \mathcal{z}^{(2)}_1= 0,\quad \delta \mathcal{z}^{(1)}_2 = - \frac{\alpha _ { t}}{4\pi } \,n_c\,\cdot \,\frac{1}{\epsilon } , \nonumber \\ \delta \mathcal{z}^{(2)}_2&= \frac{\alpha _ { t}\alpha _ { s}}{(4\pi ) ^2}\,2\,n_c\,c_f\cdot \left ( \frac{1}{\epsilon ^2}-\frac{1}{\epsilon } \right ) , \end{aligned}$$\end{document}z1(1)=z1(2)=0,z2(1)=-t4nc1,z2(2)=ts(4)22nccf12 - 1,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_c=3$$\end{document}nc=3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_f=4/3$$\end{document}cf=4/3 are color factors , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon = ( 4-d)/2\,$$\end{document}=(4-d)/2 in dimensional reduction , and the coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ { t}$$\end{document}t entering the one - loop counterterm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{z}^{(1)}_2$$\end{document}z2(1 ) is in turn renormalized in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . finally , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta ^{(2)}$$\end{document}tan(2 ) in eqs . ( 5)(7 ) denotes the two - loop part of the counterterm for the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. the choice of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan implies that , in our approximation , its counterterm can be expressed via the wfr counterterms:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{~\delta \tan \beta ^{(\ell ) } } { \tan \beta } = \frac{1}{2}\,\left ( \delta \mathcal{z}^{(\ell ) } _ 2 -\delta \mathcal{z}^{(\ell ) } _ ( 5)(7 ) include both 1pi two - loop contributions and non-1pi contributions arising from the renormalization of the parameters entering their one - loop counterparts . since we are focusing on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to the higgs masses , we need to introduce counterterms only for the parameters that are subject to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { s})$$\end{document}o(s ) corrections , namely the top mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_t$$\end{document}mt , the stop masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{\tilde{t}_{1}}$$\end{document}mt~1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{\tilde{t}_{2}}$$\end{document}mt~2 , the stop mixing angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta _ t$$\end{document}t , the soft supersymmetry - breaking higgs - stop coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_t$$\end{document}at and the masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{\tilde{q}_i}$$\end{document}mq~i of all squarks other than the stops . the latter enter the one - loop tadpoles and self - energies of the higgs bosons via d - term - induced ew couplings , and the one - loop self - energy of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z boson via the gauge interaction . in our calculation we neglect all yukawa couplings ( and hence quark masses ) other than the top one,3 therefore none of the other squarks mix . we obtained results for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) contributions to tadpoles and self - energies assuming that the relevant quark / squark parameters are renormalized either in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr or in the os scheme . formulas for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dros shift of the parameters in the top / stop sector can be found , e.g. , in appendix b of ref . , while the shifts for the remaining squark masses can be obtained by setting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{t}}=\theta _ t=0$$\end{document}mt=t=0 in the corresponding formulas for the stop masses . ( 5)(7 ) are constructed to give finite entries in the inverse - propagator matrix of the scalars . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1/\epsilon ^2$$\end{document}1/2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1/\epsilon $ $ \end{document}1/ poles in the right - hand sides of eqs . ( 5)(7 ) cancel out . in principle , the two - loop contributions to the higgs inverse propagator given in eqs . ( 5)(7 ) must be combined with a full calculation of the corresponding one - loop contributions , and used to determine the physical higgs masses by solving directly eq . however , as will be discussed in the next section , the computing times required for the evaluation of momentum - dependent two - loop integrals are not negligible . a numerical search for the solutions of eq . ( 2 ) could significantly slow down the codes for the calculation of the higgs masses , making them unsuitable for extensive phenomenological analyses of the mssm parameter space . it is therefore convenient to compute the higgs masses in two steps , with a procedure similar to the one discussed in refs . ( 2 ) including in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2 ) the full one - loop corrections plus the dominant two - loop corrections of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { b}\alpha _ { s})$$\end{document}o(bs ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{o}(\alpha _ { t}^2+\alpha _ { t}\alpha _ { b}+\alpha _ { b}^2)$$\end{document}o(t2+tb+b2 ) computed in the approximation of vanishing external momentum . from feynhiggs we obtain the scalar masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_h^2$$\end{document}mh2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_{{\scriptscriptstyle h}}^2$$\end{document}mh2 , and an effective mixing angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document} which diagonalizes the loop - corrected scalar mass matrix at vanishing external momentum . our full results for the scalar masses are then obtained by adding to the results of feynhiggs the momentum - dependent parts of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections:11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } m^2_{h,{\scriptscriptstyle h } } = \overline{m}^2_{h,{\scriptscriptstyle h } } ~+~ ( \delta m^2_{h,{\scriptscriptstyle h}})^{\alpha _ { t}\alpha _ { s},\,p^2 } ~+~ ( \delta m^2_{h,{\scriptscriptstyle h}})^{\alpha \alpha _ { s}}. \end{aligned}$$\end{document}mh , h2=mh , h2+(mh , h2)ts , p2+(mh , h2)s.concerning the former , we have12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left ( \delta m^2_{h}\right ) ^{\alpha _ { t}\alpha _ { s},\,p^2}&= c^2_{\beta -\overline{\alpha } } \,\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}\left ( m_a^2\right ) - s^2_{\overline{\alpha } } \ , \delta \pi _ { 11}^{(2)}\left ( \overline{m}_h^2\right ) \nonumber \\&\quad + s_{2\overline{\alpha } } \,\delta \pi _ { 12}^{(2)}\left ( \overline{m}_h^2\right ) - c^2_{\overline{\alpha } } \,\delta \pi _ { 22}^{(2)}\left ( \overline{m}_h^2\right ) , \end{aligned}$$\end{document}mh2ts , p2=c-2aa(2)ma2-s211(2)mh2+s212(2)mh2-c222(2)mh2,13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left ( \delta m^2_{{\scriptscriptstyle h}}\right ) ^{\alpha _ { t}\alpha _ { s},\,p^2}&= s^2_{\beta -\overline{\alpha } } \,\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}\left ( m_a^2\right ) - c^2_{\overline{\alpha } } \ , \delta \pi _ { 11}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \nonumber \\&\quad - s_{2\overline{\alpha } } \,\delta \pi _ { 12}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) - s^2_{\overline{\alpha } } \,\delta \pi _ { 22}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) , \end{aligned}$$\end{document}mh2ts , p2=s-2aa(2)ma2-c211(2)mh2-s212(2)mh2-s222(2)mh2,where we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \pi ( p^2 ) \,\equiv \ , \pi ( p^2)-\pi ( 0)$$\end{document}(p2)(p2)-(0 ) , and retain only the real and finite part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) contributions to the two - loop self - energies . for what concerns the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) ( 5)(7):14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left ( \delta m^2_{h}\right ) ^{\alpha \alpha _ { s}}&= s^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{11}\nonumber \\&\quad - s_{2\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{12 } + c^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{22},\end{aligned}$$\end{document}mh2s = s2m2mh211s - s2m2mh212s+c2m2mh222s,15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left ( \delta m^2_{{\scriptscriptstyle h}}\right ) ^{\alpha \alpha _ { s}}&= c^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{11}\nonumber \\&\quad + \ , s_{2\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{12 } + s^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{22 } , \end{aligned}$$\end{document}mh2s = c2m2mh211s+s2m2mh212s+s2m2mh222s , where again we take the real part of all two - loop self - energies . . proposes an alternative two - step procedure to include the momentum - dependent parts of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections in feynhiggs , differing from the one outlined above only by higher - order effects . finally , a comment is in order about the dependence of the corrections to the higgs masses on the wfr constants.4 in principle , the predictions for the physical higgs masses at a given order in the perturbative expansion should not depend directly on the wfr constants ( although they could still depend indirectly on them via the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan counterterm ) . indeed , if the two - loop contributions to the inverse - propagator matrix are computed with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2$$\end{document}p2 equal to the tree - level scalar masses and then rotated to the mass - eigenstate basis via the tree - level mixing angle , so that the computation is performed strictly at the two - loop level , the terms in eqs . ( 5)(7 ) that depend explicitly on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) drop out of the mass corrections \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m^2_{h,{\scriptscriptstyle h}}\,$$\end{document}mh , h2 . on the other hand , if the loop - corrected scalar masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_h^2$$\end{document}mh2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_{{\scriptscriptstyle h}}^2$$\end{document}mh2 and the effective mixing angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document} are used , as in eqs . ( 12)(15 ) above , or if the zeroes of the inverse - propagator matrix are determined numerically , the corrections to the scalar masses retain a dependence on the wfr counterterms . in our calculation we adopt a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for the wfr ; therefore the terms involving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) are purely divergent and cancel out against other divergent terms in the individual entries of the inverse - propagator matrix , hence they do not show up in eqs . if , however , one adopts a non - minimal definition of the wfr , higgs - mass corrections computed as in eqs . ( 14 ) and ( 15 ) will contain non - vanishing terms that depend on the finite part of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) . albeit formally of higher order in the perturbative expansion , these terms can be numerically relevant when the loop - corrected scalar masses differ significantly from their tree - level values ( as is the case for a sm - like scalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h with mass around 125 gev ) . the computation of the two - loop corrections to the neutral mssm higgs masses considered in this paper requires the knowledge of the tadpole and self - energy diagrams entering eqs . while the strategy for the computation in the zero - momentum approximation is well known , the evaluation of the self - energies with arbitrary external momentum is more involved . we illustrate in this section the details of our calculation , which we performed in a fully automated way . the relevant diagrams are generated with feynarts , using a modified version of the original mssm model file that implements the qcd interactions in the background field gauge . the diagrams contributing to the vacuum polarization of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z boson are contracted with a suitable projector in order to single out their transverse part . the color factors are simplified with a private package and the dirac algebra is handled by form . the computation is performed in dimensional reduction , which we can implement in this case by generating the diagrams in dimensional regularization and replacing , in each diagram involving an internal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d - dimensional gluon , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^{\mu \nu } \rightarrow g^{\mu \nu } + g^{\hat{\mu } \hat{\nu } } $ $ \end{document}gg+g^^ ( where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^{\hat{\mu } \hat{\nu } } $ $ \end{document}g^^ is the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\epsilon $ $ \end{document}2-dimensional metric tensor ) in order to include the corresponding \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon $ $ \end{document}-scalar contribution . we are then left with feynman integrals of the form16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int d^dk_1 d^dk_2 \frac{\left ( k_1 ^ 2\right ) ^\alpha \left ( k_2 ^ 2\right ) ^\beta ( k_1\cdot p)^\gamma ( k_2\cdot p)^\delta ( k_1\cdot k_2)^\eta } { d_1^{a_1 } d_2^{a_2 } d_3^{a_3 } d_4^{a_4 } d_5^{a_5 } } , \end{aligned}$$\end{document}ddk1ddk2k12k22(k1p)(k2p)(k1k2)d1a1d2a2d3a3d4a4d5a5,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha , \ldots , \eta , \ , a_1 , \ldots , a_5$$\end{document}, ,,a1, ,a5 are positive ( or zero ) integer exponents and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_i$$\end{document}di s are defined as\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } d_1&= k_1 ^ 2 - m_1 ^ 2 , \ , d_2 = ( k_1-p)^2 - m_2 ^ 2 , \ , d_3 = k_2 ^ 2 - m_3 ^ 2,\\ d_4&= ( k_2-p)^2 - m_4 ^ 2 , \ , d_5 = ( k_1-k_2)^2 - m_5 ^ 2 . \end{aligned}$$\end{document}d1=k12-m12,d2=(k1-p)2-m22,d3=k22-m32,d4=(k2-p)2-m42,d5=(k1-k2)2-m52.integrals belonging to the class above are in general not linearly independent of each other . when the scalar products in the numerator are expressed in terms of the denominators , powers of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_i$$\end{document}di present in the original integral might cancel against a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_i$$\end{document}di in the numerator , possibly generating a feynman integral in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_i$$\end{document}di does not appear , i.e. in which the corresponding line has been shrunk to a point . for given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_i$$\end{document}ai s and high enough \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha , \ldots , \eta $ $ \end{document}, , , some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_i$$\end{document}di s may acquire negative exponents . ( 16 ) therefore reduces to the evaluation of a number of integrals of the form17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int \frac{d^dk_1 d^dk_2}{d_1^{n_1 } d_2^{n_2 } d_3^{n_3 } d_4^{n_4 } d_5^{n_5 } } , \end{aligned}$$\end{document}ddk1ddk2d1n1d2n2d3n3d4n4d5n5,where the exponents \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_i\in \mathbb { z}$$\end{document}niz . in the present case , one has to evaluate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal { o}(300)$$\end{document}o(300 ) different feynman integrals . there exists a convenient procedure for dealing with such large numbers of different feynman integrals in a more efficient way than their direct evaluation . dimensionally regularized integrals , at arbitrary loop order and with arbitrary number of external legs , satisfy identities of the ibp type [ 54 , 55 ] . these identities are linear relations that connect integrals with different sets of exponents \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\{n_1,\ldots , n_5\}$$\end{document}{n1, ,n5}. after a set of independent integrals , the mis , has been identified , all the remaining integrals can then be reduced to linear combinations of the mis , the coefficients being rational functions of the masses , the kinematic invariants and the space - time dimension \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d . one practical advantage of such a procedure is its divide and conquer spirit . on the one hand few mis encode the analyticity properties ( singularities , thresholds , branch cuts ) of the problem under consideration . on the other hand , the evaluation of the large number of different feynman integrals entering a computation is reduced to a problem of linear algebra , which can easily be automated , if the mis are known . we perform the reduction to mis with the public code reduze [ 70 , 71 ] , which implements the laporta algorithm and produces the ibp identities relevant to our case . the evaluation of the mis is in general a complicated problem and can proceed via different techniques , like the integration in the feynman , schwinger or mellin a remarkable consequence of the aforementioned ibp relations is that the mis obey linear systems of first - order differential equations ( des ) in the kinematic invariants , which provide an alternative means for their computation [ 73 , 74 ] . finding the analytic solution of the des for arbitrary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d is possible only in some simple cases . in more general cases the mis are expanded in powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon = ( 4-d)/2$$\end{document}=(4-d)/2 , giving rise to a ( generally coupled ) system of des for the expansion coefficients . in the limit of vanishing external momentum , two - loop self - energies become two - loop vacuum diagrams , which reduce via ibp to linear combinations of only one genuine two - loop mi and products of the one - loop one - propagator mi . two - loop self - energies with arbitrary external momentum , in the general case with five different masses in the loops , reduce via ibp to linear combinations of 30 mis . the finite part of such mis can be expressed in terms of four functions , in addition to the well - known one - loop mis.5 as already mentioned , analytic solutions for such functions have been derived only for special patterns of up to two internal masses ( only one function is known in a particular case with three different masses ) . on the other hand , the diagrams that in our approximation contribute to the self - energies entering eqs . ( 5)(7 ) require the knowledge of mis with up to four different masses , the most complicated ones being those involving simultaneously \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_t^2,{m}_{\tilde{t}_1}^{2},{m}_{\tilde{t}_2}^{2}$$\end{document}mt2,mt~12,mt~22 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{\tilde{g}}^{2}$$\end{document}mg~2 . in our computation we rely on the package tsil , which implements ( besides all the analytically known cases ) the numerical solution of the des for the two - loop self - energy mis . [ 4751 ] is based on the fact that the value at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2=0$$\end{document}p2=0 ( or the expansion for small \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2$$\end{document}p2 in the case of logarithmically divergent integrals ) is known for each function and can be used to build the set of initial conditions needed for the solution of the des . in the computation of the self - energies entering eqs . ( 5)(7 ) we need to evaluate the corresponding mis at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2=m_z^2,m_a^2,m_h^2,m_h^2$$\end{document}p2=mz2,ma2,mh2,mh2 . given that we include the contribution of light quarks to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}$$\end{document}zz ( in the approximation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_q=0$$\end{document}mq=0 ) and that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma is a free parameter , it is clear that the way thresholds are handled in the numerical evaluation of the mis is of particular relevance . in the des approach , the physical two- and three - particle thresholds show up , together with the pseudothresholds , as poles in the coefficients of the des . tsil overcomes the numerical instabilities related to such poles by displacing the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2$$\end{document}p2-integration contour in the upper half - plane when the momentum is above the smallest ( pseudo)threshold . the evaluation at ( or very close to ) the ( pseudo)thresholds is performed through a variant of the algorithm , which is slightly less efficient but ensures reliable results in such critical cases . as an example , the time needed on an intel core i7 - 4650u cpu for the evaluation of the complete set of mis , for any of the mass patterns entering the self - energies , ranges between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$5 \times 10^{-4}\,s$$\end{document}510 - 4s and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$8 \times 10^{-2}\,s$$\end{document}810 - 2s , the latter being the typical time for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{p^2}$$\end{document}p2 close or equal to the heavy stop pair threshold and to the three - particle ( pseudo)thresholds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{\tilde{t}_i}+m_{\tilde{g}}\pm { { m}_{t}}$$\end{document}mt~i+mg~mt . in ref . the relative accuracy of tsil is claimed to be better than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{-10}$$\end{document}10 - 10 for generic cases , or worse in cases with large mass hierarchies . being tsil a package dedicated to the evaluation of the mis for two - loop self - energy diagrams , it is not surprising that its speed and accuracy prove much better than those quoted in ref . , where the general - purpose package secdec is used and , in the most complicated case , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$100\,s$$\end{document}100s are needed in order to reach a relative accuracy of at least \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{-5}$$\end{document}10 - 5 close to a threshold . in this section we assess the numerical impact of the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and of the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections on the predictions for the neutral higgs - boson masses in the mssm . we focus on six benchmark scenarios introduced in ref . , which identify regions in the mssm parameter space compatible with the current bounds from susy - particle searches and with the requirement that the predicted value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh agrees , within the theoretical uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pm 3~\mathrm{gev}$$\end{document}3gev estimated in refs . [ 78 , 79 ] , with the mass of the sm - like higgs boson discovered at the lhc.6 in our numerical examples we adopt the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme described in sect . 2 . the sm input parameters are chosen as the pole top mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_t = 173.2~\mathrm{gev}$$\end{document}mt=173.2gev , the running bottom mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_b(m_b ) = 4.2~\mathrm{gev}$$\end{document}mb(mb)=4.2gev , the fermi constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_f = 1.16639 \times 10^{-5}~\mathrm{gev}^{-2}$$\end{document}gf=1.1663910 - 5gev-2 , the strong gauge coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ s({{m}_{{\scriptscriptstyle z } } } ) = 0.118$$\end{document}s(mz)=0.118 , and the pole gauge - boson masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle z}}}= 91.1876~\mathrm{gev}$$\end{document}mz=91.1876gev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle w}}}= 80.385~\mathrm{gev}$$\end{document}mw=80.385gev . to compute the scalar masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}^2_{h,{\scriptscriptstyle h}}$$\end{document}mh , h2 and the effective mixing angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document} entering the corrections in eqs . we use default values for all settings with the exception of runningmt = 0 , i.e. the top mass in the radiative corrections is identified with the pole mass ( to match the renormalization conditions imposed both in our os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr calculation and in the one of ref . ) . by default , the renormalization scale associated to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition of the higgs wfr and of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan is fixed as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu _ { { \scriptscriptstyle r}}=m_t$$\end{document}r = mt . in fig . 1 we present our predictions for the lightest - scalar mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h$$\end{document}mh in the six benchmark scenarios . we choose \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}= 500~\mathrm{gev}$$\end{document}ma=500gev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 , so that the lightest scalar \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 sm - like , the bound on its tree - level mass is saturated , and the corrections controlled by the bottom yukawa coupling , which we do not compute beyond the approximations of feynhiggs , are not expected to be particularly relevant . for each scenario mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_{h}$$\end{document}mh , obtained from feynhiggs without additional corrections ; the middle bar includes the effect of the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , i.e. the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(\delta m_h^2)^{\alpha _ ( 12 ) ; finally , the lower bar represents our final result for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h$$\end{document}mh , and includes the effects of both the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections , i.e. the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(\delta m_h^2)^{\alpha \alpha _ { s}}$$\end{document}(mh2)s defined in eq . 1predictions for the mass of the lightest scalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h in the six benchmark scenarios of ref . , for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}=500~\mathrm{gev}$$\end{document}ma=500gev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 . for each scenario , the three bars show : the unperturbed mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_h$$\end{document}mh computed with feynhiggs 2.10.2 ( upper ) , the inclusion of the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections ( middle ) and the additional inclusion of the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections ( lower ) . from top to bottom , the considered scenarios are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^{\mathrm{max}}$$\end{document}mhmax ( red ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}^\mathrm{mod+}$$\end{document}mhmod+ ( blue ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}^\mathrm{mod-}$$\end{document}mhmod- ( green ) , light stop ( turquoise ) , light stau ( purple ) , tau - phobic ( orange ) predictions for the mass of the lightest scalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h in the six benchmark scenarios of ref . , for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}=500~\mathrm{gev}$$\end{document}ma=500gev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 . for each scenario , the three bars show : the unperturbed mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{m}_h$$\end{document}mh computed with feynhiggs 2.10.2 ( upper ) , the inclusion of the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections ( middle ) and the additional inclusion of the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections ( lower ) . from top to bottom , the considered scenarios are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^{\mathrm{max}}$$\end{document}mhmax ( red ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}^\mathrm{mod+}$$\end{document}mhmod+ ( blue ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}^\mathrm{mod-}$$\end{document}mhmod- ( green ) , light stop ( turquoise ) , light stau ( purple ) , tau - phobic ( orange ) figure 1 shows that , in all considered scenarios , the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections can shift the prediction for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h$$\end{document}mh by several hundred mev each ( the largest shifts , of about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pm 1$$\end{document}1 gev , occur in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{mod-}$$\end{document}mhmod- scenario ) . however , in all of our examples the two classes of corrections happen to be similar to each other in magnitude , and to enter the prediction for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h$$\end{document}mh with opposite signs . as a result , their combined effect is always fairly small , less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } 2 we illustrate the impact of the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections on the prediction for the lightest - scalar mass as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , and in fig . 3 we do the same for the heaviest - scalar mass . in each figure , the mssm parameters for the left plot are chosen as in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax benchmark scenario of ref . , while for the right plot they are chosen as in the modified light - stop scenario . in each plot , the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , while the solid lines include both the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections . the red lines were obtained with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 5$$\end{document}tan=5 while the blue lines were obtained with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 . 2corrections to the lightest - scalar mass as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 5$$\end{document}tan=5 ( red ) and for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 ( blue ) . the other mssm parameters are chosen as in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario ( left ) or as in the light - stop scenario ( right ) . the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , the solid lines include both the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) correctionsfig . 3same as fig . 2 for the corrections to the heaviest - scalar mass corrections to the lightest - scalar mass as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 5$$\end{document}tan=5 ( red ) and for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 20$$\end{document}tan=20 ( blue ) . the other mssm parameters are chosen as in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario ( left ) or as in the light - stop scenario ( right ) . the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , the solid lines include both the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) 2 for the corrections to the heaviest - scalar mass figure 2 shows that the corrections to the lightest - scalar mass are negligible at low values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , but they become larger and essentially independent of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma as the latter increases . the transition to this decoupling regime where the lightest scalar has sm - like couplings and its mass is insensitive to the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma is sharper for larger values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. in both the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax and light - stop scenarios , the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) effects decrease \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh by 300400 mev at large \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma . however , as already seen in fig . 1 , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) effects enter the prediction for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh with comparable magnitude but opposite sign , significantly reducing the total size of the correction . figure 3 shows that for low values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , where the heaviest scalar is the one with sm - like couplings , the corrections to its mass are comparable to the ones that affect the lightest - scalar mass in the decoupling region . on the other hand , for larger values of the pseudoscalar mass where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}\approx { { m}_{{\scriptscriptstyle a}}}$$\end{document}mhma the corrections to the heaviest - scalar mass show a series of spikes , related to the opening of real - particle thresholds in diagrams that involve a virtual gluon . the first spike is visible in correspondence with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}= 2\,{{m}_{t}}$$\end{document}ma=2mt in the plot on the left for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario . more - pronounced spikes ( note the different scale on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y$$\end{document}y axis ) are visible in correspondence with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}= 2\,m_{\tilde{t}_1}$$\end{document}mh=2mt~1 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}= m_{\tilde{t}_1}+m_{\tilde{t}_2}\,$$\end{document}mh = mt~1+mt~2 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}= 2\,m_{\tilde{t}_2}\,$$\end{document}mh=2mt~2 in the plot on the right for the light - stop scenario . analogous spikes would appear at larger values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario , where the stops are heavier . we stress that our results are not reliable in the vicinity of these thresholds : the two - loop correction to the heaviest - scalar mass is actually divergent there , and the height of the spikes in the plots carries no physical meaning . a more sophisticated analysis , taking into account the widths of the virtual particles in the loops as well as non - perturbative qcd effects , would be necessary around the thresholds , but it is beyond the scope of our calculation . figure 3 also shows that , in the decoupling region and away from the thresholds , the corrections to the heaviest - scalar mass amount at most to a few hundred mev , and they decrease in size with increasing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. moreover , the effect of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections is negligible ( the dashed and solid lines are practically overlapping in the plots ) . inspection of our analytic formulae shows that , in the decoupling limit , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh are suppressed by one or two powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan , whereas the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections contain unsuppressed contributions proportional to the square of the superpotential higgs - mass parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu $ $ \end{document}. while corrections of this size might be considered negligible in comparison with the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh itself , they are not entirely irrelevant when compared to the difference \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}-{{m}_{{\scriptscriptstyle a}}}$$\end{document}mh - ma , which can be of the order of a few gev and is the quantity of interest when a large physical mass for the pseudoscalar is taken as input in the calculation . the way we compute the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to the entries of the inverse - propagator matrix for the neutral scalars allows for a relatively easy comparison with earlier calculations . we first renormalize all the relevant parameters in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , i.e. we introduce minimal counterterms that , by definition , subtract only powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1/\epsilon , $ $ \end{document}1/ , multiplied by coefficients that should be polynomial in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr-renormalized masses and couplings . in a second step , we convert our results to the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme adopted in feynhiggs , replacing the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr top / stop parameters entering the one - loop part of the corrections with the corresponding os parameters plus the finite one - loop shifts given in ref . . as a first obvious check , we took the limit of vanishing external momentum in the scalar self - energies entering the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and we compared our results with those in ref . , finding full agreement . we also successfully compared the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections at vanishing external momentum with the results of an independent calculation based on the effective - potential techniques of ref . . note , however , that this comparison does not cover the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) contributions to 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 self - energy . concerning the latter , we checked that we can reproduce the result of ref . for the subset of two - loop diagrams that involve only quarks and a gluon , taking into account the fact that ref . we then compared our results for the momentum - dependent corrections with those of ref . , where the two - loop calculation of the higgs masses was performed entirely in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . as mentioned in sect . 1 , the higgs - mass corrections in ref are organized in a different way with respect to our calculation , therefore we could compare only at the level of individual two - loop self - energies for scalars and pseudoscalars ( the two - loop self - energy for 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 was not computed in ref . ) . rotating our scalar self - energies from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(s_1,s_2)$$\end{document}(s1,s2 ) basis to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(h , h)$$\end{document}(h , h ) basis with the tree - level mixing angle defined in ref . , we reproduce perfectly the results for the top / gluon and top / stop / gluino contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \pi _ { hh}$$\end{document}hh shown in figure 2 of that paper . this provides a full cross - check of the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) contribution to the self - energy , as well as a partial check of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) contribution , restricted to diagrams involving the stop squarks ( the diagrams involving the other squarks are included in the others line in the above - mentioned figure ) . we also checked the analogous contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \pi _ { h{\scriptscriptstyle h}}$$\end{document}hh , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \pi _ { { \scriptscriptstyle h}{\scriptscriptstyle h}}$$\end{document}hh , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}$$\end{document}aa against results provided by the author of ref . , finding again perfect numerical agreement . although our calculation and the one in ref . both use tsil to compute the mis , and thus can not be considered entirely independent , the agreement in the results for the self - energies gives us confidence that the computation of two - loop feynman diagrams in terms of mis and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr subtraction of their divergences are correct in both papers . our results for the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections in the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme can in turn be compared with those of ref . . to start with , we compared our two - loop 1pi contributions to the scalar and pseudoscalar self - energies with analogous results provided by the authors of ref . , and we found agreement within the accuracy of the sector - decomposition procedure used to compute the loop integrals in that paper . the successful comparison between two sets of self - energies in which the loop integrals were evaluated with tsil and secdec , respectively , validates the results for the two - loop mis , thus reinforcing our cross - check of ref . . on the other hand , our results for the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the higgs masses , obtained by combining the 1pi diagrams with all the necessary counterterm contributions , differ significantly from the ones in ref . . considering for example the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_h^{\mathrm{max}}$$\end{document}mhmax scenario discussed in the previous section , we find that for large \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma the lightest - scalar mass is subject to a negative correction of about 350400 mev ( depending on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan ; see the left plot in fig . is also negative but quite smaller , about 5060 mev ( see the upper plot in figure 7 of that paper ) . , related to the renormalization conditions for the higgs fields and for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , the wfr counterterm for each field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h^0_i$$\end{document}hi0 can be related to the divergent part of the derivative of the scalar self - energy with respect to the external momentum:18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta \mathcal{z}^{(\ell ) } _ i = - \left [ \frac{d\ , \mathrm{re}\,\pi ^{(\ell ) } _ { ii}(p^2)}{dp^2 } \right ] ^\mathrm{div}\quad ( \ell = 1,2 ) . \end{aligned}$$\end{document}zi()=-dreii()(p2)dp2div(=1,2).indeed , when all parameters entering the one - loop part of the scalar self - energies are renormalized in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , eq . ( 18 ) leads to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr wfr counterterms given in eq . ( 9 ) , in accordance with the anomalous dimensions of the higgs fields given in refs . however , in the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme of ref . the top / stop parameters in the one - loop self - energies are renormalized os . in that case , the use of eq . ( 18 ) to determine the wfr counterterms leads to19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathcal{z}_2^{}&= 1 - \frac{~\alpha _ { t}^{{\mathrm{os}}}}{4\pi } \,n_c\,\cdot \,\frac{1}{\epsilon } ~+~ \frac{\alpha _ { t}\alpha _ { s}}{(4\pi ) ^2}\,2\,n_c\,c_f\nonumber \\&\cdot \left ( \frac{1}{\epsilon ^2}-\frac{1}{\epsilon } \right ) - \frac{\alpha _ { t}}{2\pi } \,n_c\ , \frac{\delta m_t}{m_t}\,\cdot \,\frac{1}{\epsilon } , \end{aligned}$$\end{document}z2=1-tos4nc1+ts(4)22nccf12 - 1-t2ncmtmt1,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ { t}^{{\mathrm{os}}}$$\end{document}tos is a scale - independent coupling extracted from the pole top mass , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m_t$$\end{document}mt is the finite one - loop shift for the top mass given in eq . ( b2 ) of ref . . by converting the coupling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ { t}^{{\mathrm{os}}}$$\end{document}tos in the one - loop term of eq . ( 19 ) into the corresponding \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr coupling , it is easy to see that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{z}_2^{}$$\end{document}z2 differs from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr wfr constant in eq . ( 9 ) by a finite two - loop term:20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathcal{z}_2^ { } = \mathcal{z}_2^{\,{\overline{\mathrm{dr } } } } ~+~ \frac{\alpha _ { t}}{2\pi } \,n_c\ , \frac{\delta _ \epsilon m_t}{m_t } , \end{aligned}$$\end{document}z2=z2dr+t2ncmtmt , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ \epsilon m_t$$\end{document}mt denotes the part proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon $ $ \end{document} in the top self - energy regularized with dimensional reduction:21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{\delta _ s}{4 \pi } \,c_f\ , \left\ { -\frac{3}{2}\,\ln ^2 \frac{{{m}_{t}}^2}{\mu _ { { \scriptscriptstyle r}}^2 } + 5\,\ln \frac{{{m}_{t}}^2}{\mu _ { { \scriptscriptstyle r}}^2 } - \frac{~\pi ^2}{4 } - 9 - \frac{m_{\tilde{g}}^{2}}{{{m}_{t}}^2}\right . \left ( \frac{1}{2 } \ , \ln ^2 \frac{m_{\tilde{g}}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } - \ln \frac{m_{\tilde{g}}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } + \frac{~\pi ^2}{12 } + 1 \right ) \right . \nonumber \\&+ \frac{1}{2}\left [ ~ \frac{m_{\tilde{g}}^{2}+{{m}_{t}}^2 - { m}_{\tilde{t}_1}^{2}- 2 \,{s}_{2\theta _ { t}}\,m_{\tilde{g}}\ , { { m}_{t}}}{{{m}_{t}}^2 } \,b_\epsilon ( { { m}_{t}}^2,m_{\tilde{g}}^{2},{m}_{\tilde{t}_1}^{2 } ) \right . \nonumber \\&+\left . \left . \,\frac{{m}_{\tilde{t}_1}^{2}}{{{m}_{t}}^2 } \left ( \frac{1}{2 } \ , \ln ^2 \frac{{m}_{\tilde{t}_1}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } - \ln \frac{{m}_{\tilde{t}_1}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } + \frac{~\pi ^2}{12 } + 1 \right ) \right . \right . ( \tilde{t}_1 \rightarrow \tilde{t}_2 , ~~ { s}_{2\theta _ { t}}\rightarrow -{s}_{2\theta _ { t } } ) \right ] \right\ } . \end{aligned}$$\end{document}mtmt=s4cf-32ln2mt2r2 + 5lnmt2r2-24 - 9-mg~2mt212ln2mg~2r2-lnmg~2r2+212 + 1 + 12mg~2+mt2-mt~12 - 2s2tmg~mtmt2b(mt2,mg~2,mt~12)+mt~12mt212ln2mt~12r2-lnmt~12r2+212 + 1+(t~1t~2,s2t-s2t).in the equation above \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu _ { { \scriptscriptstyle r}}$$\end{document}r is the renormalization scale associated to the higgs wfr and to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan , while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\epsilon ( s , x , y)$$\end{document}b(s , x , y ) denotes the coefficient of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon $ $ \end{document} in the expansion of the passarino veltman function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_0$$\end{document}b0 . an explicit expression for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\epsilon $ $ \end{document}b can be found , e.g. , in eq . ( 2.31 ) of the tsil manual . in the calculation of ref . , where the top / stop parameters entering the one - loop part of the corrections are directly renormalized os instead of being first renormalized in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme and then converted to the os scheme via a finite shift , the two - loop self - energies and tadpoles contain terms proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ \epsilon m_t$$\end{document}mt . such terms would drop out of the final result for the renormalized inverse - propagator matrix if eq . ( 20 ) was used to obtain the correct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for the wfr constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{z}_2^{\,{\overline{\mathrm{dr}}}}$$\end{document}z2dr , and consequently for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta $ $ \end{document}tan , but they survive if the wfr constant is defined as in eq . ( 19 ) . to prove that these terms are indeed at the root of the observed discrepancies , we modified our own calculation , using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{z}_2^{}$$\end{document}z2 as obtained from eq . ( 20 ) instead of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{z}_2^{\,{\overline{\mathrm{dr}}}}$$\end{document}z2dr and then computing a non - minimal counterterm for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan via eq . we checked that , with this modification , we reproduce exactly the corrections to the renormalized inverse propagator shown in figures 5 and 10 of ref . . we also reproduce the corrections to the scalar masses shown in figures 7 , 8 , 12 and 13 of that paper , although small discrepancies persist in the case of the heaviest scalar when its mass is above the threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}=2\,m_t$$\end{document}mh=2mt . these residual discrepancies are formally of higher order in the perturbative expansion , and they result from different approximations in the two - step procedure for the determination of the poles of the propagator ( namely , we drop the imaginary parts of the two - loop self - energies , while ref the two - loop renormalization of the higgs fields and of the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan is not performed in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme as claimed in the paper , but rather in some non - minimal scheme where the wfr counterterms and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta $ $ \end{document}tan differ from their \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr counterparts by finite , non - polynomial terms , and neither the higgs fields nor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan obey their usual renormalization - group equations ( because of the explicit scale dependence of the additional terms ) . this inconsistency should be taken into account when comparing the results of ref . with those of calculations that actually employ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definitions for the wfr and for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan. first of all , to account for the difference in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta $ $ \end{document}tan , the input value for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr-renormalized parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan should be converted to the corresponding value in the non - minimal scheme of ref . , according to22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \tan \beta ^ { } = \tan \beta ^{\,{\overline{\mathrm{dr } } } } - \frac{\alpha _ { t}}{4\pi } \,n_c\,\tan \beta ~ \frac{\delta _ ( 5)(7 ) show that the contributions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta ^{(2)}$$\end{document}tan(2 ) to the entries of the higgs mass matrix are suppressed by powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cos \beta $ $ \end{document}cos. consequently , the effect on the higgs masses arising from a two - loop difference in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tan \beta $ $ \end{document}tan is very small already for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta = 5$$\end{document}tan=5 . in fact , the bulk of the numerical discrepancy between our results and those of ref . is due to higher - order effects that are directly related to the finite shift in the wfr . as discussed at the end of sect . 2 , such effects are included in the higgs - mass corrections when the latter are computed in terms of loop - corrected higgs masses and mixing , and can become numerically relevant when the loop - corrected masses differ significantly from their tree - level values . we computed the two - loop corrections to the neutral mssm higgs masses of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) i.e. , all two - loop corrections that involve the strong gauge coupling when the only non - vanishing yukawa coupling is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_t$$\end{document}ht including the effect of non - vanishing external momenta in the self - energies . we relied on an ibps technique to express the momentum - dependent loop integrals in terms of a minimal set of master integrals , and we used the public code tsil to evaluate the latter . we obtained results for the higgs - mass corrections valid when all parameters in the one - loop part of the corrections are renormalized in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , as well as results valid in a mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme where the top / stop parameters are renormalized on - shell . our results for the scalar and pseudoscalar self - energies in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme confirm the results of an earlier calculation , ref . in addition , we obtained new results for the two - loop contributions to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - boson self - energy that involve the strong gauge coupling . , enter the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to the higgs masses when the tree - level mass matrix of the scalars is expressed in terms of the physical \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z$$\end{document}z - boson mass . we also compared our results for the momentum - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections in the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme with those of a recent calculation of the same corrections , ref . , and found disagreement . we traced the reason for the discrepancy to the fact that , contrary to what stated in ref . , in that calculation the higgs fields and the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \beta $ $ \end{document}tan are renormalized in a non - minimal scheme instead of the usual \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . when this difference is taken into account , we reproduce the results of ref . , providing in passing a cross - check of the codes used to evaluate the loop integrals in the two calculations ( i.e. , tsil and secdec , respectively ) . however , we noticed that tsil , which implements dedicated algorithms for two - loop self - energy integrals , can be a thousand times faster than a multi - purpose code such as secdec in the computation of the higgs - mass corrections . this should be taken into consideration when including the momentum - dependent corrections in phenomenological analyses of the mssm parameter space . as to the numerical impact of the corrections computed in this paper , it could at best be described as moderate . we considered six benchmark scenarios compatible with the results of higgs and susy searches at the lhc , and we found that both the momentum - dependent part of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the whole \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections can shift the prediction for the lightest - scalar mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh by several hundred mev . however , we noticed that at least in the considered scenarios the two classes of corrections enter the prediction for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{h}}$$\end{document}mh with opposite sign , and they compensate each other to a good extent . for what concerns the heaviest - scalar mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh , the impact of the new corrections is also modest , with the exception of regions around real - particle thresholds where a fixed - order calculation is not reliable anyway . the predictions for the lightest - scalar mass , as obtained from popular codes for the determination of the mssm mass spectrum , carry a theoretical uncertainty that has been estimated to be ( at least ) of the order of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pm 3$$\end{document}3 gev see , e.g. , refs . [ 78 , 79 ] and the more recent discussion in ref . . against this backdrop , the corrections presented in this paper can be considered sub - dominant , and their inclusion in public codes might seem less urgent than , e.g. , the inclusion of the dominant three - loop effects [ 41 , 42 ] or the proper resummation of large logarithms in scenarios with multi - tev stop masses [ 39 , 40 , 82 ] , both of which can shift the prediction for the lightest - scalar mass by several gev . nevertheless , one should not forget that the accuracy of the measurement of the higgs mass at the lhc has already reached the level of a few hundred mev i.e. , comparable to our sub - dominant corrections and will improve further when more data become available . if susy shows up at last when the lhc operates at 1314 tev , the higgs mass will serve as a precision observable to constrain mssm parameters that might not be directly accessible by experiment . to this purpose , the accuracy of the theoretical prediction will have to match the experimental one , making a full inclusion of the two - loop corrections to the higgs masses unavoidable .

we compute the two - loop qcd corrections to the neutral higgs - boson masses in the minimal supersymmetric standard model , including the effect of non - vanishing external momenta in the self - energies . we obtain corrections of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) , i.e. , all two - loop corrections that involve the strong gauge coupling when the only non - vanishing yukawa coupling is the top one . we adopt either the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization scheme or a mixed on - shell ( os)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme where the top / stop parameters are renormalized on - shell . we compare our results with those of earlier calculations , pointing out an inconsistency in a recent result obtained in the mixed os\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . the numerical impact of the new corrections on the prediction for the lightest - scalar mass is moderate , but already comparable to the accuracy of the higgs - mass measurement at the large hadron collider .

Introduction Neutral Higgs boson masses in the MSSM Calculation of two-loop diagrams with nonzero momentum Numerical examples Comparison with earlier calculations Conclusions

PMC3141350

while brm scales with body mass ( bm ) , several other factors seem to influence brm , such as sociability , environmental and dietary specializations , as well as energetic costs of brain tissue ( mace et al . , 1981 ) . large brains contain more neurons and neural connections , and thus have greater potential for information processing . large brains also tend to be more modular , which allows a great amount of connections between neurons ( krubitzer and kaas , 2005 ) . hence , increased brain mass and potential for neural connections may have facilitated large brained mammals to colonize complex habitats , develop sensory systems and evolved complex societies ( e.g. , budeau and verts , 1986 ) . for example , the mass - specific metabolic rate of the human brain is nine times higher than that of the body as a whole ( martin , 1981 ) . the metabolic costs by having a large brain must be paid for by a direct mother metabolic constraint or by a trade - off mechanism between brain mass and energy consumption by other functions ( e.g. , gibbons , 1998 ; pitnick et al . , 2006 ) . other energy allocations such as relative costs of flight and reproductive strategy in birds may also be reduced to shunt energy to an enlarged brain ( isler and van schaik , 2006 ) . recent examples also show that brm may become decoupled from bm over short time spans . 2009 ) found that tanganyikan cichlid bm exhibited recent bursts of rapid evolution , a process that is consistent with divergence linked to ecological specialization , while brm showed no bursts of divergence but evolved in gradual manner , consistent with energetic constraints to rapid bm change . originally , aiello and wheeler ( 1995 ) proposed that a primate is able to meet the high metabolic cost of a large brain without incurring a compensatory increase in relative basal metabolic rate ( bmr ) by decreasing the amount of other metabolically expensive tissues ( i.e. , heart , lung , kidney , liver , and gastrointestinal tract ) . a similar hypothesis was recently proposed for fish ( kaufman et al . , 2003 ) . recently , isler and van schaik ( 2009 ) using a large compilation of brain size , bm , and life history data , found evidence that an energetically costly increase in brain size has to be met by either increasing the total energy budget of a species or by compensating changes of energy allocation to other maintenance functions , such as digestion or growth and offspring production , or a combination of these . martin , 1981 , 1996 ) , for example , found that the energetic investment of the mammalian mother during the development of the fetus and during the postnatal life up to the time of weaning resulted in a weak link between bmr and brain mass . similarly , jones and maclarnon ( 2004 ) showed that for certain clades of bats , maternal investment plays an important role in the adult brain mass . while a number of studies have thus focused on these mechanisms of development , little is known about the evolutionary relationship between bmr and brm . very few studies have tested the generality of the costly brain hypotheses across multiple species with different evolutionary histories , and using phylogenetic approaches . basal metabolic rate is a fundamental parameter in comparative studies and lineages - specific exponents characterize its allometric scaling ( white et al . , 2009 ) . recently , isler and van schaik ( 2006 ) controlling both for bm and phylogentic relationships found evidence that bmr correlated with brm in large groups of mammals . however , brm explained a small % of the variation in metabolic rate at the species and family level ( 2.6 and 10.4% , respectively ) . at higher taxonomic levels , independent contrast ( ic ) revealed a significant correlation only for primates . it is possible that the use of composite phylogenies lacking resolution and accurate estimates of branch lengths ( bl ) may have obscured the underlying patterns ( e.g. , malia et al . , 2003 ) . other potential confounding we generated a novel phylogeny of wild rodents using bayesian analysis of cytochrome b sequence data . we included species where high - quality bmr , brm , and bm data are available . we then used this analysis to test the hypothesis that bmr and brm are correlated within rodents , after taking into consideration both bm and phylogeny . cytochrome b for 132 rodent species and six rabbits as outgroups ( wilson and reeder , 2005 ) were downloaded from genbank , and one sequence donated ( table a1 in appendix ) . cytochrome b was chosen as that marker has proven to be of high utility for species level phylogenetics ( may - collado and agnarsson , 2006 ; agnarsson et al . , 2010 , 2011 ) sequences were aligned using clustalx 1.83 ( thompson et al . , the preferred model for the bayesian analyses was selected with modeltest ( posada and crandall , 2001 ) using the aic criterion ( posada and buckley , 2004 ) . the best - fitting model was gtr + + i ( yang , 1994 ) . bayesian analyses were carried out using mrbayes v3.12 ( huelsenbeck and ronchist , 2001 ) with the settings as specified in agnarsson and may - collado ( 2008 ) . the markov chain monte carlo search was ran with 10,000,000 generations sampling the markov chain every 1,000 generations , and the sample points of the first 7,000,000 generations were removed ( burnin ) , after which the chain had reached stationarity . data on the log of brm and bm ( g ) , and bmr ( cmo2/h ) were used in this study ( table a1 in appendix ) . for studies comparing traits among species , such as regression analyses , it is necessary to account for phylogenetic relationships among the compared species ( felsenstein , 1985 ) . ignoring phylogenetic relationships can lead to pseudoreplication as species are not independent data points , rather independent evolutionary changes in the traits being compared are the data points , or the ic ( see felsenstein , 1985 ; may - collado et al . , 2007 ) among species and lineages . to describe the evolutionary relationship between bmr and brm we performed various phylogenetic analyses . ( i ) the pdap module in mesquite ( midford et al . , 2008 ) was used to estimate ic ( felsenstein , 1985 ) . we used bl as estimated by mrbayes testing them for statistic appropriateness using pdap . to correct for bm we regressed bmr and brm against bm and subsequently regressed the residuals from these regressions ( garland et al . , 1993 ) . if the residuals are correlated then that is consistent with a relationship among these variables ( bmr and brm ) , that is independent of the bm of , and phylogenetic relationship among , species ( e.g. , may - collado et al . , 2007 ) . ( ii ) to evaluate the correlated evolution among bmr , brm , and bm , we assess the phylogenetic effect on the trends in character relationships between taxa ( i.e. , the observed pattern ) using the best model of evolution that was found for each character . to do this we evaluated the significance of the relationships between the pair of characters using a measure of correlated evolution ( corr ) in a bayesian framework implemented in bayestrait 1.0 ( pagel and meade , 2007 ) , assessing the probability of positively correlated ( corr > 0 ) and negatively correlated evolution ( corr < 0 ) . as the null hypothesis we used a model in which the covariance between characters was set to zero ( i.e. , complete character independence , corr = 0 ) , and the alternative hypothesis was , then , the observed covariance between characters ( pagel , 1999a , 1999b ) . if the null hypothesis was rejected ( i.e. , a significant historical relationship between characters exists ) , then we concluded that the phylogenetic relationship and the models of evolution of the characters influence the observed patterns , and we corroborate the hypothesis of correlated evolution between bmr , brm , and bm . we used a bayesian approach based on maximum likelihood with 10 test per tree and estimating pagel ( 1999a , b ) escalated phylogenetic parameters ( table a2 in appendix ) . the sign test was used for statistical comparisons ( zar , 1996 ) with statistica 6.0 ( statsoft , 2001 ) . the novel phylogeny finds support for the monophyly of each currently recognized taxonomical bat family with the exception that heteromyidae contains geomyidae , and one species of muridae , sigmodon hispidus , groups with cricetidae ( figure a1 in appendix ) . the phylogeny overall agrees well with recent rodent phylogenies at higher levels ( e.g. , jansa and weksler , 2004 ; montgelard et al . , 2008 ) and thus represents an reasonable hypothesis for to study the evolution of characters ( e.g. , pagel and harvey , 1988 ) independent contrast revealed associations between bm and bmr ( p < 0.0001 ; r = 0.77 ) , and with brm ( p < 0.0001 ; r = 0.85 ) , and between bmr and brm ( p < 0.0001 ; r = 0.71 ) . when using a single value to represent species mass we also found significant correlation between bmr and brm , after accounting for bm . when using a single individual weight to represent the species , bmr explained 9.7% of the variation in brm ( p = 0.0003 ; r = 0.097 ; figure 1a ) . two extreme outliers affected the regression and removing these outliers resulted in much stronger regression ( p < 0.0001 ; r = 0.20 ; figure 1b ) . the outliers represent 3 species of small rodents ( cricetidae , arvicolinae ) , that inhabit circumpolar northern hemisphere biome ( nowak , 1999 ) . these lemmings seem to have higher metabolic rate than typical rodents with similar brm , which may be related to living in extreme climates requiring higher metabolism . this demonstrates that the climate and habitat as well as other potentially confounding factors ( bm , food habits , substrate , a restriction to islands or highlands , use of torpor , and type of reproduction ) make it difficult to demonstrate a significant correlation between bmr and brm , even when it exists ( e.g. , mcnab , 2008 ) . ( a ) independent contrast regression analysis between bmr and brm residuals , corrected by bm . ( b ) ic regression analysis between bmr and brm residuals , corrected by bm and removing outliners ( myopus schisticolor , lemmus sibiricus , and l. lemmus ) . where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass . when using species mean bm , bmr explained approximately 3% of variation in brm ( p < 0.05 , r = 0.029 ) . this correlation , however , disappears when using two bm values for each species , one , the estimated species mean , to calculate residuals of brm , and the second , of the individuals used for the bmr experiments , to calculate residuals of bmr ( p > 0.05 ) . corr indicated significant correlations between all variables ( p < 0.0001 ; corr 1 ) , with the highest correlation recorded between bm and brm ( p < 0.0001 ; corr = 2.86 ; r = 0.95 ; figure 2 ) , followed by bm and bmr ( p < 0.0001 ; corr = 2.24 ; r = 0.92 ; figure 2 ) , and finally bmr and brm ( p < 0.0001 ; corr = 1.86 ; r = 0.91 ; figure 2 ) . the series of arrows in the middle of the figure indicates the model of correlated evolution between the studied characters in a bayesian framework ( for more details see materials and methods ) , where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass . each inset labeled with a lower - case letter contains : on the left - hand side a line graph of a sample of 1,000 markov chain estimations ( x - axis ) of corr ( y - axis ) between pairs of characters , where the continuous black line represents corr = 0 , and the continuous gray line indicates corr = 1 ; and on the right - hand side a histogram of the probability distribution of covariance between pairs of characters , where the black bar indicates the average value and the gray bars indicate the lower 5% percentile ( lp ) and upper 95% percentile ( up ) . in particular , the analyzed characters were ( a ) bmr and brm ( lp = 1.33 , up = 2.46 ) ; ( b ) bm and bmr ( lp = 1.50 , up = 3.12 ) ; and ( c ) bm and brm ( lp = 2.08 , up = 3.77 ) . brains are the centers of the nervous system of vertebrates , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment ( shultz , 2010 ) . although brain mass per se does not capture the complexity of brain function , there is general evidence that relative brain size roughly correlates with cognitive ability ( e.g. , barton and harvey , 2000 ) . hence the evolution of brain size is of broad interest , including what factors may favor and constrain the evolution of relatively large , modular and complex brains ( sol , 2009 ) . basal metabolic rate is influenced by a variety of factors ( bm white and seymour , 2003 ; climate lovegrove , 2000 ; demography kurta and ferkin , 1991 ) . furthermore , an increase in brm results in increased costs for maintenance and information processing ( niven and laughlin , 2008 ) . for example , karbowski ( 2007 ) found that in volume - specific cerebral glucose metabolic rate of different brain structures closely scales with brain volume . these results confirm that information processing in the brain requires large amounts of metabolic energy . here , isler and van schaik ( 2006 ) found support for this relationship across all mammals combined , and within primates ( ic p = 0.025 , r = 0.20 ) , but not within other orders , such as rodents . adjustments appear to be clade - specific , the slopes of best - fit lines for brm against bm tend to be higher in analyses of more inclusive taxa ( e.g. , orders and suborders ) and lower in analyses of less inclusive taxa ( families , subfamilies , and genera ; e.g. , finarelli and flynn , 2009 ) . we hypothesize that the discrepancy between our findings and previous studies is potentially caused by two factors . first , the use of a composite versus primary - data based phylogenies , and second , differences in accounting for bm . composite phylogenies often reflect taxonomy , not necessarily phylogeny , and typically lack accurate bl estimates , two aspects that reduce the efficiency of comparative tests . accounting for bm is a complicated problem , but we argue that using more than a single value for a species , as have prior studies , may introduce confounding variables . thus using an estimate of species bm , such as average species bm to generate brm residuals , and the bm of the individual that happened to be used to evaluate bmr to generate bmr residuals , can likely adds noise that may obscure real patterns . if , for example , the evaluation of bmr happened to have been done on an atypically small or large animal , this would strongly affect the bm - bmr residual , and could readily obscure subtle patterns across two or more variables that are both highly correlated with bm . instead using a single value , whether mean bm of a species , or the weight of a single individual seems at least to be a reasonable alternative . furthermore , species mean bm is a measure independent of the available measures for bmr and brm ( typically single individuals ) , and as such provides a relatively neutral control unlikely to result in systematic error . here , we used a novel primary - data - based phylogeny with bl estimates and single estimates of species bm ( species average or single individuals ) . considering the compendium of factors that may contribute to bmr , the up to 20% of variation explained by brm in rodents is high . clearly , though , further research is necessary to understand the interplay between these variables , and ideally accurate estimates of species means based on multiple individuals would be available for each of these variables ( e.g. , smith and jungers , 1997 ) . our study is also consistent with two evolutionary paths favoring an increase in brm across rodent species . path in which an increase in bmr correlates with an increase in brm ( figure 2a ) . alternatively , indirect and additive path in which the effect of bm on bmr allows brm to increase ( figures 2a , b ) . comparatively , the direct scaling effect of bmr on brm is the least important relationship ( figure 2a ) , but the importance of direct scaling increases when considering the indirect path . dunbar and shultz ( 2007 ) supported the indirect path scenario in primates , where bmr had a limiting effect on brm , while bm had an effect on brm through bmr . here , we corroborate the hypothesis of isler and van schaik ( 2006 ) that an increase in brain mass is accompanied by an increase in basal metabolic rate , and suggests that this pattern may be general across mammals . our findings corroborate the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur ( niven and laughlin , 2008 ) . 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 . aic , akaike information criterion ; bl , branch lengths in phylogenies ; bm , body mass ; bmr , basal metabolic rate ; brm , brain mass ; corr , measure of correlated evolution between characters in a bayesian framework ; gtr , general time reversible models of nucleotide substitution ; ic , phylogenetic independent contrasts ; pdap , phenotypic diversity analysis programs .

brains are the centers of the nervous system of animals , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment . the evolution of traits that correlate with cognitive ability , such as relative brain size is thus of broad interest . brain mass relative to body mass ( bm ) varies among mammals , and diverse factors have been proposed to explain this variation . a recent study provided evidence that energetics play an important role in brain evolution ( isler and van schaik , 2006 ) . using composite phylogenies and data drawn from multiple sources , these authors showed that basal metabolic rate ( bmr ) correlates with brain mass across mammals . however , no such relationship was found within rodents . here we re - examined the relationship between bmr and brain mass within rodentia using a novel species - level phylogeny . our results are sensitive to parameter evaluation ; in particular how species mass is estimated . we detect no pattern when applying an approach used by previous studies , where each species bm is represented by two different numbers , one being the individual that happened to be used for bmr estimates of that species . however , this approach may compromise the analysis . when using a single value of bm for each species , whether representing a single individual , or available species mean , our findings provide evidence that brain mass ( independent of bm ) and bmr are correlated . these findings are thus consistent with the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur .

Introduction Materials and Methods Results Discussion Concluding Remarks Conflict of Interest Statement Abbreviations

PMC3692193

histological studies have shown that the inferior alveolar nerve typically courses through the mandible as one major trunk with branches extending to apices of the teeth . however , there are multiple smaller branches of the inferior alveolar nerve ( ian ) running roughly parallel to the major trunk such bifid canals are seen on panoramic and cone beam computed tomography ( cbct ) images . patients with bifid canals are at greater risk of inadequate anesthesia or difficulties with jaw surgery . even trifid mandibular canal has been reported . the major importance of location and configuration of mandibular canal variations is in the lower jaw surgeries , such as extraction of an impacted third molar , dental implant treatment , and sagital split ramus osteotomy . there are many causes of failure of ian blocks , including poor technique , anatomical variations , the presence of an acute infection , inability to enter the needle to the appropriate site or a reduced pain threshold . this case study focuses on one example of an anatomical variation : bifid mandibular canal ( bmc ) . when we reviewed the literature regarding bmc , we encountered only a few case reports that had two mental foramens . the following case report describes a patient with bilateral bmcs with two mental foramens by using cbct . a 47-year - old man was referred to his clinicians with persistent dull pain , 1 year after retrograde root canal treatment of tooth#24 . bifid mandibular canal with different patterns were located in each side of the jaw . while two mandibular canals with two distinct mandibular nerves ended to two different mental foramens in the left side [ figure 1 ] , in the right side the conjunction of two different mandibular canals ended to an isolated mental foramen [ figure 2 ] . cbct images of right mandibular canal , a : panoramic view , b : coronal view , c : sagittal - oblique view , d : crosssectional view . in the right side , the mandibular canal ended to two mandibular foramens and only one mental foramen cbct images of left mandibular canal , a , b : cross - sectional image , c : panoramic view , d : lateral sagittal , e : coronal view . in the left side , two mandibular canals including two mandibular nerves opened to two mental foramens the occurrence of bifid mandibular canal has been reported to be 0.35% by sanchis et al . , 0.8% by grover , 0.9% by nortje et al . , and 0.95% by langlais et al . reported that mandibular canal of specimen cadavers is not visible in 36.1% of panoramic radiographs . also , lindh et al . reported that the mandibular canal of specimen cadavers is clearly visible in almost 25% of panoramic radiographs . thus it was considered that there was a limitation in the observation of the mandibular canal by means of panoramic radiography . nowadays by the accurate new diagnostic equipments such as cbct , this statistics may increase . reported four cases of bmc by using cbct images that only two of them can be detected by panoramic radiography . cbct has enabled dentists to visualize the anatomy of the mandibular canal and ramified canals better than past . according to these finding and our observation , we suggest that cbct is more useful than conventional radiography such as panoramic images in detection of bmc . the first included bmcs extending to the area surrounding the third molar or to the tooth itself ; the second included bmcs arising from the same foramen but forming two separate canals which rejoined to form a single canal anteriorly ; the third type included a combination of the first two types ; and the last type included two radiographically separate canals with separate origins that eventually fused into a single canal anteriorly . nevertheless , the fact that , as opposed to the upper canals , the duplicate canals on the left side extended into different mental foramen allows us to consider this case as a new division . it is believed that one of the reasons that previous studies could not identify this type was using conventional radiographies and subsequently failing in detection narrow second mental foramen . the most common and first complication of bifid canal may appear in performing mandibular nerve block . the responsible nerve maybe long buccul or accessory ian . conventionally , anesthesia of the ipsilateral lip , chin , and teeth is indicative of an effective ianb . if a patient experiences only soft tissue anesthesia around the injection site , but not of the ipsilateral lip or chin , then a problem with local anesthesia technique is likely to be the cause of the failure . however , if there is soft tissue anesthesia of the lips and chin but not the teeth , one should consider anatomical variation . when dentists encounter this situation , if their problem is only inadequate anesthesia they should use another technique for local anesthesia . but if the surgical process must be done in this region , the anatomical variation of ian must be evaluated with diagnostic images . the best diagnostic imaging modality is cbct that has considerable benefits , such as low dose , appropriate cost , and well visual quality . the most common and first complication of bifid canal may appear in performing mandibular nerve block . the responsible nerve maybe long buccul or accessory ian . conventionally , anesthesia of the ipsilateral lip , chin , and teeth is indicative of an effective ianb . if a patient experiences only soft tissue anesthesia around the injection site , but not of the ipsilateral lip or chin , then a problem with local anesthesia technique is likely to be the cause of the failure . however , if there is soft tissue anesthesia of the lips and chin but not the teeth , one should consider anatomical variation . when dentists encounter this situation , if their problem is only inadequate anesthesia they should use another technique for local anesthesia . but if the surgical process must be done in this region , the anatomical variation of ian must be evaluated with diagnostic images . the best diagnostic imaging modality is cbct that has considerable benefits , such as low dose , appropriate cost , and well visual quality . bifid mandibular canal may exist in each patient . to manage this anatomic variation , dentists should choose appropriate images and local anesthesia technique .

one of the normal interesting variations that we may encounter in the mandible is bifid mandibular canal . this condition can lead to difficulties when performing mandibular anesthesia or during extraction of lower third molar , placement of implants , and surgery in the mandible . therefore diagnosis of this variation is sometimes very important and necessary .

INTRODUCTION CASE REPORT DISCUSSION When should bifid canal be suspected? How should dental approaches be considered to efficient anesthesia? CONCLUSION

PMC3129082

a 75 year old woman presented to the emergency room with a seven months history of increased fatigability , loss of appetite , weight loss and progressive abdominal distension . on examination the patient was markedly pale , asthenic , dehydrated and febrile and had altered mental status . her general physical examination revealed anemia and pedal edema without any lymphadenopathy . on inspection abdomen was distended , without any visible vessel , scar or sinus . a periumblical bluish violet swelling 5 cm in diameter was also noted ( figure 1 ) . on palpation a firm liver edge 4 cm below the right costal margin with a hard periumblical swelling was also found . the cardiovascular system was normal except for a systolic flow murmur at the apex and base . investigations revealed a low hemoglobin of 3% , microcytic hypochromic type with a reticulocyte count of 5% and no abnormal cells on peripheral blood smear . kidney function tests , electrolytes , blood sugar , urine examination and septic screen ( blood , urine and asitic fluid cultures ) were normal . ascites and pleural taps revealed a lymphocytic exudate without malignant cells and adenosine deaminase in a suspect range . ultrasound of the abdomen revealed ascites with an enlarged liver , normal pancreas , gall bladder , adnexae and no para - aortic lymphadenopathy . fine needle aspiration cytology ( fnac ) of the umbilical nodule done showed metastatic deposits of well - differentiated adenocarcinoma ( figure 2 ) . the patient succumbed to massive rectal bleeding and despite all measures could not be resuscitated . a visible periumblical bluish violet swelling suggestive of a sister mary joseph nodule metastatic deposits of a well differentiated adenocarcinoma documented by fnac umbilical tumors are relatively rare and can be classified as benign or malignant.1 malignant tumors can be primary or metastatic tumors . sister mary joseph nodule is used to describe a malignant umbilical tumor usually associated with advanced metastasizing intra - abdominal cancer and generally indicating a poor prognosis . this sign was first identified by sister mary joseph ( 1856 - 1939 ) who as a surgical assistant to dr william james mayo drew attention to the presence of a hard umbilical nodule in a patient being prepared for surgery in 1928 . sister mary joseph nodule in the 11 edition of his textbook demonstrations of physical signs in clinical surgery in 1949 . the nodule usually presents as a firm , indurate often vascular swelling which may be fissured or ulcerated and may have serous , mucinous , purulent or bloody discharge . the nodule has been described as white , bluish violet and brownish red and is occasionally pruritic . it is usually irregular in shape , generally painless when palpated except if the overlying skin has ulcerated . it is typically less than 5 cm in diameter but occasionally enlarges enough to form a protruding tumor . patients with sister mary joseph nodule present with a number of clinical symptoms consistent with intra - abdominal cancer including epigastric pain , abdominal distension , weight loss , nausea , ascites and bleeding per rectum.2 the sign of sister mary joseph nodule has been extensively described in literature . the evaluation of an umbilical mass should be directed by suspicion of its being a metastatic deposit keeping in mind its potential to be either a primary malignant umbilical lesion or a benign disease . in a patient with a known malignancy an umbilical mass represents a spread or seeding of the primary tumor and thus can influence therapeutic decision making.3 the most common origins of sister mary joseph nodule are gastrointestinal ( 52% ) , gynecologic ( 28% ) , stomach ( 23% ) and ovarian ( 16% ) carcinomas . about 15 - 29% of all cases have an unknown origin ( as depicted by the presented case ) and 3% originate from the thoracic cavity . primary tumors in many other sites like gall bladder , uterus , liver , endometrium , small intestine , fallopian tube , appendix , cervix , penis , prostrate , urinary bladder , breast , lung and kidneys have also been reported to cause sister mary joseph nodules . histology of the metastatic umbilical tumor usually reveals adenocarcinoma but rare reports of umbilical metastasis from sarcomas , mesotheliomas and melanomas have also been seen.24 ct scan ( abdomen and chest ) and fine needle aspiration cytology ( fnac ) of the tumor are invaluable in the diagnosis of sister mary joseph nodule and help to exclude a primary benign umbilical neoplasm . in 14 - 33% of cases , umbilical metastases lead to the diagnosis of previously occult neoplasms.5 in 40% of patients with a known neoplasm the nodule was an early sign of relapse.6 spread of metastatic carcinoma to the umbilical region has been hypothesized to occur by either contiguous spread of peritoneal cancer , hematogenous spread through arterial and venous systems or lymphatic spread ( mainly pancreatic carcinoma ) with extension along ligaments of embryonic origin ( round ligament of liver , urachus , vitello intestinal duct reminant and the obliterated vitelline artery).67 the presence of a sister mary joseph nodule verified histological signifies advanced metastatic carcinoma and a poor prognosis with a survival time of 10 months ( range 2 - 17 months ) and inoperability . treatment of established sister mary joseph nodule is palliative as wide excision , surgery and radiotherapy have all proved ineffective . nevertheless a sister mary joseph nodule is a time tested , honored clinical sign emphasizing the importance of a careful physical examination of the abdomen . ihd examined , planned the work up , executed the investigative profile and wrote the manuscript .

sister mary joseph nodule or sister mary joseph sign refers to a palpable nodule bulging into the umbilicus as a result of metastasis of a malignant cancer in the pelvis or abdomen . a rare case of sister mary joseph nodule , manifesting as ascites , cachexia and bleeding per rectum , is presented without any primary tumor despite extensive search for the same .

Case Report Discussion Authors Contributions

PMC3076110

iridoschisis is a rare condition in which the iris stroma is cleaved in two portions and is defined as a separation of the anterior iris stroma from the posterior stroma and muscle layers . the anterior layer splits into strands , and the free ends float freely in the anterior chamber.1 the condition was first described in 1922 by schmitt when he presented a case with detachment of the anterior iris layer.2 the term iridoschisis was first introduced by loewenstein and foster in 1945 , when they described a case of end - stage glaucoma in an old woman who had an inferior localized cleavage of the iris into two layers , with the anterior fibrils of the iris waving in the anterior chamber.3 an 80-year - old woman was referred to the emergency unit of our ophthalmology department where she presented with a history of acute and persistent pain in the left eye and temple , accompanied by nausea and vomiting during the previous 24 hours . the clinical examination showed that the best - corrected visual acuity of the affected eye was finger count ( at 1.5 m ) , and the intraocular pressure at the time of admission was 54 mmhg . the conjunctiva was intensely red , with corneal injection , and mild edema was seen in the corneal stroma . the anterior chamber was remarkably shallow and showed loose ends of degenerated anterior stromal leaf fibrils floating freely in the aqueous . the iris showed typical features of iridoschisis , ie , stromal separation with cystic iris elevation in the inferior half and forward bowing of the iris ( figure 1 ) . the lens showed mature cataract which hindered visualization of the fundus and evaluation of any optic disc alterations . slit - lamp examination of the fellow eye revealed similar features , with a shallow anterior chamber and iridoschisis concerning the inferior half of the tissue ( figure 2 ) . the patient was free from a history of ocular trauma or of heritable ocular disease . conjunctivitis occurring at any time of the year with no mucopurulent secretion or pruritus , affecting either eye during an attack . the patient was diagnosed as having acute angle - closure glaucoma , and was receiving acetazolamide 250 mg three times daily , cosopt twice daily , isoptocarpine every two hours , intravenous mannitol ( 1 g / kg body weight stat ) , and oral pain - relieving drugs . on the third day after admission , the intraocular pressure of the affected eye was regulated at 14 mmhg and the administration of acetazolamide and cosopt was gradually discontinued . slit - lamp biomicroscopy showed that the cornea was no longer edematous and gonioscopy was therefore possible . we observed that there was a closed angle ( shaffer grade 01 ) for 270 and scattered peripheral anterior synechiae in the superior quadrant of both eyes . laboratory investigations showed a normal full blood count , prolonged prothrombin time , and increased inr . there were slightly increased values for urea , uric acid , and c - reactive protein ( 6.03 mg / l ) . the erythrocyte sedimentation rate was 58 mm for the first hour . observing that the lesion was in the iris stroma , we wanted to exclude whether this could be attributed to a connective tissue disease , so did an immunoserologic profile examination . the values for rheumatoid factor , antinuclear antibodies , antimitochondrial antibodies , antineutrophil cytoplasmic antibodies , and anticardiolipin antibodies were all within normal range . these immunoserologic assays are part of the standard procedure used in our screening protocol for exclusion of autoimmune connective tissue disease . on the fifth day after her admission , when intraocular pressure measurements were within normal limits and showed no fluctuation , and the cornea and anterior chamber presented no inflammatory activity , the patient was ready to leave the clinic with a regimen for topical antiglaucoma treatment ( isoptocarpine twice daily ) . the patient was extensively informed about the association of iridoschisis and the appearance of glaucoma , and she was advised to have frequent ophthalmologic examination , monitoring of intraocular pressure , and assessment for any progression of glaucomatous changes in both eyes . the anterior layer is usually divided into a loose mixture of large number of pigmented and white atrophic strands running in all directions , but mostly from the periphery to the collarette.4 larger free sections may contain blood vessels . it has been reported by fluoroiridography that there is normal perfusion of blood vessels , from the inner pupillary margin to the outer iris of the affected sectors.5 histopathologic study of the iris showed tissue fibrosis and atrophy and evidence of several small clefts in the substance . the vessels did not look abnormal , although the endothelial lining of some of them might be swollen.6 occasionally , separation at the posterior stroma might occur with irregular pigmentation of the pigment layers.7 electron transmission microscopy of the iris showed significant thinning of the stroma and a diminished number of collagen fibrils in the affected area , whereas the appearance of the stromal blood vessels and nerves was normal.7 corneal changes were uncommon and , if present , degenerated corneal endothelial cells were localized over the area of iridoschisis , where the strands of degenerated iris may come into contact with the corneal endothelium.7 the causative agent of iridoschisis is not yet identified . lowenstein and foster suggested that cleavage of the iris in this particular plane may have an anatomic basis and that there has to be an atrophic effect of lytic substances in the aqueous , originating from a glaucomatous condition , or that the condition could be attributed to senile changes.3 albers and klein also ascribe the changes in the iris to the aging process and they propose that , as the sclerosis of blood vessels increases in the anterior iris stroma , a shearing action tears the tissue between the anterior and posterior sections during constriction and dilatation of the iris.6 bojer suggests that iridoschisis is a form of essential iris atrophy in the elderly.8 progressive iris atrophy begins in the third decade of life ; iridoschisis , on the other hand , is seen mainly after the seventh decade of life . other authors have shown that both the vasculature and its perfusion are normal in iridoschisis , in contrast with the vasculature of essential iris atrophy.5,7 loewenstein et al have proposed that iridoschisis might be the result of trauma , and they assumed that a post - traumatic peak in intraocular pressure , which shears along the dilator fibers , could lead to splitting of the anterior and posterior iris stroma.9 an important aspect of iridoschisis is the frequent association with glaucoma . about two - thirds of reported cases of iridoschisis were seen in association with glaucoma.7,1012 salmon and murray studied a number of patients with iridoschisis and coexistent primary angle - closure glaucoma to determine the clinical features of the condition and to examine the relationship of iridoschisis to primary angle - closure glaucoma . their study suggests that iridoschisis is an unusual manifestation of iris stromal atrophy , and results from intermittent or acute elevation of intraocular pressure . they proposed that primary angle - closure glaucoma should be excluded in patients who present with iridoschisis.13 romano et al report that iridoschisis precedes the angle - closure episode.11 shima et al presented a case of iridoschisis and plateau iris configuration evidenced by ultrasound biomicroscopy.14 the patient had narrow angles and scattered peripheral anterior synechiae in both eyes . ultrasound biomicroscopy showed anteriorly positioned ciliary body , slit - like narrowing of the iridocorneal angle , and a flat iris in all four quadrants in both eyes . this case points to the idea that iridoschisis might be a part of a general anterior anatomic malformation of ocular structures , together with plateau iris configuration . iridoschisis might also possess a familial character , because there is a report of a family with iridoschisis , narrow anterior chamber angle , and presenile cataract . the author proposes that family members should be screened for iridoschisis , as well as for associated ocular abnormalities.15 iridoschisis is reported to be associated with lens subluxation , either anterior subluxation pushing the iris forward , shallowing the anterior chamber and causing angle - closure glaucoma,16 or posterior subluxation into the vitreous cavity.17 a subluxated lens rubbing against the iris may be a mechanical precipitating factor in the development of iridoschisis.16 there have been a number of cases reported to have iridoschisis and interstitial keratitis due to syphilis.1820 patients with congenital syphilis , apart from showing systemic features of congenital disease , presented with interstitial keratitis , ghost vessels , and iridoschisis involving predominantly the inferior half of the iris , and some of the patients had open - angle glaucoma . corectopia and ectropion uvea were absent.21 no peripheral anterior synechiae or fibrovascular membranes were seen in patients with open - angle glaucoma . neither interstitial keratitis nor iridoschisis had a suppurative component , and both were presumably immunologic in their pathogenesis.18 in the differential diagnosis of iridoschisis are the two other main iris stromal anomalies , namely , the iridocorneal endothelial syndrome and neurocristopathy of the iris and cornea ( axenfeld - rieger syndrome ) . diagnosis is made on clinical appearance , age of onset , laterality , and whether the condition is congenital , progressive , or with coexistent glaucoma.5,22,23 the age of onset for iridocorneal endothelial syndrome is the third or fourth decade and is unilateral and progressive , while iridoschisis appears later , usually in the sixth to seventh decade , and is generally bilateral , symmetrical , and progressive . on the other hand , axenfeld - rieger syndrome is present at birth , has a congenital character , and is bilateral , asymmetrical , and nonprogressive . in iridoschisis , the iris displays stromal splitting without any holes , is sectorial , is mainly inferiorly located , with anterior stromal atrophy and strands floating in the anterior chamber . the pupil is round and reactive to light and accommodation , and there are usually no corneal changes , unless anterior iris strands reach to the endothelium . in iridocorneal endothelial syndrome , stromal atrophy of the iris is observed , with or without holes , and peripheral anterior synechiae extending to schwalbe s line are present . the pupil may be ectopic , polycoria may even be observed , and the cornea manifests abnormal endothelium with pleomorphism and cell loss . in axenfeld - rieger syndrome , there is mild stromal thinning of the iris , atrophy , and hole formation , and peripheral anterior synechiae are observed on the anteriorly displaced schwalbe s line . the pupil may be irregular , and the cornea presents with endothelial changes related to peripheral anterior synechiae.1,2225 according to the aforementioned clinical entities , our patient had the differential characteristics of iridoschisis , although the exclusion of a diagnosis of iridocorneal endothelial syndrome or axenfeld - rieger syndrome can be sometimes difficult . an 80-year - old patient presented with an episode of acute angle - closure glaucoma and iridoschisis . the finding of iridoschisis is a warning for the ophthalmologist to exclude any association with glaucoma , because two - thirds of patients with iridoschisis have glaucoma . it is also possible that a patient may develop glaucoma in the future , so standard glaucoma tests ( including visual acuity , visual field assessment , tonometry , gonioscopy , biomicroscopy , and assessment of the optic nerve head ) and regular follow - up are required .

iridoschisis is a rare condition that consists of the separation of the anterior mesodermic layer of the iris . in more than two - thirds of cases it is associated with glaucoma . we report the case of an 80-year - old patient who had bilateral iridoschisis and presented with acute angle - closure glaucoma . the patient was free from a history of ocular trauma or of heritable ocular disease . chronic open - angle glaucoma , as well as intermittent angle - closure glaucoma , should be excluded in all patients with iridoschisis , and regular follow - up should be established upon diagnosis of the entity .

Introduction Case presentation Discussion Conclusion

PMC4261946

in eukaryotes , the basic repeating unit of chromatin is the nucleosome , consisting of 147 base pairs of dna wrapped around an octamer of histone proteins . the octamer contains two copies of each of the core histones , h2a , h2b , h3 , and h4 . histone proteins are dynamically post - translationally modified to regulate a wide array of nuclear processes , including transcription , dna damage repair , and signaling pathways . most of this post - translational modification ( ptm ) occurs on the n - terminal histone tails that protrude from the nucleosomal surface . of the core histones , h3 and h4 undergo the most extensive modifications mass spectrometry ( ms ) has emerged as a powerful tool to characterize histone ptms as it can accurately identify and quantify all histone ptm profiles in an unbiased manner . ms can also identify novel modifications as well as multiple modifications on a single peptide . most histone ptm analysis by ms has been conducted on high - resolution mass analyzers , such as orbitraps , because they provide sufficient mass accuracy to differentiate two very common , nearly isobaric ptms : acetylation and trimethylation , 42.0106 and 42.0470 da , respectively . for example , the ultrahigh - resolution fourier transform ion cyclotron resonance mass spectrometer ( fticr ms ) has a resolving power ( > 10 ) that can differentiate peptides with almost identical mass ( m / z < 0.001 ) , and can therefore easily discriminate trimethylation and acetylation based on mass alone . lower - resolution mass analyzers , such as linear ion traps , are unable to resolve this small mass difference . however , other techniques can be used to differentiate these modifications on lower - resolution mass analyzers , such as ms / ms spectra in combination with retention time information or synthetic peptide coelution information . the hunt lab demonstrated that the h3 917 aa peptide bearing k14ac or k9me3 eluted 17 min apart based on a 240 min gradient ( 0 to 100% b ) , suggesting that relative retention time can be used to distinguish the modifications . subsequently , the freitas group utilized this shift in retention time to demonstrate that trimethylated and acetylated peptides can be distinguished on low - resolution mass spectrometers based on their relative retention times . they validated their peak assignments by running the same sample on a high - resolution mass spectrometer to resolve the mass difference on the ms1 level and also performed ms / ms for further validation . one way to accomplish this task is to use heavy isotope labeled methyl and/or acetyl donors so that the modifications are no longer isobaric . however , it is relatively time - consuming to wait for the light modifications to turn over . another option is to spike in heavy labeled synthetic peptides , which will then coelute with the modified peptide of interest , but will be distinguishable based on its unique mass . used synthetic peptide coelution to determine that a potential new modification on a histone peptide ( h2at15ac ) was falsely assigned . neutral losses can also be used to lend confidence to identity assignments for isobaric peptides . trimethylated peptides undergo signature neutral losses during collision induced dissociation ( cid ) fragmentation , and the resulting mass shifts observed in ms / ms spectra can be used to validate methylation sites . fragmentation of acetylated peptides does not result in neutral losses but does produce immonium ions at m / z 143.1 which can be used to verify the presence of acetylated resides . since lower - resolution instruments are less expensive , easier to maintain , and somewhat more ubiquitous compared to high - resolution instruments , we aimed to determine if lower - resolution mass analyzers are as capable of robust and accurate histone ptm identification and quantification as high - resolution mass analyzers using several of the above - described techniques . to this end , we compared the performance of a low - resolution linear ion trap ( ltq velos pro ) and a high - resolution ion trap orbitrap hybrid instrument ( ltq - orbitrap velos pro ) in comprehensive ptm analysis of histones , one of the most extensively modified proteins in eurkaryotes . most modifications have a large enough mass difference to be resolved on lower - resolution mass analyzers , and so one of the biggest challenges when using low - resolution detectors for histone ptm analysis is differentiating trimethylation from acetylation . we first determined the optimal scan mode on the ltq velos pro based on three criteria : ( 1 ) reproducibility of relative peptide abundance measurements , ( 2 ) resolution of peptides in higher charge states ( e.g. , + 2 and + 3 charges ) , and ( 3 ) the number of ms1 and ms2 scans obtained per run . from this analysis , we determined that enhanced scan mode has the optimal trade - off between resolution and number of scans , while providing very consistent quantification . we then compared the performance of the ltq velos pro on enhanced scan mode to the orbitrap velos pro using the same criteria listed above as well as mass accuracy . the orbitrap velos pro was able to resolve the mass difference between acetylation and trimethylation , while the ltq velos pro could not . however , these modifications could be accurately quantified on the ltq velos pro using either retention time information or stable isotope - labeled synthetic peptide spike - in standards . as such , we conclude that while high - resolution mass analyzers are ideally suited for histone ptm analysis , low - resolution detectors are also adequate . hela s3 cells were gown in suspension as described previously and harvested using standard protocols . nuclei were resuspended in 0.4 m nacl buffer containing 1 mm dtt , 0.3 mm aebsf , and 10 mm sodium butyrate ( 10:1 buffer : pellet by volume ) and were incubated at 4 c with shaking for 30 min . the nuclei were pelleted at 3000 g for 5 min at 4 c , and the supernatant was removed by decanting . the pellet was resuspended in 2.5 m nacl containing 1 mm dtt , 0.3 mm aebsf , and 10 mm sodium butyrate ( 5:1 by volume ) . an equal volume of cold 0.4 n h2so4 was added slowly , and the nuclei were incubated at 4 c with shaking for 2 h. the nuclei were pelleted at 3400 g for 5 min , and proteins were precipitated from the supernatant with tca as previously described . when needed , offline reverse - phase high - performance liquid chromatography ( rp - hplc ) was used to further purify histone proteins as described previously . acid - extracted total histones or rp - hplc - purified histones were chemically derivatized with propionic anhydride and digested using trypsin ( promega ; substrate / protease ratio of 20:1 ) as described previously . histone peptides were subsequently desalted for ms analysis using homemade c18 stage tips as described previously . briefly , 93 synthetic tryptic histone peptides containing heavy - labeled amino acids were synthesized containing the most common ptm profiles . the peptides were purified by rp - hplc and combined to a final concentration of 0.27 pmol/l / peptide in water . the synthetic peptide library was added to a total acid - extracted histone sample in a ratio of 100 fmol of synthetic peptides:1 g of histone . a fused silica microcapillary column ( 75 m i.d . ) was packed in - house with reprosil - pur c18 resin ( 3 m , dr . the column was fitted to a commercial fused silica emitter with a 10 m tip ( new objective ) . small aliquots ( 1.5 g ) of a single histone h4 sample were loaded onto the column by an eksigent nanolc as-2 autosampler . peptides were separated using an eksigent nanolc 2d plus system hplc across a 76 min multistep gradient : 2% buffer b for 1 min ( a , 0.1% formic acid in water ; b , 0.1% formic acid in acetonitrile ) , 2% to 30% b in 55 min , 30% to 98% b in 15 min , 98% b for 10 min , 98% b to 2% b in 30 s , 2% b for 9.5 min ( equilibration ) at a constant flow of 250 nl / min . the hplc was coupled to a linear quadrupole ion trap ( ltq velos pro , thermo scientific ) mass spectrometer operating in zoom , enhanced , normal , or turbo scan modes ( see table 1 for scan rate information ) . a full ms spectrum was obtained followed by 6 data - dependent ms / ms acquisitions for the top six most abundant ions using cid fragmentation ( collision energy of 40 , activation q of 0.25 , and activation time of 10 ms ) . three technical replicates were obtained for each scan mode . based on a 76 min gradient ( 2% b for 1 min ; 2% to 30% b in 55 min ; 30 to 98% b in 15 min ; 98% b for 10 min ; 98% to 2% b in 30 s ; 2% b for 9.5 min ) at 250 nl / min flow rate using an eksigent nanolc ultra loading pump . aliquots ( 1.5 g ) of the same acid - extracted histone sample with synthetic peptides were run on two separate mass spectrometers : a linear ion trap ( ltq velos pro ) and a hybrid linear ion trap column was used on both instruments ( packed in - house with 3 m c18 resin ) fitted with a fused silica emitter with a 10 m tip ( new objective ) . the sample was loaded using an eksigent nanolc 2d plus system hplc for the ltq instrument while a thermo easy nlc 1000 was used to load the samples for the hybrid ltq - orbitrap instrument . the same multistep gradient was used for both instruments , consisting of 2% b for 1 min , 2 to 30% b in 40 min , 30% to 98% b in 15 min , 98% b for 10 min , with the exception that the eksigent nanolc gradient had an additional 10 min equilibration step at 2% b. data acquisition for both instruments was broken into 3 segments , 14 , 26 , and 16 min long , respectively . in the first segment , a full ms was obtained followed by 9 data dependent ms / ms acquisitions of the top nine most abundant ions from the full ms . in the second segment , a full ms was obtained , followed by 5 targeted ms / ms acquisitions ( m / z : 528.30 , 570.84 , 754.93 , 761.94 , and 768.95 ) and 5 data dependent ms / ms acquisitions of the top 5 most abundant ions from the full ms . in the third segment , a full ms was acquired followed by 10 data dependent ms / ms acquisitions of the top 10 most abundant ions . all full ms acquisitions for the orbitrap velos pro were obtained in the orbitrap in profile mode ( resolution : 60,000 at m / z 400 ) , while ms / ms acquisitions were obtained in the ion trap . cid fragmentation was used to fragment ions ( collision energy of 40 , activation q of 0.25 , activation time of 10 ms ) . we used an in - house developed algorithm to identify and quantify ptms for the ltq - orbitrap velos pro data , as described in wu et al . the algorithm uses ms , ms / ms , and retention time information to accurately identify and provide relative abundances of ptm profiles for tryptic histone peptides . relative abundance is obtained by integrating the single ion chromatogram of a particular modified peptide in all occupied charge states and dividing it by the total ion current for that peptide in all of its modified forms . the relative abundances of three technical replicates were averaged to obtain the final estimation of relative abundance . all ltq data were manually quantified because they are not compatible with the aforementioned algorithm . monoisotopic raw abundance of each peptide was manually extracted from xcalibur qual browser , based on the area underneath extracted ion chromatograms at the full ms level . all detectable charge states were considered , typically [ m + h ] , [ m + 2h ] , and [ m + 3h ] as previously described . the ltq velos pro mass spectrometer can operate in four different scan modes that have varying scan rates : turbo , normal , enhanced , and zoom ( rates listed in table 1 ) . the scan rate affects several important properties , including reproducibility of peptide abundance measurements , resolution , and number of scans collected per run . the optimal scan mode will vary depending on the specific application , and , as such , we sought to determine which scan mode is optimal for histone ptm analysis based on their ability to resolve doubly and triply charged histone peptides while still allowing for the maximal number of scans and consistent quantification . to our knowledge , this study represents the first comprehensive analysis of different scan modes for histone ptm analysis on a low - resolution mass spectrometer . three technical replicates of a single histone h4 sample were analyzed for this experiment . the histone sample was purified from hela cells treated with butyrate , a histone deacetylase inhibitor , therefore containing more pronounced and combinatorial acetylation profiles . the sample was derivatized using our propionic anhydride methodology as it increases the hydrophobicity of the histone peptides , allowing for better retention on c18 resin , and also prevents trypsin cleavage after lysine . to reduce variance due to instrument setup , the sample replicates were run using identical chromatographic gradients ( delivered by different hplcs ) and the same analytical column within 2 days . since small changes in ptm abundances can have large biological implications , it is important to run samples for ptm analysis on an instrument that generates highly reproducible results . therefore , any observed differences can be attributed to biological phenomena rather than technical variances . as such , we investigated the reproducibility of measuring the relative abundances of modifications for a typical example peptide from histone h4 ( amino acids 417 ; sequence : gkggkglgkggakr ) , for each scan mode . relative abundances were obtained by integrating the single ion chromatogram ( sic ) in ms1 spectra of a particularly modified peptide in all occupied charge states and normalizing the signal by dividing it by the total ion current of that peptide in all of its modified forms , as we and others have performed for many years now . the relative abundance values obtained on each scan mode were found to have a fairly small standard deviation , indicating that each mode produces highly reproducible abundance measurements ( figure 1 ) . however , some scan modes produce different relative peptide abundance measurements compared to the other scan modes , with turbo scan mode being the most variable . this variability indicates that turbo scan mode may lead to inaccuracies in abundance measurements , possibly due to the presence of multiple species in a given m / z range that can not be resolved . the tri- and tetra - acetylated peptides have larger standard deviations than the other modified forms of the peptide , which is more common for low - abundance peptides . reproducibility of relative peptide abundance measurements for h4(417aa ) peptides on each ltq velos pro scan mode . the relative abundance of each modified form of the peptide was calculated from three technical replicates of the same purified h4 sample . error bars represent standard deviation from average relative peptide abundances . * p < 0.05 . both resolution and scan rate are important factors affecting accuracy in peptide and ptm identification . obtaining adequate resolution is vital for identifying and quantifying the ptm profiles present in a sample . if the resolution is too low , the charge state of the peptides can not be determined , and the identity can not be confidently assigned . ion trap mass analyzers scan and record the m / z range by increasing the radio frequency ( rf ) voltage applied to the electrode over time to eject peptide ions of increasing m / z from the trap to be detected . fast scan rates result in poor resolution because the ions of a given m / z do not have enough time to fully eject before the rf increases , causing peak widening . the resolution obtained in the four different ltq velos pro scan modes is shown in figure 2a for an example peptide , diacetylated h4 417 + 2 ( [ m + 2h ] = 761.939 ) . the fastest scan rate , turbo , is completely unresolved , and the isotopes can not be distinguished at all . however , the zoom , enhanced , and normal scan modes have sufficient resolution to reliably assign charge states and are therefore amenable to this type of analysis . resolution of diacetylated h4 417 on each ltq velos pro scan mode . mass spectra of ( a ) doubly charged ( [ m + 2h ] = 761.939 ) and ( b ) triply charged ( [ m + 3h ] = 508.295 ) peptides are displayed . the resolution ( m / m ) of the monoisotopic peak ( c ) resolution of the peptides from panels a and b as a function of scan rate . as the charge state increases , it becomes more difficult to resolve the isotopes of a given peptide . propionylated tryptic histone peptides generally occupy lower charge states ( + 1 to + 3 ) . figure 2b displays the resolution of the triply charged example peptide ( [ m + 3h ] = 508.295 ) . zoom and enhanced modes can resolve the charge state , while normal and turbo scan modes do not fully resolve the peptide . therefore , zoom and enhanced scan modes are better options for histone ptm analysis on a low - resolution mass spectrometer . figure 2c illustrates the relationship between scan rate and resolution for the example peptide in both charge states . one important note is that scan rate affects the amount of data that can be collected during an ms analysis . zoom has the best resolution , but is also the slowest scan rate and therefore does not collect as many full ms and ms / ms spectra as enhanced scan mode ( table 1 ) . since some modified peptides have low abundances ( e.g. , k18me1 , k23me1 ) , it may be advantageous to sacrifice higher resolution for an increased number of scans . as such , we recommend using enhanced mode for analyzing histone ptms as it has sufficient resolution to resolve isotopes for double and triply charged peptides , allows reproducible quantification , and allows for almost 20% more ms2 scans compared to zoom mode . high - resolution mass analyzers have been at the forefront in histone ptm analysis , despite the fact that low - resolution mass analyzers are less expensive and generally more accessible . one of the main reasons for using a high - resolution detector is that it can resolve the mass difference between trimethylation and acetylation modifications ( 42.0470 and 42.0106 da , respectively ) , which occur at high frequency on histone proteins . however , it is still possible to differentiate these ptms on a low - resolution detector using a combination of retention time and ms / ms information . furthermore , krey et al . demonstrated that the low - resolution ltq and ltq velos pro mass spectrometers can quantify protein abundance as accurately as high - resolution orbitrap mass spectrometers over a range of protein concentrations . as such , low - resolution mass analyzers could potentially be useful in analyzing histone ptm profiles , however to our knowledge no side - by - side comparison between low- and high - resolution mass analyzers has been published yet for comprehensive analysis of histones , which contain one of the most complicated ptm profiles of any eukaryotic protein . we therefore compared the performance of the ltq and orbitrap mass analyzers to determine if low - resolution mass analyzers are adequate for this type of complicated analysis . to conduct this comparison , we used a single sample containing total acid - extracted histones from butyrate - treated hela cells , which were subsequently propionylated and digested with trypsin . propionylated , heavy isotope - labeled synthetic peptides were spiked in the hela histone peptides to aid in identification of endogenous histone peptides during data analysis , as will be described in more detail later . we analyzed three technical replicates of this sample on each instrument and compared the resulting reproducibility of relative peptide abundance values , resolution , and mass accuracy . the ltq velos pro was operated in enhanced scan mode as this was determined to be optimal for histone ptm analysis . the runs were conducted within 3 days on the same analytical column , instrument method , and gradient to reduce differences due to instrument setup . one drawback of using the orbitrap velos pro mass analyzer is that the scan rate is inherently slower than that of the ltq velos pro mass analyzer . table 2 compares scan rate information for the ltq velos pro and the orbitrap velos pro . the scan numbers refer to the number of scans collected in the first three segments ( see experimental section ) . the orbitrap velos pro collected an average of 873 full ms scans and 8,784 ms / ms scans , while the ltq velos pro collected 1,189 and 11,703 , respectively ( table 2 ) . thus , while high - resolution detectors allow for better mass accuracy and consequently more facilitated data analysis , low - resolution detectors collect a larger amount of information even when operated on one of the slowest scan modes . this trade - off of reduced accuracy for increased ms and ms / ms scans could be desirable for the identification and quantification of rare or low - level ptms . the orbitrap data is based on a 66 min gradient that omits the final equilibration step ( 2% b for 1 min ; 2% to 30% b in 55 min ; 30 to 98% b in 15 min ; 98% b for 10 min ) at 250 nl / min flow rate using a thermo easy nanolc hplc . as mentioned previously , it is important that the instrument used for ptm analysis generate highly reproducible abundance measurements . as such , we compared the reproducibility of calculated relative peptide abundances obtained on the ltq velos pro and the orbitrap velos pro instruments based on three technical replicates each . we only analyzed h3 and h4 because they are the most modified histone proteins and are therefore the most challenging to analyze . the results , as shown in figure 3 , demonstrate that the standard deviation in relative abundance measurements is very small on both instruments , and results are very comparable between both instruments ( p = 0.09 ; paired t test comparing standard deviations between the two instruments ) , indicating that both instruments are highly reproducible . reproducibility of relative peptide abundance measurements obtained on the ltq velos pro and the orbitrap velos pro . ( a , b ) each point represents a particular modified form of either ( a ) an h3 peptide or ( b ) an h4 peptide , while the color of the point indicates the identity of the peptide . the black line indicates perfect correlation between relative peptide abundance values on the two instruments . the dashed line is a linear regression for all data points on the plot ( pearson , ( a ) r = 0.878 , ( b ) r = 0.839 ; spearman , ( a ) r = 0.853 , ( b ) r = 0.883 ) . the modified peptides shown in the figure include the following : ( a ) 38 , unmodified , k4me1 ; 917 , unmodified , k9me1 , k9me1k14ac , k9me3k14ac , k9me2 , k9me3 , k9 or k14ac , k9me2k14ac ; 1826 , unmodified , k18ac or k23ac ; 2740 , unmodified , k36me1 , k36me2 , k27me1 , k27me2 , k27me3 , k27me2k36me1 , k27me1k36me2 , k27me1k36me3 , k27me1k36me1 ; ( b ) 417 , unmodified , monoacetylated , and diacetylated ; 2023 , unmodified , k20me1 , k20me2 . each point represents a particular modified form of the h3 917 peptide , listed in panel b. the blue points represent the data before normalization as shown in panel b , and the red points represent the data after normalization to the synthetic peptide standards . the dashed blue line is a linear regression fit of the data before normalization ( r = 0.579 ) , and the red dotted line is a linear regression fit of the data after normalization ( r = 0.979 ) . since the same sample was analyzed on both instruments , we would expect the calculated relative abundance measurements on each instrument to be similar . the results indicate that while in general this is true , some of these values differ between the two instruments ( figure 3a , b , as evidenced by deviation from the black line that indicates perfect correlation ) . however , a linear regression fit to all data points for h3 or h4 peptides demonstrates a high degree of correlation between the values obtained on each instrument ( pearson , r = 0.878 for h3 and r = 0.834 for h4 ; spearman , r = 0.853 for h3 and r = 0.883 for h4 ) , indicating that the instruments produce highly similar abundance measurements . it is also important to note that the measurements are relative , so an error in the abundance of one modified form of a peptide affects the relative abundance measurement of all other forms of that peptide due to the way the data is normalized . although the correlation of peptide abundance measurements between the two instruments is relatively high overall , some peptides do not correlate well . the peptide with the lowest correlation between instruments was the h3 917 peptide ( pearson : r = 0.579 ) . low correlation is likely a result of the major differences in how the instruments collect data . there were no common features between modifications or peptides that did not correlate well . to correct for differences in instrument data acquisition , the relative peptide abundance measurements of the endogenous peptides can be normalized to those of the synthetic peptide standards , which are present in equal concentrations . to normalize the data , a normalization factor was calculated for each form of the peptide by dividing the expected relative abundance measurements of the synthetic peptides by the observed relative abundance measurement of the synthetic peptides . the average raw abundance values for the endogenous peptides were then multiplied by the respective normalization factor , and the relative peptide abundances were calculated from these corrected values . our group has previously used this method to correct for differences in ionization efficiencies between differently modified histone peptides during quantification . figure 3c plots the relative abundances of the h3 917 peptides before and after normalization to the synthetic peptide standards . after normalization , the abundance measurements obtained on the two instruments are very highly correlated ( pearson : r = 0.979 ) , indicating that the ltq velos pro measures histone ptm abundances as accurately as the orbitrap velos pro . we calculated the average mass accuracy of the ltq velos pro and the orbitrap velos pro based on two example peptides , h3 1826 and h4 417 , in different modified forms to gauge what mass tolerance can be used to identify peptides . the mass accuracy for the ltq velos pro ranges from 53.0 to 193 ppm , with an average of 129 ppm , and the mass accuracy for the orbitrap velos pro ranges from 0.00 to 6.62 ppm , with an average of 2.01 ppm ( table 3 ) . using a mass tolerance of 10 ppm , the orbitrap velos pro can differentiate trimethylation and acetylation , while the ltq velos pro , using a mass tolerance of 150 ppm , can not , as has been noted by others . this is demonstrated in figure 4 , which shows the chromatograms of different modified forms of the histone h3 917 peptide . for the orbitrap velos pro , the trimethylated and acetylated peptide ( row 4 and 5 , respectively ) have a large enough mass difference under the given mass tolerance to be unequivocally assigned to the single peak , while the ltq velos pro can not resolve these peptides . the resolution of the ltq velos pro is high enough to differentiate all of the other common ptms , such as mono- and dimethylation and multiply modified peptides such as k9me1k14ac ( figure 4 , rows 13 ) . chromatographic information for h3 917 obtained on ltq velos pro or ltq - orbitrap velos pro . the mass tolerance used for selection is 150 ppm for the ltq velos pro and 10 ppm for the ltq - orbitrap velos pro . in each row , labeled 1 to 7 , the mass of the peptide bearing a specific modification was specified . rows 1 to 5 represent endogenous peptides while rows 6 and 7 represent heavy - labeled synthetic peptides . the acetylated and trimethylated peptides can still be distinguished on the ltq , however , by using relative retention time information and high - resolution ms / ms data as has been noted by the freitas group . however , this group did not derivatize their samples with propionic anhydride and did not use this technique to quantify peptides , and so we decided to apply this method to quantify histone ptms for the instrument comparison . synthetic peptides eliminate the need to perform high - resolution ms to validate peak assignments . the retention time of acetylated peptides is later than their corresponding methylated peptides because acetyl groups are more hydrophobic than trimethyl groups ( figure 4 , row 4 versus 5 ) . since the peptides are propionylated in this study , the di- and trimethylated peptides are more hydrophilic than the unmodified peptide . di- and trimethylation prevent propionylation so k9 is propionylated in the unmodified peptide and is not propionylated in the di- and trimethylated peptides . since propionyl groups are more hydrophobic than di- and trimethylation , the unmodified peptide is more hydrophobic and elutes later ( figure 4 , row 3 and 4 compared to 1 ) . heavy - labeled synthetic peptides bearing a trimethyl or acetyl group at the residue of interest can also be used to validate peak assignments . similar methods have been described by others , but there have been no studies using heavy - labeled synthetic peptides to differentiate nearly isobaric ptms . the synthetic peptides have the same retention time as the unlabeled endogenous histone peptides containing the same modifications , as c or n isotopes do not influence retention on c18 columns , but impart a unique mass . therefore , we can determine which ambiguous peak contains a trimethylated or acetylated peptide by seeing which coelutes with the synthetic peptide bearing the respective modification . in figure 4 , synthetic peptides were used to differentiate trimethylated and acetylated peptides . rows 6 and 7 show the chromatograms for the h3 917 synthetic peptides bearing k9me3 or k14ac , respectively . the k14ac synthetic peptide ( row 7 ) elutes under the k14ac peptide peak in the endogenous trace ( row 5 ) , and the k9me3 synthetic peptide ( row 6 ) elutes under the corresponding k9me3 peak in the endogenous trace ( row 4 ) . therefore , low - resolution detectors can be used to accurately quantify and identify histone ptm profiles when supplemented with synthetic peptides . the results of the current study show for the first time that low - resolution mass analyzers , such as the ltq velos pro , are adequate for comprehensive ptm analysis although the data analysis is more easily facilitated on high - resolution instruments . the reproducibility of obtained relative peptide abundances is equally high on the ltq velos pro as the orbitrap velos pro . although the ltq velos pro is not able to resolve the small mass difference between acetylation and trimethylation , other orthogonal lines of evidence can be used to accurately identify the peak containing each modified peptide . as such , high - resolution does not seem to be absolutely required for histone ptm analysis .

mass spectrometry ( ms ) is a powerful tool to accurately identify and quantify histone post - translational modifications ( ptms ) . high - resolution mass analyzers have been regarded as essential for these ptm analyses because the mass accuracy afforded is sufficient to differentiate trimethylation versus acetylation ( 42.0470 and 42.0106 da , respectively ) , whereas lower - resolution mass analyzers can not . noting this limitation , we sought to determine whether lower - resolution detectors are nonetheless adequate for histone ptm analysis by comparing the low - resolution ltq velos pro with the high - resolution ltq - orbitrap velos pro . we first determined that the optimal scan mode on the ltq velos pro is the enhanced scan mode with respect to apparent resolution , number of ms and ms / ms scans per run , and reproducibility of label - free quantifications . we next compared the performance of the ltq velos pro to the ltq - orbitrap velos pro using the same criteria for comparison , and we found that the main difference is that the ltq - orbitrap velos pro is able to resolve the difference between acetylation and trimethylation while the ltq velos pro can not . however , using heavy isotope labeled synthetic peptide standards and retention time information enables confident assignment of these modifications and comparable quantification between the instruments . therefore , lower - resolution instruments can confidently be utilized for histone ptm analysis .

Introduction Experimental Section Results and Discussion

PMC4324538

ethiopia has one of the worst maternal mortality ratios in the world , at 673 per 100,000 ( range 54810).1 concealed within this figure is a marked urban / rural split . the 2011 ethiopian demographic health survey carried out by the ministry of health showed that in the capital , addis ababa , 82% of mothers deliver in a health facility whereas in the amhara and afar districts that are largely rural , the rates are 10% and 7% , respectively.2 there are many problems involved in overcoming this . for example , in the amhara region of ethiopia with a population of over 20 million people there were 16 hospitals in 2010 and only six of these could reliably deliver emergency obstetric care . lack of facilities combined with costs that were cheap in global terms ( us$5 ) , but prohibitively expensive for the local population , poor roads and communications , most women can not attend a hospital for their delivery . as a result only 10% of women in ethiopia receive help from a skilled delivery assistant with some estimates of 96% women delivering at home in some areas of ethiopia.3 the ethiopian government has tried some programs to increase the human resources to deliver emergency obstetric care ( emoc ) in those remaining hospitals . the current scheme is a 3-year master of surgery course available not only to medical doctors , but also to health officers . the master of surgery course not only covers emergency obstetric management , but also emergency surgery . however , it will be many years before the shortage in labor is resolved due to the number of candidates being trained versus the number required and the length of time it takes to train each candidate . in the meantime , the amhara regional health bureau of ethiopia requested overseas help to overcome the labor shortage by placing volunteer teams of obstetricians and midwives in rural hospitals to help provide the service and also train local staff members in emoc . the objective of the study reported here was to assess the impact of placing volunteer medical teams within the ethiopian government hospital system in targeted hospitals on maternal morbidity and mortality , before , during , and after the intervention . it is a government district hospital that was opened in the year 2000 to service a population of 1.2 million people . these are fresh university graduates who have no surgical training or experience . before the placement of volunteers there was no emoc service and those women needing some emergency or operative intervention had the option of returning home or being referred to the regional referral hospital in the town of barhirdar , 120 kilometers away along an unsealed road . there was one hospital ambulance that charged 400 birr ( or about us$30 ) to take them to barhirdar , or an unreliable bus service . the expense of both meant that most women in a critical condition were sent home . the population , knowing that there was very little help to be offered at mota hospital generally did not present to hospital in labor . the hospital had many other problems : no running water , a temperamental generator to cover frequent power shortages , shortage of staff , no blood transfusion service , and a poor supply of drugs to the pharmacy . volunteer teams of one obstetrician and one volunteer midwife were recruited from holland , australia , india , and america . they would stay for periods of 24 months , usually 3 . over the 3 years they were self - funded with respect to air travel , visas , living expenses , and health insurance . accommodation was provided at the hospital , medical registration was organized with the amhara national regional state health bureau , and internal transport was provided by the program . the first volunteer team arrived at mota in may 2010 and the last left in june 2013 . a supervisory visit was made by a volunteer in january 2014 and then in may 2014 . the role of the volunteers was to establish and provide an emergency obstetric service , including full operative and emergency care along with blood transfusion service . they were also to supervise and train local staff . a retrospective analysis of monthly hospital records from january 2009 until april 2013 was done , with 2009 being the control year . since the placement of the volunteer teams of obstetricians and midwives within mota hospital began in may 2010 , the intervention took place during only some months of the year , so this year was excluded from the comparison . data were collected from the birth registry book and entered into a microsoft excel spreadsheet at the end of each month and the raw data analyzed from excel . outcome measures were the number of deliveries before , during , and after the 3-year intervention period , the maternal mortality rate , and maternal morbidity rate , with the latter assessed by the number of referrals of obstetric fistula patients to a nearby specialist fistula repair centre . also analyzed were the instrumental and operative delivery rates , number of women needing referral during labor , and perinatal mortality . the placement of volunteers was phased out when the regional health bureau was able to staff the target hospital sufficiently to provide the service without outside assistance . there was no ethical board at mota hospital at the time of our study , and as this was a review of cases , permission to do the study was granted by the current medical director . there was a 40% increase in the number of women delivering within the hospital from 738 in 2009 to over 1,000 in both 2011 and 2012 ; there was a slight proportional decrease in the number of assisted vaginal deliveries ( table 1 ) . there was an obvious increase in the number of deliveries by cesarean section as there was no service provided in 2009 ( 0.0% ) and the cesarean rate was 8.7% and 10.7% for 2011 and 2012 , respectively . there was a large decrease in the number of patients referred to the referral hospital in barhirdar for further treatment : from 79 ( 9.3% ) pregnant women in 2009 to only one in 2011 and 2012 . this referral was at a time when there was no anesthesia cover for a short period of time and the patient needed an operative delivery . , it is estimated that there were 17 maternal deaths within the hospital itself ( or 2.4% of women delivering ) , but there were at least 100 patients referred to barhirdar or sent home in extremis during 2009 as there was nothing the hospital could offer the patients . many women died en route to barhirdar or after arrival and most women who went home died . estimating that up to 50% of women transferred or sent home died would make approximately 60 maternal deaths that year , or one in 14 women presenting to the hospital ( 738 deliveries plus 100 women turned away ; 60 deaths from 838 women ) . this figure could easily be higher and is thought to be a conservative estimate . compared with 2009 , in 2011 and 2012 there were no women turned away from the hospital and only one transfer with 15 maternal deaths over the 2 years from 2,043 deliveries this works out to be less than one in every 157 women who presented for delivery at mota . the number of perinatal deaths was not recorded in 2009 , so comparison was not possible . most of these were either stillbirths with the woman presenting late in labor with the fetus already deceased in utero on arrival , or other unavoidable deaths often associated with a long labor , uterine rupture , and sepsis . the number patients with obstetric fistula referred from mota hospital for fistula repair in a specialist unit in barhirdar in 2009 was eleven , as per mota hospital records ( 1.5% of women delivering ) . from ab s experience of working as the surgeon at the barhirdar fistula hospital during those years , on average , four fistula patients would present per month , a total of up to 48 women , most of whom delivered in mota hospital so the real figure is likely to be higher . after the intervention , over the 2 years , twelve ( 0.5% of hospital deliveries ) presented at mota hospital with an obstetric fistula , none of whom had their fistula develop within the hospital , but already had the injury on arrival in labor . the regional health bureau placed a local health officer with emoc training in mota hospital in january 2013 and the volunteers worked alongside the health officer before leaving . after withdrawing the full time volunteer presence and leaving the service in the hands of the local health officer , monitoring visits were made six and 11 months later . the number of deliveries was maintained at the same level as when the volunteers had left . no maternal deaths and few stillbirths were recorded , although it was unclear if adequate records had been maintained for accurate comparison of the latter two parameters . there have been many different efforts to try to combat maternal morbidity and mortality in the developing world , from introducing birth preparedness schemes,4 to getting women to assess health facilities during labor,5 and even providing financial assistance and motivation.6,7 the ultimate aim of these interventions has been to get women to deliver within a health institution . however , there has been some question as to whether it is worthwhile encouraging women to access health services if the quality of the health services is poor.8,9 other groups have thus focused on brief periods of intensive training to improve this , but the impact of short - term training has been questioned.10,11 it is certainly our personal experience that short - term training , although enthusiastically taken on board , makes little impact on changing practices on the ground . it appears more long - term investment into training over years is what slowly brings change , which was the aim of the program reported here . as expected , establishment of an emoc service at mota has had positive benefits for the women who are able to attend the hospital for antenatal care and delivery , most particularly in relation to maternal death , which fell from a conservatively estimated 1 in 14 in 2009 to 1 in 157 women delivering within the hospital during 2011 and 2012 . many of the maternal deaths were high - risk patients who had not attended any antenatal services and arrived at the hospital in labour , in extremis from surrounding rural areas . it was interesting to note that a further increase in women coming to the hospital to deliver did not occur in the second year of activity in mota this has been taken to indicate that even though there are undoubtedly thousands more women in the region who could benefit from delivering in mota , they live too far away to walk there , are too poor to afford public transport , or live too far away from a road . the women who came for delivery at mota were probably only from the immediate surrounds and it is likely the women from more remote areas remained at home to deliver . the benefits the volunteers brought were not only the introduction of a service and training of all staff , including cleaners , but also an improvement in infrastructure . the volunteers certainly felt ownership of the project and raised money from their home countries to fix the water well ; replace water piping ; purchase a new water pump to ensure a water supply to the hospital ; and buy other surgical instruments , supplies , and training equipment . the other impact was on the volunteers themselves , many of whom have returned for their second or third term of volunteering while two others have committed to 12 years of volunteering at other sites having had their the budget for the intervention was low ; initial funds were used to upgrade hospital accommodation for the volunteers and in - country transport to get volunteers to the hospital . the project budget was also used to purchase new delivery equipment and an ultrasound unit , repair a water well , purchase a new pump for the well , and repair plumbing to ensure a water supply to the hospital . although this is a single example of one site in ethiopia , the intervention had a very positive impact on maternal morbidity and mortality . the analysis was limited by rough , incomplete records being interpreted for 2009 and for the period of 2013 after the volunteers left . although the follow - up after the 3-year intervention is only at 11 months at this stage , we have seem a sustainable improvement in services and significant impact on maternal morbidity and mortality for very little overall financial input . we believe it has been a cheap , effective strategy to commence an emoc service in an area that has never had one and we believe it is also sustainable , as the service , now successfully started , has been handed over to the responsible ethiopian health bureau . similar projects utilizing volunteers have been undertaken in four other hospitals in ethiopia and two in tanzania with similar success . placement of volunteer teams of obstetrician and midwife volunteers had a positive and ongoing impact on the women of the area of mota in terms of both maternal morbidity and mortality . it was cheap , is easily replicable , and if volunteers can be found , can easily be introduced into other areas .

objectivesto determine the impact of volunteer obstetricians and midwife teams on obstetric services in a rural hospital in ethiopia.methodsthe intervention was undertaken in mota district hospital , a rural hospital in the amhara region of ethiopia , which is the only hospital for 1.2 million people . before the placement of volunteer teams it had a rudimentary basic obstetric service , no blood transfusion service , and no operative delivery . the study prospectively analyzed delivery data before , during , and after the placement of volunteer obstetrician and midwife teams . the volunteers established emergency obstetric care , and trained and supervised local staff over a 3-year period . measurable outcomes consisted of the number of women delivering , the number of referrals of pregnant women , the number of maternal deaths , and the number of referrals of obstetric fistula patients.resultswith the establishment of the service the number of women attending hospital for delivery increased by 40% . in the hospital maternal mortality decreased from 7.1% to < 0.5% , and morbidity , as measured by number of obstetric fistulae , decreased from 1.5% deliveries to 0.5% over the 3-year intervention period . the improvements were sustained after handing the project back to the government.conclusionthe placement of volunteer teams was an effective method of decreasing maternal mortality and morbidity .

Introduction Materials and methods Results Discussion Conclusion

PMC4868230

a throwing event is a sports competition that displays a player s ability to throw an object , such as a shot put , discus , javelin , or hammer . to achieve a high level of performance in this sport , total physical fitness , including explosive muscle power , flexibility , agility , and coordination is required1 . in particular , core and low extremity muscles are considered to be important muscles for creating a greater rotatory force and maximum speed2 , 3 . the core muscle is found to improve the performance of athletes by increasing balance , as well as trunk and lower extremity strength in various sports , including short - distance running , volleyball , and golf , as core muscle training exercises enhance the stability of the lumbus and spine balance by increasing the musculus transversus abdominis contraction4,5,6,7,8,9 . thus , the core exercise program , which maximizes the mobility and stability of the core muscles , is greatly emphasized for athletes11 . although previous studies examined the effect of core exercises on isokinetic muscle functions in various sports5,6,7,8 , 12 , only limited studies are available to establish an effective and practical muscle training program focusing on throwing events in particular . therefore , this study aimed to investigate the effect of peculiar complex core balance training on isokinetic muscle function of the knee joint and lumbus to provide fundamental data for establishing a training program that improves the performance and prevention of injury by developing the core and low extremity muscles . a total of ten high school athletes , who have been performing in throwing events for over five years , participated in this study . the subjects were randomly divided into two groups : the experimental group ( n=5 ) and the control group ( n=5 ) . the experimental group underwent peculiar complex core balance training ( 60 min / three times a week/16 weeks ) while the control group did not participate in such training . all subjects were educated about the content and purpose of the experiment and provided written informed consent before beginning the study . a peculiar complex core balance training program was established based on previous studies13 with two sports training and conditioning experts , and three current throwing coaches ( table 1table 1.peculiar complex core balance training programstage 1 ( week 14 ) programcore exercisesling exercisestraight leg lifts , twisted waist oblique crunches , butt raises , shoulder - to - knee leg overs , side to side planks , plank with hip drops , around the world plank , bridge plank , straight plank , mountain climbers , side plank hip abduction with a twist , spiderman plank , side planks with reachsuspended plank , suspended single plank , suspended double pike , reverse , climber , outsiders , roll - outs , bicep curl , push curl , single - leg lunge , hamstring curl , hip raise , oblique twist , tricep extension , rear deltoid , captain america workout intensity = rpe 1314 , 10 rep 3 set ; duration = 50 min ; rest = 1 min between setstage 2 ( week 510 ) programcore exercisesling exercisesky reachers , sit - up , feet up crunch , bicycle pausers , fast bicycles , extended reverse crunch , compound push up , burpees , dynamic push up , leaping lunges , bring knee to elbow on the way down , ski abs , plank squatsbicep curl , push curl , single - leg lunge , hamstring curl , hip raise , oblique twist , tricep extension , rear deltoid , captain america workout , suspended crunch 1 & 2 , pendulum 1 & 2 , standing body crunch , standing oblique twist , suspended oblique crunchintensity = rpe 1314 , 15 rep 3 set ; duration = 60 min ; rest = 1 min between setstage 3 ( week 1116 ) programcore exercisesling exercisesingle rowers , supermans , oppo - raisers , bird dogs , throw downs , hip to hip , hand walk - outs , , bicycle pausers , plank push up , side plank rotation , side planks reach throughs , tricep dip oblique crunches , hip thrust , grass hoppers , russian twist , bicycle crunchbicep curl , push curl , single - leg lunge , hamstring curl , hip raise , oblique twist , tricep extension , rear deltoid , captain america workout , suspended crunch 1 , suspended crunch 2 , pendulum 1 , pendulum 2 , standing body crunch , standing oblique twist , suspended oblique crunchintensity = rpe 1314 , 20 rep 3 set ; duration = 60 min ; rest = 1 min between set ) . the training consisted of 16 weeks of mat , medicine ball , and sling training , focusing on the development of core muscles with three stages , including basic balancing and muscle adaptation ( 4 weeks ) , kinetic balancing and muscle development ( 6 weeks ) , and complex training of kinetic balancing and muscle development ( 6 weeks ) . the intensity of training was decided by rating the perceived exertion ( rpe)14 . in this study all subjects underwent measurement of the lower extremity ; lumbus muscular strength was measured by isokinetic measurement equipment ( cyber-770 , lumax . all experiments were thoroughly reviewed and approved by the institutional review board of daegu university . the means and standard deviations of the two groups were calculated , and the analysis of covariance ( ancova ) was conducted to examine the differences between the groups and between measurement time points . the results of the ancova indicate a significant effect of peculiar complex core balance training and difference between the experimental and control groups . first , in terms of the change of isokinetic strength of the knee joint , ancova [ between - subjects factor : group ( experimental , control ) ; covariate : time ( pre - post ) ] revealed the main effects of group and time in all measurements ( p<0.05 ) , excluding extension of muscle power ; the interaction between group and time in extension of muscle power was also significant ( p<0.05 ) ( table 2table 2.result of ancova for knee joint strength and muscle powertypeexperimental groupcontrol group strength ( nm)lextensionpre254.614.2258.121.6post268.417.3260.219.7**flexionpre207.011.6198.311.2post224.913.6201.218.3*rextensionpre268.420.3257.421.6post270.719.1256.396.0**flexionpre181.618.3189.614.2post210.121.3194.415.7*muscle power ( nm)lextensionpre57.214.154.110.5post72.112.156.911.2**flexionpre145.210.1143.516.3post158.213.8143.817.1*rextensionpre68.117.269.810.1post75.116.370.120.7**flexionpre156.210.1158.109.1post162.209.3158.417.3*values are meansd . significantly different ( * * p<0.01 , * p<0.05 ) from the experimental group . significantly different ( p<0.05 ) between pre and post in the experimental group . significant interaction ( p<0.05 ) between group and time in the experimental group ) . values are meansd . significantly different ( * * p<0.01 , * p<0.05 ) from the experimental group . significantly different ( p<0.05 ) between pre and post in the experimental group . significant interaction ( p<0.05 ) between group and time in the experimental group secondly , regarding the change of isokinetic strength of the lumbus , ancova [ between - subjects factor : group ( experimental , control ) ; covariate : time ] revealed the main effects of group and time in all measurements ( p<0.05 ) ; the interaction between groups and time in flexion of strength ( p<0.05 ) and extension of muscle power ( p<0.05 ) were also significant ( table 3table 3.results of ancova for lumbus strength and muscle powertypeexperimental groupcontrol groupstrength ( nm)extensionpre297.121.8288.222.3post317.119.3291.322.0**flexionpre334.217.7335.220.1post359.219.1339.219.2*muscle power ( nm)extensionpre354.122.6361.220.2post369.118.0368.111.2**flexionpre321.114.2319.220.2post342.220.1324.116.3*values are meansd . significantly different ( * * p<0.01 , * p<0.05 ) from the experimental group . significant interaction ( p<0.05 ) between group and time in the experimental group ) . therefore , the isokinetic muscle function of the knee joint and the lumbus in the experimental group was significantly increased compared to the control group . values are meansd . significantly different ( * * p<0.01 , * p<0.05 ) from the experimental group . significantly different ( p<0.05 ) between pre and post in the experimental group . core exercises mostly include the raising of legs and arms in the supine position to create resistance in the abdomen and waist . core muscle training also helps athletes develop and maintain total body balance . throwing , in particular , requires the creation of a unified movement by connecting various steps of positions to maximize the performance , and the core muscles are especially important1 . strong core muscle training is especially important to improve the distribution of kinetic efficiency to the whole body15 . core muscle training improves the following fitness components : muscle strength , endurance , agility , speed , balance , and the nervous system , including the vestibular system and the proprioceptive system4 , 16,17,18 . in this study , peculiar complex core balance training significantly increased the isokinetic strength of the knee joint and lumbus . this result is consistent with previous studies , which suggest a positive effect of core muscle training on the lower extremity and lumbus muscle reinforcement5,6,7,8 , 12 . according to yang s study12 , a 12-week core training increased isokinetic muscle power of the knee joint and lumbar in short - distance athletes . kim and chung5 conducted a 10-week core exercise program for athletes , and found lumbar strength increased . a study by shin , kim , and park6 showed that a 4-week core exercise program had a positive impact on lumbar strength in golfers . han , kim , and hyun7 reported that a 12-week specificity core balance training increased the isokinetic muscular functions of the knee joint and lumbar in high school volleyball players . moreover , song and hong s study8 indicated that an 8-week core strengthening program had a positive impact on knee extensor and flexor muscular strength in college baseball players . in conclusion , this study found a positive effect on peculiar complex core balance training on the isokinetic muscle functions of the knee and lumbus in throwing event athletes . therefore , core balance training would improve the performance of throwing event athletes by improving balance and distribution of kinetic energy through the arms and legs . hence , establishing an effective core training program that incorporates a mat , medicine ball , and sling training , as this study has utilized , is required for athletes . it is also worthy to note that this kind of core balance training prevents injury as well . although the results of this study indicated that peculiar complex core balance training develops the isokinetic muscle function in throwing event athletes , more specific and diverse research is necessary to identify how exercises can be integrated into an ideal physical activity program that caters to each athlete s own characteristics ( e.g. , age , gender , exercise career , and etc . ) and type of sport .

[ purpose ] this study aimed to investigate the effect of peculiar complex core balance training on the isokinetic muscle function of the knee joint and lumbus to provide fundamental data for establishing a training program that focuses on improving the performance and prevention of injury by developing the core and low extremity muscles . [ subjects and methods ] the participants in this study included a total of ten high school athletes involved in a throwing event for over five years . the subjects were randomly divided into two groups : the experimental group ( n=5 ) and the control group ( n=5 ) . the experimental group underwent peculiar complex core balance training . [ results ] according to the analysis of covariance , there was a significant effect of peculiar complex core balance training . therefore , the isokinetic muscle function of the knee joint and lumbus in the experimental group participating in peculiar complex core balance training was significantly increased compared to the control group . [ conclusion ] it is concluded that peculiar complex core balance training had a positive effect on the isokinetic muscle function of the knee and lumbus in throwing event athletes .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2827790

a 46-year - old caucasian man presented with a 10-year history of mild gait ataxia and undirected vertigo after fast head movements . he had a past medical history of resection of a thyroid adenoma and also for benign polyposis of the sigmoid colon . at the age of 41 years physical inspection revealed megalocephaly , congenital facial asymmetry and left thenar aplasia . at the latest presentation , the neurological examination showed minimal intention tremor , gait ataxia without visual compensation , an undirected imbalance on romberg 's test and bradydiadochokinesia . with approval of the local ethic committee and with the patient 's informed written consent , mri examinations of the brain were performed on a 1.5 t scanner ( avanto , siemens medical solutions , erlangen , germany ) in combination with using a vendor supplied 12-channel receive - only head coil and then mri examinations of the brain were done on a 7 t scanner ( magnetom 7 t , siemens medical solutions , erlangen , germany ) in combination with an 8-channel transmit - and - receive head coil ( rapid biomed , wuerzburg , germany ) . the gradient - echo and turbo spin echo sequences were performed to obtain the axial proton density ( pd ) , t2 , t2 and susceptibility weighted images ( swi ) , which were optimized for each field strength ( table 1 ) . in addition , proton ( h ) mr spectroscopy ( mrs ) was performed at 1.5 t . the spectroscopic data was acquired from the patient 's cerebellar lesion using a single - voxel , point - resolved technique ( te = 135 ms ; tr = 1500 ms ) . the resulting prominent resonances representing choline ( cho ) , creatine ( cr ) and n - acetylaspartate ( naa ) within the lesion were compared to the mirror image voxels on the white matter of the normal contralateral hemisphere . spectral post - processing was performed using the software provided by the mri system manufacturer ( siemens syngo , vb 15 , siemens medical solutions , erlangen , germany ) . for 11 years , repeated 1.5 t mri examinations revealed a slowly growing , non - enhancing tumor mass in the left cerebellar hemisphere with preservation of the gyral pattern . thus , the present study was done without administration of contrast media . on mri at 1.5 t and 7 t , the posterior fossa tumor ( 493432 mm in size ) appeared mainly hyperintense on the t2-weighted images ( fig . 1a ) and iso - hypointense on the proton density images ( not shown ) . the characteristic striated pattern of the lesion was best displayed on the t2-weighted images at both field strengths ( fig . the tumor caused descensus of the cerebellar tonsils , but any obstructive supratentorial hydrocephalus was absent . due to their high sensitivity for paramagnetic substances like deoxyhemoglobin , the swi and t2 weighted images revealed thin veins running deep between the thickened folia of the cerebellar lesion in great detail ( fig . the 7 t swi minimal intensity projection ( mip ) images depicted thin vessel branches as small as 250 m , whereas the 1.5 t swi mip images could only resolve larger vessels to a size of 450 m . compared to the 1.5 t swi images ( fig . 1c ) the medial displacement and compression of the dentate nucleus by the tumor were much better registered on 7 t swi images ( fig . the h - mrs at 1.5 t demonstrated a reduction in naa and a prominent lactate peak . contrary to other previous reports , the cho , cr and the resulting cho / cr ratio were slightly elevated in the lesion and the myoinositol ( mi ) levels were not changed ( 3 , 4 ) . thus far , neurosurgical therapy and histopathological examination have not been performed because the lesion exerted only mild compression of the iv ventricle without any hydrocephalus . a dna analysis revealed a heterozygous mutation in exon 5 of the pten gene ( chromosome 10 q23 ) , and this supported the diagnosis of ldd ( c. 388c > t ; p. arg130x ) . for 11 years , repeated 1.5 t mri examinations revealed a slowly growing , non - enhancing tumor mass in the left cerebellar hemisphere with preservation of the gyral pattern . thus , the present study was done without administration of contrast media . on mri at 1.5 t and 7 t , the posterior fossa tumor ( 493432 mm in size ) appeared mainly hyperintense on the t2-weighted images ( fig . 1a ) and iso - hypointense on the proton density images ( not shown ) . the characteristic striated pattern of the lesion was best displayed on the t2-weighted images at both field strengths ( fig . the tumor caused descensus of the cerebellar tonsils , but any obstructive supratentorial hydrocephalus was absent . due to their high sensitivity for paramagnetic substances like deoxyhemoglobin , the swi and t2 weighted images revealed thin veins running deep between the thickened folia of the cerebellar lesion in great detail ( fig . images depicted thin vessel branches as small as 250 m , whereas the 1.5 t swi mip images could only resolve larger vessels to a size of 450 m . compared to the 1.5 t swi images ( fig . 1c ) the medial displacement and compression of the dentate nucleus by the tumor were much better registered on 7 t swi images ( fig . the h - mrs at 1.5 t demonstrated a reduction in naa and a prominent lactate peak . contrary to other previous reports , the cho , cr and the resulting cho / cr ratio were slightly elevated in the lesion and the myoinositol ( mi ) levels were not changed ( 3 , 4 ) . thus far , neurosurgical therapy and histopathological examination have not been performed because the lesion exerted only mild compression of the iv ventricle without any hydrocephalus . a dna analysis revealed a heterozygous mutation in exon 5 of the pten gene ( chromosome 10 q23 ) , and this supported the diagnosis of ldd ( c. 388c > t ; p. arg130x ) . lhermitte - duclos disease or dysplastic cerebellar gangliocytoma is a slowly enlarging mass within the cerebellar cortex , and patients with this malady present with headaches , occlusive hydrocephalus , cranial nerve palsies , gait ataxia and other symptoms of cerebellar dysfunction ( 1 ) . beside some pediatric cases , the majority of patients are diagnosed in the third or fourth decade of life without a gender predilection . histopathologically , ldd is characterized by regional enlargement of the cerebellar stratum granulosum , an absence of the purkinje cell layer and progressive hypertrophy of the granular cell neurons with increased myelination of their axons in the expanded molecular layer ( 5 ) . mri has proven to be the best imaging modality for revealing the characteristic appearance of ldd , and mri often enables physicians to make the diagnosis of ldd even without histopathological confirmation ( 3 , 4 , 6 - 10 ) . the striated pattern of ldd is a result of the close apposition of the thickened cerebellar folia that are lacking their secondary arborization . a non - enhancing , unilateral cerebellar mass in a middle - aged patient , which is characterized by a ' tiger - striped ' pattern of hyperintensity on the t2-weighted mr images and this respects the cerebellar margins , is typical of ldd ( 8) . to the best of our knowledge , this is the first reported case of ldd that has been examined by 7 t mri ultrahigh field mri systems ( 7 t ) with their increased signal - to - noise ratio ( snr ) and higher sensitivity to susceptibility contrast . these systems are currently being tested for clinical applications and they allow for imaging anatomical structures with thinner sections , larger matrices and reduced acquisition times . however , the clinical challenges associated with 7 t mri include higher specific absorption rates ( sar ) , non - uniformity of the transmitted radiofrequency field , non - homogeneity of the static magnetic field , increased susceptibility artifacts and potential physiological side - effects . moving from 1.5 t to 7 t mri , the t2 is shortened and the susceptibility contrast of paramagnetic substances ( e.g. deoxyhemoglobin , neuromelanin , iron ) is significantly amplified , which enables a superior illustration of the venous vasculature and cerebellar nuclei . 1.5 t swi was found to be helpful for detecting deep running veins around the thickened folia of ldd ( 3 ) . 7 t swi can demonstrate in greater detail veins that are even smaller than the voxel size , due to the associated paramagnetic effect , than the corresponding 1.5 t images ( fig . 1c - f ) because the sensitivity of mri scanners for paramagnetic substances increases in proportion to the applied magnetic field . the deoxyhemoglobin in these veins helps to resolve the outer most layer of the ldd , which consists of the outer molecular layer , the leptomeninges and the associated abnormal vessels . ( 3 ) and kulkantrakorn et al . ( 7 ) reported good correlation of this mri pattern with the histological specimen . beside the characteristic striated pattern on the t2-weighted images , these abnormal veins surrounding the thickened folia on the swi images seem to be another unique feature of ldd . beside the preoperative identification of ldd , the higher resolution of 7 t swi their anatomical relation is of clinical importance for planning surgical procedures because lesions affecting the cerebellar nuclei are associated with more severe symptoms than are cortical lesions . h - mrs demonstrated a prominent lactate peak , suggesting increased glycolysis and the high metabolism of this ldd lesion ( 4 , 9 ) . decreased levels of cho , cr , naa and mi and the chr / cr are normally found on the side affected by the ldd ( 9 ) . the slightly increased cho levels in our patient suggested increased demyelination and membrane turnover , whereas the decreased cho levels in the lesion suggested a non - neoplastic etiology of this lesion ( 4 , 9 ) . making the preoperative diagnosis of ldd via mri obviates the need for biopsy and this allows surgeons to plan an appropriate therapy , which consists of decompression of the posterior fossa by total surgical resection . tumor resection has not yet been performed in our patient due to the mild clinical symptoms . in conclusion , as seen on mri , a non - enhancing , ' tigers striped ' cerebellar lesion with unilateral hemispheric expansion and preservation of the gyral pattern should be considered specific for ldd and these findings often allow making the preoperative diagnosis . 7 t mri more precisely reveals the known morphology and microstructure of this tumor entity than can 1.5 t mri . especially , 7 t swi enables the identification of the outermost layer of ldd due to its inherent venous vasculature , and 7 t swi better displays the iron containing dentate nuclei . hence , 7 t mri is expected to become a valuable tool for studying the cerebral microvascularity and tumor angiogenesis not only for ldd , but also for other resectable brain tumors .

lhermitte - duclos disease ( ldd ; dysplastic cerebellar gangliocytoma ) is a rare hamartomatous lesion of the cerebellar cortex and this was first described in 1920 . ldd is considered to be part of the autosomal - dominant phacomatosis and cancer syndrome cowden disease ( cs ) . we examined the brain of a 46-year - old man , who displayed the manifestations of cs , with 7 tesla ( t ) and 1.5 t mri and 1.5 t mr spectroscopy ( 1h - mrs ) . we discuss the possible benefits of employing ultrahigh - field mri for making the diagnosis of this rare lesion .

CASE REPORT Imaging Findings DISCUSSION

PMC4152626

saprolegnia parasitica is a fish pathogenic oomycete ( water mould ) , belonging to the saprolegniales order and known to infect a wide range of fish , amphibians , and crustaceans relevant to aquaculture and to aquatic ecosystems ( van west et al . it causes saprolegniosis , a disease characterised by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish ( van west 2006 ; schornack et al . gene transformation technology has been developed but its efficiency is , at present , limited to a restricted number of oomycete species ( judelson et al . 1991 ; whisson et al . 2005 ; judelson & ah - fong 2009 ) . attempts to successfully establish transformation protocols for some oomycetes have had , in some cases , little or no success . an alternative way to functionally characterise genes , which is independent of a stable transformation protocol , can be the use of rna - interference ( rnai ) . this technique was successfully developed for transient gene silencing of several genes in phytophthora infestans ( whisson et al . 2005 ; grenville - briggs et al . 2008 ; walker et al . 2008 ) . in the current study we performed detailed experiments to investigate whether the rnai - technique can also be employed to silence genes in s. parasitica . to select a suitable gene we searched the genome of s. parasitica of strain cbs223.65 ( jiang et al . 2013 ) and found a gene ( sprg_01728 ) that encodes for a putative tyrosinase ( sptyr ) , which is highly expressed in sporulating mycelium . tyrosinases are mono - oxygenases ( monophenol , o - diphenol : oxygen oxidoreductases , ec 1.14.18.1 ) and bifunctional enzymes , catalysing the o - hydroxylation of monophenols and subsequent oxidation of o - diphenols to quinones ( fig 1 ) . in the catalytic cycle , molecular oxygen is used as an electron acceptor leading to subsequent reduction of oxygen to water ( westerholm - parvinen et al . these enzymes are crucial not only in the biosynthesis of pigments such as melanin but also in the biosynthesis of other phenol polymers such as lignin , flavonoids , and tannins ( obata et al . melanins are negatively charged and high molecular hydrophobic compounds . as a result of oxidative polymerisation of phenolic compounds melanin they are insoluble in both aqueous and organic solvents and consequently difficult to study biochemically and biophysically ( casadevall et al . 2000 ) . many of the dark pigments found in nature are considered melanins ( wheeler & bell 1988 ) and in microorganisms , melanin can be found in the extracellular or intracellular matrix , melanised cells of the fungal human pathogen cryptococcus neoformans were shown to possess a thick layer of melanin in the cell wall ( wang et al . ( 2002 ) demonstrated that melanin in the opportunistic plant pathogen alternaria alternata , is located in the septa and outer walls of conidia ( carzaniga et al . 2002 ) . in other plant pathogenic fungi like colletotrichum lagenarium and verticillium dahliae melanin has been found in layers within the cell wall and deposited as granules at the surface of the cell wall ( nosanchuk & casadevall 2003 ) . the production of melanin has also been associated with virulence in several different microorganisms such as the pathogenic fungi c. neoformans and paracoccidioides brasiliensis and the bacterial pathogen pseudomonas aeruginosa ( nosanchuk & casadevall 2003 ) . also , strains that do not produce melanin are unable to form functional appressoria and seem to lose their pathogenicity ( forrest 1990 ; takano et al . melanin can also act as a protective agent against environment insults and it can bind to diverse drugs and chemicals and maintain cellular integrity ( hill 1992 ) . melanins have a great affinity towards metal particles and react readily with free radicals protecting the organism against oxidants such as hypochlorite and permanganate ( jacobson et al . 1994 ; nyhus et al . 1997 ) but also against the oxidative burst of activated host effector cells ( nosanchuk & casadevall 2003 ) . moreover they are less susceptible to microbicidal peptides and defensins produced by phagocytic cells . the suggested mechanism of action is the absorption of the microbicidal peptide by melanin in such a way that it can not reach its target ( nosanchuk & casadevall 2003 ) . currently it is unclear whether oomycete tyrosinases are involved in melanin production , however it has been proposed that melanin is formed and is involved in the formation of reproductive organs and spores , in virulence , and in protection after physical damage ( lerch 1983 ) . indeed , during microscopic analysis of sporangial development we noticed also that the sporangia from s. parasitica are slightly darker than the mycelia . therefore we decided to investigate whether the tyrosinase gene is involved in melanin production by silencing the gene via rnai . the strain of saprolegnia parasitica cbs 223.65 was maintained on 4 % ( w / v ) potato dextrose agar ( oxoid ) at 18 c . mycelium from saprolegnia parasitica strain cbs 223.65 was grown in pea broth ( 125 g of boiled and filtered peas per litre ) for 2 d at 24 c , washed with sterile distilled water and collected in a 50 ml polypropylene tube ( greiner ) . for each 1 ml of mycelium a 3 ml solution of 10 mg ml cellulase ( sigma ) and 5 mg ml of glucanase ( novozyme ) diluted in 0.5 m sorbitol was prepared and added to the corresponding mycelium . the mixture was placed on a shaking platform for 90 min at room temperature ( rt ) to allow enzymatic cell wall degradation . the resulting protoplasts were filtrated using a 70 m cell strainer ( fisherbrand ) and washed three times with 5 ml sorbitol ( 0.5 m ) by centrifugation ( 1200 g , 4 c , 5 min ) eliminating enzyme residues . the protoplasts were finally resuspended in 5 ml sorbitol ( 0.5 m ) and two samples were counted two times each using a haemocytometer . to test protoplast regeneration ability 1000 protoplasts were inoculated into 20 ml pea broth and incubated at 18 c for 24 h. mycelium from saprolegnia parasitica strain cbs 223.65 was grown in pea broth for 4 d at 24 c , washed with sterile distilled water and left in 10 ml of sterile 1:1 tap : tank water to induce sporulation ; the sporulating mycelium was then collected in a 2 ml tube with 50 l glass beads , acid washed , and immediately frozen in liquid nitrogen . the rna extraction was performed using the rneasy mini kit ( qiagen ) according to manufacturer 's protocol for fungi and plants with some modifications . briefly , 600 l of lysis buffer ( rlt ) buffer was added to each sample which was frozen in liquid nitrogen , mycelium was disrupted using a fast prep machine ( four times , 6 ms for 45 s ) , samples were maintained on ice . afterwards the samples were treated as described in the manufacture 's protocol . to exclude dna contamination the samples were subjected to a dnase treatment using the turbo dnase kit ( ambion ) according to manufacturer 's protocol . the quantity and purity of the rna was determined using the nanodrop and the quality verified by running 1 g of rna in 1 % ( w / v ) agarose gel . subsequent cdna was produced using the first strand cdna synthesis kit ( fermentas ) according to manufacturer 's protocol . a pcr using tyr - t7 primers ( 5-gtaatacgactcactatagggagcagctgatgttgtagagc-3 and 5- gtaatacgactcactataggggatcccgtactgggactact-3 ) was carried out using the cdna as template . the dsrna from the green fluorescent protein encoded by gfp gene was obtained by performing colony pcr from escherichia coli transformed with pgfph ( ah - fong & judelson 2011 ) . positive colonies were grown overnight in 5 ml lb medium supplemented with 100 mg l ampicillin . plasmid isolation and purification was carried out using the plasmid midi kit ( fermentas ) following the manufacturer 's protocol . afterwards gfp - dsrna and sptyr - dsrna were obtained using megascript kit ( ambion ) according to manufacturer 's protocol . three tubes were incubated at rt for 15 min : one tube with 10 l lipofectin ( invitrogen ) and 10 l gfp - dsrna , a second tube with 10 l lipofectin and 10 l sptyr - dsrna and a third tube with 10 l lipofectin and 10 l of sterile rnase free water . subsequently 10 l of protoplasts solution containing 1 10 protoplasts / ml was added to each tube and incubated at 18 c for 16 h. each experimental condition was then diluted in 200 ml pea broth and distributed in 2 ml aliquots in 24-well plates and incubated overnight at 24 c . the mycelium of regenerated protoplasts ( of treated and non - treated with dsrna ) was then inoculated in pda ( potato dextrose agar , oxoid ) plates and incubated at 24 c overnight before further processing . for all specific primers were designed for each gene using the primer3 tool following the manufacturer 's guidelines for primer design . the constitutively expressed gene tubulin ( tub ) was used as an endogenous control in all qpcr reactions . a master mix was prepared containing per well : 25 l lightcycler 480 sybr green i master ( roche ) , 2 l of each 10 mm primer [ tyr 3acctcttctacggtcagca5 and 3aggttgtgctagtggatcgg5 , tub 5-aggagatgttcaagcgcgtc-3 and 5-gatcgttcatgttggactcggc-3(van west et al . the standard error from all qpcr reactions was calculated for all individual lines tested and used to determine the confidence intervals . sporulating mycelium from each individual line was ground with liquid nitrogen and added to 5 ml phosphate buffer 100 mm ph 6.5 containing : 500 l pmsf 1 mm , 850 l sorbitol 0.65 m , protease inhibitor edta free ( roche ) . samples were centrifuged ( 10 min , 2100 g , 4 c ) and the supernatant ( crude extract ) collected into 15 ml tubes . in new 1.5 ml tubes , 500 l acetate buffer 50 mm ph 5.0 , 200 l l - dopa 3.2 mm , 20 l dmf 2 % ( w / v ) and 220 l mbth 5 mm were mixed and incubated 5 min at 37 c . thereafter 100 l of crude extract was added and incubated for a further 30 min at 37 c . absorbance was measured at 505 nm and the activity determined using the equation below ( winder 1994 ) : rate ( nmol min ) = a505 t 10/29 10/30 , where t = time . samples were centrifuged 10 min at 2100 g and 4 c and supernatant transferred to a new tube . a standard curve was made using concentrations of commercial melanin ( sigma ) ranging between 0 and 2 g l. sporulating mycelium of control and putatively silenced lines were fixed in 3 % glutaraldehyde in 0.1 m phosphate buffer ( pb ) ph 7.4 for 24 h. afterwards samples were washed and stored in 0.1 m pb ph 7.4 until further processing . for transmission electron microscopy ( tem ) analysis , samples were subsequently processed in an automated routine tissue processor leica em tp ( leica microsystems , vienna , austria ) comprising following steps . samples were post - fixed in 1 % ( v / v ) osmium tetroxide ( oso4 ; aqueous solution ; code o004 ; taab , england , uk ; batch nr 70150 ) for 1 h preceded by three 5-min washes with pb and three 5-min washes with distilled water and an extra wash step for 30 min with distilled water . next , samples were dehydrated in increasing concentrations of ethanol ( 30 % , 70 % and 95 % , and 100 % ( v / v ) ; 30 min each ) , followed by three incubations in acetone for 1 h. samples were then incubated in increasing concentrations of epoxy / acetone ( 1/1 , 6/1 , and 100 % epoxy ) for 1 , 6 and 24 h respectively before embedding the samples in labelled capsules with freshly prepared resin , leaving the resin to polymerise for 24 h at 60 c . ultra - thin sections ( 7080 nm ) were cut with a leica em uc6 ultramicrotome ( leica microsystems , vienna , austria ) and mounted on 200-mesh uncoated copper grids . 705631095 , laurylab , france ) and 0.5 % lead citrate ( ultrastain2 , ref . 70553022 , laurylab , france ) on an automated contrasting instrument leica em ac20 ( leica microsystems , vienna , austria ) . finally the grids were analysed at 80 kv using a jem-1400 plus ( jeol , tokyo , japan ) transmission electron microscope equipped with an amt ultravue camera . bioinformatic analysis of the saprolegnia parasitica genome , ( broad institute website ) , uncovered a gene that encodes a putative tyrosinase ( jiang et al . 2013 ) that , according to rna sequence data , is highly expressed in sporulating mycelium . to verify this , quantitative expression analysis by real - time quantitative pcr of the different life stages was carried out demonstrating that the expression of sptyr gene is 30 fold higher in sporulating mycelium compared to other s. parasitica life stages , confirming the initial rna sequence data ( de bruijn et al . one of the most efficient ones is gene silencing , where gene expression can be completely or partially abolished . since the tyrosinase enzyme was expected to be involved in pigment formation , a decrease in gene expression would cause a visible change in the phenotype of the dsrna treated individual lines . for transient gene silencing we used rnai protocols that had already been established for phytophthora infestans ( judelson et al . 2005 ) and optimised it for silencing in s. parasitica . in order to maximize the change in gene expression of a silenced gene it is necessary to know the optimum time of silencing . the time line for this phenomenon differs according to the organism of study . to unravel the specific silencing timeline for s. parasitica , a time course rnai assay was conducted for 12 d ( fig 2 ) and gene expression levels assessed by qpcr . the results of this experiment suggested that at 8 d after introduction of dsrna into the cells , gene expression of sptyr is down regulated the most in the majority of the individual lines tested . five out of six individual lines ( ty1 ty5 ) , treated with sptyr - dsrna ( fig 2 ) had less than 20 % gene expression when compared with the control lines . individual line ty6 was silenced within the time frame ; however , the most significant decrease in gene expression occurred on day 9 post - rnai . these results suggest that the window of opportunity for gene silencing in s. parasitica cbs 223.65 is about 89 d after dsrna uptake , which is much earlier than has been found for p. infestans where the maximum silencing was observed between 12 and 15 d after dsrna uptake ( whisson et al . with such a short timeframe and depending on the life stage where the gene of interest is most expressed , the amount of material from each individual line for further analysis is reduced . after establishing the optimum time period for gene silencing in s. parasitica four rnai assays were performed in order to optimise and increase the reproducibility of the technique . a final , fifth , optimised rnai was conducted and ten individual lines treated with gfp - dsrna ( control ) and ten individual lines treated with sptyr - dsrna were analysed by qpcr on the eighth day after dsrna uptake to determine the level of silencing . out of the ten individual lines treated with sptyr - dsrna , seven were silenced with gene expression levels reduced between 25 and 80 % of the levels of individual lines treated with gfp - dsrna ( fig 3 ) . the same individual lines used for gene expression analysis were also tested for tyrosinase activity using a spectrophotometric assay . tyrosinase activity in the individual lines that showed most silencing was decreased when compared to the control ( fig 4 ) , while individual lines that did not present sptyr - silencing or were at a low silencing level maintained a tyrosinase activity that was similar to the control lines . the lowest levels of tyrosinase activity were measured in the lines with the highest percentage of gene silencing . additionally , melanin content of each individual line was determined spectrophotometrically and the concentration extrapolated from a standard curve ( fig 5a ) . the most silenced lines ( lines ty1 to ty5 ) possessed less melanin when compared with the individual lines treated with gfp - dsrna ( fig 5b ) . these results demonstrate that silencing the sptyr gene affects the melanin production and demonstrate that the pigments present in s. parasitica contain melanin . furthermore , microscopic analysis revealed some altered sporangium morphology in the most - silenced lines when compared with gfp - silenced lines and the wild type strain . sptyr - silenced lines seem to have less pigmentation , smaller sporangia and also abnormally shaped sporangia ( fig 6 ) . we observed these differences in both young and mature sporangia ( data not shown ) . the effect of tyrosinase silencing on sporangial morphology may be due to the pigments being deposited in or close to the cell wall , helping the structure thickness and shape . however , we can not infer that this altered morphology is solely due to the silencing of sptyr gene . for example it is possible that a pleiotropic effect , resulting in altered sporangial morphology , was obtained due to sptyr - silencing . this has been described before when an rna - helicase of p. infestans was silenced with rnai ( walker et al . transmission electron microscopy ( tem ) was used to study the effect of sptyr - silencing on sporangium morphology in more detail . young sporangia were analysed from both gfp - dsrna - treated lines ( control ) and sptyr - silenced lines after induction of sporulation . no significant differences in the cellular structures and organelles of control and sptyr - silenced sporangia were observed . in both control and sptyr - silenced lines , the sporangial cell wall of the control lines and the weakly sptyr - silenced lines are more electron dense than the most sptyr - silenced lines ( fig 8) . these observations would suggest that melanin is located within the cell wall , which has been demonstrated in some true fungi ( wang et al . abnormally shaped sporangia were also observed in sptyr - silenced lines using tem , whereby elongated and even triangular shaped sporangia were formed ( data not shown ) . in both types of sporangia the increase in vacuoles could also explain the altered morphology of the sporangia observed with the light microscope . the tyrosinase gene , sptyr , of saprolegnia parasitica is integral to the melanin biosynthetic pathway of this oomycete . after silencing the tyrosinase gene , microscopical analysis suggest that melanin is located in an electron - dense layer in the cell wall of sporangia of s. parasitica , since the electron dense layer was absent in the most silenced lines . with this work , we have demonstrated for the first time that transient gene silencing through rnai is a feasible method to functionally characterise genes in s. parasitica .

here we describe the first application of transient gene silencing in saprolegnia parasitica , a pathogenic oomycete that infects a wide range of fish , amphibians , and crustaceans . a gene encoding a putative tyrosinase from s. parasitica , sptyr , was selected to investigate the suitability of rna - interference ( rnai ) to functionally characterize genes of this economically important pathogen . tyrosinase is a mono - oxygenase enzyme that catalyses the o - hydroxylation of monophenols and subsequent oxidation of o - diphenols to quinines . these enzymes are widely distributed in nature , and are involved in the melanin biosynthesis . gene silencing was obtained by delivering in vitro synthesized sptyr dsrna into protoplasts . expression analysis , tyrosinase activity measurements , and melanin content analysis confirmed silencing in individual lines . silencing of sptyr resulted in a decrease of tyrosinase activity between 38 % and 60 % , dependent on the level of sptyr - expression achieved . the sptyr - silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology . moreover , developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines , treated with gfp - dsrna . in conclusion , the tyrosinase gene of s. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in s. parasitica .

Introduction Material and methods Results and discussion Conclusions

PMC3767330

atropisomeric biaryls constitute an important structural element of many natural products , biologically active compounds , and chiral ligands . despite significant importance of the synthesis of atropisomeric biaryls ( even in the racemic form ) , their syntheses based on the common cross - coupling reaction such as suzuki - miyaura ( sm ) , negishi , stille , or hiyama are still rare what could be explained by easily recognised problems associated with creation of sterically hindered multiply ortho - substituted aryl - aryl bonds . herein , we propose an alternative approach to axially chiral biaryls by selective functionalisation of prochiral substrates , unsubstituted at one or two of the four present ortho positions . thus , introduction of an additional ortho substituent into the biaryls already bearing 2 or 3 of them eventually restrains free rotation around the single aryl - aryl bond and creates a pair of atropoisomers ( scheme 1 ) . such functionalisation could be achieved by classical methods if the position of the functionalisation is unambiguously defined by the substitution pattern , or it could be performed in a catalytic manner , mediated by the transition metal ( tm ) complex and directed by proper functional groups [ 2 , 3 ] . therefore , main impact was made on the synthesis of prochiral precursors of atropisomeric compounds possessing proper functionality for selective introduction of the fourth ortho substituent . herein , we would like to present a simple guide for selection of an optimal approach to the synthesis of prochiral biaryls which next could be used in the synthesis of racemic atropisomeric compounds or in their atroposelective synthesis . we have concentrated on the creation of a small library of prochiral biaryls using a combinatorial approach to the sm reactions between several arylboronic acids and arylhalides as well as using other complementary methods . for example , as a result of cross - coupling of nine substrates ( both boronic acids and bromoarenes ( table 1 ) ) , twenty biaryls could be theoretically obtained . in the case of the preparation of simple tri - ortho - substituted biaryls , the syntheses of the individual coupling components could be based on the modified known procedures . the majority of aromatic boronic acids could be obtained by the reaction of organometallic compounds such as grignard reagents , organolithium , and organozinc ones with trialkyl borates . some boronic acid derivatives could also be obtained by the direct c h activation protocol with utilisation of diboro- and hydridoboro - aromatics [ 8 , 9 ] . these syntheses are suitable for the production of large quantities of boronic acids in a relatively simple manner . for example , desired 2-methoxyphenylboronic acid ( 5 ) was obtained by the approach involving the grignard reagent in 73% isolated yield starting from 2-bromoanisole ( 12 ) ( scheme 2 ) . the alternative organolithium approach could be demonstrated by the synthesis of 2-(n , n - diethylcarbamyloxy)phenylboronic acid ( 7 ) obtained by tri - isopropyl borate quench of a suitable organolithium reagent formed in the direct ortho lithiation in 81% isolated yield ( scheme 3 ) . according to the standard procedure the solution of n - buli then , it was chilled down to 76c , mixed with tri - isopropyl borate and slowly quenched by phenyl diethylcarbamate ( 15 ) . after that the reaction mixture was allowed to warm up to rt and a base was neutralised with the saturated solution of nh4cl , the formed arylboronic acid 7 was extracted with dcm , dried with mgso4 , and eventually purified by flash column chromatography . in the case of difficulties in purification of some boronic acids , they could be easily converted to the corresponding pinacolborates ( available from crude boronic acids in a high yielding reaction with pinacol ) , which usually undergo rapid chromatographic purification . importantly , the obtained chromatographically pure pinacolborates ( e.g. , 17 , scheme 4 ) could be used in the cross - coupling reaction with the same efficiency as unprotected boronic acids . there is also a large number of these compounds commercially available at reasonable prices . nevertheless , in some cases , it is more economical to synthesise them by means of one of the many available methodologies . for example , 2-bromo--picoline ( 10 ) was rapidly obtained in that reaction from an in situ formed heteroaryl diazonium bromide in 80% isolated yield ( scheme 5 ) . derivatisation of simple aromatic halides can provide a number of diversified substrates for the cross - coupling reactions . for example , the reactions of 2-bromophenol ( 19 ) with simple derivatisation agents such as diethylcarbamoyl chloride ( 20 ) or pivaloyl chloride ( 21 ) ran in dmf at 0c and catalysed by n - methylimidazole ( nmi ) or dmap lead to 2-bromophenyl diethylcarbamate ( 11 ) and 2-bromophenyl pivalate ( 9 ) in 86% and 72% yields , respectively ( scheme 6 ) . obtained halides 9 , 10 , 11 , and 19 were used as precursors of prochiral biaryls possessing in the ortho position to aryl aryl bond a functional group potentially useful for coordination of transition metals . therefore , the desired activation of c h bond and directing effects of the planned transition metal - catalysed reactions at a remote biaryl position as delineated in scheme 1 could be expected . probably the simplest way to biaryls is via sm coupling of boronic acid with aryl halides . the major limitation of the sm coupling reactions is difficulty in the creation of sterically hindered biaryls possessing more than two ortho substituents . at the same time the synthesis of prochiral ( doubly or triply ortho - substituted biaryls ) can be usually performed in high yields . one of the model prochiral biaryls , 3-methyl-2-(4-methylphenyl)pyridine ( 25 ) , was obtained in 48% yield in the reaction of 4-methylphenylboronic acid ( 3 ) with 2-bromo--picoline ( 10 ) ( scheme 7 ) . this biaryl precursor could be used in the synthesis of chiral atropisomeric compounds 26 in the reaction mediated by tm complexes directed by a lone electron pair of nitrogen and run through the ch activation step . the application of pinacoloborates in the sm reaction is well recognised and is frequently utilized in cases where the corresponding boronic acids are difficult to purify . nevertheless , the direct comparison of efficiency of utilising in coupling reactions boronic acids and the corresponding boranates is very rare . the synthesis of 3-methyl-2-(2-methoxyphenyl)pyridine ( 27 ) shown in scheme 8 provides such an example . the 2-bromo--picoline ( 10 ) undergoes the sm reaction with 2-methoxyphenylboronic acid ( 5 ) or corresponding pinacol ester ( 17 ) under palladium - catalysed conditions with the yields depending on conditions and substrates used . in order to optimise the reaction conditions several different solvents , bases , catalysts , and additives were tested ( table 2 ) . the best yield ( table 2 , entry 8) was obtained when 2-methoxyphenylboronic ( 5 ) acid was utilised in dmf / methanol mixture with tetrakis(triphenylphosphine)palladium(0 ) as the catalyst . of many bases used , only potassium phosphate monohydrate secured good yields . the data collected in table 2 confirmed also a crucial role of water and a strong inorganic base of low nucleophilicity required to achieve reasonable yield in sm reaction . as mentioned previously , obtained prochiral biaryl compound 27 could be used in the synthesis of atropisomeric compound 28 by substitution of hydrogen atom in the second ortho position with any bulky group ( scheme 8) . similarly , prochiral 3-methyl-2-(1-naphthyl)pyridine ( 29 ) was prepared by coupling of 1-naphtylboronic acid ( 6 ) with 2-bromo--picoline ( 10 ) in 74% yield ( scheme 9 ) and could be used for the synthesis of atropisomeric compound 30 by substitution of hydrogen in position 2 or 8 of the naphthalene ring directed by a lone electron pair of nitrogen . based on naphthylphenyl core biaryl 31 ( 2-(naphthalen-1-yl)phenyl diethylcarbamate ) , possessing a directing metalation group ( dmg ) , different from nitrogen , was obtained under similar reaction conditions by coupling of 1-naphthylboronic acid ( 6 ) and 2-bromophenyl diethylcarbamate ( 11 ) in 74% yield . in this case , diethylcarbamate is the group - directing substitution of hydrogen in the position 2 or 8 of naphthalene ring ( scheme 10 ) . in many cases there is no reason to create biaryls by coupling of two monoaryl compounds because of availability of easy to functionalise biaryls . for example , commercially available 2,2-biphenol ( 33 ) could be used as a substrate for synthesis of chiral 3,3-disubstituted biphenol derivatives . not only 33 but also its derivatives with the dmg groups such as carbamates could be used for the synthesis of atropisomeric biaryls ( scheme 12 ) . thus , the reaction of 2,2-biphenol with diethylcarbamoyl chloride ( 10 ) led to biphenyl-2,2-diyl bis(diethylcarbamate ) ( 34 ) in 67% yield . 2-hydroxybiphenyl-2-yl diethylcarbamate ( 35 ) , which could be used as another prochiral substrate , was also isolated as a side product of this reaction in 10% yield only ( scheme 11 ) . mixed o - pivaloyl , o - carbamoylobiphenol ( 39 ) was obtained in reaction of 35 with the stoichiometric amount of 2,2-dimethylpropanoyl chloride ( 21 ) in 82% yield . the obtained product 39 could be used as a precursor of the synthesis of chiral biaryls in the reaction directed by either pivaloyl or carbamoyl function ( scheme 12 ) . a less popular but still powerful method of aryl aryl bond formation by meyers reaction [ 14 , 15 ] could be successfully used when the substitution pattern does not allow to achieve acceptable yield in the tm - mediated cross couplings . for example , for oxazole and oxazoline ortho - substituted halogenoarenes as well as boronic acids , the tm catalysed couplings proved to be difficult because of strong interaction of those heterocyclic substituents with the catalysts . desired oxazole and oxazoline ortho - substituted biaryls could be however accessible by the meyers reaction utilising the ortho - methoxy - substituted aryloxazolines ( and some other ortho - methoxy - substituted aromatics ) in good yields ( scheme 13 ) . 2-(2-methoxyphenyl)-4,4-dimethyl-4,5-dihydro-1,3-oxazole ( 8) undergoes the ipso nucleophilic substitution reaction when treated with 2-methoxyphenylmagnesium bromide ( 41 ) and forms prochiral biaryl 42 in 94% yield . obtained products 42 may be used for the synthesis of chiral compound 43 by substitution of hydrogen with the bulky group possible in the c h - activated reaction directed by oxazoline ( scheme 13 ) . to demonstrate a possibility of transformation of prochiral biaryl into the atropisomeric ones , we carried out direct arylation reaction directed by a lone electron pair of nitrogen atom . thus , the reaction of 3-methyl-2-(p - methylphenyl)pyridine ( 25 ) with 4-bromoanisole ( 44 ) led to a mixture of mono- and disubstituted products 45 , and 46 ( scheme 14 ) . obtained monosubstituted product 45 , could be used in the next arylation synthesis with different halogenoarenes ( other than 4-meoc6h4 ) to accomplish doubly ortho arylated biaryl . high yield in the monosubstitution reaction was obtained when [ rucl2(p - cymene)]2 was utilised as a catalyst precursor with no phosphorus ligands added ( table 3 , entry 7 ) . when tris(pentafluorophenyl)phosphine was used in combination with [ rucl2(p - cymene)]2 to form a catalyst the formation of disubstituted product 46 in 28% yield was observed ( table 3 , entry 6 ) . aryl coupling approach to atropisomeric biaryls based on direct and directed by certain dmgs arylation running through the c h activation reaction step . of the dmgs promoting aromatic c h activation reactions , perhaps the most powerful are sp - hybridised nitrogen , carbonyl group , secondary amine , and , rarely , aromatic hydroxyl group . nevertheless , other functionalities such as nitro group , carbamate , and carboxylate groups as well as those possessing a lone electron pair at a proximal heteroatom , could play a role of a dmg in transition metal mediated c the extension of library of dmgs on new functional groups will create an additional opportunity for the synthesis of atropisomeric biaryls . in summary , we have demonstrated a general approach to the synthesis of prochiral biaryls by several complementary methods . the selection of the method was based on the analysis of the availability of starting materials and desirable substitution pattern of the target products . the assumption that easily available prochiral biaryls could be the perspective substrates in the synthesis of atropoismer compounds was confirmed in model direct and directed by the nitrogen lone electron pair transition metal mediated arylation of 2-arylopirydines ran though the ch activation reaction step . all suzuki coupling reactions were carried out under argon atmosphere using oven - dried glassware and the dry solvent . the products were purified by distillation or flash column chromatography ( merck silica gel 60 ( 230400 mesh ) ) . hnmr : spectra were recorded on bruker avance 300 in cdcl3 ; chemical shifts are given in ppm relative to tms , coupling constants ( j ) in hz . attenuated total reflection ir spectra were recorded on ftir nicolet 8700 a spectrometer and measured in cm . the hrms ( esi ) measurements were performed on shimadzu lcms - it - tof instrument . hplc study was performed on a merck reversed - phase column : 250 4 mm , 5 m , eluted by methanol / water . all commercially available substrates were used as received , and all known self - made substrates were examined by comparison with authentic commercial samples . 4-methylphenylboronic acid ( 4)a dried 500 ml flask equipped with a magnetic stirrer , dropping funnel , and reflux condenser was charged with magnesium turnings ( 1.1 equiv . , 80.6 mmol ) , next , flask was argonated by vacuum / argon triple exchange and a solution of a few crystal of iodine in 10 ml thf was added to activate of the magnesium . , 73 mmol ) in dry thf ( 100 ml ) was added dropwise for a period of 1 hour while the reaction mixture was stirred and heated to maintain a gentle reflux . after the additional 1 h refluxing solution was cooled down to 78c and trimethylborate ( 22 ml , 183 mmol , 2.5 equiv . ) in dry thf ( 85 ml ) was slowly added . reaction was quenched with saturated aqueous solution of nh4cl ( 70 ml ) and then thf was removed under reduced pressure . the precipitated crystalline 4 was filtered , washed with cold water and next few times with ether diethyl , dried under vacuum . 256263c ) . a dried 500 ml flask equipped with a magnetic stirrer , dropping funnel , and reflux condenser was charged with magnesium turnings ( 1.1 equiv . , 80.6 mmol ) , next , flask was argonated by vacuum / argon triple exchange and a solution of a few crystal of iodine in 10 ml thf was added to activate of the magnesium . , 73 mmol ) in dry thf ( 100 ml ) was added dropwise for a period of 1 hour while the reaction mixture was stirred and heated to maintain a gentle reflux . after the additional 1 h refluxing solution was cooled down to 78c and trimethylborate ( 22 ml , 183 mmol , 2.5 equiv . ) in dry thf ( 85 ml ) was slowly added . reaction was quenched with saturated aqueous solution of nh4cl ( 70 ml ) and then thf was removed under reduced pressure . the precipitated crystalline 4 was filtered , washed with cold water and next few times with ether diethyl , dried under vacuum . 256263c ) . 4-methoxyphenylboronic acid ( 3 ) was prepared in a similar way as 4yield 17.9 g , 56% , mp 202c , ( lit . 202204c ) . 2-methoxyphenylboronic acid ( 5 ) was prepared in a similar way as 4yield 17.6 g , 73% , mp 99c , ( lit . 105c ) . 1-naphtylboronic acid ( 6 ) was prepared in a similar way as 4yield 21.1 g , 85% , mp 217219c , ( lit . , n - diethylcarbamoyloxy)phenylboronic acid ( 7 ) was obtained in two steps ( 1)a 100 ml flask equipped with a magnetic stirrer was charged with phenol ( 53.2 mmol , 1 equiv . ) , 50 ml acetonitrile , 4.5 ml n - methylimidazole , triethylamine ( 79.8 mmol , 1.5 equiv . ) , and diethylcarbamoyl chloride ( 14 ) ( 79.8 mmol , 1.5 equiv . ) . the flask was closed with a tight ptfe stopper and heated at 100c for 24 h. after that time , the reaction solution was cooled down to room temperature and the solvents were removed under reduced pressure . 100 ml water was added to a residue , and product was extracted with diethyl ether ( 90 ml ) , washed with water ( 80 ml ) and 5% aq . solvent was evaporated in vacuum and the remaining residue was distilled under the reduced pressure of 1 mmhg , the product was collected in fraction at about 100c . h nmr ( 300.33 mhz , cdcl3 ) : = 1.231.27 ( m , 6h ) 3.403.47 ( m , 4h ) 7.127.23 ( m , 3h ) 7.347.40 ( m , 2h ) . 100 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with dipa ( 10.2 mmol , 1.5 equiv . ) and 30 ml thf next cooled down to 30c followed by 1.6 m solution of n - buli in hexane ( 10.2 mmol , 1.5 equiv . ) was slowly added . the lda solution was stirred for 30 min at 30c and then cooled down to 78c . a solution of tri - isopropyl borate ( 10.2 mmol , 1.5 equiv . ) in 10 ml thf was injected to the reaction mixture followed by a solution of 15 ( 6.8 mmol , 1 equiv . ) in 10 ml thf was slowly added . after addition , the reaction mixture was allowed to warm to room temperature and was quenched with saturated aqueous solution of nh4cl ( 60 ml ) . thf was removed under reduced pressure , and the product was extracted from residue with dichloromethane ( 60 ml ) . nahco3 ( 3 20 ml ) , next with water ( 2 20 ml ) and dried with mgso4 . solvent was evaporated in vacuum , and the remaining pure product 7 was collected . yield 3.96 g ( 81% , mp 162165c ) . h nmr ( 300.33 mhz , cdcl3 ) : = 1.041.13 ( m , 6h ) 3.223.32 ( m , 4h ) 7.02 ( d , j = 8.05 , 1h ) 7.187.23 ( m , 1h ) 7.337.39 ( m , 1h ) 7.857.88 ( m , 1h ) , c nmr ( 62.90 mhz , cdcl3 ) : = 11.45 , 12.03 , 40.09 , 40.52 , 74.84 , 75.34 , 75.86 , 117.54 , 123.20 , 128.44 , 133.60 , 153.62 ( lit . ) . ( 1)a 100 ml flask equipped with a magnetic stirrer was charged with phenol ( 53.2 mmol , 1 equiv . ) , 50 ml acetonitrile , 4.5 ml n - methylimidazole , triethylamine ( 79.8 mmol , 1.5 equiv . ) , and diethylcarbamoyl chloride ( 14 ) ( 79.8 mmol , 1.5 equiv . ) . the flask was closed with a tight ptfe stopper and heated at 100c for 24 h. after that time , the reaction solution was cooled down to room temperature and the solvents were removed under reduced pressure . 100 ml water was added to a residue , and product was extracted with diethyl ether ( 90 ml ) , washed with water ( 80 ml ) and 5% aq . solvent was evaporated in vacuum and the remaining residue was distilled under the reduced pressure of 1 mmhg , the product was collected in fraction at about 100c . h nmr ( 300.33 mhz , cdcl3 ) : = 1.231.27 ( m , 6h ) 3.403.47 ( m , 4h ) 7.127.23 ( m , 3h ) 7.347.40 ( m , 2h ) . 100 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with dipa ( 10.2 mmol , 1.5 equiv . ) and 30 ml thf next cooled down to 30c followed by 1.6 m solution of n - buli in hexane ( 10.2 mmol , 1.5 equiv . ) was slowly added . the lda solution was stirred for 30 min at 30c and then cooled down to 78c . a solution of tri - isopropyl borate ( 10.2 mmol , 1.5 equiv . ) in 10 ml thf was injected to the reaction mixture followed by a solution of 15 ( 6.8 mmol , 1 equiv . ) in 10 ml thf was slowly added . after addition , the reaction mixture was allowed to warm to room temperature and was quenched with saturated aqueous solution of nh4cl ( 60 ml ) . thf was removed under reduced pressure , and the product was extracted from residue with dichloromethane ( 60 ml ) . nahco3 ( 3 20 ml ) , next with water ( 2 20 ml ) and dried with mgso4 . solvent was evaporated in vacuum , and the remaining pure product 7 was collected . yield 3.96 g ( 81% , mp 162165c ) . h nmr ( 300.33 mhz , cdcl3 ) : = 1.041.13 ( m , 6h ) 3.223.32 ( m , 4h ) 7.02 ( d , j = 8.05 , 1h ) 7.187.23 ( m , 1h ) 7.337.39 ( m , 1h ) 7.857.88 ( m , 1h ) , c nmr ( 62.90 mhz , cdcl3 ) : = 11.45 , 12.03 , 40.09 , 40.52 , 74.84 , 75.34 , 75.86 , 117.54 , 123.20 , 128.44 , 133.60 , 153.62 ( lit . ) . a 100 ml flask equipped with a magnetic stirrer was charged with phenol ( 53.2 mmol , 1 equiv . ) , 50 ml acetonitrile , 4.5 ml n - methylimidazole , triethylamine ( 79.8 mmol , 1.5 equiv . ) , and diethylcarbamoyl chloride ( 14 ) ( 79.8 mmol , 1.5 equiv . ) . the flask was closed with a tight ptfe stopper and heated at 100c for 24 h. after that time , the reaction solution was cooled down to room temperature and the solvents were removed under reduced pressure . 100 ml water was added to a residue , and product was extracted with diethyl ether ( 90 ml ) , washed with water ( 80 ml ) and 5% aq . solvent was evaporated in vacuum and the remaining residue was distilled under the reduced pressure of 1 mmhg , the product was collected in fraction at about 100c . h nmr ( 300.33 mhz , cdcl3 ) : = 1.231.27 ( m , 6h ) 3.403.47 ( m , 4h ) 7.127.23 ( m , 3h ) 7.347.40 ( m , 2h ) . a 100 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with dipa ( 10.2 mmol , 1.5 equiv . ) and 30 ml thf next cooled down to 30c followed by 1.6 m solution of n - buli in hexane ( 10.2 mmol , 1.5 equiv . ) was slowly added . the lda solution was stirred for 30 min at 30c and then cooled down to 78c . a solution of tri - isopropyl borate ( 10.2 mmol , 1.5 equiv . ) in 10 ml thf was injected to the reaction mixture followed by a solution of 15 ( 6.8 mmol , 1 equiv . ) in 10 ml thf was slowly added . after addition , the reaction mixture was allowed to warm to room temperature and was quenched with saturated aqueous solution of nh4cl ( 60 ml ) . thf was removed under reduced pressure , and the product was extracted from residue with dichloromethane ( 60 ml ) . nahco3 ( 3 20 ml ) , next with water ( 2 20 ml ) and dried with mgso4 . solvent was evaporated in vacuum , and the remaining pure product 7 was collected . yield 3.96 g ( 81% , mp 162165c ) . h nmr ( 300.33 mhz , cdcl3 ) : = 1.041.13 ( m , 6h ) 3.223.32 ( m , 4h ) 7.02 ( d , j = 8.05 , 1h ) 7.187.23 ( m , 1h ) 7.337.39 ( m , 1h ) 7.857.88 ( m , 1h ) , c nmr ( 62.90 mhz , cdcl3 ) : = 11.45 , 12.03 , 40.09 , 40.52 , 74.84 , 75.34 , 75.86 , 117.54 , 123.20 , 128.44 , 133.60 , 153.62 ( lit . ) . 2-methoxyphenylpinacolborate ( 17)a 50 ml flask equipped with a magnetic stirrer was charged with 2-methoxyphenylboronic acid ( 5 ) ( 13.1 mmol , 1 equiv . ) , 30 ml of thf , pinacol ( 16 ) ( 15.8 mmol , 1.2 equiv . ) , and nh4cl ( 2.6 mmol , 0.2 equiv . ) then heated at 40c for 24 hours . after that time , thf was removed under reduced pressure and product was crystallised from petroleum ether . yield 2.31 g ( 73% ) , mp 8082c ( lit . [ 10 , 24 ] 8081c ) . a 50 ml flask equipped with a magnetic stirrer was charged with 2-methoxyphenylboronic acid ( 5 ) ( 13.1 mmol , 1 equiv . ) , 30 ml of thf , pinacol ( 16 ) ( 15.8 mmol , 1.2 equiv . ) , and nh4cl ( 2.6 mmol , 0.2 equiv . ) then heated at 40c for 24 hours . after that time yield 2.31 g ( 73% ) , mp 8082c ( lit . [ 10 , 24 ] 8081c ) . 2-bromo--picoline ( 10)a 100 ml flask equipped with a magnetic stirrer and thermometer was charged with 40% aqueous solution of hbr ( 39.5 ml ) , cooled down to 10c , and 2-amino--picoline(18 ) ( 80 mmol ) was slowly added . the temperature was kept below 0c while br2 ( 0.23 mmol ) was added over a period of 2 h. after that , a solution of nano2 ( 0.2 mol ) in h2o ( 20 ml ) was slowly added at the same temperature , and the mixture was stirred for next 30 minute . after that solution of naoh ( 0.75 mol ) in h2o ( 30 ml ) and solid koh ( 90 mmol ) were added . after 1 hour of stirring products were extracted with diethyl ether . the organic phase was separated and dried with mgso4 . solvent was evaporated in vacuum and the remaining residue was distilled to give 8.16 g ( 80% ) of 10 . a 100 ml flask equipped with a magnetic stirrer and thermometer was charged with 40% aqueous solution of hbr ( 39.5 ml ) , cooled down to 10c , and 2-amino--picoline(18 ) ( 80 mmol ) was slowly added . the temperature was kept below 0c while br2 ( 0.23 mmol ) was added over a period of 2 h. after that , a solution of nano2 ( 0.2 mol ) in h2o ( 20 ml ) was slowly added at the same temperature , and the mixture was stirred for next 30 minute . after that solution of naoh ( 0.75 mol ) in h2o ( 30 ml ) and solid koh ( 90 mmol ) were added . after 1 hour of stirring products were extracted with diethyl ether . the organic phase was separated and dried with mgso4 . solvent was evaporated in vacuum and the remaining residue was distilled to give 8.16 g ( 80% ) of 10 . 2-bromophenyl pivalate ( 9)a 100 ml flask equipped with a magnetic stirrer charged with 2-bromophenol ( 19 ) ( 11.6 mmol , 1 equiv . ) , 20 ml dichloromethane , 1.1 ml n - methylimidazole , and triethylamine ( 1.76 g , 17.4 mmol , 1.5 equiv . ) was cooled down to 0c , and pivaloyl chloride ( 21 ) ( 17.4 mmol , 1.5 equiv . ) was added . the mixture next was stirred for 2 hours at room temperature , the solvents were removed under reduced pressure , and a residue was diluted with water . the product was extracted with diethyl ether and washed with aqueous 1 m hcl , saturated solution of nahco3 and water again . the organic phase was separated and dried with mgso4 . solvent was evaporated in vacuum and the remaining residue was distilled to give 2.73 g ( 90% ) of 9 . h nmr ( 300.33 mhz , cdcl3 ) : = 1.43 ( sc , 9h ) 7.107.15 ( m , 2h ) 7.287.37 ( m , 1h ) 7.607.63 ( m , 1h ) . ( lit ) . a 100 ml flask equipped with a magnetic stirrer charged with 2-bromophenol ( 19 ) ( 11.6 mmol , 1 equiv . ) , 20 ml dichloromethane , 1.1 ml n - methylimidazole , and triethylamine ( 1.76 g , 17.4 mmol , 1.5 equiv . ) was cooled down to 0c , and pivaloyl chloride ( 21 ) ( 17.4 mmol , 1.5 equiv . the mixture next was stirred for 2 hours at room temperature , the solvents were removed under reduced pressure , and a residue was diluted with water . the product was extracted with diethyl ether and washed with aqueous 1 m hcl , saturated solution of nahco3 and water again . the organic phase was separated and dried with mgso4 solvent was evaporated in vacuum and the remaining residue was distilled to give 2.73 g ( 90% ) of 9 . h nmr ( 300.33 mhz , cdcl3 ) : = 1.43 ( sc , 9h ) 7.107.15 ( m , 2h ) 7.287.37 ( m , 1h ) 7.607.63 ( m , 1h ) . 2-bromophenyl diethylcarbamate ( 11 ) was prepared in a similar way as 9 , but dmf was used as a solvent instead of dcmyield 1.26 g ( 86% ) , bp 125c at 1 mmhg . 3-methyl-2-(4-methylphenyl)pyridine ( 25)a 50 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with 4-methoxyphenylboronic acid ( 3 ) ( 21.8 mmol , 2 equiv . ) , 2-bromo--picoline ( 10 ) ( 10.9 mmol , 1 equiv . ) , k3po4h2o ( 43.6 mmol ) , 30 ml dmf , and [ pd(pph3)4 ] ( 0.27 mmol ) . the flask was closed with a tight ptfe stopper and heated at 130c for 24 h. the reaction mixture was allowed to cool down to room temperature and dmf was removed under reduced pressure . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / isopropanol ( 9/1 ) as an eluent . yield 1 g ( 48% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 2.38 ( sc , 3h ) 2.42 ( sc , 3h ) 7.16 ( q , j = 4.76 , 2.93 hz , j = 4.76 hz , 1h ) 7.45 ( d , j = 8.05 hz , 2h ) 7.28 ( d , j = 7.68 hz , 2h ) 7.58 ( d , j = 7.50 hz , 1h ) 8.54 ( d , j = 4.76 hz , 1h ) . a 50 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with 4-methoxyphenylboronic acid ( 3 ) ( 21.8 mmol , 2 equiv . ) , 2-bromo--picoline ( 10 ) ( 10.9 mmol , 1 equiv . ) , k3po4h2o ( 43.6 mmol ) , 30 ml dmf , and [ pd(pph3)4 ] ( 0.27 mmol ) . the flask was closed with a tight ptfe stopper and heated at 130c for 24 h. the reaction mixture was allowed to cool down to room temperature and dmf was removed under reduced pressure . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / isopropanol ( 9/1 ) as an eluent . yield 1 g ( 48% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 2.38 ( sc , 3h ) 2.42 ( sc , 3h ) 7.16 ( q , j = 4.76 , 2.93 hz , j = 4.76 hz , 1h ) 7.45 ( d , j = 8.05 hz , 2h ) 7.28 ( d , j = 7.68 hz , 2h ) 7.58 ( d , j = 7.50 hz , 1h ) 8.54 ( d , j = 4.76 hz , 1h ) . yield 435 mg ( 66% ) h nmr ( 300.33 mhz , cdcl3 ) : = 2.18 ( sc , 3h ) 3.78 ( sc , 3h ) 6.98 ( d , j = 8 , 2 hz , 1h ) 7.06 ( t , j = 7.5 , 8.5 hz , 1h ) 7.20 ( q , j = 4.7 , 2.9 , 4.8 hz , 1h ) 7.29 ( dd , j = 1.8 , 4.6 , 1.8 hz , 1h ) 7.367.42 ( m , 1h ) 7.57 ( d , j = 8.6 hz , 1h ) 8.53 ( d , j = 5.8 hz , 1h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 19.39 , 55.87 , 111.24 , 121.20 , 122.61 , 125.30 , 129.91 , 130.93 , 137.8 , 145.94 , 147.02 ( lit . ) . h nmr ( 300.33 mhz , cdcl3 ) : = 2.18 ( sc , 3h ) 3.78 ( sc , 3h ) 6.98 ( d , j = 8 , 2 hz , 1h ) 7.06 ( t , j = 7.5 , 8.5 hz , 1h ) 7.20 ( q , j = 4.7 , 2.9 , 4.8 hz , 1h ) 7.29 ( dd , j = 1.8 , 4.6 , 1.8 hz , 1h ) 7.367.42 ( m , 1h ) 7.57 ( d , j = 8.6 hz , 1h ) 8.53 ( d , j = 5.8 hz , 1h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 19.39 , 55.87 , 111.24 , 121.20 , 122.61 , 125.30 , 129.91 , 130.93 , 137.8 , 145.94 , 147.02 ( lit . ) . 2-(1-naphthyl)phenyl diethylcarbamate ( 29 ) was prepared in a similar way as 25yield 483 mg ( 74% ) ; h nmr ( 300.33 mhz , cdcl3 ) : = 0.371.36 ( m , 6h ) 2.623.54 ( m , 4h ) 7.077.66 ( m , 9h ) 7.857.89 ( m , 2h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 13.42 , 41.41 , 122.19 , 123.45 , 125.65 , 126.05 , 126.32 , 126.77 , 127.03 , 127.83 , 128.18 , 128.34 , 129.07 , 129.61 , 131.92 , 133.54 , 148.27 , 151.64 , 159.83 ; ir ( neat ) = 3050 , 3000 , 1992 , 1892 , 1832 , 1587 , 1568 , 1505 , 1468 , 1433 , 1388 , 1254 , 1218 , 1197 , 1178 , 1133 , 1108 , 1016 , 971 , 918 , 873,788 , 685 , 657 , 626 , 572 , 550 , 443 ; hrms ( esi ) : m / z = 220.1125 [ c16h13n+h ] , m / z ( teor . ) yield 483 mg ( 74% ) ; h nmr ( 300.33 mhz , cdcl3 ) : = 0.371.36 ( m , 6h ) 2.623.54 ( m , 4h ) 7.077.66 ( m , 9h ) 7.857.89 ( m , 2h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 13.42 , 41.41 , 122.19 , 123.45 , 125.65 , 126.05 , 126.32 , 126.77 , 127.03 , 127.83 , 128.18 , 128.34 , 129.07 , 129.61 , 131.92 , 133.54 , 148.27 , 151.64 , 159.83 ; ir ( neat ) = 3050 , 3000 , 1992 , 1892 , 1832 , 1587 , 1568 , 1505 , 1468 , 1433 , 1388 , 1254 , 1218 , 1197 , 1178 , 1133 , 1108 , 1016 , 971 , 918 , 873,788 , 685 , 657 , 626 , 572 , 550 , 443 ; hrms ( esi ) : m / z = 220.1125 [ c16h13n+h ] , m / z ( teor . ) 3-methyl-2-(1-naphthyl)pyridine ( 31 ) was prepared in similar way as 25yield 781 mg ( 74% ) ; h nmr ( 300.33 mhz , cdcl3 ) : = 2.11 ( sc , 3h ) 7.30 ( dd , j = 4.94 , 2.93 , 4.94 hz , 1h ) 7.417.68 ( m , 4h ) 7.59 ( t , j = 6.95 , 8.23 hz , 1h ) 7.67 ( d , j = 7.68 hz , 1h ) 7.93 ( d , j = 8.23 hz , 2h ) 8.64 ( d , j = 4.39 hz , 1h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 19.72 , 122.96 , 125.79 , 125.85 , 126.25 , 126.72 , 126.82 , 128.73 , 128.78 , 131.85 , 131.98 , 132.98 , 134.14 , 138.30 , 147.37 , 158.96 ; ir ( neat ) = 3058 , 2973 , 2933 , 2874 , 1712 , 1592 , 1509 , 1471 , 1457 , 1414 , 1379 , 1316 , 1272 , 1259 , 1200 , 1151 , 1096 , 1045 , 956 , 937 , 802 , 778 , 751 , 618 . hrms ( esi ) : m / z = 320.1630 [ c21h21no2+h ] , m / z ( teor . ) = 320.1645 , diff . yield 781 mg ( 74% ) ; h nmr ( 300.33 mhz , cdcl3 ) : = 2.11 ( sc , 3h ) 7.30 ( dd , j = 4.94 , 2.93 , 4.94 hz , 1h ) 7.417.68 ( m , 4h ) 7.59 ( t , j = 6.95 , 8.23 hz , 1h ) 7.67 ( d , j = 7.68 hz , 1h ) 7.93 ( d , j = 8.23 hz , 2h ) 8.64 ( d , j = 4.39 hz , 1h ) ; c nmr ( 62.90 mhz , cdcl3 ) : = 19.72 , 122.96 , 125.79 , 125.85 , 126.25 , 126.72 , 126.82 , 128.73 , 128.78 , 131.85 , 131.98 , 132.98 , 134.14 , 138.30 , 147.37 , 158.96 ; ir ( neat ) = 3058 , 2973 , 2933 , 2874 , 1712 , 1592 , 1509 , 1471 , 1457 , 1414 , 1379 , 1316 , 1272 , 1259 , 1200 , 1151 , 1096 , 1045 , 956 , 937 , 802 , 778 , 751 , 618 . hrms ( esi ) : m / z = 320.1630 [ c21h21no2+h ] , m / z ( teor . ) = 320.1645 , diff . biphenyl-2,2-diyl bis(diethylcarbamate ) ( 34)a 100 ml flask equipped with a magnetic stirrer was charged with 2,2-biphenol ( 33 ) ( 26.8 mmol , 1 equiv . ) , dimethylformamide ( 30 ml ) , n - methylimidazole ( 2.5 ml ) , triethylamine ( 5.4 g , 53.7 mmol , 2.2 equiv . ) and diethylcarbamoyl chloride ( 20 ) ( 7.3 g , 2.2 equiv . ) . the flask was closed with a tight ptfe stopper and heated at 100c for 24 h. after that time the reaction solution was cooled down to room temperature and the solvents were removed under reduced pressure . 100 ml water was added to a residue and the product was extracted with diethyl ether ( 90 ml ) , washed with water ( 80 ml ) , and 5% aq . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 9/1 ) as an eluent . yield 6.87 g ( 67% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 0.861.03 ( m , 12h ) 3.113.21 ( m , 8h ) 7.187.38 ( m , 8h ) . a 100 ml flask equipped with a magnetic stirrer was charged with 2,2-biphenol ( 33 ) ( 26.8 mmol , 1 equiv . ) , dimethylformamide ( 30 ml ) , n - methylimidazole ( 2.5 ml ) , triethylamine ( 5.4 g , 53.7 mmol , 2.2 equiv . ) and diethylcarbamoyl chloride ( 20 ) ( 7.3 g , 2.2 equiv . ) . the flask was closed with a tight ptfe stopper and heated at 100c for 24 h. after that time the reaction solution was cooled down to room temperature and the solvents were removed under reduced pressure . 100 ml water was added to a residue and the product was extracted with diethyl ether ( 90 ml ) , washed with water ( 80 ml ) , and 5% aq . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 9/1 ) as an eluent . yield 6.87 g ( 67% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 0.861.03 ( m , 12h ) 3.113.21 ( m , 8h ) 7.187.38 ( m , 8h ) . 2-hydroxybiphenyl-2-yl diethylcarbamate ( 35 ) was also obtained in this reaction leading to 34 in 0.79 g ( 10% ) yield h nmr ( 300.33 mhz , cdcl3 ) : = 0.921.04 ( m , 6h ) 3.183.23 ( m , 4h ) 6.926.95 ( m , 1h ) 6.987.01 ( m , 1h ) 7.12 ( dd , j = 2.20 , 6.22 , 1.28 , 1h ) , 7.227.29 ( m , 2h ) 7.33 ( d , j = 3.48 hz , 1h ) 7.35 ( sc , 1h ) 7.427.47 ( m , 1h ) ; ir ( neat ) = 3260 , 3061 , 2976 , 2935 , 2875 , 2707 , 1683 , 1606 , 1573 , 1506 , 1483 , 1442 , 1426 , 1379 , 1364 , 1275 , 1197 , 1165 , 1118 , 1095 , 1047 , 1006 , 966 , 938 , 842 , 818 , 748 , 618 , 574 , 496 , 432 ; hrms ( esi ) : m / z = 308.1240 [ c17h19no3+na ] , m / z ( teor . ) h nmr ( 300.33 mhz , cdcl3 ) : = 0.921.04 ( m , 6h ) 3.183.23 ( m , 4h ) 6.926.95 ( m , 1h ) 6.987.01 ( m , 1h ) 7.12 ( dd , j = 2.20 , 6.22 , 1.28 , 1h ) , 7.227.29 ( m , 2h ) 7.33 ( d , j = 3.48 hz , 1h ) 7.35 ( sc , 1h ) 7.427.47 ( m , 1h ) ; ir ( neat ) = 3260 , 3061 , 2976 , 2935 , 2875 , 2707 , 1683 , 1606 , 1573 , 1506 , 1483 , 1442 , 1426 , 1379 , 1364 , 1275 , 1197 , 1165 , 1118 , 1095 , 1047 , 1006 , 966 , 938 , 842 , 818 , 748 , 618 , 574 , 496 , 432 ; hrms ( esi ) : m / z = 308.1240 [ c17h19no3+na ] , m / z ( teor . ) 2-pivaloyloxy-2-n , n - diethylcarbomoyloxybiphenyl ( 39)a 100 ml flask equipped with a magnetic stirrer charged with 0.3 g 2-hydroxybiphenyl-2-yl diethylcarbamate ( 35 ) ( 1 mmol , 1 equiv . ) , 1.8 ml dimethylformamide , 0.1 ml n - methylimidazole , and 0.2 ml triethylamine ( 5.4 g , 53.7 mmol , 2.2 equiv . ) was cooled down to 0c , and pivaloyl chloride ( 21 ) ( 126.7 mg , 1 mmol , 1 equiv . ) was added . the mixture next was stirred for 2 hours at room temperature ; the solvents were removed under reduced pressure and a residue was diluted with water . the product was extracted with diethyl ether and washed with aqueous 1 m hcl . solvent was evaporated in vacuum , and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 99/1 ) as an eluent . yield 318 mg ( 82% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 0.871.03 ( m , 15h ) 3.123.22 ( m , 4h ) 7.087.11 ( m , 1h ) 7.197.40 ( m , 7h ) . ir ( neat ) = 2973 , 2933 , 2874 , 1749 , 1715 , 1471 , 1414 , 1380 , 1367 , 1270 , 1250 , 1227 , 1198 , 1153 , 1110 , 1045 , 1010 , 959 , 938 , 897 , 749 . hrms ( esi ) : m / z = 392.1855 [ c22h27no4+na ] , m / z ( teor . ) = 392.1832 , diff . ( lit . ) . a 100 ml flask equipped with a magnetic stirrer charged with 0.3 g 2-hydroxybiphenyl-2-yl diethylcarbamate ( 35 ) ( 1 mmol , 1 equiv . ) , 1.8 ml dimethylformamide , 0.1 ml n - methylimidazole , and 0.2 ml triethylamine ( 5.4 g , 53.7 mmol , 2.2 equiv . ) was cooled down to 0c , and pivaloyl chloride ( 21 ) ( 126.7 mg , 1 mmol , 1 equiv . ) was added . the mixture next was stirred for 2 hours at room temperature ; the solvents were removed under reduced pressure and a residue was diluted with water . the product was extracted with diethyl ether and washed with aqueous 1 m hcl . solvent was evaporated in vacuum , and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 99/1 ) as an eluent . yield 318 mg ( 82% ) . h nmr ( 300.33 mhz , cdcl3 ) : = 0.871.03 ( m , 15h ) 3.123.22 ( m , 4h ) 7.087.11 ( m , 1h ) 7.197.40 ( m , 7h ) . ir ( neat ) = 2973 , 2933 , 2874 , 1749 , 1715 , 1471 , 1414 , 1380 , 1367 , 1270 , 1250 , 1227 , 1198 , 1153 , 1110 , 1045 , 1010 , 959 , 938 , 897 , 749 . hrms ( esi ) : m / z = 392.1855 [ c22h27no4+na ] , m / z ( teor . ) = 392.1832 , diff . 2-(2-methoxybiphenyl-2-yl)-4,4-dimethyl-4,5-dihydro-1,3-oxazole ( 42)a dried schlenk equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with the 1 m thf solution of grignard reagent 41 ( 3.4 mmol , 3 equiv . ) then 2-(2-methoxyphenyl)-4,4-dimethyl-4,5-dihydro-1,3-oxazole ( 8) ( 1.1 mmol , 1 equiv . ) was added and the reaction mixture was heated overnight at 80c . the reaction was allowed to cool down to room temperature and it was quenched with saturated aqueous solution of nh4cl ( 3 ml ) . the solvents were removed under reduced pressure , 100 ml of water was added to a residue and products were extracted with diethyl ether . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 99/1 ) as an eluent . yield 299 mg ( 94% ) of product . h nmr ( 300.33 mhz , cdcl3 ) : = 1.27 ( sc , 6h ) , 3.75 ( sc , 3h ) , 3.78 ( sc , 2h ) , 6.896.92 ( m , 1h ) , 7.02 ( t , j = 7.50 , 7.50 hz , 1h ) , 7.247.41 ( m , 4h ) , 7.477.52 ( m , 1h ) , 7.857.88 ( m , 1h ) . a dried schlenk equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with the 1 m thf solution of grignard reagent 41 ( 3.4 mmol , 3 equiv . ) then 2-(2-methoxyphenyl)-4,4-dimethyl-4,5-dihydro-1,3-oxazole ( 8) ( 1.1 mmol , 1 equiv . ) was added and the reaction mixture was heated overnight at 80c . the reaction was allowed to cool down to room temperature and it was quenched with saturated aqueous solution of nh4cl ( 3 ml ) . the solvents were removed under reduced pressure , 100 ml of water was added to a residue and products were extracted with diethyl ether . solvent was evaporated in vacuum and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 99/1 ) as an eluent . yield 299 mg ( 94% ) of product . h nmr ( 300.33 mhz , cdcl3 ) : = 1.27 ( sc , 6h ) , 3.75 ( sc , 3h ) , 3.78 ( sc , 2h ) , 6.896.92 ( m , 1h ) , 7.02 ( t , j = 7.50 , 7.50 hz , 1h ) , 7.247.41 ( m , 4h ) , 7.477.52 ( m , 1h ) , 7.857.88 ( m , 1h ) . 4-methoxy-5-methyl-1,1:2,1-terphenyl ( 45 ) , 4-methoxy-3-(4-methoxyphenyl)-5-methyl-1,1:2,1-terphenyl ( 46)a 50 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with [ rhcl(cod)]2 ( 3 mol , 2.5 mol% ) , tris(pentafluorophenyl)phosphine ( 2%-mol ) , and 2 ml n - methylpyrrolidone ( nmp ) . the reaction mixture stirred for 30 minute at 70c , cooled down to room temperature and to solution of formed catalyst 3-methyl-2-(4-methylphenyl)pyridine ( 25 ) ( 1.4 mmol , 1 equiv . ) , p - bromoanisole ( 44 ) ( 2.8 mmol , 2 equiv . ) , cs2co3 ( 2.7 mmol , 2 equiv . ) and 1 ml nmp were added . the solution was heated at 160c for 24 h , after that the reaction mixture was cooled down to room temperature . the products were extracted with diethyl ether and washed with water . the organic phase was separated and dried with mgso4 . solvents were evaporated in vacuum , and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 6/1 ) as an eluent to afford 45 ( 290 mg , 64% ) and 46 ( trace).45 : h nmr ( 300.33 mhz , cdcl3 ) : = 2.47 ( sc , 3h ) , 3.74 ( sc , 6h ) , 6.666.69 ( m , 4h ) , 6.897.28 ( m , 9h ) , 8.29 ( d , j = 4.76 , 1h ) . ir ( neat ) = 3002 , 2953 , 2923 , 2858 , 2835 , 1608 , 1581 , 1568 , 1515 , 1496 , 1460 1441 , 1423 , 1380 , 1290 , 1245 , 1177 , 1137 , 1111 , 1066 , 1051 , 1031 , 989 , 827 , 788 , 597 , 566 , 520 . hrms ( esi ) : m / z = 290.1535 [ c20h19no+h ] , m / z ( teor . ) = 290.1539 , diff . 46 : h nmr ( 300.33 mhz , cdcl3 ) : = 1.76 ( sc , 3h ) 2.46 ( sc , 3h ) 3.76 ( sc , 3h ) 6.71 ( d , j = 8.78 , 2h ) 7.04 ( d , j = 8.78 , 2h ) 7.087.12 ( m , 1h ) 7.227.32 ( m , 5h ) 8.508.52 ( m , 1h ) . ir ( neat ) = 2957 , 2928 , 2835 , 1710 , 1607 , 1576 , 1510 , 1460 , 1441 , 1421 , 1401 , 1379 , 1290 , 1243 , 1176 , 1109 , 1031 , 830 , 793 , 731 , 595 , 568 , 548 , 530 . hrms ( esi ) : m / z = 396.1958 [ c27h25no2+h ] , m / z ( teor . ) = 396.1958 , diff . a 50 ml flask equipped with a magnetic stirrer was argonated by vacuum / argon triple exchange and charged with [ rhcl(cod)]2 ( 3 mol , 2.5 mol% ) , tris(pentafluorophenyl)phosphine ( 2%-mol ) , and 2 ml n - methylpyrrolidone ( nmp ) . the reaction mixture stirred for 30 minute at 70c , cooled down to room temperature and to solution of formed catalyst 3-methyl-2-(4-methylphenyl)pyridine ( 25 ) ( 1.4 mmol , 1 equiv . ) , p - bromoanisole ( 44 ) ( 2.8 mmol , 2 equiv . ) , cs2co3 ( 2.7 mmol , 2 equiv . ) and 1 ml nmp were added . the solution was heated at 160c for 24 h , after that the reaction mixture was cooled down to room temperature . the products were extracted with diethyl ether and washed with water . the organic phase was separated and dried with mgso4 . solvents were evaporated in vacuum , and the remaining residue was purified by column chromatography on silica gel using hexane / acetone ( 6/1 ) as an eluent to afford 45 ( 290 mg , 64% ) and 46 ( trace).45 : h nmr ( 300.33 mhz , cdcl3 ) : = 2.47 ( sc , 3h ) , 3.74 ( sc , 6h ) , 6.666.69 ( m , 4h ) , 6.897.28 ( m , 9h ) , 8.29 ( d , j = 4.76 , 1h ) . ir ( neat ) = 3002 , 2953 , 2923 , 2858 , 2835 , 1608 , 1581 , 1568 , 1515 , 1496 , 1460 1441 , 1423 , 1380 , 1290 , 1245 , 1177 , 1137 , 1111 , 1066 , 1051 , 1031 , 989 , 827 , 788 , 597 , 566 , 520 . hrms ( esi ) : m / z = 290.1535 [ c20h19no+h ] , m / z ( teor . ) = 290.1539 , diff . 46 : h nmr ( 300.33 mhz , cdcl3 ) : = 1.76 ( sc , 3h ) 2.46 ( sc , 3h ) 3.76 ( sc , 3h ) 6.71 ( d , j = 8.78 , 2h ) 7.04 ( d , j = 8.78 , 2h ) 7.087.12 ( m , 1h ) 7.227.32 ( m , 5h ) 8.508.52 ( m , 1h ) . ir ( neat ) = 2957 , 2928 , 2835 , 1710 , 1607 , 1576 , 1510 , 1460 , 1441 , 1421 , 1401 , 1379 , 1290 , 1243 , 1176 , 1109 , 1031 , 830 , 793 , 731 , 595 , 568 , 548 , 530 . hrms ( esi ) : m / z = 396.1958 [ c27h25no2+h ] , m / z ( teor . ) = 396.1958 , diff . 45 : h nmr ( 300.33 mhz , cdcl3 ) : = 2.47 ( sc , 3h ) , 3.74 ( sc , 6h ) , 6.666.69 ( m , 4h ) , 6.897.28 ( m , 9h ) , 8.29 ( d , j = 4.76 , 1h ) . ir ( neat ) = 3002 , 2953 , 2923 , 2858 , 2835 , 1608 , 1581 , 1568 , 1515 , 1496 , 1460 1441 , 1423 , 1380 , 1290 , 1245 , 1177 , 1137 , 1111 , 1066 , 1051 , 1031 , 989 , 827 , 788 , 597 , 566 , 520 . hrms ( esi ) : m / z = 290.1535 [ c20h19no+h ] , m / z ( teor . ) = 290.1539 , diff . 46 : h nmr ( 300.33 mhz , cdcl3 ) : = 1.76 ( sc , 3h ) 2.46 ( sc , 3h ) 3.76 ( sc , 3h ) 6.71 ( d , j = 8.78 , 2h ) 7.04 ( d , j = 8.78 , 2h ) 7.087.12 ( m , 1h ) 7.227.32 ( m , 5h ) 8.508.52 ( m , 1h ) . ir ( neat ) = 2957 , 2928 , 2835 , 1710 , 1607 , 1576 , 1510 , 1460 , 1441 , 1421 , 1401 , 1379 , 1290 , 1243 , 1176 , 1109 , 1031 , 830 , 793 , 731 , 595 , 568 , 548 , 530 . hrms ( esi ) : m / z = 396.1958 [ c27h25no2+h ] , m / z ( teor . )

general approach to the synthesis of prochiral precursors of chiral atropisomeric biaryls based on several complementary methods has been developed . biaryls were obtained in good to excellent yields depending on their structure and selected method of synthesis . furthermore , we demonstrate a possibility of utilisation of the obtained compounds possessing 2 or 3 ortho substituents around the aryl - aryl bond in direct and directed arylation reaction leading through transition metal - mediated c h bond activation to atropisomeric compounds .

1. Introduction 2. Combinatorial Approach 3. Synthesis of Prochiral Biaryls 4. Application of Prochiral Compounds in the Synthesis of Atropisomeric Biaryls 5. Conclusion 6. Experimental

PMC4609870

physical exercise has long been known to be effective in the treatment and prevention of many physical conditions such as type 2 diabetes , hypertension , obesity , dyslipidemia , and cardiovascular disease [ 13 ] . furthermore , exercise has been found to reduce symptoms of depression and anxiety [ 47 ] . studies of healthy older adults have shown positive effects of exercise on measures of cognitive function [ 8 , 9 ] . research into the effects of physical activity on enhancing cognitive / academic abilities in children shows some promise . however , the findings are still fairly limited and more randomized , controlled trials are needed [ 1012 ] . similarly , there is some evidence that physical activity can improve cognition or prevent mental decline in people with neurological and neurodegenerative disorders . the overall results , however , remain inconclusive due to differences in methodologies and quality of studies [ 1316 ] . physical exercise after acquired brain injury ( abi ) has received attention as a cost - effective , noninvasive , and practicable rehabilitation tool . preclinical research has shown that post - abi exercise can increase cerebral growth factor levels [ 1721 ] , reduce apoptosis - related processes [ 2224 ] , promote neurogenesis , neuronal survival , and regeneration [ 2528 ] , reduce lesion size [ 29 , 30 ] , modulate inflammatory responses , reduce astrocytosis [ 32 , 33 ] , and improve cerebral blood flow [ 34 , 35 ] . however , less is known about the potential effects of exercise on cognitive recovery after abi . cognitive dysfunctions after brain injury , such as memory , attentional , and executive function impairments , are common and can negatively affect work performance , social competencies , and experienced quality of life . in this paper , the preclinical research investigating the effects of post - abi exercise on cognitive recovery will be systematically reviewed . within brain injury rehabilitation , several factors ( e.g. , timing , repetition , intensity ) have been shown to be of importance for promoting brain plasticity mechanisms and enhancing recovery outcome . such factors are also believed to be essential when using exercise as a cognitive rehabilitation tool . in the following , parameters that are believed to play a role in the efficacy of exercise , including type of exercise , starting point , and dose - related issues , will be examined . relevant research studies were found using the search terms exercise ( and ) animal model ( or ) rodent ( or ) rat ( and ) traumatic brain injury ( or ) cerebral ischemia ( or ) brain irradiation , all in all 9 search strings . the searches were performed in february of 2014 on the pubmed , scopus , and psycinfo databases , providing a total of 2308 hits . articles were then selected using the following inclusion criteria : in english.animal model based.employing adult animals ( rat models : min . 55 g).animals were subjected to acquired brain injury ( abi ) in their adult life , either through mechanical injury , neurotoxic injection , irradiation , or induction of cerebral ischemia.exercise was used as an intervention / treatment tool after cerebral injury ( habituation to the exercise apparatuses prior to injury was accepted).the exercise regimens consisted of a general motor activation of all of the animals ' extremities ( i.e. , running , swimming ) . sole training of a single muscle group or extremity ( i.e. , forced limb use , grip training ) was not included.the effects of the exercise intervention could be clearly distinguished from effects of nonexercise interventions if such were also investigated.studies contained at least one measure of cognitive and/or emotional function after ( or during ) exercise treatment . studies solely investigating motor abilities ( i.e. , balance tests , physical strength tests ) or neural / molecular mechanisms were excluded . animals were subjected to acquired brain injury ( abi ) in their adult life , either through mechanical injury , neurotoxic injection , irradiation , or induction of cerebral ischemia . exercise was used as an intervention / treatment tool after cerebral injury ( habituation to the exercise apparatuses prior to injury was accepted ) . the exercise regimens consisted of a general motor activation of all of the animals ' extremities ( i.e. , running , swimming ) . sole training of a single muscle group or extremity ( i.e. , forced limb use , grip training ) was not included . the effects of the exercise intervention could be clearly distinguished from effects of nonexercise interventions if such were also investigated . studies contained at least one measure of cognitive and/or emotional function after ( or during ) exercise treatment . studies solely investigating motor abilities ( i.e. , balance tests , physical strength tests ) or neural / molecular mechanisms were excluded . examination of the references in these articles did not uncover further publications that fulfilled the inclusion criteria . of the 22 papers , 14 used rats , five used mice , and three used gerbils as their experimental subjects . all used male animals except two ( see table 1 ) . regarding type of brain injury , eight were ischemia models ( common carotid artery occlusion , middle cerebral artery occlusion , and photothrombosis ) , five used cortical impact injury , four used fluid percussion injury , one used closed head injury equipment , another used neurotoxic injection , and three used gamma irradiation . experimental groups fell into four types of exercise : nonmotorized running wheel exercise ( nine studies ) , motorized treadmill exercise ( 11 studies ) , motorized running wheel ( one study ) , swimming in a circular pool ( one study ) , and swimming or running wheel exercise ( one study ) . cognitive measures applied in these studies were spatial learning / retention paradigms administered in a water maze ( 12 studies ) or in a barnes maze ( one study ) , visual discrimination and retention in a water maze ( one study ) , object recognition tests ( three studies ) , an object location test ( one study ) , conditioning based learning paradigms ( seven studies ) ( i.e. , contextual fear learning , step - down avoidance task , passive avoidance task , stop - signal reaction time task , conditioned learning in a y - maze ) , open field tests ( three studies ) , and tail suspension tests ( two studies ) . some studies used more than one test . within animal model based research , exercise is often differentiated into voluntary or forced paradigms . in voluntary paradigms , the animals are given a choice between movement and inactivity while having access to the exercise apparatus . in forced exercise , exercise in a nonmotorized running wheel allows animals to exercise at their own accord , while motorized treadmill running / running wheel exercise and swimming exercise do not offer such movement autonomy . the following section examines whether the type of exercise ( voluntary or forced ) exerts differential effects on cognitive recovery after abi . subjected rats to lateral fluid percussion injury ( lfpi ) immediately followed by 12 days of running wheel exercise ( 7 of those days prior to cognitive testing ) , a diet high in docosahexaenoic acid ( dha ) , or both . they found that lfpi exercised animals did significantly better in a spatial learning task in a water maze ( as shown by reduced latency to find a platform ) in comparison to nonexercised lfpi animals kept on a normal diet . furthermore , injured rats on the combined exercise and dha diet significantly outperformed all other lfpi groups . molecular analysis showed increased levels of dha , acox1 , and 17-hsd4 ( enzymes involved in dha metabolism ) , sir2 ( involved in mitochondrial function ) , ipla2 ( molecules involved in membrane homeostasis ) , p - trkb ( bdnf receptor ) , and lower levels of 4-hhe ( marker for lipid peroxidation ) in the groups subjected to either exercise or the dha diet ( compared to controls ) and a further increase / decrease in the combined group . the combined group also showed increased stx-3 ( also involved in membrane homeostasis ) and brain derived neurotrophic factor ( bdnf ) levels . the study indicates that early initiated voluntary exercise and/or the dha diet can positively affect cognitive recovery after tbi , possibly through counteracting membrane damage and coordinating dha metabolism . . found that animals exposed to lfpi and early initiated exercise ( from post - injury day 0 ) performed significantly worse on a spatial acquisition task in a water maze than all other groups , including an lfpi group starting exercise at a later point in time ( at postinjury day 14 ) and lfpi nonexercised controls . animals exercised later performed at the level of the sham operated animals . during a retention test ( probe trial ) the late exercise and sham groups showed increased hippocampal levels of the transcriptional regulator phosphorylated cyclic amp response element - binding protein ( pcreb ) and bdnf . moreover , there was a positive correlation between bdnf - levels and the amount of exercise . no bdnf - increase was seen in the early exercised group , which also showed lower levels of synapsin - i ( involved in synaptic vesicle clustering and release ) and creb . also finding detrimental effects , crane et al . subjected animals to a cortical contusion injury immediately followed by a 7-day running wheel exercise regimen . in a complex stop - signal reaction time task ( a conditioning - based learning task requiring either inhibition or execution of a learned behavior depending on stimuli given ) , the exercised animals performed significantly worse for the first five test days compared with both the nonexercised injured animals and the sham animal groups . however , after a week of testing , the exercised animals returned to their baseline levels . the contused , exercised animals showed larger inflammatory responses ( more gfab and iba1 positive cells ) in the cortex and hippocampus , respectively . all contused groups had fewer surviving cells ( less dap1 positive cells ) in the cortex , hippocampus , mediodorsal nucleus of the thalamus , and corpus callosum compared to the nonexercised , sham animals . the conflicting results of the two first studies are somewhat surprising , as they use similar models and setups . discrete differences , for example , in the duration of the exercise protocol , could potentially account for these divergent findings . while the studies by griesbach et al . and crane et al . both found detrimental effects of early exercise on cognitive performance , it appears that these effects are transient , as the animals seem to catch up during the rather short - time course of task acquisition . subjected c57/bl6 mice to middle cerebral artery occlusion ( mcao ) followed by a one week postinjury break . subsequently , the animals were exercised for 39 days in either running wheels or by swimming in a circular pool before starting a spatial acquisition task in a water maze . this was not the case for the swimming group , whose performance did not differentiate from the nonexercised mcao group . progenitor cell survival in the dentate gyrus and pcreb levels were increased in the mcao running wheel group compared to the control group . this suggests that different exercise types can affect cognitive recovery differently and that voluntary exercise initiated after the first postinjury week can induce functional recovery and help promote cell survival . however , in another study by piao et al . starting exercise 1 week after injury did not produce similar results . examining the timing effects of a 4-week running wheel regimen after controlled cortical impact injury ( cci ) , animals were exercised beginning either 1 week ( early ) or 5 weeks ( late ) postinjury . the study found that the late exercised cci - group had a significantly reduced latency to find the platform in a spatial water maze learning task and better retention of the task compared to a nonexercised cci - group . in a reversed platform test , a test of cognitive flexibility , they found that the late exercised animals showed a significant improvement compared to both the early exercised cci - group and the nonexercised cci - group . retention of the reversed platform task was significantly better in the late exercised cci - animals compared to the nonexercised cci - group . there were no differences between the early initiated group and the nonexercised cci - group on any of the above parameters . in a novel object recognition task , the late exercised cci - animals spent significantly longer time exploring a new object than both the early exercised cci - group and a nonexercised cci - group , indicating improved short - term memory abilities ; in fact their exploration time was at the level of uninjured , nave animals . there were no group differences in locomotor activity in an open field test . in a tail suspension test , all cci - groups showed increased immobility times regardless of exercise status , suggesting more pronounced behavioral despair due to injury . furthermore , the late exercised cci - group had a reduced lesion size compared to the early exercised cci - group and the nonexercised cci - group . there were time - dependent increases and decreases in different microglia activation markers : il-1 levels ( a proinflammatory marker ) increased in the early exercise group in week 5 after cci and levels reduced in the late exercise group in postinjury week 9 ( both compared to the nonexercised cci - group ) . there were also increased levels of il-6 ( a proinflammatory marker ) and il-10 ( an anti - inflammatory marker ) in the late exercise group in week 9 . cortical ( ipsilateral ) galectin-3 and c1qb levels ( microglial activation markers ) were increased in the early exercised animals , while they were reduced in the late exercised group together with levels of gp91phox and p22phox ( membrane components of nadph oxidase enzyme ) . late exercise increased hippocampal creb gene expression , bdnf , and igf-1 ( insulin - like growth factor 1 ) levels and increased neurogenesis and cell survival in the late exercise group ( but not in the early exercise group ) . the study concludes that the improved cognitive performance in the group subjected to late exercise is possibly due to a more optimal coordination / balance of microglia expression and increased growth factor levels . similar to studies initiating exercise immediately after abi , starting a voluntary exercise paradigm one week after injury produces conflicting results , suggesting that the exercise type ( voluntary versus forced ) is not the only factor determining the efficacy of exercise . as already mentioned , griesbach et al . found improved cognitive performance in animals exercised 14 days after injury . in a later study , griesbach et al . reproduced this finding . animals exercised 14 days after lfpi acquired a spatial learning task in a water maze significantly faster than nonexercised lfpi animals . in addition , they reached six out of seven criterion scores ( e.g. , reaching a platform from 10 sec down to 4 sec ) significantly faster than the nonexercised lfpi group . however , mbdnf and creb levels were higher in the lfpi exercised animals than in the lfpi control animals . this also held true for the exercised sham animals , who showed increased levels of synapsin - i . when blocking trkb receptors in lfpi animals , the exercise - induced increase in mbdnf was reduced . [ 39 , 42 ] suggest that voluntary exercise started at a later stage ( 14 days ) is beneficial for cognitive recovery , possibly through upregulation of bdnf and down - stream effectors of synaptic transmission . subjected female c57bl/6 mice to whole brain irradiation ( 5 gy , single dose ) . the animals were then given access to running wheels outside of their home cages for 812 hours a day , starting 1 month after irradiation . prior to initiation of exercise , all animals were tested in a spatial learning and retention test in a barnes maze as well as a tail suspension test . after 6 weeks of wheel running , the animals were once again tested in the barnes maze ( both 3 and 4 months after irradiation ) and in the tail suspension test ( 2.5 months after irradiation ) . results from the first testing period in the barnes maze ( preexercise ) showed no differences between the groups : the sham animals learned the task by day 2 , the irradiated animals by day 3 . the tests performed after exercise showed that irradiated , exercised animals had longer latency to complete the task on the first test - day compared to all other groups ; however , by day 3 there were no differences . the irradiated , sedentary animals did not exhibit target quadrant preferences in retention trials ( which they did during the first test period ) . however , the irradiated , exercised animals spent more time in the target quadrant , indicating improved memory . there were no differences between the groups in immobility times in the tail suspension test at any test point . histology showed no differences in dentate gyrus size in any of the groups . running elevated the number of hippocampal brdu and neun positive cells ( markers of newborn cells and mature neurons , resp . ) in the irradiated animals . levels of proinflammatory cytokines ( tumor necrosis factor- ( tnf- ) , interferon- ( ifn- ) , and interleukin-6 ( il-6 ) ) were elevated in the irradiated groups ( compared to shams ) . levels of igf were increased in the irradiated , exercised animals compared to irradiated , sedentary animals . levels of bdnf and vegf ( vascular endothelial growth factor ) were decreased in all irradiated animals ( both exercised and nonexercised ) . however , running partially restored vegf - levels in the irradiated , exercised animals . the study shows that later - initiated voluntary running can prevent memory decline at a later stage in irradiation - exposed animals . showing similar positive recovery effects of late - initiated voluntary exercise after brain irradiation , winocur et al . twenty - five days after irradiation approximately half the animals were allowed to exercise in running wheels in their home cages . after two weeks of running , all animals were tested in a visual discrimination task in a water maze , followed by either a high - interference task ( an unsolvable task ) or a low - interference task ( demanding no visual discrimination for task solution ) . there were no differences between the groups in acquiring the visual discrimination task . in the retention task , the irradiated animals had the most errors ; this was especially pronounced in the animals that had previously performed the high - interference task . further analysis showed that irradiated , exercised animals in the high - interference group performed significantly better in the retention test than the irradiated , sedentary animals in the low - interference group . the exercised , sham animals performed better in the retention test than the sedentary sham animals regardless of interference group affiliation . analysis of hippocampal dcx and ki67 positive neurons ( neurogenesis markers ) showed that irradiation decreased their levels , but running increased the levels in all exercised groups . the authors conclude that neurogenesis is a part of the mechanism that controls memory interference , as suppressing neurogenesis disrupts retention in the high - interference groups . however , this effect can be diminished by promoting neurogenesis through exercise . while three studies found cognitive improvement in animals starting exercise 25 days after injury or later [ 31 , 43 , 44 ] , clark et al . administered running wheel exercise between 114 and 142 days after gamma irradiation of the hippocampal area of both male and female c57bl/6j mice . they found that 54 days of wheel exercise did not have an effect on spatial learning and retention in a water maze in the gamma radiated group compared with a nonexercised radiated group . running did , however , have a positive effect on sham operated animals . in a contextual fear conditioning test , running increased freezing time ( indicating increased memory of a formerly presented painful stimulus ) ; however , this was regardless of radiation status . in other words , in the spatial tasks no effects of exercise were found in the irradiated animals , yet running did improve performance in the conditioning task in all exercise groups . running increased hippocampal neurogenesis exercise counteracted radiation - induced reductions in neurogenesis , neuronal differentiation , and glia cell levels . five studies [ 31 , 39 , 4244 ] found positive effects of later initiated voluntary exercise on measures of spatial learning and retention . however , this was not the case in the study by clark et al . , who waited 3 - 4 months with exercise administration . this opens the question whether there is a window of rehabilitation opportunity that closes after certain amount of time has passed . other factors could also account for the conflicting results such as different injury types and duration of exercise . interestingly , running affected performance positively in the fear conditioning task in the clark et al . all in all , the above research shows a somewhat mixed picture of using voluntary exercise in cognitive rehabilitation after abi . later starting points ( from 14-days post - injury ) however , further research is needed to determine if exercise interventions can be administered too late to produce cognitive improvements . moreover , caution should be taken in making general recommendations based on such a limited and methodologically diverse set of studies . the results indicate that the voluntary aspect of exercise is not the sole determinant of effect ; other variables such as starting point and duration may also play a significant role . fourteen studies have looked into the effects of forced exercise on cognitive recovery after abi . itoh et al . subjected rats to cci injury followed by a 7-day treadmill exercise regimen beginning one day after injury . in acquisition and retention of a spatial task in a water maze , the lesioned , exercised animals did significantly better than the lesioned , nonexercised animals ; the former performing to the functional level of the sham animals . there was a significant reduction in lesion size in the exercised group compared to the controls . additionally , there was a significant reduction in ssdna immunopositive cells ( a marker of apoptosis ) around the damaged cortical area in the exercised group on postinjury days 1 , 3 , and 7 , an increase in the number of neun positive cells , and a reduction in gfap positive cells ( marker for astrocytes ) 7 days after tbi compared to the lesioned , nonexercised control group . this suggests that early initiated forced exercise can improve cognitive function while reducing apoptosis and impacting the glial scarring . . looked at effects of both pre- and postinjury treadmill exercise in a bilateral common carotid artery occlusion ( ccao ) rat model . the postinjury trained group started exercising 24 hours following surgery and continued for 12 weeks , 3 days a week . they found that all exercised groups , including the postinjury exercised group , did significantly better on three of the five testing days in acquisition of a spatial task in a water maze compared to a lesioned , nonexercised group . this pattern was also seen in a retention ( probe trial ) test and in a working memory test in a water maze . there were no differences between groups in levels of free radicals or sod ( superoxide dismutase , an antioxidant enzyme ) levels . however , there were heightened hippocampal lipoperoxidation ( evaluated by tbars test ) and thiol - levels ( antioxidants ) in the lesioned , nonexercised group compared to the other groups . like the study by itoh et al . , this study shows that early initiated forced exercise can positively affect cognitive recovery , potentially through reducing oxidative damage by regulation of antioxidant levels . after 24 hours , the animals began exercising at either a low or a high - intensity ( speed ) on a treadmill for 14 days . they found that the lesioned , low - intensity group had shorter latencies on three out of the four testing days in a spatial learning task in a water maze ( compared to the lesioned , high - intensity group and a lesioned , nonexercised control group ) . furthermore , the low - intensity paradigm increased levels of hippocampal bdnf , synapsin - i ( contralaterally ) , and psd-95 ( membrane scaffolding protein ) as well as the dendritic complexity ( measured by sholl analysis ) and the number of dendritic spines compared to the control group . there were higher levels of corticosterone ( stress - hormone ) in the high - intensity group compared to the controls . the study is interesting as it investigates the effects of exercise intensity on cognitive measures . while both groups initiated exercise 24 hours after injury , only the low - intensity group showed positive cognitive effects concomitant with increases in plasticity - related proteins and dendrite development . furthermore , the high - intensity group displayed higher levels of stress - hormone , which may have inhibited the efficacy of exercise . in another study investigating the effects of different exercise intensities , shen et al . subjected rats to cci immediately followed by two different intensities of treadmill exercise for 14 days . they found that the lesioned , low - intensity group performed better on two out of the four days of the acquisition part of a spatial task in a water maze compared to the high - intensity group and a lesioned , nonexercised control group . the low - intensity group also showed better retention than the control group . on a neurological deficit score all cci animals did worse than the sham animals , but they all improved by day 6 post - tbi . bdnf and phosphorylated creb measurements showed higher levels in the contralateral hippocampus in the low - intensity group compared to the control group . there were no differences in measurements of synapsin - i and creb in any of the groups . while the above studies suggest that early forced exercise can promote cognitive recovery , these results are not unchallenged . . found that animals exposed to lfpi and 18 days of treadmill exercise initiated the day following injury differed in neither spatial acquisition nor retention tasks in a water maze compared to a lesioned , nonexercised group . they found increased bdnf mrna levels in ca1 and ca3 in the exercised , lesioned animals compared to lesioned , sedentary animals . however , the left neocortex ( ipsilaterally to the injury ) was significantly smaller than the right neocortex in the nonexercised , lesioned animals compared to the exercised , lesioned animals . these results are in contrast to many of the above studies , as they fail to find cognitive effects of early initiated forced exercise , but do find bdnf and some histological effects of exercise . were echoed by song et al . , who used photothrombosis to induce cerebral stroke in rats . one day after injury the animals were swim - exercised in a circular pool for 4 weeks ( a total of 20 days ) , or given acetyl - l - carnitine ( alc ) injections , or both . they found no significant differences in any of their treatment groups compared to lesioned controls on acquisition of a spatial task in a water maze tested the first , second , and fourth week after injury . hippocampal sod - levels were increased in all treatment groups compared to the lesioned controls ; these were significantly higher in the combined ( exercise + alc - injection ) group in comparison to the other groups . mda - levels ( related to lipid peroxidation ) were reduced in the treatment groups compared to the controls . histologically , there were an increased number of cells in ca3 in the treatment groups compared to the controls . exercised gerbils on treadmills either 12 , 24 , 48 , or 72 hours after ccao for 1 up to 3 days . in an open field , animals exercised 12 hours after injury showed a decreased number of field crossings and an increase in grooming ( indicating increased anxiety and stereotyped behavior ) compared to a nonlesioned , nonexercised group . all ccao animals showed a reduced time spent on a rotarod compared with the nonlesioned , nonexercised group . there were a decreased number of cells in ca1 and striatum in the group exercised after 12 hours compared to the group exercised after 24 hours . this study indicates that very early initiated short - duration exercise ( 12 hours after injury ) can lead to increased anxiety - like behavior and cell death , while exercise starting 24 hours ( or later ) does not induce these emotional responses . exercise doses were decreased with later initiation points ( down to a single 15 min session ) , making it difficult to decipher starting point effects from dose - related effects in the exercised groups . . found that treadmill exercise for 10 days resulted in longer latencies ( i.e. , better short - term memory for a noxious stimulus ) in a step - down avoidance task than in a nonexercised , lesioned group . they also found reduced levels of tunel positive and caspase-3 positive cells ( markers for apoptosis ) in the lesioned , exercise group compared with the lesioned , nonexercised group . cell proliferation was increased in the nonexercised , lesioned group and the exercised , sham group , but not in the lesioned , exercise group . the authors hypothesized that this finding might be due to reduced cell death in the exercised group , reflected in less cell proliferation . in a later experiment , using the same injury model but a longer exercise regimen ( 4 weeks , starting on the first postinjury day ) , sim et al . found that the lesioned , exercised animals did better than the nonexercised lesioned group in the step - down avoidance task . the exercised , lesioned group presented with fewer tunel and caspase-3 positive cells than the nonexercised , lesioned group . the studies indicate that exercise might protect the brain from neuronal cell death , which could play a part in the functional recovery . interestingly , this finding goes for both a shorter and longer duration exercise paradigm at the same running speed . chen et al . exposed rats to hippocampal injury via unilateral kainic acid injection to the ca1 area . starting on the second postinjury day , the animals were exercised in a motorized running wheel for seven consecutive days at one of three different intensities : light , moderate , and heavy . the animals were then tested in a conditioning ( pain - avoidance ) learning task in a y - maze for one session of 20 trials . the study found that lesioned animals that had been exercised at moderate intensity performed significantly better in the learning task than nonexercised , lesioned animals , as well as showing significantly higher numbers of brdu positive cells . there were no learning or brdu staining differences between the other lesioned , exercised groups and the nonexercised , lesioned animals . furthermore , a positive correlation between learning and brdu positive labelled cells in the dentate gyrus was found , indicating that neurogenesis may have supported the functional recovery . positive recovery effects of second day postinjury initiation were also found by kim et al . . using electromagnetic contusion in rats followed by 10 days of treadmill exercise , they found that lesioned , exercised animals had shorter latency times in a step - down avoidance test than a lesioned , nonexercised group , indicating better ( short - term ) memory for a noxious stimulus in the exercised group . measurements of hippocampal dna fragmentation ( a marker for apoptosis ) , caspase-3 , and bax ( pro - apoptosis molecules ) showed reduced levels in the lesioned , exercised group compared to the lesioned , nonexercised group . levels of blcl2 ( antiapoptosis molecules ) were increased in the lesioned , exercised group compared to the control group . besides improvement in short - term memory , the study , like those by sim et al . [ 53 , 54 ] , shows effects on markers of apoptosis further supporting the assumption that enhanced functional recovery after exercise could be mediated by regulation of neuronal cell death mechanisms . chen et al . compared the timing of treadmill exercise initiated two days ( early , for either 7 or 14 days ) or nine days after injury ( late , for 7 days ) in a closed head injury mouse model . in an object recognition task , they found that the early initiated groups spent significantly more time exploring a new object compared to a lesioned , nonexercised group indicating better memory for the previously encountered object . the late initiated group and the nonexercised , lesioned group spent less time exploring the new object than the sham animals . furthermore , early exercise hindered progressive cell loss in the cortex and the hippocampus to a larger extent than in the late exercised group . early exercise boosted neurite regeneration in the early postinjury stages , but late exercise only hindered later stage cell loss . early exercise for 14 days restored the lesion - induced reduction in bdnf and mkp-1 ( an anti - inflammation marker ) . when animals were given triptolide ( a mkp-1 synthesis inhibitor ) neither this nor cognitive recovery was seen ; however , there were positive effects on neuronal loss and neuroinflammation . these findings show that different starting points can generate different outcomes in short - term memory , cell survival , and plasticity - related protein levels . starting exercise on the fourth day after injury , shimada et al . subjected rats to left mcao followed by one of two different treadmill intensities ( low or high ) for 28 days . in both an object recognition and an object location task , the low - intensity group spent more time exploring the novel object / newly placed object than the lesioned , nonexercised control group . the high - intensity group explored less than the low - intensity group . in a passive avoidance test , both exercise groups showed longer latencies than the controls , indicating that exercise resulted in better memory for noxious stimuli . both intensity groups had increased number of neurons in the dentate gyrus compared with the controls and the shams ; this was higher in the ipsilateral dentate gyrus in the low - intensity group versus the high - intensity group . in the ipsilateral dentate gyrus , map-2 levels ( microtubule - associated protein 2 ) were increased in the low - intensity group compared to the controls . map-2 was lower in the high - intensity group compared with the low - intensity group and the shams ipsilaterally in all examined hippocampal areas . contralaterally , the levels were lower in ca1 and ca3 in the high - intensity group than in the low - intensity group . [ 48 , 49 , 55 ] underlining the potential differential effects of varying exercise intensities in the early stages of recovery . as in the studies by shih et al . and , this study shows that low - intensity forced exercise is able to produce cognitive recovery effects after abi ; however , in this study there were also positive effects of high - intensity exercise on one of the cognitive parameters ( passive avoidance ) . two studies have begun forced exercise 1 week after abi or later . the previously mentioned study by luo et al . compared a forced exercise protocol ( swimming ) with a voluntary exercise protocol starting one week after mcao . ( see above ) did not find any effects of exercise starting 9 days after injury on cognitive parameters . this opens the question as to whether there exists a window of opportunity for rehabilitation via forced exercise that closes after a certain time point . compared to what appears to be the case regarding voluntary exercise , this window may be substantially smaller . the studies of forced exercise , like those of voluntary exercise , show a somewhat conflicting , pattern of outcome . though only one study shows detrimental effects ( in one group ) on anxiety - related behavior , the above studies generally show that especially early forced exercise can lead to improvement in animals exposed to low or moderate intensity exercise . one may therefore ask whether exercise needs to be maintained at a certain intensity level in order to produce cognitive gains . neither of the two studies using swimming exercise produced cognitive recovery effects . however , more studies are needed to determine whether this is a result of the type of exercise or protocol related issues . unfortunately , only two studies investigated effects of exercise starting later than a week , leaving us with limited knowledge about the effects of forced exercise initiated at a later stage . as already described , the above research varies in the time points of exercise initiation . of the 22 studies included in this overview , we find that 16 studies had experimental groups starting exercise from postinjury days 04 , while eight studies had experimental groups starting exercising at the earliest from postinjury day 7 . examining common traits or dissimilarities of the early intervention groups with positive or no effects does not render a clear picture . almost all early initiation studies with positive effects of exercise on cognition use forced exercise paradigms . however , as early initiation voluntary exercise studies are much fewer in number , this might be a paradigm bias . moreover , the studies vary on most parameters including types of injury and animal as well as exercise duration and intensity . the three studies showing groups with adverse effects [ 39 , 40 , 52 ] started exercising the animals immediately after injury , that is , within the first 24 hours . however , positive cognitive outcomes using very early exercise have also been reported ( see above ) . later initiation studies are fewer and with starting points spanning from 1 week to almost 4 months after injury ; it is difficult to obtain a coherent picture . studies with groups starting 7 to 9 days after injury [ 31 , 41 , 57 ] showed either positive and/or no effects , and a 14 day postinjury start showed cognitive improvement effects in two studies [ 39 , 42 ] . starting at even later time points showed some variability : starting 2530 days post - abi induced positive effects in three studies [ 31 , 43 , 44 ] , while an approximately 4-month postinjury start generated both an improvement and no effects depending on the cognitive measure . thus it would appear that later initiated exercise , in most cases , can promote cognitive recovery . it is quite surprising that we know relatively little about the cognitive effects of exercise starting relatively late post - tbi . in clinical rehabilitation settings exercise is often initiated in later recuperation stages , when patients are stabilized and able to perform physical activities . it therefore seems clinically relevant to further investigate the potential effects of late - initiation exercise . exercise dose encompasses many variables including total length of intervention ( how many days ) , session duration ( how many minutes ) , distribution ( how often ) , distance moved ( how far ) , and intensity ( how fast ) . in the 22 studies included in this review , the total length of intervention varied considerably , ranging from 1 day to almost 4 months . regarding individual session durations , most of the voluntary exercise studies gave the animals unlimited access to the running wheels , that is , 24 hour access . the forced exercise paradigm sessions lasted between 5 min and 1 hour ; eight of those studies used 30 min session durations . by and large , the animals exercised / or had access to exercise apparatuses on a daily basis throughout the intervention period except in two studies that distributed the intervention somewhat differently [ 47 , 51 ] , as well as one study that exercised animals twice daily . average group distances and/or intensities are not stated in all studies ( see table 1 ) . minute and often vary within individual exercise sessions or over days / weeks . in some cases , exercise duration ( number of minutes ) is increased over a period of days . while such graduation of intensity or duration of exercise might in itself be an important rehabilitative factor , the individual protocols vary too much for meaningful comparisons to be carried out . when calculating mean daily / session distances over the total duration of exercise , they range between 97.2 m and 8.4 km . such a wide variation is also found in the total exercise distances over time ( i.e. , total distance over all exercise days / sessions ) that range from 150 m to 313.2 km . four studies explicitly examined the cognitive effects of different exercise intensities [ 48 , 49 , 55 , 58 ] . interestingly , all of these studies found that the low or moderate exercise intensity groups produced positive results , while the higher intensity groups did not produce any results ( or only produced results in one test ) ( see above ) . this indicates that intensity is indeed an important factor when using exercise as a cognitive rehabilitation tool . while it would appear that average doses up to around 250 m daily in many cases produce positive results [ 31 , 48 , 49 , 53 , 54 , 56 , 58 ] , this is not always the case [ 31 , 52 , 55 ] . the picture becomes more blurred when using higher daily doses . in the studies specifically looking into exercise intensities , daily doses exceeding an average of 320 m daily in other cases [ 43 , 44 , 46 , 47 , 57 ] average session distances of 320 m and above improved cognitive recovery , but this was in some cases contingent upon other variables such as starting point . in some studies , doses exceeding 320 m daily did not produce any results on the spatial tasks [ 45 , 50 ] or had detrimental effects . study , the moderate exercise group ( that showed positive recovery effects ) ran 180 m twice daily , making the individual exercise trials fall below the 320 m mark ( but the total daily running distance was slightly above ) . however , their heavy intensity group ran 324 m twice daily ( to a total of 648 m ) and did not show recovery effects . one may therefore ask whether total running distances are a good dose measure , or whether the intensity of individual training trials are of more importance for cognitive recovery . although an unresolved matter , this could be another explanatory factor for the differential results in studies examining voluntary running effects , where intensity and duration of individual running bouts are not experimentally controlled . all in all , the substantial variations in exercise protocols among the studies make it difficult to make general dose recommendations . while it does appear that dose , duration , and intensity are important factors for cognitive recovery , more systematic research looking into these aspects and how they interact with other variables such as starting point is needed to elucidate this further . while many neural mechanisms behind the effects of exercise are being investigated , special attention has been given to neurotrophic factors , in particular bdnf . bdnf is highly expressed in the cortex and hippocampus and is involved in many neural processes including neuronal differentiation and survival , as well as axonal path - finding . furthermore , the relationship between forced exercise and stress - hormone levels has garnered considerable interest . in the following these topics will be investigated further in relation to exercise type , timing , and intensity . in relation to exercise type , a special focus has been placed on the connection between exercise and the release of stress - hormones , as forced exercise is believed to be more stressful than voluntary exercise . . found that early stage postinjury forced exercise elevated corticosterone and acth levels in lfpi animals . neither exercise regimens elevated bdnf - levels . in another experiment starting exercise at a later stage , griesbach et al . found that forced exercise stimulated the corticotrophic axis in all animals . bdnf - levels were unaffected by forced exercise , yet they were elevated in all rats exposed to voluntary exercise . in two other studies [ 42 , 62 ] the same lab also found an increase in bdnf - levels as a result of voluntary exercise . similarly , ke et al . found that voluntary exercise improved motor function and elevated bdnf - levels , an effect not seen in the group exposed to forced exercise , although these animals did present higher levels of corticosterone . wong - goodrich et al . did not see any exercise - related bdnf - changes in their late voluntary paradigm ; however , they did find that the intervention improved cognition in their irradiated animals . several studies using forced exercise after tbi have found bdnf - elevations [ 21 , 57 , 6365 ] , indicating that forced exercise paradigms can increase bdnf - levels after injury . using both forced and voluntary exercise , ploughman et al . found that corticosterone levels were elevated in all exercise groups but were highest in animals exposed to forced exercise running at greater speed or duration . exercise did not increase bdnf , igf-1 , or synapsin - i in the ischemic hemisphere . furthermore , they found that voluntary exercise decreased serum levels of igf-1 and increased hippocampal levels of igf-1 in the ischemic hemisphere . ( see above ) , no differences in stress - hormone levels were found between the treadmill exercised and nonexercised groups . found that forced exercise created a rapid , but more short - lived bdnf - increase compared to voluntary exercise . the group exposed to forced exercise also showed increased levels of corticosterone in several brain regions . thus , it appears that forced exercise does lead to elevated stress - hormone levels . when it comes to impact on bdnf - levels , the picture is more unclear . it seems that the type of exercise ( voluntary or forced ) can not solely account for variation in neurotrophic factor levels , but other factors such as timing and intensity are also key players . how stress - hormones and neuroplasticity - related proteins are affected by exercise , how they interact , and , importantly , what consequences this has for functional recovery remain to be resolved . as discussed above , though the efficacy of forced exercise on cognitive parameters is inconclusive , this poses the question of whether elevations in stress - hormones during physical activity are necessarily harmful when it comes to the recovery of cognitive functions . like exercise type , some research indicates that starting point affects bdnf - levels after abi . early exercise initiation ( defined here from day 06 postinjury ) has been shown to elevate bdnf - levels in several studies [ 21 , 50 , 57 , 62 , 63 , 65 ] ; in some cases this elevation is also dependent upon exercise intensity [ 48 , 49 ] ( see below ) or type of exercise [ 20 , 67 ] ( see above ) . in other cases , early exercise did not affect bdnf - levels [ 38 , 39 , 60 ] . later post - abi exercise ( defined here from postinjury day 7 and onwards ) has also been shown to produce bdnf - elevations [ 31 , 39 , 42 ] , in some cases this is dependent on type of exercise or injury severity . a few studies have found that later initiated exercise did not produce bdnf - elevations [ 31 , 43 , 57 ] . these studies indicate that both early and later initiated exercise regimens can increase bdnf - levels in some cases . whether such bdnf - elevations are part of the neural processes mediating cognitive recovery is still unclear . did not find bdnf - elevations after early initiated exercise and this group also showed delayed learning . wu et al . found no bdnf - effects either but did see improvements in their cognitive measure after early initiated exercise . reversely , hicks et al . found bdnf - elevations after early initiation , but no cognitive effects . chen et al . found elevated bdnf - levels ( in their early initiated group running for 14 days ) as well as a cognitive improvement . also found both bdnf - elevations and cognitive improvements ( in their low - intensity running groups ) . initiating exercise at later points , griesbach et al . wong - goodrich et al . found no exercise - related bdnf - level changes in their irradiated animals ; they did , however , find a cognitive improvement . it seems that the relationship between bdnf - responses and cognitive recovery outcome at different exercise initiation points is still largely unresolved . currently , there are a limited number of studies investigating this , underlining a need for additional research . not many studies have investigated the relationship between bdnf - levels and exercise intensity in post - abi exercise . found hippocampal bdnf - elevations ( contralaterally ) in their low - intensity exercise groups concomitant with cognitive improvement . in a study by ploughman et al . , rats were subjected to focal stroke using endothelin - i . after 4 days of recovery , the animals were given either a 30 min or a 60 min walk in a motorized running wheel ( both 11 m / min ) , a 30 min run in a motorized running wheel ( 14 m / min ) , or a 12-hour voluntary run in a ( nonmotorized ) running wheel . the animals in the 30 min motorized walking group and the voluntary running group had increased hippocampal bdnf - levels ( in the noninjured hemisphere ) compared to noninjured , nonexercised animals . furthermore , the 30 min walking group showed increased bdnf - levels in the intact sensorimotor cortex compared to the 60 min walking group and nonexercised animals . placed together , these studies indicate that exercise of a lower intensity can increase bdnf - levels in areas contralaterally to the inflicted injury . however , intensity and duration of intervention ( and thereby total distance run ) vary between the studies , restricting what overall information can be derived regarding the relationship between bdnf and post - abi intensity parameters . the above studies provide some information as to the effects of exercise on cognition in the brain injured individual . they also stress some of the parameters that are important for the efficiency of this intervention . however , there are still many unresolved issues . voluntary and forced exercise paradigms vary on parameters of choice of movement and , potentially , level of stress - hormone activation . the studies included in this review also reveal other differences between the two exercise paradigms . most of the voluntary paradigms allow animals access to the exercise apparatus in their home environment , while the forced paradigms require moving and handling of the animals to initiate ( and sometimes prompt ) exercise . whether such environmental differences can affect the outcome in terms of cognitive recovery will have to be clarified in the future . in all but two of the studies using voluntary exercise , most of the forced exercise studies do not report housing conditions ; however those that do have animals pair or group housed . . found that both single and group housed male rats had corticosterone elevations due to running . when exposing these animals to additional stressors , the socially isolated animals showed decreased neurogenesis compared to the controls . in another study using female rats , leasure and decker found that social isolation suppressed the cell - proliferation effects of exercise that were seen in group housed animals . furthermore , there was a correlation between brdu+ cells and the running distance in the group housed animals , but not in the single housed animals . in a study looking into the emotional effects of housing , berry et al . found that single housing triggered anxiety and depression - like behaviors in the animals , increased hpa - axis reactivity , and reduced bdnf - levels . such findings indicate that housing - paradigms ( and animal gender ) can influence the effects of exercise as well as emotional reactivity . whether single housing would also influence cognitive performance in animals subjected to abi it is therefore relevant to investigate whether cognitive effects in exercise studies using single housing are related to the exercise intervention per se , boredom - factors due to isolation , or other variables . . animals in the voluntary paradigms have 24-hour access to the exercise apparatuses ( except in one study ) and can administer their treatment when they choose and in the intensity and duration that they prefer . this is a marked difference from forced exercise paradigms that mainly offer single exercise bouts of limited duration ( up to 1 hour ) under controlled running speeds . these differences underline that paradigms of voluntary and forced exercise vary on many variables that can affect the cognitive ( and neural ) outcome . epidemiological research shows that premenopausal women have decreased risk of stroke compared to age - matched males as well as to postmenopausal women . animal studies have shown that female hormones regulate and protect against a variety of pathological processes associated with stroke . both estrogen and progesterone have been shown to have neuroprotective effects after stroke in animal models [ 73 , 74 ] . this indicates that gender - specific hormonal environments can influence the recovery outcome after brain injury . however , all but two of the studies discussed in this review use male animals ( see table 1 ) , leaving us with very limited data about the effects of exercise in the traumatized female brain . the type and severity of injuries in the above studies are quite different . some types of injury cause more focal tissue damage ; others are more wide - spread and diffuse in nature . the studies using traumatic brain injury models ( i.e. , an external force afflicting the brain ) use different techniques , impact velocities , and depths of compression . the models inducing injury by irradiation use different doses and afflict different cerebral areas . as different types of brain injury and injury severities can cause different injury patterns , both in terms of tissue responses as well as their spatial and temporal occurrence , this can also affect the efficacy of the employed exercise protocols . much research has shown that the same brain injury method can induce significantly different cerebral ( and behavioral ) responses depending on the rodent strain / stock used [ 7690 ] . even animals of the same stock , but purchased from different breeders , have been shown to differ in their cerebral responses when exposed to the same injury [ 9193 ] . thus , strain / stock choice is an important factor to take into account when assessing brain injury outcomes as well as the efficacy of treatment interventions . in the 22 studies included in this review , however , due to the considerable procedural differences in performing the same brain injury ( see above ) as well as substantial interstudy variations in the exercise protocols and outcome measures , meaningful comparisons of the studies on the basis of strain are very difficult to make . further research is needed to elucidate the effects of strain on post - abi exercise on cognitive recovery . the applied cognitive tests are generally brief , limiting our knowledge to mainly short - term learning effects . potential long - term effects of exercise have not been examined in any of the studies , leaving us with little knowledge as to whether the observed cognitive effects are lasting or transient . furthermore , the majority of studies use tests that motivate learning through avoidance , that is , the ability to avoid an unwanted stimulus ( escaping water or a previously presented painful stimulus ) . testing animals in nonavoidance based tasks would help to clarify whether the outcome is related to the treatment or the method of testing . another discussion related to the cognitive tests pertains to the individual test protocols and setups . while many of the studies used spatial acquisition tasks in a water maze , the individual testing protocols were very varied , in terms of both number of acquisition trials and sessions . such differences could potentially affect the learning outcome if some animals were to be trained more intensively than others . furthermore , the visual surroundings when performing spatial acquisition tasks ( i.e. , the number and salience of visual cues as well as their distance to the animals ) have been shown to be of importance for both the neural substrate and cognitive mechanisms of task solution in rodents [ 9597 ] . it is generally taken for granted that different cognitive tasks reflect different neural substrates and cognitive mechanisms . however , within what is generally considered the same cognitive tasks , various experimental and/or test setups can also vary with respect to the underlying neural and cognitive mechanisms [ 98 , 99 ] . consequently , what may superficially appear to be the same cognitive test may result in different cognitive recovery effects of a given exercise protocol ; even minor variations in experimental setups can be essential . thus , it appears that research in this area would benefit greatly from more homogenous use of cognitive tests / setups to facilitate comparisons between labs and help eliminate test protocol differences as a source of variation when assessing the effects of exercise on cognition . as already mentioned , exercise is often used in the treatment of depression and anxiety - related disorders . however , whether these impairments are primarily psychosocially or neurobiologically founded , transient or enduring , is still debated . though some of the above studies have included tests of emotional behavior in their experimental protocols , we still know very little about how post - tbi exercise affects emotional states , and how this potentially affects cognitive performance . knowing more about the relationship between injury - related emotional and cognitive problems will help to further clarify when ( and in what way ) exercise promotes cognitive recovery after abi . in this review we have examined the effects of exercise on cognitive measures after acquired brain injury in animal models . although there is cause for optimism in using exercise as a rehabilitation tool in the treatment of cognitive sequelae after abi , research in this area is still fairly limited . however , what distinguishes these cases from others that do not produce effects ( or have adverse effects ) remains unclear . considerable variations in models and experimental protocols , including differences in animal strains , injury type , exercise type , post - injury starting point , dose - related differences , and cognitive measures , should presently warrant caution in making general protocol recommendations . more research is needed to clarify these issues as well as the potential long - term effects of postinjury exercise .

the objective of the present paper is to review the current status of exercise as a tool to promote cognitive rehabilitation after acquired brain injury ( abi ) in animal model - based research . searches were conducted on the pubmed , scopus , and psycinfo databases in february 2014 . search strings used were : exercise ( and ) animal model ( or ) rodent ( or ) rat ( and ) traumatic brain injury ( or ) cerebral ischemia ( or ) brain irradiation . studies were selected if they were ( 1 ) in english , ( 2 ) used adult animals subjected to acquired brain injury , ( 3 ) used exercise as an intervention tool after inflicted injury , ( 4 ) used exercise paradigms demanding movement of all extremities , ( 5 ) had exercise intervention effects that could be distinguished from other potential intervention effects , and ( 6 ) contained at least one measure of cognitive and/or emotional function . out of 2308 hits , 22 publications fulfilled the criteria . the studies were examined relative to cognitive effects associated with three themes : exercise type ( forced or voluntary ) , timing of exercise ( early or late ) , and dose - related factors ( intensity , duration , etc . ) . the studies indicate that exercise in many cases can promote cognitive recovery after brain injury . however , the optimal parameters to ensure cognitive rehabilitation efficacy still elude us , due to considerable methodological variations between studies .

1. Introduction 2. Inclusion Criteria 3. Voluntary or Forced? 4. Sooner or Later? 5. Easy Does It? 6. Post-ABI Exercise and Brain-Derived Neurotrophic Factor (BDNF) 7. General Considerations 8. Conclusion

PMC5119809

twenty - nine participants ( 16 females and 13 males ) aged 67.70 6.07 participated in this study . participants were recruited from various pa programs that focused on older adults ( ages 60 + ) in the community . height , weight , and waist circumference were assessed in light clothing and without shoes to the nearest 0.5 cm and 0.1 kg , respectively . each participant completed a physical activity readiness questionnaire ( par - q ) . a positive response to one or more of the seven par - q questions excluded a participant from the study . the par - q is widely used as a screening tool for all pa participation . the par - q is used in canada and has been adopted widely throughout the united states ( adams , 1999 ; thomas , reading , & shephard , 1992 ) . prior to participation , all participants had the research study and its potential risks and benefits explained fully before providing written informed consent . = standard deviation ; cm = centimeters ; kg = kilograms ; bmi = body mass index ; kgm = kilogram per meters squared ; ms = meters per second . the dual mode actical accelerometer ( respironics inc . , bend , oregon ) , yamax sw-200 ( yamax corporation , tokyo , japan ) , omron hj-112 ( omron healthcare , vernon hills , illinois ) , and walk4life elite ( walk4life , inc , plainfield , illinois ) were used to estimate steps registered by participants . the yamax sw-200 is a spring - lever pedometer that includes a step counter , which accurately counts steps while walking , hiking , jogging , or running . a more detailed description of the yamax sw-200 and its functions have been provided in previous research ( crouter , schneider , & bassett , 2005 ) . the yamax sw-200 pedometer has also been the pedometer of choice when examining step count accuracy in older adults who either reside in assisted - living facilities or are community - dwelling ( bergman et al . , 2008 ; cyarto et al . , 2004 ; grant et al . , 2008 ; storti et al . , the walk4life elite is a spring - lever pedometer that includes a step counter , activity time , and distance displayed in miles walked . the omron hj-112 is a piezoelectric pedometer that records number of steps during walking and jogging activities . a more detailed description of the omron hj-112 and its functions have been described in previous research ( hasson , haller , pober , staudenmayer , & freedson , 2009 ) . very limited research is available on the walk4life elite and omron hj-112 step count accuracy during walking at low treadmill speeds and no research is available involving community - dwelling older adults . all pedometers used in this study were checked for calibration using a shake test ( vincent & sidman , 2003 ) . these pedometers have been some of the more commonly used in recent research ( abel et al . , 2011 ; bassett et al . , 2008 ; feito , bassett , & thompson , 2012 ; hasson et al . , 2009 ) . bend , oregon ) is a dual mode accelerometer ( registers accelerometer counts and step counts ) that uses a piezoelectric accelerometer mechanism . a more detailed description of the actical and its functions has been described in previous research ( esliger et al . the few studies that are available on the dual mode actical step count function ( johnson , meltz , hart , schmudlach , clarkson , & broman , 2014 ; esliger et al . 2007 ; feito , bassett , & thompson , 2012 ; feito , bassett , thompson , & tyo , 2012 ) have not included older adults ( age 60 ) . the actical accelerometers used in this study were checked using manufacturer - recommended hardware and software , and calibrated if necessary . at the end of each test , accelerometer data were downloaded using manufacturer - recommended hardware and software . the actical step counts and three pedometer step counts were recorded while walking on an indoor track and walking on a motor - driven treadmill at various speeds . directly observed step counts ( recorded with a hand - tally device ) served as the criterion . the testing took place over the course of 6.69 3.4 days , and each person wore the same pair of shoes for all trials . all devices were worn concurrently on an elastic belt on their right waist / hip , following the manufacturer s recommendations for placement on the body , during testing . participants were first asked to complete a 200-m walk around an indoor track ( field setting ) . participants walked at a self - selected pace while two investigators using hand - tally counters walked behind the participant , to avoid influencing pace . the amount of time it took to complete the 200-m walk was measured to calculate each participant s self - selected pace ( schneider , crouter , lukajic , & bassett , 2003 ; storti , pettee , brach , talkowski , richardson , & kriska , 2007 ) . at the end of the 200-m walk , the participants were told to stand still , and the number of steps detected by each pedometer was then recorded . participants then walked on a treadmill at three different speeds : 0.67 ms , 0.894ms , and 1.12 ms for 5 min at each speed with treadmill gradient set at 0 . these treadmill speeds have been used previously to examine activity monitor accuracy in community - dwelling older adults ( grant et al . , 2008 ) and healthy adults ( crouter , schneider , karabulut , & bassett , 2003 ; feito , bassett , & thompson , 2012 ; le masurier , lee , & tudor - locke , 2004 ) . the participants received instructions for walking on the treadmill and were allowed time to adapt to the various walking speeds . before each bout , participants stood still ( straddling the treadmill belt ) for a 2-min washout period . this was performed to ensure that any steps detected by the accelerometer before the official bout were not considered in the analysis ( le masurier & tudor - locke , 2003 ) . the 2-min washout period was repeated between each bout and after the last one . at the end of each bout , the number of steps detected by each pedometer was recorded and the units were reset to zero before the subsequent bouts . observed steps were counted by two researchers , using hand - tally counters , with the average of the two being recorded if counted steps differed . one - way within - participants repeated measure analyses of variance ( anovas ) were used to assess significant differences between actual steps taken and estimated step counts registered by the activity monitors for all four conditions ( self - selected pace walking and three treadmill walking speeds ) . post hoc analyses for the anova procedures were performed if significance were found using pairwise comparisons with bonferroni adjustments . the one - way anova for treadmill walking speed at 1.12 ms only used 28 participants data due to 1 participant unable to walk at this speed . mean absolute percent error ( mape ) was calculated between observed steps and actical / pedometer - determined steps mape = ( [ actical / pedometer steps observed steps ) / observed steps ] 100and was used as another outcome measure . a smaller mape represents better accuracy , and less than 3% is considered acceptable pedometer accuracy ( bassett et al . , 2008 ) . altman plots were also used to demonstrate level of agreement between criterion measures and estimated step counts registered by the activity monitors during self - selected pace walking and walking at three different treadmill speeds ( bland & altman , 1986 ) . statistical analyses were performed using spss 18.0 ( statistical package for the social sciences , inc , chicago , illinois ) . for all analyses , a p value < .05 was used to indicate statistical significance . means and standard deviations were reported for descriptive data . twenty - nine participants ( 16 females and 13 males ) aged 67.70 6.07 participated in this study . participants were recruited from various pa programs that focused on older adults ( ages 60 + ) in the community . height , weight , and waist circumference were assessed in light clothing and without shoes to the nearest 0.5 cm and 0.1 kg , respectively . each participant completed a physical activity readiness questionnaire ( par - q ) . a positive response to one or more of the seven par - q questions excluded a participant from the study . the par - q is widely used as a screening tool for all pa participation . the par - q is used in canada and has been adopted widely throughout the united states ( adams , 1999 ; thomas , reading , & shephard , 1992 ) . prior to participation , all participants had the research study and its potential risks and benefits explained fully before providing written informed consent . = standard deviation ; cm = centimeters ; kg = kilograms ; bmi = body mass index ; kgm = kilogram per meters squared ; ms = meters per second . bend , oregon ) , yamax sw-200 ( yamax corporation , tokyo , japan ) , omron hj-112 ( omron healthcare , vernon hills , illinois ) , and walk4life elite ( walk4life , inc , plainfield , illinois ) were used to estimate steps registered by participants . the yamax sw-200 is a spring - lever pedometer that includes a step counter , which accurately counts steps while walking , hiking , jogging , or running . a more detailed description of the yamax sw-200 and its functions have been provided in previous research ( crouter , schneider , & bassett , 2005 ) . the yamax sw-200 pedometer has also been the pedometer of choice when examining step count accuracy in older adults who either reside in assisted - living facilities or are community - dwelling ( bergman et al . , 2008 ; cyarto et al . , 2004 ; grant et al . , 2008 ; storti et al . , the walk4life elite is a spring - lever pedometer that includes a step counter , activity time , and distance displayed in miles walked . the omron hj-112 is a piezoelectric pedometer that records number of steps during walking and jogging activities . a more detailed description of the omron hj-112 and its functions have been described in previous research ( hasson , haller , pober , staudenmayer , & freedson , 2009 ) . very limited research is available on the walk4life elite and omron hj-112 step count accuracy during walking at low treadmill speeds and no research is available involving community - dwelling older adults . all pedometers used in this study were checked for calibration using a shake test ( vincent & sidman , 2003 ) . these pedometers have been some of the more commonly used in recent research ( abel et al . , 2011 ; bassett et al . , 2008 ; feito , bassett , & thompson , 2012 ; hasson et al . , 2009 ) . bend , oregon ) is a dual mode accelerometer ( registers accelerometer counts and step counts ) that uses a piezoelectric accelerometer mechanism . a more detailed description of the actical and its functions has been described in previous research ( esliger et al . , 2007 ; heil , 2006 ) . the few studies that are available on the dual mode actical step count function ( johnson , meltz , hart , schmudlach , clarkson , & broman , 2014 ; esliger et al . 2007 ; feito , bassett , & thompson , 2012 ; feito , bassett , thompson , & tyo , 2012 ) have not included older adults ( age 60 ) . the actical accelerometers used in this study were checked using manufacturer - recommended hardware and software , and calibrated if necessary . at the end of each test the actical step counts and three pedometer step counts were recorded while walking on an indoor track and walking on a motor - driven treadmill at various speeds . directly observed step counts ( recorded with a hand - tally device ) served as the criterion . the testing took place over the course of 6.69 3.4 days , and each person wore the same pair of shoes for all trials . all devices were worn concurrently on an elastic belt on their right waist / hip , following the manufacturer s recommendations for placement on the body , during testing . participants were first asked to complete a 200-m walk around an indoor track ( field setting ) . participants walked at a self - selected pace while two investigators using hand - tally counters walked behind the participant , to avoid influencing pace . the amount of time it took to complete the 200-m walk was measured to calculate each participant s self - selected pace ( schneider , crouter , lukajic , & bassett , 2003 ; storti , pettee , brach , talkowski , richardson , & kriska , 2007 ) . at the end of the 200-m walk , the participants were told to stand still , and the number of steps detected by each pedometer was then recorded . participants then walked on a treadmill at three different speeds : 0.67 ms , 0.894ms , and 1.12 ms for 5 min at each speed with treadmill gradient set at 0 . these treadmill speeds have been used previously to examine activity monitor accuracy in community - dwelling older adults ( grant et al . , 2008 ) and healthy adults ( crouter , schneider , karabulut , & bassett , 2003 ; feito , bassett , & thompson , 2012 ; le masurier , lee , & tudor - locke , 2004 ) . the participants received instructions for walking on the treadmill and were allowed time to adapt to the various walking speeds . before each bout , participants stood still ( straddling the treadmill belt ) for a 2-min washout period . this was performed to ensure that any steps detected by the accelerometer before the official bout were not considered in the analysis ( le masurier & tudor - locke , 2003 ) . the 2-min washout period was repeated between each bout and after the last one . at the end of each bout , the number of steps detected by each pedometer was recorded and the units were reset to zero before the subsequent bouts . observed steps were counted by two researchers , using hand - tally counters , with the average of the two being recorded if counted steps differed . one - way within - participants repeated measure analyses of variance ( anovas ) were used to assess significant differences between actual steps taken and estimated step counts registered by the activity monitors for all four conditions ( self - selected pace walking and three treadmill walking speeds ) . post hoc analyses for the anova procedures were performed if significance were found using pairwise comparisons with bonferroni adjustments . the one - way anova for treadmill walking speed at 1.12 ms only used 28 participants data due to 1 participant unable to walk at this speed . mean absolute percent error ( mape ) was calculated between observed steps and actical / pedometer - determined steps mape = ( [ actical / pedometer steps observed steps ) / observed steps ] 100and was used as another outcome measure . a smaller mape represents better accuracy , and less than 3% is considered acceptable pedometer accuracy ( bassett et al . , 2008 ) . altman plots were also used to demonstrate level of agreement between criterion measures and estimated step counts registered by the activity monitors during self - selected pace walking and walking at three different treadmill speeds ( bland & altman , 1986 ) . statistical analyses were performed using spss 18.0 ( statistical package for the social sciences , inc , chicago , illinois ) . for all analyses , a p value < .05 was used to indicate statistical significance . means and standard deviations were reported for descriptive data . there was no statistical difference between any of the activity monitors step counts and actual steps taken during self - selected pace walking , wilks s = 0.76 , f(4 , 25 ) = 2 , p = .13 , and all mape values were acceptable ( 2.62% ) . during treadmill walking at 0.67 ms , all the activity monitors step counts were significantly different from actual steps taken , wilks s = 0.16 , f(4 , 25 ) = 33.58 , p < .001 . post hoc analysis revealed that the three pedometers and accelerometer step counts were significantly different ( all p < .001 ) and all mape were unacceptable ( 38.06% ) . during treadmill walking at 0.894 ms , activity monitors step counts were significantly different from actual steps taken , wilks s = 0.27 , f(4 , 25 ) = 16.55 , p < .001 . post hoc analysis revealed that the omron hj-112 pedometer step counts were the only ones not statistically different from actual steps taken ( p = .16 ) , and the mape was 3.5% . the yamax sw-200 and walk4life pedometers step counts were both significantly different at p < .001 , while the actical accelerometer step counts were significantly different at p = .002 . during treadmill walking at 1.12 ms speed , activity monitors step counts were significantly different from actual steps taken , wilks s = 0.42 , ( 4 , 24 ) = 8.24 , p < .001 . post hoc analysis revealed that the yamax sw-200 step counts were significantly different from actual steps taken ( p = .012 ) and had an unacceptable mape value = 11.48% . all other devices step counts were not significantly different from actual steps taken and had acceptable mape values < 2.58% . post hoc power estimates = 1 for anovas examining differences between actual steps taken and steps registered by activity monitors at treadmill walking speeds 0.671 ms and 0.894 ms . post hoc power estimates were .99 and .92 for anovas examining differences between actual steps taken and steps registered by activity monitors at treadmill walking speed 1.12 ms and self - selected pace walking , respectively . table 2 presents the mape values for all activity monitors during self - selected pace and treadmill walking . altman plots for each activity monitors step count results for self - selected pace and treadmill walking . table 3 presents the estimated step counts registered for all activity monitors during self - selected pace and treadmill walking . mean absolute percentage error ( mape ) in self - selected pace and treadmill walking . note . om = omron hj-112 model ; ym = yamax sw-200 model ; w4l = walk4life elite model ; ms = meters per second on treadmill . altman plots depicting error scores ( actual steps estimated step ) for the yamax sw-200 during ( a ) self - selected pace , ( b ) treadmill walking at 0.67 ms , ( c ) treadmill walking at 0.894 ms , and ( d ) treadmill walking at 1.12 ms . note . altman plots depicting error scores ( actual steps estimated step ) for the walk4life elite during ( a ) self - selected pace , ( b ) treadmill walking at 0.67 ms , ( c ) treadmill walking at 0.894 ms , and ( d ) treadmill walking at 1.12 ms . note . altman plots depicting error scores ( actual steps estimated step ) for the omron hj-112 during ( a ) self - selected pace , ( b ) treadmill walking at 0.67 ms , ( c ) treadmill walking at 0.894 ms , and ( d ) treadmill walking at 1.12 ms . note . altman plots depicting error scores ( actual steps estimated step ) for the actical during ( a ) self - selected pace , ( b ) treadmill walking at 0.67 ms , ( c ) treadmill walking at 0.894 ms , and ( d ) treadmill walking at 1.12 ms . note . step counts registered by four different devices , for overground walking at a self - selected pace ( ssp ) and treadmill walking at three speeds . note . om = omron hj-112 model ; ym = yamax sw-200 model ; w4l = walk4life elite model ; = standard deviation ; ms = meters per second on treadmill . the purpose of this study was to examine the step count function of pedometers ( spring - lever vs. piezoelectric ) and an accelerometer in a group of community - dwelling older adults during self - selected pace and treadmill walking in comparison with actual steps taken . all activity monitors performed exceptionally well for walking around an indoor track at self - selected pace . there was no statistical difference between the activity monitors step counts and actual steps taken during self - selected pace walking and all mape values were < 3% . ( 2008 ) for the yamax sw-200 who reported < 2% mape values in a group of community - dwelling older adults ( mage = 72 ) with similar self - selected pace values during an outdoor walking protocol . conversely , cyarto et al . ( 2004 ) reported higher mape values for the yamax in a group of community - dwelling older adults ( mage = 71 ) walking a 13-m course with self - selected pace values ranging from 0.95 ms to 1.61 ms . ( 2008 ) also reported higher mape values for the yamax sw-200 in a group of community - dwelling older adults ( mage = 79 ) during a 100-step walking test . the reported self - selected pace values ranged from < 0.80 ms to > 1.0 ms , which were slower than the values in the current study . 2008 ) reported a low relationship between the yamaxsw-200 and actual steps taken over a 161-m walking trial by older adults ( mage = 77 ) living in assisted - living facilities . the reported average self - selected pace of participants in their study was much slower than the participants in the current study . this supports the notion that older adults who reside in an assisted - living facility walk at a slower pace than community - dwelling older adults . cyarto et al . also demonstrated that community - dwelling older adults walk at a faster pace when compared with older adults residing in a nursing home . ( 2004 ) also reported acceptable mape values for the walk4life elite pedometer for self - selected pace walking in adults . ( 2011 ) found that the omron hj-112 did not have superior step counting accuracy at self - selecting walking speed in adults . the actical accelerometer self - selected walking results from this study were similar to those of authors ( 2014 ) who reported no differences between actual steps and actical - recorded steps while walking around a track in young adults . previous studies that have examined the actical step count function in children and adults in a controlled setting ( treadmill walking ) have shown it to perform exceptionally well at walking speeds > 1.12 ms and 2.19 ms ( authors , 2014 ; colley et al . , 2013 ; esliger et al . 2007 ; feito , bassett , & thompson , 2012 ; feito et al . , 2012 ) . our results suggest that this is also true for older adults in a free - living setting as well . the actical accelerometer , walk4life elite , and omron hj-112 pedometers performed well for walking on a motorized treadmill at 1.12 ms speed , and mape values were < 3% . the actical treadmill walking at 1.12 ms results from this study are supported by feito , bassett , and thompson ( 2012 ) and feito et al . ( 2012 ) , who reported no differences between actual steps and actical - recorded steps while walking on a treadmill at 1.12 ms in normal and overweight adults . previous research ( crouter et al . , 2003 ; hasson et al . , 2009 ; melanson et al . , 2004 ) has shown the walk4life elite and omron hj-112 pedometers to accurately detect steps at this treadmill speed in adults . 2011 ) reported that the omron hj-112 did not accurately detect steps at this treadmill speed in adults . our results support previous findings regarding the yamax sw-200 susceptibility in registering steps while walking at this treadmill speed in adults and older adults ( colley et al . , 2013 ; feito , bassett , & thompson , 2012 ; grant et al . , 2008 ; horvath , taylor , marsh , & kriellaars , 2007 ; melanson et al . , 2004 ) . the actical step count function did not perform well while walking on a motorized treadmill at 0.67 ms and 0.894 ms speeds and had unacceptable mape values . ( 2012 ) , who reported a significant difference between actual steps and actical - recorded steps while walking on a treadmill at 0.67 ms in adults . previous studies have demonstrated the actical step count function susceptibility to walking at similar slow speeds on a treadmill for children and adults . it appears that regardless of age group , the actical step count function may be susceptible to registering steps at slow treadmill walking speeds 1.05 ms ( authors , 2014 ; colley et al . these walking speeds may be indicative of older adults who reside in assisted - living facilities or nursing homes ( bergman et al . , 2008 ; cyarto et al . , all pedometer - registered steps were significantly different from actual steps during treadmill walking at 0.67 ms and had unacceptable mape values . ( 2008 ) , who reported a significant difference for the yamax sw-200 pedometer in older adults at the same treadmill walking speeds . this relationship has also been previously shown in the adult population as well for both the yamax sw-200 and walk4life elite pedometers ( colley et al . , 2013 ; crouter et al . , 2003 ; feito , bassett , & thompson , 2012 ; melanson et al . , 2004 ) . during treadmill walking at 0.894 ms , the omron hj-112 pedometer was the only activity monitor with registered steps not significantly different from actual steps and an mape value slightly above acceptability ( 3.5% ) . this is the first study to demonstrate this relationship in older adults , and it is supported by authors ( 2014 ) who reported a similar relationship in young adults at this treadmill walking speed . these results support previous research that indicated that piezoelectric pedometers are more accurate in detecting steps at slower walking speeds in normal , overweight , and obese adults when compared with spring - lever pedometers ( crouter et al . , 2005 ; melanson et al . , 2004 this walking speed was indicative of some older adults , who resided either in assisted - living facilities or nursing homes ( bergman et al . the yamax sw-200 and walk4life elite pedometer - registered steps were significantly different from observed steps during treadmill walking at 0.894 ms . results are inconclusive for the walk4life elite pedometer at this treadmill speed in adults ( crouter et al . , 2003 ; melanson et al . , 2004 ) and the yamax sw-200 has shown similar susceptibility in both the older adult and adult populations ( authors , 2014 ; colley et al . , 2013 ; feito , bassett , & thompson , 2012 ; grant et al . , 2008 ; horvath et al . to gain a better understanding of the relationship between pa and health for the aging population , epidemiologists , exercise scientists , clinicians , and behavioral researchers must rely on objective measures of pa with supportive evidence specific to that population of interest . all activity monitors provided valid estimates of step counts during self - selected pace walking . the walk4life elite ( spring - lever ) , omron hj-112 ( piezoelectric ) , and actical accelerometer provided valid estimates of step counts walking at a constant speed of 1.12 ms . the omron hj-112 pedometer was the only monitor that provided valid estimates of step counts walking at a constant speed of 0.894 ms . it is recommended that pedometer and accelerometer be used in tandem ( if feasible ) to capture ambulatory behavior in community - dwelling older adults walking at this pace if the focus is on capturing steps , intensity , duration , and frequency of pa ( authors , 2014 ; hooker et al . , none of the activity monitors provided valid estimates of step counts walking at a constant speed of 0.67 ms , which may be indicative of older adults residing in an assisted - living facility . it is recommended that a different device be used to capture ambulatory behavior in this group such as the step watch 3 ( bergman et al . , 2008 ) . this study provides some preliminary evidence of validity for the actical accelerometer , walk4life elite , and omron hj-112 pedometers step count function in community - dwelling older adults where studies have been distinctly lacking .

objective : to examine the step count accuracy of activity monitors in community - dwelling older adults . method : twenty - nine participants aged 67.70 6.07 participated . three pedometers and the actical accelerometer step count functions were compared with actual steps taken during a 200-m walk around an indoor track and during treadmill walking at three different speeds . results : there was no statistical difference between activity monitors step counts and actual steps during self - selected pace walking . during treadmill walking at 0.67 ms1 , all activity monitors step counts were significantly different from actual steps . during treadmill walking at 0.894ms1 , the omron hj-112 pedometer step counts were not significantly different from actual steps . during treadmill walking at 1.12 ms1 , the yamax sw-200 pedometer steps were significantly different from actual steps . discussion : activity monitor selection should be deliberate when examining the walking behaviors of community - dwelling older adults , especially for those who walk at a slower pace .

Method Participants Equipment Protocol Statistical Analysis Results Discussion Conclusion

PMC2902016

nowadays , the african region still faces the wors health conditions in comparison with the rest of the world . thirty - two african countries are among the world 's forty - eight least developed nations while literature reports that the 90% of the eleven million of children ( up to 5 years age ) dying every year for neonatal disorders , diarrhea , acute respiratory infections and malaria are found in the poorest counties worldwide . poverty , underdevelopment and un - nourishment together with wars , drought , lack of health and educational services are some of the more relevant health determinants in the region . since common risk factors , such as social , economic , and political ones , are worldwide recognized both for some of the most common noncommunicable diseases and oral diseases , awareness is generally rising about the need to integrate general health action plans with oral health interventions . dental caries and periodontal disease prevalence seems not to be so severe in africa as in the rest of the world while oral disease pattern is not homogeneous across the region and need to be assessed in each community . in 2006 , thorpe highlighted the importance of having reliable data about the oral health profile of different communities across africa as a prerequisite to develop and improve effective intervention programmes to address oral health needs . the major challenges for the near future will be to turn knowledge and experience of disease prevention into action . clear disparities widened the gap between poor and rich countries . in may 2007 , the who general assembly ( wha60.17 resolution ) claimed for considering mechanism to provide coverage of the population with essential oral health care to promote the availability of oral health services that should be directed towards diseases prevention and health promotion for poor and disadvantage , countries . for these countries it is needed to consider , especially for schoolchildren , the development and implementation of preventive programmes , as a part of activities in health promoting schools , aiming to introduce healthy lifestyle and self - care practices in children [ 6 , 7 ] . burkina faso , previously known as upper volta , is a low - income country in west africa having 50% of the population living with less then 1 us$ per day , characterized by poor sanitary conditions and lack of services . in burkina faso , as well as in other african countries , a national oral health system is developing , and health promotion and oral disease prevention are the main goals health authorities are promoting . in order to plan community oral health promotion programmes , although the dmft is reported to be rather low ( 0.7 at 12 years old and 1.9 at 18 years ) , periodontal diseases are extensively diffused and oral manifestation of aids , gangrenous infections ( anug ) and noma still represents a serious concern [ 4 , 9 ] . the oudalan province is one of the 45 provinces of burkina faso , located in the northern part of burkina faso , and it is included within sahel , the semi - arid belt running across sub - saharan africa . oudalan is divided into 5 departments : deou , gorom - gorom , markoye , oursi and tin - akof . almost 400 kilometers far from ouagadougou , burkina faso capital city , there is the municipality of gorom gorom , a small town surrounded by tens of villages and gatherings in the savannah . no current oral health services are available both for town and villages inhabitants and the first available dental clinic is 60 kilometres faraway . consequently in oudalan province , the population has a poor or lack access to dental care . people suffer with tooth pain , often with no opportunity for relief , without making a long and costly journey . dental visits are mainly performed for symptomatic reasons and not for restorative reasons . in order to set up the needs for intervention and to plan oral health prevention and care programmes , this paper aims to describe the epidemiological oral health status and behaviour in children living in the municipality of gorom - gorom , oudalan province , sahel region , burkina faso . this survey included the most relevant subgroups of the population ( always 12 years age included ) according to the world health organization . the survey was carried out as a cross - sectional study , during two months , in 2008 in children living in the municipality of gorom gorom , oudalan province , sahel region , burkina faso ( figure 1 ) . the municipality counts about 80,000 inhabitants living in 82 villages and in gorom gorom capital . the fluoride concentration was carried out using an orion model 96.09 fluoride ion selective electrode and an orion model 900200 double junction plastic body ag / cl reference electrode with orion model 290 mv digital meter . fluoride content was quite high , with a mean value of 1.7 0.6 ppm . no direct previous contact had been with a dental worker before for the children population , but traditional healer serves for people oral care needs . children aged 5 - 6 and 12 years from 11 schools and 9 villages of the municipality were included in the survey . the 5 - 6 year group is relevant for the examination of deciduous teeth while the 12 year for the permanent ones . an authorization for the survey was obtained both from national health , educational authorities and local administrators . during a school meeting , parents or guardians were informed about the aim of the survey , describing the dental visit procedure and explaining the questionnaire items ; after all , their verbal agreement was obtained . all children 's parents or guardians consented to clinical examination and oral interview . in schools the collection was conducted in the presence of teachers and in rural villages in the presence of the local chief . the sample size was 692 children ; 334 females ( 48.3% ) and 358 males ( 51.7% ) . the subjects were then categorized for age into two groups : 338 subjects aged 5 - 6 years ( 48.8% ) and 354 subjects aged 12 years ( 51.2% ) . when it was not possible , due to the lack of data collection ( i.e. remote villages and gatherings ) , children with a deciduous full - arch were considered 5 years old , those with first permanent molars not yet fully erupted ( under occlusal edge ) 6 years old , those with second permanent molars not completely yet erupted 12 years old . the month before the beginning of the study , the examiner took part to a calibration session organized in the who collaboration centre of milan . forty subjects ( 20 for each age group ) were re - examined after 72 hours by the examiner . the visits were performed on a simple bed or school table using a plain mirror , a who - cpi probe , paper hand towels , and gauzes to remove debris from around the teeth . the instruments were first washed in a conventional manner with soap and then sterilized with a solution of paracetic acid ( parasafe ) . an artificial head - light source was used . the examination setting was always arranged according to the local condition and avoiding natural light sources and crowding . general information about the patient like age , gender , ethnic group , and living conditions was collected . the recorded clinical conditions were dental caries experience ( dmft / dmft ) , gingival condition ( presence of plaque and/or calculus ) , and fluorosis following who indication . dental caries was registered when decay at the dentinal lesion level was found ; gingival conditions were scored into three classes following the community periodontal index : score 0 ( healthy ) , score 1 ( gingival bleeding at probing ) , and score 2 ( calculus ) . cleanliness of the hands was recorded as follows : score 1 ( clean ) , score 2 ( dirty ) and score 3 ( crusted ) as a proxy of hygiene condition of the subject . oral health related behaviours were collected by a questionnaire directed to children and parents or guardians . it was divided into different items : oral hygiene habits ( tooth - brushing habit , use of a toothbrush or a chewing stick , use of fluoridated toothpaste ) and dietary habits ( number of meal per diem ) . caries distribution according to age was calculated as mean standard errors ( se ) . caries prevalence was calculated as the number of subjects with dt > 0 and dmft > 0 compared to the whole sample . odds ratio ( or ) was calculated between caries prevalence and other variables ( age , gender , fluorosis , and cleanliness of hands ) as well as for periodontal condition ( with gender , caries experience , cleanliness of hands , and oral hygiene habit ) . no score for missing or filled tooth was assigned , so data reported only caries activity ( dt ) prevalence . in the two age groups ( 5 - 6 and 12 years ) , dt ( mean se ) was 1.5 0.1 and 0.1 0.3 , while dt ( mean se ) was 0.2 0.3 and 0.5 0.6 . table 1 describes the entire caries experience by age , gender , fluorosis , and cleanliness of hands as count ( percent ) and odds ratio ( or ) for each category . according to dean 's index , 61 children ( 8.8% ) showed no sign of fluorosis or it was uncertain ; for 343 children ( 49.7% ) it was very mild , for 213 ( 30.9% ) it was mild , for 72 ( 10.4% ) it was moderate or severe ( table 2 ) . overall , 37.9% ( 150 of 5 - 6 years and 112 of 12 years ) of children showed healthy gingival condition . plaque was scored in 27.8% of cases ( 107 of 5 - 6 years and 85 of 12 years ) , while calculus in 34.4% of cases ( 81 of 5 - 6 years and 157 of 12 years ) . children reported having 3 meals per day in 97.1% of the sample ( 672 ) and 2 meals per day in 2.9% ( 20 children ) . children were reported to brush their teeth in 35.7% ( 247 ) of the sample : 7.1% ( 49 children ) with a toothbrush and 28.6% ( 198 children ) with a chewing stick . table 3 shows an association between community periodontal index scores and toothbrushing habit and chewing stick usage , respectively . no association was found between cpi scores and gender , caries experience and cleanliness of hands , respectively ( p > .05 ) ( data not in table ) . significant trends in proportion ( p < .05 ) of community periodontal index , across categories of exposure , were found for these two variables . nowadays , the usual etiologic approach to tooth decay or periodontal disease is revised considering social , cultural , environmental determinants as well as oral health systems orientation . many more other studies have been carried out on oral health determinants and today oral pathology is considered a social exclusion pointer . oral health is today an unfulfilled need for most people worldwide especially for those belonging to the most disadvantaged groups of society both in low and high income countries . the burden of oral disease is growing due to a lack of proper community oral health programmes . the evidence for a link between sociobehavioural risk factors and dental decay was pointed out , underlining a social gradient in caries prevalence [ 14 , 15 ] . this model highlights three distal determinants for oral health : health systems and oral health services , sociocultural risk factors and environmental risk factors . the aim of this study was to provide an overview of the oral health condition of children in the region of burkina faso in order to assist an intervention of oral health promotion . few epidemiological data are available for oral health condition in burkina faso as a whole ; the latest ones date back to the 90s and reported general poor oral health hygiene , significant prevalence of periodontal diseases , and generally low prevalence of dental decays ( with higher levels in urban areas ) . no specific data for the oudalan province was found at the moment of this survey nor conducted and no previous contact has had been with a dental worker for the children population . this situation points out the urgent need to implement integrated primary oral health care and oral health services that meet the needs of the local population as suggested by who 's global strategies . the main finding of this study was the low caries prevalence ( more than three quarters of the children were without obvious caries caries free ) probably linked to the quite high concentration of fluoride in well water . similarly , the high fluoride concentration is the cause of the high level of fluorosis observed . the absence of filled or missing for caries scored teeth ( m / f score ) highlights the absence of accessible services even for urgent treatments . comparing with other data collected in southwest burkina faso in 1999 , scoring caries prevalence at 38% of 6 year - aged children , this study reports a very lower rate . the majority of children resulted to have 3 meals per day : diet is not varied , poor in quantity , and low in calories . the most common food is a milk soup , millet , and sugar , badly affecting their growth . cariogenic snacks are not available except for a minority of children living in gorom gorom , while it is a common habit to drink sweet herb brew . only a few children from gorom gorom reported to use a fluoridated toothpaste due to a lack of availability as well as for general high cost , as reported in literature [ 18 , 19 ] . the use of a toothbrush is quite common in town , while it is almost absent in villages . a common habit of the population is to use a chewing stick from acacia tree ; a statistically significant association has been found between periodontal condition and chewing habit . no clear significance was found between periodontal condition and cleanliness of hands , while it was found for caries experience . less than fifty percent of children have clean hands considering that drought and lack of water are common . moreover , this survey was conducted during the dry season when highly severe diseases as acute and chronic intestinal infections spread and put life of children at serious risk . the present study maybe the typical limitation of a cross - sectional survey , like the low number of participants with respect to total population . . however , in this report the rate of participants was high and this is the first report about children oral condition living in this area . from a public health point of view this study supports recommendation from the most authoritative international organizations claiming the urgent need to change policies and strategies for oral health . in may 2007 , the assembly of the world health organisation declared that the economic burden of oral diseases is expected to rise rapidly globally , above all , among disadvantaged and poor population groups , at least until programmes for the prevention of oral disease are promoted . moreover , as reported by the lancet in 2009 , training more dentists and building dental clinics the western curative model of care is costly and unrealistic in most low - income and middle - income countries . primary health care approach ( basic and sustainable care , community participation , appropriate technology , maternal and childhood health focus and prevention integrated with educational and social sectors ) is the only realistic one to address oral health needs for communities at any latitude of the world . a good first step to improve the oral condition of the oudalan children should be that local authorities would accept and organize a dental service based on basic package of oral care ( bpoc ) developed by the who collaborating centre of nijmegen . although it is recognised that communities and the circumstances in which african populations live are widely different , to win the many challenges ahead for africa , it is important to build up strategies to provide equitable and universal access to oral health care services and to promote preventive oral health programs in order to significantly reduce the impact of oral diseases in african populations .

aim . in order to set up the needs for intervention and to plan oral health prevention and care programmes , this paper aims to describe the oral health status and behaviour in children living in the municipality of gorom - gorom , burkina faso . design . the sample size was 692 children , 334 females ( 48.3% ) and 358 males ( 51.7% ) . clinical and oral health related behaviours were collected . results . 83.4% of the children were caries - free . fluorosis was recorded in 41.3% of the sample , while only 37.9% of children showed healthy gingival condition . toothbrushing was reported by 35.7% of children . a statistically significant association was found between caries experience and cleanliness of hands . community periodontal index was statistically associated to toothbrushing and chewingstick use . conclusion . as suggested by who 's global strategies , integrated primary oral health care and services meeting the dental needs of the local population are necessary for children living in this area of africa .

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

PMC4522295

plasmodium vivax ( p. vivax ) is the second widespread malaria species that causes disease in human and imposes sizeable socioeconomic burden and public health difficulty on many countries , particularly in asia and south and central america ( 1 ) . although , p. vivax is responsible for more than 50% of malaria morbidity outside sub - saharan africa , but little consideration for control and research has been dedicated to this parasite ( 2 ) . at present , about 2.8 billion people universally are at risk of p. vivax infection and occurrence of the disease is nearly 132391 million cases each year ( 3 ) . in a nutshell , in malaria life cycle , being bit by female anopheles mosquitoes , sporozoites are transferred to human and infection is initiated . within hepatocytes , the sporozoites reproduce and increase in quantity to thousands of merozoites , which attack the red blood cells ( rbcs ) in the next stage . in the infected rbcs , the small ring shape changes through trophozoite to schizont , in which it bursts and liberates more merozoites ( 4 ) . up to now , some problems such as non cultivable nature of p. vivax , caused limitations in the study of its molecular biology , but genetic diversity and population organization of this parasite have become more clear by sequencing and genetic engineering including cloning methods ( 5 ) . several p. vivax antigens have been proposed as detectable potential candidates ( 6 ) , among which c - terminal region of pvmsp-1 has high expression on the mature merozoites surface as well as mass protein production , that it has been proven to take part in the parasite invasion to the erythrocyte . specific antibodies against msp-1 , have been particularly shown react to the c - terminal region ( 33-kda and 19-kda sub fragments ) ( 79 ) . antigenic variation is one of the limitative agents in antibody detection that could be due to haplotype variations on target gene . it can ultimately affect the protein conformation , affinity of antibody - antigen and serological test results . little information is available regarding the genetic polymorphism of msp-142 among iranian p. vivax population . in this study , in order to assess the nature of iranian p. vivax isolate , molecular diversity of cloned pvmsp-142 kda gene was analyzed . in this study , 11 isolates of chabahar district ( sistan and balouchestan province ) that had p. vivax infection symptoms were chosen and sequenced in order to surveying their similarities . one out of 11 mentioned isolates which had highest homology was selected to cloning process . . the parasite genomic dna was extracted from the whole blood by genomic dna extraction kit , dng plus ( cinna gene , iran , dn8118c ) using the kit manufacturer s guidelines . continuously , genomic dna quantity and quality was controlled by electrophoresis on 0.8% agarose gel and a biophotometer ( ependorf ) at 260 and 280 nm . amplification from 42 kda partial regions of pvmsp-1 gene was done which included 19 kda and 33kda fragments . the primers were designed on the basis of the sequence of pvmsp-142 kda gene , ( genbank : accession no : dq907673 ) . the following primers were used for sequencing : msp1.42f ( 25mer ) ( 5-ggatccgaccaagtaacaacgggag-3 ) , msp1.42r ( 25mer ) ( 5-gaattccaaagagtggctcagaacc-3 ) . the pcr was performed in a pcr tube containing : 200 ng ( 1.5l ) extracted dna as template , 20 pmol ( 0.25 l ) of each primer , 7.5 l of pcr master mix 2x that contained taq and 5.5 l of ddh2o . the objective gene was amplified for 30 cycles ( initial denaturation at 96 c for 5 min , 96 c for 30s , 58 c for 30s and 72 c for 1 min and final extension for 20 min ) subsequently , the pcr product was controlled on 1% agarose gel against a standard dna ladder ( fermentase co. ) . pcr products that consisted of a single specific band can be directly ligated to t - vectors . however , the removal of the impurities such as primers and nonspecific products by gel separation causes increase in the percentage of colonies containing the correct inserts . thermo - stable dna polymerase which does not preferentially add a 3-a at the ends of the pcr products is essential to removal of that enzyme by gel separation . purification and recovery of the dna from agarose gel will be explained in the next sections . purified dna fragment was inserted into ptz57r / t that provided by the following protocol . any plasmid could be selected to gratifies our requirements including the plasmid contains a unique blunt - end restriction site in the multiple cloning site ( in this study , the ecor v site of the ptz57r plasmid was used that has blue / white color selection ) . digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing : 25 l plasmid dna ( 2g ) , 4 l of 10x ecor v buffer , ( 10 unit ) 1 l of ecor v ( fermentase co. ) and dd h2o up to 40l . then it was mixed by gentle vortex , centrifuged and incubated at 37 c for 2 hours . in the next step , 2 l of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed . thereafter it was incubated to 65 c by water bath for 10 min at the end of digestion ( fig . a : lane1 : intact plasmid ptz57r , lane2 : digested plasmid ptz57r by ecorv , lane3 : size marker : 1 kb . b : lane1 : recombinant plasmid , lane2 : intact plasmid , lane3 : size marker1 kb . c : lane1 : ( a : separated pcr product from digested recombinant plasmid ) , lane2 : intact recombinant plasmid , lane3 : size marker1 kb taq polymerase and dttp were used to add a 3-t to the blunt - ended plasmid . briefly , the making t - overhangs was performed in a 200 l pcr tube containing : 40 l blunt - ended plasmid dna , 5 l of 10 x pcr buffer ( mgcl2 free ) , 2.5 l of mgcl2 ( 250 mm ) , 0.5 l of dttp ( 100 mm ) , 2 l of taq polymerase ( 5 u/l ) . it was mixed by gentle vortex , centrifuged shortly and incubated at 72 c for 2 h. t - vectors were separated from the self - ligated and concatemerized plasmid dna on a 1% agarose gel by 0.5 g / ml of ethidium bromide . the dna bands were visualized by a hand - held long wave ( 365 nm ) uv light . the gel slice transferred to a 2 ml micro centrifuge tube , and then t - vectors were purified and recovered by silica bead dna gel extraction # k0513 ( fermentas co. ) via the kit manufacturer s guidelines . the density of the purified t - vectors was determined by running 1 l of the t - vector in compared to standard dna ladder in a 1% agarose gel at 5 volts / cm for 45 min and then compared the comparative brilliantness of the bands . 1:3 molar ratio of t - vector to insert dna was recommended , but the amount of a - tailed dna solution in a 10 l ligation should not be more than 2 l . in a sterile 0.5 ml micro centrifuge tube , added : 2 l t - vector , 2 l of a - tailed dna , 1 l of 10 x ligation buffer , 1 l of t4 dna ligase ( 23 u/l ) and 4 l of ddh2o . it was mixed gently , centrifuged briefly and incubated at 14 c for overnight ( fig . before transformation , luria - bertani ( lb ) agar plates contained 100 g / ml of ampicillin , were prepared and spreaded with 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) and isopropylthio--d - galactoside ( iptg ) . for each ligation reaction , 50 l aliquoted of frozen competent cells ( top 10 ) was thawed on ice and added 2 l of the ligation reaction to the competent cells and mixed gently by stirring with the pipette tip . the cells were incubated on ice for 30 min then heats shocked for 30 s in the 42 c water bath , and then were immediately placed on ice for 2 min . the transformed cells were spreaded on each labeled lb - ampicillin plate with x - gal and iptg . the positive colonies that had white color were identified either by restriction enzyme and screening by pcr method after transformation . pcr screening was carried out with same primers that be used for pcr amplification for insert production . for screening by restriction enzyme method , the insert was released by digestion with two unique restriction enzymes ( with cutting sites , ecor1 : gaattc and bamh1 : ggatcc ) from the multiple cloning sites , and the insert size was confirmed by agarose gel electrophoresis ( fig . pcr product of pvmsp-1 42 kda was directly analyzed on abi prismtm 310 automated sequencer to determine p. vivax variation ( strain / haplotype ) in the infected patient . both directions of our sequences were aligned and edited using sequencher tm 4.0.4 software for pc ( gene codes corporation ) and mega 5 software for phylogenetic analysis and then were compared with some genbank sequences for their similarity ( 10 ) . a phylogenetic hypothesis of p. vivax was generated with pvmsp-1 42 kda using the nonparametric bootstrapping , neighbor joining method . in this study , 11 isolates of chabahar district ( sistan and balouchestan province ) that had p. vivax infection symptoms were chosen and sequenced in order to surveying their similarities . one out of 11 mentioned isolates which had highest homology was selected to cloning process . . the parasite genomic dna was extracted from the whole blood by genomic dna extraction kit , dng plus ( cinna gene , iran , dn8118c ) using the kit manufacturer s guidelines . continuously , genomic dna quantity and quality was controlled by electrophoresis on 0.8% agarose gel and a biophotometer ( ependorf ) at 260 and 280 nm . amplification from 42 kda partial regions of pvmsp-1 gene was done which included 19 kda and 33kda fragments . the primers were designed on the basis of the sequence of pvmsp-142 kda gene , ( genbank : accession no : dq907673 ) . the following primers were used for sequencing : msp1.42f ( 25mer ) ( 5-ggatccgaccaagtaacaacgggag-3 ) , msp1.42r ( 25mer ) ( 5-gaattccaaagagtggctcagaacc-3 ) . the pcr was performed in a pcr tube containing : 200 ng ( 1.5l ) extracted dna as template , 20 pmol ( 0.25 l ) of each primer , 7.5 l of pcr master mix 2x that contained taq and 5.5 l of ddh2o . the objective gene was amplified for 30 cycles ( initial denaturation at 96 c for 5 min , 96 c for 30s , 58 c for 30s and 72 c for 1 min and final extension for 20 min ) subsequently , the pcr product was controlled on 1% agarose gel against a standard dna ladder ( fermentase co. ) . pcr products that consisted of a single specific band can be directly ligated to t - vectors . however , the removal of the impurities such as primers and nonspecific products by gel separation causes increase in the percentage of colonies containing the correct inserts . thermo - stable dna polymerase which does not preferentially add a 3-a at the ends of the pcr products is essential to removal of that enzyme by gel separation . purification and recovery of the dna from agarose gel will be explained in the next sections . purified dna fragment was inserted into ptz57r / t that provided by the following protocol . any plasmid could be selected to gratifies our requirements including the plasmid contains a unique blunt - end restriction site in the multiple cloning site ( in this study , the ecor v site of the ptz57r plasmid was used that has blue / white color selection ) . digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing : 25 l plasmid dna ( 2g ) , 4 l of 10x ecor v buffer , ( 10 unit ) 1 l of ecor v ( fermentase co. ) and dd h2o up to 40l . then it was mixed by gentle vortex , centrifuged and incubated at 37 c for 2 hours . in the next step , 2 l of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed . thereafter it was incubated to 65 c by water bath for 10 min at the end of digestion ( fig . a : lane1 : intact plasmid ptz57r , lane2 : digested plasmid ptz57r by ecorv , lane3 : size marker : 1 kb . b : lane1 : recombinant plasmid , lane2 : intact plasmid , lane3 : size marker1 kb . c : lane1 : ( a : separated pcr product from digested recombinant plasmid ) , lane2 : intact recombinant plasmid , lane3 : size marker1 kb taq polymerase and dttp were used to add a 3-t to the blunt - ended plasmid . briefly , the making t - overhangs was performed in a 200 l pcr tube containing : 40 l blunt - ended plasmid dna , 5 l of 10 x pcr buffer ( mgcl2 free ) , 2.5 l of mgcl2 ( 250 mm ) , 0.5 l of dttp ( 100 mm ) , 2 l of taq polymerase ( 5 u/l ) . it was mixed by gentle vortex , centrifuged shortly and incubated at 72 c for 2 h. t - vectors were separated from the self - ligated and concatemerized plasmid dna on a 1% agarose gel by 0.5 g / ml of ethidium bromide . the dna bands were visualized by a hand - held long wave ( 365 nm ) uv light . the gel slice transferred to a 2 ml micro centrifuge tube , and then t - vectors were purified and recovered by silica bead dna gel extraction # k0513 ( fermentas co. ) via the kit manufacturer s guidelines . the density of the purified t - vectors was determined by running 1 l of the t - vector in compared to standard dna ladder in a 1% agarose gel at 5 volts / cm for 45 min and then compared the comparative brilliantness of the bands . . 1:3 molar ratio of t - vector to insert dna was recommended , but the amount of a - tailed dna solution in a 10 l ligation should not be more than 2 l . in a sterile 0.5 ml micro centrifuge tube , added : 2 l t - vector , 2 l of a - tailed dna , 1 l of 10 x ligation buffer , 1 l of t4 dna ligase ( 23 u/l ) and 4 l of ddh2o . it was mixed gently , centrifuged briefly and incubated at 14 c for overnight ( fig . before transformation , luria - bertani ( lb ) agar plates contained 100 g / ml of ampicillin , were prepared and spreaded with 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) and isopropylthio--d - galactoside ( iptg ) . for each ligation reaction , 50 l aliquoted of frozen competent cells ( top 10 ) was thawed on ice and added 2 l of the ligation reaction to the competent cells and mixed gently by stirring with the pipette tip . the cells were incubated on ice for 30 min then heats shocked for 30 s in the 42 c water bath , and then were immediately placed on ice for 2 min . the transformed cells were spreaded on each labeled lb - ampicillin plate with x - gal and iptg . the positive colonies that had white color were identified either by restriction enzyme and screening by pcr method after transformation . pcr screening was carried out with same primers that be used for pcr amplification for insert production . for screening by restriction enzyme method , the insert was released by digestion with two unique restriction enzymes ( with cutting sites , ecor1 : gaattc and bamh1 : ggatcc ) from the multiple cloning sites , and the insert size was confirmed by agarose gel electrophoresis ( fig . pcr product of pvmsp-1 42 kda was directly analyzed on abi prismtm 310 automated sequencer to determine p. vivax variation ( strain / haplotype ) in the infected patient . both directions of our sequences were aligned and edited using sequencher tm 4.0.4 software for pc ( gene codes corporation ) and mega 5 software for phylogenetic analysis and then were compared with some genbank sequences for their similarity ( 10 ) . a phylogenetic hypothesis of p. vivax was generated with pvmsp-1 42 kda using the nonparametric bootstrapping , neighbor joining method . in fig . 1 , intact plasmid ptz57r , digested plasmid ptz57r by ecorv and digested recombinant ptz57r/ msp by bamhi and eco - ri restriction enzymes and expected insert band are presented . as fig . 2 shows an 1124 bp band has been demonstrated from extracted genomic dna by pcr . lane 1 : pv msp-142 kda gene was nearly 1124bp long , lane 2 : size marker 1 kb the yield of pcr was cloned in ptz57r and then sequenced . in the iranian p. vivax isolate , as compared to the reference sal i sequence , 38 snp of pvmsp-142 kda , were identified . as fig . 4 shows , msp-1 42 kda phylogenetic tree based on bootstrap - neighbor joining method in iranian sequenced isolate ( msp 42-yyxxxxxx - iran 01 ) and other recorded sequences in genbank . all of the mutations were localized in the pvmsp-133kda , while there was almost no variation in the msp-119 kda . 2 out of 38 mutations ( haplotype ) were novel in the 545 and 573 positions as compared to the homogeneous sequences from other surveyed countries ( fig . 3 , 4 ) . bootstrap neighbor - joining consensus tree of pv - msp-142 kda data set by mega5 . sequence of iranian isolate is shown for msp 42-yyxxxxxx - iran 01 in clad of indian and japonica sequences ( accession nos : eu 430479.1 , af435635.1 ) sequence alignment of pv msp-142 kda gene in p. vivax isolated from iran and correlated part of the isolated genes from other countries in research on diagnostic potential for p. vivax malaria parasite subunits , the focus is on the c - terminal region of p. vivax msp-1 . as fig . 5 depicts , generally , msp1 is manufactured as a big protein ( 200 kda ) which is connected to the cell membrane by a glycophosphatidylinositol ( gpi ) anchor in the carboxyl terminus region ( 11 ) . msp1 undergoes a series of proteolytic maturation changes at the same time as the merozoite unleashed from ( rbc ) and generates four polypeptide fragments of nearly 83 , 30 , 38 , and 42 kda from the n - terminus to c - terminus ( 12 ) , which stays as interconnected parts on the cell surface by the anchored c - terminal segment ( p42 ) ( 13 ) . new rbcs are invaded rapidly by attachment of free merozoites induces second in a series cleavage of the p42 peptide to generate p33 , which is shed together with the previous fragments and p19 , abides by anchoring to the membrane of parasite as it invades the cell ( 14 , 15 ) . high plentifulness and important function on the cell surface , probably caused msp1 to be a principal object of the host immune system and antibodies are identified on different regions of this protein ( 16 ) . antibodies that recognize the c - terminal region of plasmodium falciparum msp-1 , inhibit the invasion of merozoites into the host erythrocytes in vitro and immunization of experimental animals with msp-119 kda also confers protective immunity ( 17 , 18 ) . these findings demonstrate that msp-142 kda is a promising candidate antigen for blood stage vaccine development and diagnostic kits . panel ( a ) shows primary processing , and ( b ) shows secondary processing in this study , the pv msp-142 kda was cloned , sequenced , and subsequently compared with homogeneous genes that previously had been recorded in genbank . 38 snp of pvmsp-142 kda , were recognized using the sequencher 4.0.4 and mega 5 programs . pacheco et al . in indonesia tried to show the genetic diversity of the 42 kda fragment of msp-1 antigen in p. vivax . they found that the msp-133 kda fragment exhibits greater genetic diversity than the msp-119 kda generally . previous observations confirmed that the msp-119 kda fragment is more conserved than the msp-133 kda fragment ( 19 ) . in present study , polymorphism of p. vivax msp-133 kda is not evenly spreaded , indeed there is a region of 70 bp in msp-133 kda where a clear excess of non - synonymous is observed in the overall msp-133 kda results , while there is closely no significant variation in the msp-119 kda . two out of 38 mutations were new haplotypes ( 545 and 573 positions ) in the 33 kda fragment as compared to the homogeneous sequences from other countries including japan , turkey , usa , india , sumatra , vietnam , indonesia , riometa , china , france and reference sequence from sal - i ( 20 ) ( fig . 3 ) . due to the differentiation of msp1 amino acid sequences between plasmodium spp . , it is necessary to use recombinant msp-1 to detection antibody from p. vivax in iranian cases ( 21 ) . compared the p. vivax msp-3 gene with ssrrna gene as genetic markers for the parasite detection . according to their results the sensitivity of ssrrna gene was higher than the pvmsp-3 gene ( 100% vs. 95% ) . they concluded that the pvmsp-3 gene can not be a suitable marker for detection of p. vivax in blood sample by pcr ( 22 ) . one of the prominent problems in diagnostic kits is genetic polymorphisms that encoding on this region , within and between the p. vivax population . therefore , polymorphism examination of this gene on disparate territories could be helpful in improving diagnostic kits based on antibody detection in malarious areas of iran . shahbazi and colleagues in iran assessed the genetic structure of p. vivax population by sequence analysis of the merozoite surface protein 3 ( msp-3 ) gene . moreover , their phylogenetic analysis did not show any significant geographical branching ( 23 ) . studied the p. vivax dihydrofolate reductase ( pvdhfr ) mutations among 50 blood samples of symptomatic patients from 4 separated geographical regions of south - east iran . they reported point mutations at residues 57 , 58 , 61 , and 117 by using the pcr - rflp method . polymorphism at positions 58r , 117n , and 117 t of pvdhfr gene has been found in 12% , 34% , and 2% of isolates , respectively . one of the endemic zones for p. vivax throughout the world is iran from which there are reported cases annually ( 25 ) . asymptomatic carriers of p. vivax among treated patients have been shown as a major reservoir of parasites and maintenance of high levels of transmission in malarious areas of iran ( 26 ) . although genetic polymorphisms in the regions of msp-1 in iranian p. vivax isolates has been previously analyzed , but little information is available regarding the genetic polymorphism of msp-142 kda among iranian p. vivax isolates . in this study , the genetic polymorphism of msp-142 kda in an iranian isolate was analyzed for a better understanding of the nature of iranian p. vivax . thus , it is significant to investigate the molecular character of the parasite placed within iran and comparing with the same species in other regions . predicted protein sequence of cloned pvmsp-142 kda gene obviously has a high degree of identity ( 97% ) with strong homology to the pv msp-142 kda gene of p. vivax . asian isolates specially japan and india also have sequence similarity ( 95% ) pv msp-142kda gene from sal - i isolate ( as reference sequence ) . in general , diversity of anopheles mosquitoes , presence of several plasmodium species , biotic interactions and hybrid / mixed infections can affect the genotyping variation of plasmodium in low to high degree . this might result in parasite pathogenicity , host specificity , establish and persist of infection , different clinical manifestation , transmission dynamics and antigenicity of infection . the cloned pvmsp-142 kda gene can be used as a strong candidate for diagnostic kits based on antibody , antigen detection and vaccine development .

background : carboxy - terminal 42 kda region of plasmodium vivax merozoite surface protein-1 is considered as an important antigen in blood stage . since , this region has been observed to be polymorphic among isolates of p. vivax , it is significant to survey on different regions of this antigen in various areas of the world.methods:in the present study , the genetic diversity of cloned pvmsp-142 kda gene from an iranian patient is analyzed . parasite dna was extracted from a p. vivax - infected patient in iran . the region of pvmsp-142 kda was amplified by pcr , cloned into ptz57r / t vector and then sequenced.results:sequencing of cloned pvmsp-142 kda gene clearly has a high degree of homology ( 95% ) with reference sal - i sequence and also with the homogeneous sequences from some studied countries ( 97% ) . thirty eight snps ( single nucleotide polymorphism ) were identified in cloned pvmsp-142 kda gene which the mutations had localized in the 33 kda fragment ( pvmsp-133 kda ) , while there was nearly no variation in the 19 kda fragment ( pvmsp-119 kda ) . 2 out of 38 mutations were found as to be novel haplotypes.conclusion:high similarity of cloned pvmsp-142 kda gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of p. vivax in malarious neighboring countries of iran and around the world .

Introduction Materials and Methods Preparation of insert DNA T-Vector preparation Digesting plasmids with Blunt-End restriction enzyme Making T-overhangs from the Blunt-Ended plasmid vector Separation, purification and recovery of T-vectors on agarose gel Quantification and storage of the purified T-vector Ligation Transformation of the ligated vector DNA sequencing and phylogenetic analysis Results Discussion Conclusion

PMC3517856

despite the significant progress in the therapy of hematological malignancies there are several problems that impede the use of the polychemotherapy for treatment of this group of diseases . the first one is the high intrinsic toxicity of multiple chemotherapies [ 1 , 2 ] that may lead to the multiple - organ failure in particular liver dysfunction . the next is the strengthening of multidrug resistance ( mdr ) of tumor cells by cytostatic agents that are often characterized by the mdr phenotype even at baseline [ 35 ] . these two problems can augment each other and can significantly reduce the efficiency of antineoplastic therapy . the resistance of tumor cells to chemotherapy can be a result of various processes : from active atp - dependent transport of cytotoxic agents out of the cells executed by p - glycoprotein , the member of subfamily b of abc - transporters [ 6 , 7 ] , to disorders in the apoptosis , mutations or downregulation of p53 gene , impairing its proapoptotic function , and/or overexpression of bcl-2 gene causing insensitivity of cells to proapoptotic stimuli [ 810 ] . studies of pharmacokinetics of antitumor drugs suggest that they undergo biotransformation in the liver with the formation of toxic metabolites , which may cause hepatocyte damage following hepatic dysfunction [ 11 , 12 ] . hepatic dysfunction is one of the main factors limiting full adherence to the chemotherapy regimen by patients , because the development of complications in this organ due to chemotherapy can lead to lethal outcomes during cytostatic treatment . although the researcher pays much attention to mdr phenotype acquiring by the tumor in the course of the treatment and separately to the influence of polychemotherapy onto the liver , little is known about the cumulative hepatotoxic effects evoked by cytostatics and by the tumor with mdr phenotype enhanced after exposure to multiple courses of chemotherapy . herein , we studied the combined toxic action of polychemotherapy by chop protocol , three components ( cyclophosphamide , doxorubicin , and vincristine ) of which are the substrates for abc - transporters and the impact of upregulated mdr of lymphosarcoma rls40 on the liver of experimental animals using two treatment schemes . in the course of the treatment under scheme 1 the liver of tumor - bearing animals was subjected to the only one course of chop while tumor was consequently subjected to four courses of treatment . it was achieved by the retransplantation of tumor cells from the animals received one course of chop , to the healthy animals with intact livers . then the animals were subjected to the next course of chop while the tumor cells at the same time were exposed to the second course . as a result four courses of polychemotherapy for the tumor were done but the liver has been exposed every time to the only one course . this experimental model is artificial , because it has no analogue in patients in clinical practice . we have developed this treatment scheme to evaluate the strengthening of the mdr of tumor and its contribution to the liver toxicity . in the course of the treatment under scheme 2 tumor - bearing mice were exposed to three subsequent courses of chop . this experimental model represents the real picture in clinical practice in cancer patients undergoing multiple courses of chemotherapy . our data show that the toxic effect of polychemotherapy onto the liver is accelerated by the upregulated mdr of lymphosarcoma rls40 , and altogether they cause the increase in the destructive changes of the hepatocytes and the overall reduction in the regeneration capacity of the liver . rls40 cell line was obtained from rls by selection of rls tumor cells in vitro in medium supplemented with vinblastine at concentrations ranged from 5 to 40 nm and displayed high resistance to a wide range of cytostatics . rls40 cells were maintained in the iscove 's modified dulbecco 's medium ( imdm ) supplemented with 10% fetal bovine serum ( fbs ) and 1% antibiotic antimycotic solution ( 10.000 g / ml streptomycin , 10.000 iu / ml penicillin , and 25 g / ml amphotericin ; icn , germany ) in the presence of 40 nm vinblastine in a humidified atmosphere containing 5% co2 at 37c and were regularly passaged to maintain the exponential growth . rls40 in ascitic form is permanent and maintained in cba mice by regular passaging of tumor cells . in order to generate ascites tumors from primary cell culture 2 10 cells / ml in 0.2 ml of the saline buffer were intraperitoneally injected into the abdominal cavities of cba mice . for generating solid tumors from ascites intramuscular injections of 5 10 cells / ml in 0.1 ml of solution were performed into the right thigh muscle of cba mice . the study was carried out on 1014-week - old male cba / lacsto ( hereinafter , cba ) mice bred at the institute of cytology and genetics ( siberian branch , russian academy of sciences , novosibirsk , russia ) . the mice had free access to food and water and the daylight conditions were normal . all animal procedures were carried out in accordance with approved protocol and recommendations for proper use and care of laboratory animals ( european communities council directive 86/609/cee ) . solid tumors rls40 were induced via intramuscular injection of ascitic rls40 cells ( 5 10 cell / ml , 0.1 ml per mouse ) suspended in the saline buffer into the right thighs of mice . on day 7 after tumor transplantation each mouse bearing rls40 was assigned to one of the two groups ( n = 15 per group ) : the first group , control , received the saline buffer intraperitoneally ; the second group received standard combination of cytostatics ( chop protocol ) . the cytostatics were dissolved in the saline buffer directly before use and injected into the caudal vein in doses corresponding to 1/5 of ld50 : cyclophosphamide ( 50 mg / kg ) , doxorubicin ( 4 mg / kg ) , and vincristine ( 0.1 mg / kg ) ( lens - farm ) . prednisolone ( 5 mg / kg ) ( nycomed ) was intraperitoneally injected daily for 5 days . on day 7 after chop ( day 14 after tumor transplantation ) at the 1st passage and on day 10 after chop ( day 17 after tumor transplantation ) at the subsequent passages , the mice were sacrificed by cervical dislocation under ether narcosis and tumor tissues were used for the preparation of cell suspension . a portion of this suspension was used for the retransplantation ( passages ) into intact animals ( n = 30 ) ( 5 10 cell / ml ; 0.1 ml per mouse ) ; another portion was used for the preparation of primary cell culture and evaluation of gene expression profile . on day 7 after retransplantation of the tumor cells a portion of mice were subjected to the 2nd course of chop ( n = 15 ) ; the control animals were left without treatment ( n = 15 ) . in total , four passages of tumors were carried out in mice followed by polychemotherapy . material for gene expression analysis was collected on day 7 after chop at the first passage of the tumor and on day 10 after chop during the other passages of the tumor . material for histological analysis was collected on days 1 , 3 , and 7 after the treatment at the first passage of the tumor , on days 7 and 10 at the second and the third passages , and on days 7 , 10 , and 14 at the fourth passage of the tumor . 1014-week - old male cba mice were assigned into 3 groups ( n = 45 per group ) . for generating of solid tumors rls40 cells ( 10 cells / ml , 0.1 ml per mouse ) were transplanted intramuscularly into the right thighs of the animals of groups 1 and 2 . after tumor transplantation group 2 ( tumor - bearing mice ) and group 3 ( mice without tumor ) received a standard combination of cytostatics ( chop protocol , see above ) . group 1 ( rls40-bearing mice ) was left without treatment . on day 7 after chop ( day 14 after tumor transplantation ) , a portion of the mice were sacrificed by cervical dislocation under ether narcosis and tumor tissues were used for the preparation of primary cell culture . the remaining mice both with the transplanted tumor and without the tumor received 2nd and 3rd courses of chop with an interval of 7 days . on day 7 after 2nd and 3rd courses of chop ( days 21 and 28 of tumor development ) mice were sacrificed . material for histological study was collected on days 1 , 3 , and 7 after each course of chemotherapy . material for gene expression analysis was collected on day 7 after each course of chemotherapy . the tumor size was determined every other day using caliper measurements in three perpendicular dimensions . tumor volumes were calculated as v = ( /6 length width height ) . the inhibition of tumor growth was estimated as follows : [ ( mean tumor weightcontrol mean tumor weightexperiment)/mean tumor weightcontrol ] 100% . isolation of primary cell culture from rls40 solid tumor was carried out by separation of cell suspension obtained after homogenization of solid tumors through lymphocyte separation medium ( lsm ) . the cells were cultured in imdm supplemented with 10% fetal bovine serum ( fbs ) , 1% antibiotic antimycotic solution ( 10.000 g / ml streptomycin , 10.000 i.u./ml penicillin , and amphotericin 25 g / ml ; icn , germany ) at 37c and 5% co2 for 1 week until analysis . rna concentration in the samples was measured by absorbance at 260 and 280 nm using a bio - mate 3 ( thermo electron corporation ) spectrophotometer . the primers and rt - pcr conditions were described earlier [ 13 , 15 ] . -actin - specific primers were added to the reaction mixture at the third and the first cycles , respectively . bcl-2- and p53-specific products were amplified for 31 cycles ; -actin - specific primers were added to the reaction at the sixth cycle . amplification was performed as follows : initial step 95c for 5 min , then 24 , 26 or 31 cycles of 94c for 1 min , 57c for 1 min , and 72c for 1 min . pcr products were analyzed by electrophoresis in 8% polyacrylamide gel using tbe1 as running buffer , visualized by ethidium bromide staining , photographed in uv light , and densitometrically quantified using gel - pro analyzer 4.0 . the data were presented as a ratio of specific gene expression level to -actin expression level . the levels of tnf- , il-1 , il-6 , ifn- , and ifn- in the blood serum of mice bearing rls40 without any treatment and treated with two courses of chop were measured using mouse tnf - alpha , il-1-alpha , il-6 , and ifn - gamma colorimetric elisa kits ( thermoscientific , usa ) according to the manufacture 's protocols . the level of ifn- was measured using mouse interferon alpha ( mu - ifn- ) elisa kit ( thermoscientific , usa ) . for morphological and morphometric analysis , tumor and liver samples of experimental animals were fixed in 10% neutral - buffered formaldehyde , routinely processed and embedded in paraffin [ 16 , 17 ] . paraffin sections ( up to 5 m ) were stained with hematoxylin and eosin , microscopically examined and scanned . stereological quantification was performed by point counting , using closed test system at a magnification 400 . used test system has 16 straight - line segments and 25 testing points in a testing area equal to 1.16 10 m and was applied for microscopic examination and morphometric measurements [ 1820 ] . morphometric analysis of the liver was performed and the volume densities ( vv ) of normal liver parenchyma , hepatic lymphoma infiltration , hepatocytes with degenerate , and necrotic changes ( destructive changes = degeneration + necrosis ) were evaluated . the volume density ( vv ) representing the volume fraction of tissue occupied by each compartment was determined from the points lying over these structures and calculated using the following formula : vv = ( pstructure / ptest ) 100% , where pstructure denotes the number of points over the structure and ptest represents the total number of test points , 25 in this case . numerical density of binuclear hepatocytes ( nv ) indicating the number of cells in the unit of tissue volume was determined by counting the number of binuclear cells within the test area ( grid with a square of 1.16 10 m ) . immunohistochemical staining of p - glycoprotein ( pgp ) ( ab 3364 , abcam , england ) in tumor and liver sections was carried out according to the manufacture 's protocol [ 21 , 22 ] . index of pgp staining used for assessment of staining intensity of tumor sections was calculated as follows : in(pgp ) = ( number of pgp+ cells / total cell number ) 100% , where the total cell number was at least 100150 cells for each tumor sample [ 23 , 24 ] . the intensity of pgp staining of the liver sections was scored on a scale of 03 + , in which 0 was no staining , 1 + weak positive staining , 2 + moderate positive staining , and 3 + strong positive staining . the data were statistically processed using the student 's t - test ( two - tailed , unpaired ) ; p < 0.05 was considered statistically significant . we used a tumor model with a proved predisposition to the strengthening of mdr status after cytostatic exposure - murine lymphosarcoma rls40 which is related to human diffuse large b - cell lymphoma . rls40 was obtained by selection of rls tumor cells in vitro in medium supplemented with vinblastine at concentrations ranged from 5 to 40 nm and exhibits high resistance to a wide range of cytostatics . parental rls tumor cell line is characterized by an increased expression of mdr1b and bcl-2 genes and decreased level of p53 gene . rls40 tumor is resistant to apoptosis induction and is characterized by the upregulated mdr1a/1b genes ' expression . the mdr of rls40 corresponds to the tumor status in patients after chemotherapy or to the status of a tumor initially resistant to standard chemotherapy protocols used as the first line therapy in the majority of hematological malignancies . the aim of our study was to investigate the effect of polychemotherapy ( single and multiple ) onto aggravation of the liver toxicity and the impact of the enhanced mdr of tumor into observed hepatotoxicity . for these purposes two experimental schemes were used ( figure 1 ) . in the treatment using scheme 1 mice with intramuscularly transplanted rls40 cells were exposed to polychemotherapy in chop regimen on day 7 after transplantation ( the 1st tumor passage ) . in 7 days after the 1st course of chop tumor cells were retransplanted into healthy animals and the 2nd course of chop was done ( the 2nd tumor passage ) ( figure 1 , scheme 1 ) . in 10 days after the 2nd course of chop tumor cells were retransplanted into healthy animals and the 3rd course of chop was done ( the 3rd tumor passage ) . similarly the 4th tumor passage was carried out ( figure 1 , scheme 1 ) . rls40 undergone the same passages of retransplantation in the absence of chop was used as a control . this experimental model which is artificial as there is no counterpart in patients in clinical practice was developed to evaluate the impact of upregulated mdr of the tumor into hepatotoxicity in the absence of the damaging effect of multiple chemotherapies onto the liver . as the result of the treatment using scheme 1 tumor was subjected to several courses of chop ( from 1 to 4 , in total 4 ) , the liver was subjected to only one chop exposure . during the treatment using scheme 2 a group of mice with intramuscularly transplanted rls40 cells was exposed to three subsequent courses of polychemotherapy in chop regimen ( figure 1 , scheme 2 ) . on day 7 after tumor transplantation mice were exposed to chop ( the 1st course ) ; the 2nd and the 3rd courses were done with the interval of 7 days . a group of mice with rls40 without exposure to chop and a group of mice without tumor but exposed to chop were used as controls . this experimental model represents the real picture in clinical practice in patients undergoing multiple chemotherapies and allows us to study the effect of both multiple courses of polychemotherapy and deepening mdr of tumor onto hepatotoxicity . levels of expression of the genes involved in the formation of multidrug resistance were estimated to confirm the enhancement of mdr of tumor after multiple chemotherapies . as expected after the treatment both at scheme 1 and scheme 2 the increase in expression of these genes was observed . at baseline rls40 cells were characterized by high expression levels of mdr1a , mdr1b , and p53 genes and low expression level of bcl-2 gene ( figure 2 ) that correlated with the data obtained earlier [ 13 , 15 ] . the treatment under scheme 1 resulted in 1.3- and 2.2-fold increases of the expression levels of mdr1b and bcl-2 genes , respectively , ( figure 2(a ) , right panel , and figure 2(c ) , left panel ) . the treatment under the scheme 2 caused 2.8- and 2-fold increases of the expression of these genes , respectively , ( figure 2(b ) , right panel , and figure 2(d ) , left panel ) . however , the treatment under scheme 1 and scheme 2 differently affected the expression level of p53 gene ( figures 2(c ) and 2(d ) , right panels ) . after the treatment under scheme 1 after two and four courses of polychemotherapy the expression levels of p53 gene in rls40 cells increased 1.2- and 1.6-fold , respectively , in comparison with the baseline . after the treatment under scheme 2 after the 1st and the 2nd courses a 4.4-fold drop of the expression level of p53 gene was seen in comparison with the baseline , but after the 3rd course gene expression it was slightly increased again . neither the treatment under scheme 1 nor the treatment under scheme 2 has an effect on the expression level of mdr1a gene ( figures 2(a ) and 2(b ) , left panels ) , which was in consistent with earlier data that this gene poorly responded to the treatment [ 13 , 15 ] . alteration in the expression of mdr1b , bcl-2 , and p53 genes after the treatment that has been observed both at the artificial experimental conditions and at real - life situation of mdr status enhanced due to multiple chemotherapies in hematological patients which indicates not only the strengthening of mdr , but also abnormalities of the apoptosis mechanism [ 2628 ] . as the p - glycoprotein level on the surface membrane of tumor cells displays mdr of tumor [ 2931 ] we estimated pgp representation on the surface of the tumor before and after the treatment . it is seen that about 30% of rls40 cells were initially pgp positive ( pgp+ ) ( figure 3(a ) ) . as expected multiple chemotherapies resulted in the significant increase in pgp level on the membrane of tumor cells . the treatment under scheme 1 caused the significant increase in the number of pgp+ cells in tumor : from 30% at baseline to 55.5 1.5% after the 1st course and to 88.2 1.5% after the 4th course of chop ( figure 3(b ) ) . the treatment under scheme 2 also resulted in the increase in pgp level on the membrane of tumor cell : after the 1st and the 2nd courses of chop the number of pgp+ cells was similar ( 60.9 0.5% and 63.3 0.5% , resp . ) , but after the 3rd the number of pgp+ cells significantly increased up to 85.9 1% . dynamics of tumor growth in mice exposed to the chop treatment according to scheme 1 and scheme 2 were approximately the same regardless of the treatment regimen ( figure 4 ) . reliable inhibition of rls40 tumor growth occurred upon the treatment both with scheme 1 and scheme 2 , though rls40 is not entirely regressed ( figures 4(a ) and 4(b ) ) . the inhibition of tumor growth depends on the number of treatment courses : after the 1st course of chop at scheme 1 the inhibition of tumor growth was 36.6% , after the 2nd 58.7% ( figure 4(a ) ) . the 3rd and the 4th courses of chop did not result in more pronounced inhibition of tumor growth : the inhibition rates were 50.5% and 45.5% , respectively , ( figure 4(a ) ) . after the treatment under scheme 2 , reasonably similar effects were observed independent of the number of chop courses and the inhibition of tumor growth was approximately 50% . ( figure 4(b ) ) . in the serum of mice bearing rls40 without any treatment the increase in ifn- ( up to 200 pg / ml ) and il-6 ( up to 460 pg / ml ) in comparison with healthy animals was observed . two courses of chop treatment resulted in a strong activation of inflammatory response that should be considered as a negative event and an increase of interferons was regarded as a positive immunomodulatory activity . we observed statistically significant 8-fold increase in il-1 ( up to 240 pg / ml ) , 2-fold increase in il-6 ( up to 830 pg / ml ) , 20-fold increase in tnf- ( up to 110 pg / ml ) , 7.5-fold increase in inf- ( up to 1500 pg / ml ) , and 9-fold increase in inf- ( up to 90 pg / ml ) . several factors can contribute to hepatotoxicity including endogenous intoxication caused by primary tumor node , metastatic infiltration of the liver , polychemotherapy itself and induced cytokine storm , and enhanced mdr of tumor . since the basic idea of our work was the assumption that the upregulation of mdr of the tumor negatively affects the liver , we evaluated morphological changes and regeneration capacity of the liver depending on the number of the polychemotherapy courses affected the liver and the enhancement of mdr of the tumor . diffuse and focal infiltration of the liver by tumor cells was detected in tumor - bearing mice both without any treatment and subjected to the cytostatic treatment independently from the experimental scheme . volume density of metastasis in the liver on day 17 after tumor transplantation reached 21 4% in the control group and 18 3% after the treatment under scheme 1 but this difference was statistically insignificant . in the case of scheme 2 hepatic lymphoma infiltration progressively increased and reached up to 30 5% of the entire liver parenchyma to day 24 after tumor transplantation both in groups after treatment and without it . upon the development of rls40 without any treatment metastatic infiltration of the liver by tumor cells was accompanied with severe destructive changes increased with tumor progression : from 19 0.9% ( on day 8 after transplantation ) to 54.2 1.3% ( on day 21 after transplantation ) . at the initial phase of tumor development alterative changes in the liver tissue were predominantly presented by degeneration of hepatocytes ( 11.4 0.7% degeneration , 7.6 0.7% necrosis ) . at a later date necrotic changes of the liver parenchyma were predominated ( 19.8 1% degeneration , 34.4 1.3% necrosis ) . the numerical density of binuclear hepatocyte decreased 1.5-fold ( from 0.85 0.15 to 0.55 0.1 ) , that shows a decline in the regeneration of liver tissue during tumor progression . in the early stage of tumor progression ( day 8 after transplantation ) in the group of animals with rls40 subjected to chop treatment a lesser pronounced infiltration of the liver parenchyma by tumor cells was identified , than in the control group ( tumor - bearing animals without treatment ) . however , the volume density of destructive changes increased 1.5-fold in comparison with the control ( 28.8 1% versus 19 0.9% on day 8 after transplantation ) . at the latter stage of tumor progression ( day 21 after tumor transplantation ) in the same group of animals the volume density of alterative changes represented mainly by necrosis of the liver parenchyma continued to increase , but in a lesser extent in comparison with the control ( tumor - bearing mice without any treatment ; 51.2 0.8% versus 54.2 the numerical density of binuclear hepatocytes changed insignificantly ( from 0.7 0.2 to 0.5 0.1 ) . comparison of the morphological changes developed in the liver to one and the same date at sequential passages of the tumor cells according to the scheme 1 without treatment revealed a 1.8-fold increase in necrosis in the liver parenchyma after the 4th passage in comparison with the 1st passage of the tumor without treatment , while the level of degeneration in the liver parenchyma remained unchanged ( table 1 ) . the numerical density of binuclear hepatocyte decreased 2-fold ( from 1 0.2 to 0.5 0.1 ) after the 4th passage in comparison with the 1st passage . comparison of the morphological changes developed in the liver to one and the same day after tumor transplantation and chop treatment at sequential passages of the tumor cells according to the scheme 1 revealed changes in the proportion of destructive changes in the liver of experimental animals from one passage to another . after the 1st passage ( one course of chop for the tumor and one for the liver ) the percentage of degenerative and necrotic cells was similar . while after the 4th passage ( four courses of chop for the tumor and only one for the liver ) the volume density of necrotic changes in the liver increased 1.3-fold and the volume density of degenerative change decreased 1.4-fold in comparison with the 1st passage of the tumor , which attests to the possible transformation of degeneration into necrosis ( table 1 ) . the numerical density of binuclear hepatocytes decreased 1.8-fold after the 4th passage in comparison with the 1st one ( from 0.9 0.1 to 0.5 0.1 ) . after three sequential courses of chop treatment ( scheme 2 ) both in animals with transplanted tumors and without tumors severe destructive changes in the liver parenchyma were revealed , but the dynamics of these changes in experimental groups were quite different . on day 1 after the 1st course of chop in healthy mice the volume density of destructive changes in liver parenchyma consisting predominantly of degenerative hepatocytes was 50.8 2.3% ; on day 3 and day 7 these values decreased 1.3- and 1.8-fold , respectively , ( table 2 ) . on day 1 and day 3 both after the 2nd and the 3rd courses of chop the percentage of destructive changes in liver parenchyma did not differ significantly from the value after the 1st course of chop ; however , on day 7 both after the 2nd and the 3rd courses of chop these values increased 1.7- and 1.8-fold , respectively , as compared to the 1st course of chop administered to healthy mice ( table 2 ) . on day 1 and day 3 after the 1st course of chop under scheme 2 in mice with rls40 volume densities of destructive changes in liver parenchyma were 53.8 3% and 39.8 2% , respectively . these changes were presented predominantly by degenerative hepatocytes and did not differ significantly from healthy mice exposed to chemotherapy at the same regimen . on day 7 after the 1st course of chop in tumor - bearing mice the percentage of destructive changes in liver parenchyma increased 1.6-fold in comparison with the healthy mice exposed to the same treatment . after remaining courses of chop destructive changes in liver tissue of tumor - bearing mice continued to increase progressively and as a result amounted to 85% of liver parenchyma ( table 2 ) . to evaluate the toxic effect of multiple chemotherapies and tumor with advanced mdr on the regeneration capacity of the liver the numerical density of binuclear hepatocytes was measured in the groups of experimental animals subjected to the treatment under scheme 2 with or without tumor ( figure 5 ) . maximal number of binuclear hepatocytes is observed on 1 - 2 days after any damage ( alcohol , chemotherapy , ccl4 , lps ) . hepatocyte degeneration can turn into either necrosis or regenerate and become normal hepatocytes due to the potential of binuclear hepatocytes . for all groups of animals the bell - shaped kinetics of numerical density of binuclear hepatocytes was observed : no effect on day 1 , the maximal regeneration burst was observed on day 3 , and decrease in numerical density was observed on day 7 after polychemotherapy . in the group of healthy animals subjected to polychemotherapy the regeneration capacity did not depend on the number of chop courses ( 1.55 0.3 , 1.6 0.2 , and 1.9 0.2 at the day 3 after the 1st , the 2nd , and the 3rd courses of chop , resp . ) . however , in the group of animals bearing rls40 each subsequent course of chop resulted in the progressive decrease in regeneration capacity of the liver : the numerical density of binuclear hepatocytes was 1.6 0.3 , 1.2 0.2 , and 0.7 0.15 at the day 3 after the 1st , the 2nd , and the 3rd courses of chemotherapy ( figure 5 ) . volume density of metastasis in the liver reached 30% at the end of the experiment and also contributed to the drop of regeneration capacity of the liver . during the rls40 passaging without treatment ( scheme 1 , control group ) the numerical density of binuclear hepatocytes did not change between the passages and was 1 0.15 ( day 14 after tumor transplantation ) . during the rls40 passaging with polychemotherapy ( scheme 1 , experimental group ) the numerical density of binuclear hepatocytes also did not change between the passages and was 0.8 0.1 ( day 14 after tumor transplantation , day 7 after treatment ) . the obtained data point to combined ( synergic ) toxic effects of the endogenous intoxication caused by primary tumor node , metastatic infiltration of the liver , multiple chemotherapies , and upregulated mdr of the tumor onto the liver that results in the significant destructive changes in the liver tissue and substantial reduction of the regenerative capacity of the liver . it was shown that the liver tissue of healthy mice without any treatment is characterized by pgp expression : the percentage of hepatocytes with pgp 2 + and pgp 3 + was about 40% of the entire population of liver cells . after three subsequent courses of chop ( scheme 2 ) in healthy mice the increase in pgp expression on hepatocyte membrane was detected : after the 1st course of chop the total percentage of hepatocytes with pgp 2 + and pgp 3 + was 55.8 0.5% , after the 2nd course 58.3 0.5% , and after the 3rd course 63 0.7% . therefore , several courses of chemotherapy in healthy mice resulted in a gradual and slow increase in pgp expression in the liver tissue . in contrast to healthy mice three courses of chop in tumor - bearing mice ( scheme 2 ) caused a rapid and more pronounced increase in pgp expression : after each course of chop the percentage of hepatocytes with pgp 2 + and pgp 3 + was about 70% of the entire population of liver cells . after the treatment of tumor - bearing animals under scheme 1 ( four courses of chop for the tumor and only one for the liver ) the increase in hepatocyte percentage with pgp 2 + and pgp 3 + was also detected . after the 1st tumor passage with the following cytostatic treatment percentage of hepatocyte with pgp 2 + and pgp 3 + was 57.8 0.45% , after the 2nd 59.8 0.5% , after the 3rd 63.3 0.5% , and after the 4th 64.9 0.3% ( figure 6 ) . therefore , despite the fact that the liver is exposed to only one course of chemotherapy , the upregulation of mdr of the tumor makes a contribution to the increase in pgp expression in the liver tissue after cytostatic influence . progress in antitumor therapy of hematological malignances achieved over the past decades resulted in an increase in the amount of complete remission , progression - free survival , and recovery in some cases . these results are due to the use of effective chemotherapy protocols based on the decrease in intervals between chemotherapy courses , increase in cytostatic dose , and introduction of additional chemotherapeutic agents into the protocol . however , the high toxicity of multiple courses of chemotherapy and multidrug resistance of tumor cells usually typical for hematological malignancies even at the baseline are important obstacles significantly reducing the efficiency of the treatment . the potential of antitumor chemotherapy is often limited by hepatotoxicity of cytotoxic drugs undergoing biotransformation in the liver [ 11 , 12 ] . the risk of toxic complications due to high concentrations of cytostatic metabolites is increased by tumor intoxication and metastatic lesion of the liver . toxic damage of the liver impedes the long - term treatment , reduces the efficiency of cytostatic therapy , and requires constant correction by the hepatotropic drugs in the intervals between the treatment courses . a number of factors may contribute to the toxic effects of chemotherapy ( genetically predisposed individual features of drug metabolism , the presence of comorbidity , and other factors affecting the detoxication function of the liver ) [ 36 , 37 ] . p - glycoprotein - mediated multidrug resistance of tumor may be one of these factors due to the increasing burden of cytostatics onto the liver through the drug pumping out of tumor cells . herein , we studied the combined effect of polychemotherapy ( chop protocol ) and lymphosarcoma rls40 characterized by enhanced mdr onto the liver of experimental animals using two treatment schemes . scheme 1 is an artificial and has no analogues in clinical practice . according to scheme 1 four courses of chop for tumor were completed , but only one was applied for the liver . scheme 2 reflects a real - life situation , where tumor - bearing mice were exposed to three subsequent courses of chop , so both the liver and the tumor have three chop courses . in the case of rls40 tumor model several factors can contribute to hepatotoxicity : endogenous intoxication caused by primary tumor node , metastatic infiltration of the liver , polychemotherapy itself and induced cytokine storm , and enhanced mdr of the tumor . we attempted to evaluate the contribution of the multiple chemotherapies and the strengthened mdr of the tumor to hepatotoxicity as well as the combined effect of the multiple chemotherapies and the tumor with upregulated mdr on the liver . as it was expected the enhancement of mdr of the tumor at the treatment under scheme 1 and scheme 2 multiple chemotherapies cause an additional increase in mdr1b and bcl-2 gene expression in rls40 cells that makes the tumor less sensitive to cytostatic treatment [ 27 , 38 ] . we also confirmed that multiple chemotherapies in different regimens caused the significant increase in pgp expressions on the membrane of tumor cells up to 90% of the entire tumor tissue . we evaluated the destructive changes and regeneration capacity of the liver that reflect the degree of hepatic dysfunction . morphological analysis of the liver of tumor - bearing mice both without treatment and after chop treatment revealed its diffuse and focal infiltration by tumor cells , which by itself is a liver damaging factor . at the time of tumor development without any treatment infiltration of liver tissue by tumor cells was accompanied by severe destructive changes due to both the tumor invasion and to mediated influence of the growing tumor on the host by endogenous intoxication . the livers of mice with rls40 subjected to chop are characterized by an increase in the total destructive changes , occupying more than 80% of liver parenchyma , in comparison with healthy animals without tumors subjected to chop treatment and tumor - bearing animals without any treatment . differences in the destructive changes in the liver confirm that multiple chemotherapies and tumor with upregulated mdr have a synergistic damaging effect on the liver . one of the most important characteristics of the liver is its regeneration capacity , the morphological expression of which is the numerical density of binuclear hepatocytes . chemotherapy in the absence of tumor virtually does not affect the numerical density of binuclear hepatocytes after triple exposure ( scheme 2 ) , which points to the maintenance of regeneration capacity of the liver . but the strengthening of mdr of rls40 after each subsequent course of chemotherapy ( scheme 2 ) together with metastasis progression reduces the numerical density of binuclear hepatocytes and impairs the liver regeneration capacity . treatment under scheme 1 also leads to the decrease in the regeneration capacity of the liver , but less significantly . together with the pressure of multiple chop , hepatic lymphoma infiltration and the enhancement of mdr of the tumor one of the reasons for decline of the regeneration capacity of the liver may be the synergic rising of toxic burden . it takes place because of the strengthening of multidrug resistance of the tumor that considering the large tumor mass relative to body weight leads to increased concentrations of cytostatics due to their force pumping out of tumor cells . components of chop treatment are released into the bloodstream and as a result the liver does not handle the metabolic burden . although the level of pgp in the liver increases it is not sufficient to protect hepatocytes which is confirmed by an increase in destructive changes . additionally the increase in necrotizing sites within the tumor also contributes into the toxic burden on the liver . therefore , hepatotoxic effects may become stronger with the progression of mdr of the tumor . the liver tissue itself has rich system of membrane transporters , proteins involved in the absorption , metabolism , distribution , and excretion of xenobiotics [ 40 , 41 ] , providing the maintenance and restoration of its own liver structure and this function is partly mediated by pgp [ 4244 ] . a number of chemicals , including cytotoxic drugs used in multiple dosing regimens , may cause an alteration in p - glycoprotein expression in different tissues ( liver , kidney , and intestine ) and lead to a change in drug pharmacokinetics [ 4547 ] . in our study we showed that exposure of healthy animals to chop in full regimen ( under scheme 2 , three courses ) caused a slow and gradual increase in p - glycoprotein expression on the membrane of hepatocytes . at the same time exposure of animals with rls40 to chop under scheme 2 irrespective of our data also revealed a small but reliable increase in pgp expression in the liver tissue of animals having one chop course onto the liver and four chop courses onto the tumor ( scheme 1 ) in comparison with animals having one chop course onto the liver and one chop course onto the tumor . such alteration in the expression of transporter proteins may represent a compensatory mechanism after the toxic effect of the chemotherapy onto the liver enhanced by the upregulated mdr of the tumor . the increase of the concentration of transporters in the membrane of hepatocytes may reduce the accumulation of potentially toxic chemicals [ 4951 ] . but in the used experimental models it was not sufficient to protect hepatocytes from further damage , what we observed as an increase in destructive changes . our data suggest that mdr status of a tumor may be one of the damaging factors , which contributes to toxic liver burden during polychemotherapy . progression of multidrug resistance of the tumor negatively affected the liver of the host and caused strong hepatotoxic effects manifested by the accumulation of degenerative changes of hepatocytes and the reduction in regeneration capacity of the liver . these data suggest that mdr status of the tumor at baseline may contribute to toxic liver burden and serve as a negative predictor parameter of hepatic dysfunction . the toxic effects of multiple chemotherapies onto the liver are accelerated by strengthening mdr of the tumor that should be taken into account when developing therapeutic regimens of malignancies with upregulated mdr . in this context , it is relevant to develop approaches for overcoming or reducing unfavorable toxic effects caused by polychemotherapy in combination with the upregulated mdr of the tumor . this can be achieved through the development of individual treatment protocols with detection of mdr status of the tumor prior to therapy and overcoming mdr using modern molecular gene - targeted approaches .

antitumor therapy of hematological malignancies is impeded due to the high toxicity of polychemotherapy toward liver and increasing multiple drug resistance ( mdr ) of tumor cells under the pressure of polychemotherapy . these two problems can augment each other and significantly reduce the efficiency of antineoplastic therapy . we studied the combined effect of polychemotherapy and upregulated mdr of lymphosarcoma rls40 onto the liver of experimental mice using two treatment schemes . scheme 1 is artificial : the tumor was subjected to four courses of polychemotherapy while the liver of the tumor - bearing mice was exposed to only one . this was achieved by threefold tumor retransplantation taken from animals subjected to chemotherapy into intact animals . scheme 2 displays real - life status of patients with mdr malignancies : both the tumor and the liver of tumor - bearing mice were subjected to three sequential courses of polychemotherapy . our data show that the strengthening of mdr phenotype of rls40 under polychemotherapy and toxic pressure of polychemotherapy itself has a synergistic damaging effect on the liver that is expressed in the accumulation of destructive changes in the liver tissue , the reduction of the regeneration capacity of the liver , and increasing of pgp expression on the surface of hepatocytes .

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

PMC4848045

the twentieth century witnessed a technology revolution in medicine and health through instrumentation , computer and information and communication technologies . this revolution has continued in the twenty - first century , with smart cross- and trans - disciplinary technologies and innovations directly impacting medical practice and healthcare delivery , which , in turn , have redefined the relationship between patients and healthcare providers . technology innovations and globalization have brought the world together as one global community where developing and developed economies have become more dependent and well connected than in previous times . as the overall life expectancy across the globe has increased , the global community is now facing new challenges of improving quality of life and healthcare at an affordable cost . while the exponentially rising cost of healthcare , defined as total healthcare expenditure as a percentage of a nation s gross domestic product ( gdp ) , is a critical priority challenge in developed nations , providing minimal quality healthcare to all , specifically large and sparsely distributed communities living in rural areas , is the most vital challenge to developing nations . a necessary component of affordable global healthcare is point - of - care ( poc ) healthcare technologies , and developing poc technologies will require continuous evolution of innovation in smart and portable bio - sensor , computing , information and communication technologies . a strategic study at the national institutes of health has noted ( http:// report.nih.gov/nihfactsheets/viewfactsheet.aspx?csid=112 ) : with the development of miniaturized devices and wireless communication , the way in which doctors care for patients will change dramatically and the role patients take in their own healthcare will increase . healthcare will become more personalized through tailoring of interventions to individual patients . though the challenges of providing high - quality healthcare in developing countries are different than those in developed countries , they share a common goal : to provide access to health monitoring and assessment technologies to people with limited or no healthcare facilities , or with geographically distant or difficult to physically access facilities . while developed countries may find poc technologies an effective means for reducing healthcare costs and improving efficiency , poc technologies are critical in the provision of diagnostic and monitoring healthcare needs in countries with large populations or rural areas . the developing countries in the eastern part of the globe , which account for more than two - thirds of the population of the world , face the basic challenge of providing minimal healthcare to all people living in adverse geographical or economic constraints , and also monitoring critical infectious diseases such as hiv / aids , tb , malaria , etc . given the rapidly aging populations in both developed and developing nations , it is now more critical than ever to develop collaborative synergies to explore poc health monitoring , assessment and therapeutic technologies to significantly impact global healthcare for the well - being of a healthy society . however , the implementation of poc healthcare technologies towards a tangible clinical impact poses formidable challenges in educating users . not only must users and local support staff learn about technology usage , measurement techniques , data communication and compliance , patients and family members must undergo a behavioral change to understand and accept new roles and responsibilities in keeping themselves , family members , and others healthy . the concept of self - care must be a major flagship in this education process . poc healthcare technologies ( including sensor- and biomarkers - based poc diagnostic technologies ; therapeutic and rehabilitation devices ; and information and communication technology ( ict ) with mhealth , ehealth , and health monitoring with poc decision support systems ) will directly impact patients , support staff , community center workers , and nurses , among others . all of these users will have to become comfortable , to varying degrees , with technology usage and local decision - making . in addition , physicians and business managers will also need to become conversant in the broad spectrum of data integration , mining and interpretation . the embs conference on point - of - care healthcare technologies invited scientific researchers and innovators , industry leaders , clinical experts , and policy makers from the usa , hong kong , china , japan , europe and india as plenary , keynote and panel speakers . this white paper was developed out of those presentations , as well as panel discussions , data presented , and comments made during breakout sessions , using the following multi - step methodology . the conference s first day featured plenary and keynote talks by leaders and representatives from government agencies including roderic pettigrew , md , phd , institute director , national institute of biomedical imaging and bioengineering , national institutes of health ( usa ) ; senior leaders from global industries including ibm life sciences and medtronic ( usa and india ) ; experts from clinical healthcare facilities including the cardiology department , kyushu university hospital ( japan ) and apollo hospital ( india ) ; and biomedical researchers and innovators . these talks were followed by a panel discussion led by representatives from stakeholder groups from all over the globe . both the plenary and keynote talks and the expert panel discussed current and future challenges in global healthcare . after these interactive panel discussions with over 176 researchers and stakeholders , the following steps were implemented to systematically collect insights from the various discussions generated by the conference program , including two additional panel discussions and three breakout sessions . conference attendees represented a rich spectrum of clinical practitioners , industry experts and entrepreneurs , academic researchers , and policy makers ( table 1).table 1percentage of conference attendees representing major stakeholder groups.stakeholder representation typepercentageclinical practitioners27%industry representatives and entrepreneurs23%academic researchers and students35%policy makers , federal and funding agency representatives15% this paper represents the summary of discussions , data and comments made during the conference events . 1.a questionnaire was circulated after the first panel discussion to all attendees requesting them to identify specific issues for discussion during the breakout sessions at the conference . these questions were categorized for three breakout sessions.2.three breakout sessions were held with detailed discussions on the following topics : a.breakout session 1 : priority point - of - care healthcare technology areas and global collaborationsb.breakout session 2 : patient participation and role : expectation and challengesc.breakout session 3 : pocht implementation strategies and compliance : challenges and future trends3.breakout discussions were summarized by the session leaders in consultation with designated note - takers who transcribed the sessions and presented to the entire group of conference attendees in the panel discussion 2 : reports from breakout sessions : future trends and follow - up . the discussions were summarized further in the raw data.4.the speakers from panel discussion 2 session formed a committee of authors and prepared a report that was transformed into the content of this paper . the paper went through three iterations to help ensure that it was an accurate record of the conference and that all points of view were included . a questionnaire was circulated after the first panel discussion to all attendees requesting them to identify specific issues for discussion during the breakout sessions at the conference . three breakout sessions were held with detailed discussions on the following topics : a.breakout session 1 : priority point - of - care healthcare technology areas and global collaborationsb.breakout session 2 : patient participation and role : expectation and challengesc.breakout session 3 : pocht implementation strategies and compliance : challenges and future trends breakout session 1 : priority point - of - care healthcare technology areas and global collaborations breakout session 2 : patient participation and role : expectation and challenges breakout session 3 : pocht implementation strategies and compliance : challenges and future trends breakout discussions were summarized by the session leaders in consultation with designated note - takers who transcribed the sessions and presented to the entire group of conference attendees in the panel discussion 2 : reports from breakout sessions : future trends and follow - up . the speakers from panel discussion 2 session formed a committee of authors and prepared a report that was transformed into the content of this paper . the paper went through three iterations to help ensure that it was an accurate record of the conference and that all points of view were included . , such technologies do not exist in isolation and must be integrated into and adopted by new or existing health service delivery models , be supported by sound business cases , and show demonstrated levels of improvement to patient health outcomes in terms of such metrics as quality - adjusted life years ( qalys ) as a means of quantifying disease burden . development , deployment and compliance issues related to affordable global healthcare have to be critically examined towards the creation of sound business models for their effective implementation such that they can be sustained with an economic impact . what is striking about the concept of pocht is its near universality of potential application . there is benefit to the management and treatment of most diseases hence priorities mirror the prevalence of disease in each country . there is applicability to both wealthy and developing countries with cross - fertilization of design back and forth among all nations , with both simple and advanced technologies being of use to all . both rural and urban settings would benefit greatly from poc technologies . perhaps most striking is the potential for widespread and localized screening for disease and health conditions . a simple vision of the technology might include : ( a)increasingly low - cost diagnostic tests , including those modified from existing methods through the use of novel , low - cost micro - molecular - biochemistry technology;(b)inexpensive device - based imaging and first - level analysis ( e.g. , smartphones capable of pulse oximetry , blood pressure , ekg recording and analysis or image recognition of skin disease);(c)smart device / smartphone / computer communication of data to regional or central health centers;(d)communication back to the personal care site for treatment or feedback;(e)ability to engage sophisticated healthcare treatment and diagnostic information , either by accessing human experts or clinical decision support systems . increasingly low - cost diagnostic tests , including those modified from existing methods through the use of novel , low - cost micro - molecular - biochemistry technology ; inexpensive device - based imaging and first - level analysis ( e.g. , smartphones capable of pulse oximetry , blood pressure , ekg recording and analysis or image recognition of skin disease ) ; smart device / smartphone / computer communication of data to regional or central health centers ; communication back to the personal care site for treatment or feedback ; ability to engage sophisticated healthcare treatment and diagnostic information , either by accessing human experts or clinical decision support systems . within this broad framework there are many health areas that would potentially be impacted . some of the global needs and challenges include : ( a)chronic disease management and monitoring : here , the major impact would come from the major chronic diseases , especially cardiovascular , pulmonary , neurological , geriatric , and early detection of the onset of and complications from diabetes.(b)therapeutic intervention : rehabilitation medicine and therapy would be greatly assisted by the ability to locally monitor patient progress , condition , and exercise or other health management programs . in rural areas where transport to a rehabilitation facility is prohibitive , the same logic applies in developed countries where system cost is an over - riding issue.(c)prenatal monitoring : special mention is made of the impact potential for pocht in perinatal care ; this would include routine physiological monitoring of hemoglobin , urine protein and blood pressure to detect problems at an early stage . pocht could lead to major reductions in maternal mortality rate.(d)preventative medicine through behavioral change : there is a great potential for increased feedback to the individual of key health indicators . one can imagine poor , rural areas where the introduction of paramedical personnel bringing more information coupled with simple monitoring measures of health ( blood pressure , heart rate , blood physiology ) would have significant impact on people s behavior and lives . at the other extreme , pocht technologies are already in the hands of wealthy , educated people in the form of smartphone applications for monitoring exercise there will be a continuing and rapid growth of monitoring capability for this population that already wants to change its behavior . aggressive development either private , profit - making or government could bring these behavior - changing technologies to ever - wider populations , .(e)medical information : the ability to collect medical and healthcare information locally and aggregate centrally has strong implications for epidemiology and for planning the availability of medical supplies and resources ( supply - chain management ) . a form of this already exists in the rfid of equipment for inventory within hospitals . chronic disease management and monitoring : here , the major impact would come from the major chronic diseases , especially cardiovascular , pulmonary , neurological , geriatric , and early detection of the onset of and complications from diabetes . therapeutic intervention : rehabilitation medicine and therapy would be greatly assisted by the ability to locally monitor patient progress , condition , and exercise or other health management programs . in rural areas where transport to a rehabilitation facility is prohibitive , the same logic applies in developed countries where system cost is an over - riding issue . prenatal monitoring : special mention is made of the impact potential for pocht in perinatal care ; this would include routine physiological monitoring of hemoglobin , urine protein and blood pressure to detect problems at an early stage . preventative medicine through behavioral change : there is a great potential for increased feedback to the individual of key health indicators . one can imagine poor , rural areas where the introduction of paramedical personnel bringing more information coupled with simple monitoring measures of health ( blood pressure , heart rate , blood physiology ) would have significant impact on people s behavior and lives . at the other extreme , pocht technologies are already in the hands of wealthy , educated people in the form of smartphone applications for monitoring exercise there will be a continuing and rapid growth of monitoring capability for this population that already wants to change its behavior . aggressive development either private , profit - making or government could bring these behavior - changing technologies to ever - wider populations , . medical information : the ability to collect medical and healthcare information locally and aggregate centrally has strong implications for epidemiology and for planning the availability of medical supplies and resources ( supply - chain management ) . a form of this already exists in the rfid of equipment for inventory within hospitals . there is strong consensus that several technologies are already having a huge impact on society at large , with impacts on healthcare to follow . ( i)perhaps ubiquitous is the smartphone capable of a number of diagnostic measurements , including ekg / heart rate , breathing rate , blood pressure , blood oxygen saturation , image recognition for skin disease(ii)low cost blood chemistry sensors , ranging from the more straightforward ( blood glucose ) to the highly sophisticated ( detectors for dengue fever , on chip pcr)(iii)reduced - cost versions of historically expensive technologies an example is ultrasound machines whose cost now permits wide distribution(b)communications and computational technology : smartphones put more computer power in the hands of a common person than a computer engineer dreamed of 50 years ago . the development of these technologies is practically for free as other markets are driving the cost and availability faster than the medical applications would . the goal is to harness these technologies , not develop them.(c)modeling and simulation from physiology to behavior : as more point - of - care data become available , there will be increasing need for models of increasing complexity to assist in analysis . an example is modeling the daily activity patterns of the elderly to predict medical problems . more generally , the ability to use data to anticipate individual problems is a key component of personalized medicine.(d)automated decision - making and support : we have now seen more than a decade of on - line medical diagnostic information services , which will continue to grow in sophistication . there is tremendous potential for exploiting the spectacular medical research knowledge base , advancing the practice of evidence - based medicine on a world wide scale . the initial cost is very high , but the delivery cost is very low given the communications technology . this technology can scale for the use of the expert , the general practitioner , the nurse , and the paramedical professional.(e)accessible data structures : the delivery of the benefits of pocth will be greatly assisted by enhanced accessibility of data to the broader medical community . there are a variety of competing issues to achieving this goal , including security and privacy of data , interoperability of data structures , and ownership of data . ( i)perhaps ubiquitous is the smartphone capable of a number of diagnostic measurements , including ekg / heart rate , breathing rate , blood pressure , blood oxygen saturation , image recognition for skin disease(ii)low cost blood chemistry sensors , ranging from the more straightforward ( blood glucose ) to the highly sophisticated ( detectors for dengue fever , on chip pcr)(iii)reduced - cost versions of historically expensive technologies an example is ultrasound machines whose cost now permits wide distribution perhaps ubiquitous is the smartphone capable of a number of diagnostic measurements , including ekg / heart rate , breathing rate , blood pressure , blood oxygen saturation , image recognition for skin disease low cost blood chemistry sensors , ranging from the more straightforward ( blood glucose ) to the highly sophisticated ( detectors for dengue fever , on chip pcr ) reduced - cost versions of historically expensive technologies an example is ultrasound machines whose cost now permits wide distribution communications and computational technology : smartphones put more computer power in the hands of a common person than a computer engineer dreamed of 50 years ago . the development of these technologies is practically for free as other markets are driving the cost and availability faster than the medical applications would . modeling and simulation from physiology to behavior : as more point - of - care data become available , there will be increasing need for models of increasing complexity to assist in analysis . an example is modeling the daily activity patterns of the elderly to predict medical problems . more generally , the ability to use data to anticipate individual problems is a key component of personalized medicine . automated decision - making and support : we have now seen more than a decade of on - line medical diagnostic information services , which will continue to grow in sophistication . there is tremendous potential for exploiting the spectacular medical research knowledge base , advancing the practice of evidence - based medicine on a world wide scale . the initial cost is very high , but the delivery cost is very low given the communications technology . this technology can scale for the use of the expert , the general practitioner , the nurse , and the paramedical professional . accessible data structures : the delivery of the benefits of pocth will be greatly assisted by enhanced accessibility of data to the broader medical community . there are a variety of competing issues to achieving this goal , including security and privacy of data , interoperability of data structures , and ownership of data . others will need support from governments , either directly through education as well as research and development funds , or indirectly by incentivizing the private sector . ( a)networking of practitioners : the encouragement of a growing leadership community is essential to the promulgation of pocht practice . this should come in a variety of forms : publication in scholarly journals ; publication in the medical trade press ; workshops sponsored by national medical organizations ; conferences ; social media ; and moocs ( massive online open courseware ; http://www.mooc-list.com).(b)medical and paramedical education : pocht makes possible task shifting at all levels , putting expert knowledge in the hands of generalists and general medical knowledge in the hands of paramedical personnel . there will be tremendous need for education at all levels.(c)design for all : this maxim implies that designing for the cost constraints of the developing world impacts medical products in the developed world , and vice versa . this is especially relevant in the fast - developing pocht area and a world in which innovation travels the globe nearly instantly.(d)entrepreneurship and financial support : pocht is an especially open area for entrepreneurial development , as is evidenced by rapidly developing smartphone applications , tremendous spinoffs from biomolecular science , and strong support for global health from a variety of western institutions and foundations . still , much more is needed from all sectors of society.(e)standards and harmonization : this refers to a host of properties that enable data access , storage , transmission and use ; standards for measurement and reporting of patient information ; and access to expert information and to one s own records . networking of practitioners : the encouragement of a growing leadership community is essential to the promulgation of pocht practice . this should come in a variety of forms : publication in scholarly journals ; publication in the medical trade press ; workshops sponsored by national medical organizations ; conferences ; social media ; and moocs ( massive online open courseware ; http://www.mooc-list.com ) . medical and paramedical education : pocht makes possible task shifting at all levels , putting expert knowledge in the hands of generalists and general medical knowledge in the hands of paramedical personnel . design for all : this maxim implies that designing for the cost constraints of the developing world impacts medical products in the developed world , and vice versa . this is especially relevant in the fast - developing pocht area and a world in which innovation travels the globe nearly instantly . entrepreneurship and financial support : pocht is an especially open area for entrepreneurial development , as is evidenced by rapidly developing smartphone applications , tremendous spinoffs from biomolecular science , and strong support for global health from a variety of western institutions and foundations . standards and harmonization : this refers to a host of properties that enable data access , storage , transmission and use ; standards for measurement and reporting of patient information ; and access to expert information and to one s own records . clinical impact can be realized in preventive , therapeutic and surveillance areas . in the preventive area , for example , a decline in the incidence of myocardial infarction after good control of diabetes , cholesterol and blood pressure will take years to realize . for example , the apollo hospital in india uses cardio biomarkers in early diagnosis of myocardial infarction and early transfer to the icu . it can be readily shown that poct has a favorable impact on the hospital s cardiac mortality . similarly , use of antenatal pocht kits in diagnosing early eclampsia or anemia can be easily shown to reduce maternal mortality rate . surveillance pocht is similar to preventive pocht where the actual clinical impact may be appreciable only after a few years . the major challenge would be in data acquisition , communication , and patient compliance , specifically in developing economies . the major challenge is in task shifting : shift of preliminary or pre - diagnosis responsibility at the point - of - care . other challenges include developing pathways to provide : ( a)evidence - based personalized care;(b)patient centered precision medicine;(c)preventive healthcare;(d)short - term feedback with long - term benefits;(e)cost - effectiveness , specifically in mass screening ( e.g. , diabetes and hypertension monitoring);(f)easy and intuitive devices and decision support systems . evidence - based personalized care ; patient centered precision medicine ; preventive healthcare ; short - term feedback with long - term benefits ; cost - effectiveness , specifically in mass screening ( e.g. , diabetes and hypertension monitoring ) ; easy and intuitive devices and decision support systems . the medical devices and informational communication technologies at the point - of - care also face other societal challenges for acceptance and implementation . these issues include diverse demographic and cultural backgrounds , and differences in needs due to geographical , social , and economic factors . while it makes sense to be patient - centric , empowering ptaients with point - of - care decision support systems raises concerns about legal liability and potential reduction in sensitivity . it is not yet clear how poct can be used to integrate family members and physicians into one effective healthcare team . though the challenges of providing high - quality healthcare in developing countries are different than those in developed countries , there is a common goal to provide access to health monitoring and assessment technologies to people with limited or no healthcare facilities . large developing nations with fast economic growth such as india and china are committed to providing healthcare to all , but they still face major challenges in assessing rural and underserved patients healthcare needs and in providing them with timely quality healthcare at the point - of - care . this is also a critical challenge in metropolitan areas where the hospital and primary healthcare facilities are overburdened . major challenging priority areas where healthcare is most needed for people in india , as identified by who , are : hypertension and cardiac deaths , diabetes , cancers , women s health ( specifically child birth ) and infant mortality , and neurological disorders . use of advanced point - of - care technologies , including wearable sensors , biomarkers , and mobile - communications - based education , along with health data collection and analysis , is a viable and affordable way to reach larger populations for better healthcare and compliance . the impact on quality of life from better outreach , increased affordability of quality primary care , and increased patient compliance would be tremendously positive . it is important to consider perspectives from all stakeholders , including patients , industry , healthcare providers , payers , policy makers and society as a whole ( fig . , pocht technologies must address critical issues in patient privacy , data integrity and security on one hand , but also infrastructure support and policies on the other hand . these infrastructure and policy supports will need to enable data and decision - support systems for all stakeholder groups in healthcare : patients at point - of - care , clinicians at healthcare facilities , and payers at the insurance or provider levels . all of these stakeholder groups must be willing to accept the challenges and potential errors that might impact the sensitivity and specificity of healthcare processes . in addition , clinicians and community members , such as community health workers , will need to help patients accept responsibility and accountability for their own health as they are empowered to address their health issues , illnesses , and preventive care in order to remain healthy . thus patients , families , and communities will need to learn to think in new ways about the use of technology and how to responsibly monitor their health when applying poc healthcare technologies and decision - support systems . the transformational change in defining new roles and responsibilities for patients has to come through patient - centered design , local solutions , and sensitive patient - education on poc benefits , such as significant cost - savings , reduced hospitalizations , and better personalized and preventive healthcare . the outreach to patients for poc education and monitoring may incorporate specific innovative methods such as smartphone ict and incentives from healthcare providers , payers and government . for example , many developing and developed countries are encouraging continuous monitoring of blood glucose and hypertension through innovative programs including educational camps and free or heavily discounted prices of monitoring systems . of course , the economic aspects of such initiatives in developing countries can cause critical challenges in implementation involving social and political issues . recent studies on the economic impact of using poc technologies in economically challenged nations presented in references summarize that these socio - economic and infrastructure challenges may at first overshadow the benefits of poc technologies . yet , in the long run , poc technologies with smart ict hold tremendous hope for sustainably managing resources and improving healthcare delivery in rural and developing nations . while the developed world provides quality healthcare , growing expense is a universal concern , perhaps most acutely in the usa with the most expensive healthcare system in the world . mutual collaborative efforts and networking in both developed and developing markets will allow opportunities to learn the best technologies , research , innovation , and best practices that lead to better and more affordable global solutions for quality healthcare . a collaborative meeting jointly hosted by nih and nsf with invited technology , industry , and business leaders in washington , dc to discuss resource development strategies ( e.g. , workshops ) on future collaborative research , clinical translation , and implementation infrastructure issues . develop a web portal supported by nih / nsf / embs as the resource for collaborative networking , research and development , research dissemination , workshop , and clinical translation information in pocht with active participation from academia , industry , corporate sector , hospitals , and healthcare provider facilities . an annual meeting with leaders and all stakeholders in technology innovation , development , commercialization , implementation , and clinical acceptance ( including reimbursement ) sectors should be pursued with an emphasis on the development , follow - up , and evaluation of strategic goals and global milestones .

this paper summarizes the panel discussion at the ieee engineering in medicine and biology point - of - care healthcare technology conference ( pocht 2013 ) held in bangalore india from jan 1618 , 2013 . modern medicine has witnessed interdisciplinary technology innovations in healthcare with a continuous growth in life expectancy across the globe . however , there is also a growing global concern on the affordability of rapidly rising healthcare costs . to provide quality healthcare at reasonable costs , there has to be a convergence of preventive , personalized , and precision medicine with the help of technology innovations across the entire spectrum of point - of - care ( poc ) to critical care at hospitals . the first ieee embs special topic pocht conference held in bangalore , india provided an international forum with clinicians , healthcare providers , industry experts , innovators , researchers , and students to define clinical needs and technology solutions toward commercialization and translation to clinical applications across different environments and infrastructures . this paper presents a summary of discussions that took place during the keynote presentations , panel discussions , and breakout sessions on needs , challenges , and technology innovations in poc technologies toward improving global healthcare . also presented is an overview of challenges and trends in developing and developed economies with respect to priority clinical needs , technology innovations in medical devices , translational engineering , information and communication technologies , infrastructure support , and patient and clinician acceptance of poc healthcare technologies .

Introduction Procedures and Methods Point-of-Care Healthcare Technologies (POCHT): A Paradigm Shift in Affordable Quality Global Healthcare POCHT Challenges: Technology Innovation and Priority Needs POC Healthcare Technologies Strategic Enablers POCHT Clinical Impact Concluding Remarks and Recommendations

PMC3361849

functional hallucinations are an unusual form of perceptual disorder , in which hallucinations are triggered by a stimulus in the same modality , and co - occur with it . for example , a patient may report hearing voices criticizing him every time he hears the sound of a rotating fan , and which stop when the fan is not running . their exact significance is unknown , but they have been reported in schizophrenia and other functional psychotic disorders . the treatment of positive symptoms of schizophrenia , including hallucinations , generally involves the use of antipsychotics . the following is the report of a patient diagnosed with schizophrenia , with persistent functional hallucinosis , who responded to the addition of sodium valproate . a single man , aged 30 , employed in a factory , presented to our outpatient department , in 2007 , with two years continuous illness , characterized by persistent auditory hallucinations , secondary delusions of reference , social withdrawal , and impaired occupational functioning . he also reported obsessive doubts about routine activities , such as closing doors or taps , and a compulsion to check whether he had done these properly , despite knowing that this was unnecessary . he was diagnosed to have paranoid schizophrenia and obsessive - compulsive disorder , and was treated with risperidone ( titrated up to 8 mg / day ) and fluoxetine ( titrated up to 80 mg / day ) . on the above - mentioned medications , he improved significantly , and was able to return to his job . his job involved frequent contact with machinery and motors . whenever he heard these machines running he found these distressing , and this led him to frequently avoid his work or leave it incomplete . he did not hear these voices at any other time , and did not report any recurrence of his other symptoms . his body weight was 62 kg . due to financial difficulties ( the patient was receiving the above medications free of cost from the hospital ) and his overall good response to risperidone therefore , he was given a trial of adjunctive sodium valproate , which was freely available in the hospital and had some evidence of efficacy in reducing positive symptoms , as an adjunct to antipsychotics . after obtaining the patient 's consent , sodium valproate was initiated at a dose of 600 mg / day , and gradually increased by 200 mg every week based on his response and adverse effects . at 1000 mg / day of sodium valproate , the patient reported a significant reduction in his hallucinations , and he felt that he could carry out most of his work . hence , valproate was further increased at the same rate , up to 1800 mg / day . at this dose , the patient reported feeling near - normal , and was in line for a promotion at his workplace . however , he developed significant postural tremors , which interfered with his ability to work , and valproate was reduced to 1700 mg / day . at this dose , he reported that the voices had decreased by 75% , and he scored between 2 and 3 ( mild symptoms ) on item p3 ( hallucinations ) of the positive and negative symptom scale for schizophrenia . he has remained on valproate 1700 mg / day , along with risperidone 8 mg / day and fluoxetine 80 mg / day , for the past three months , and has remained stable . his functional hallucinations still occur from time to time at work , but he is less bothered by them and does not experience any impairment . the management of patients with schizophrenia , who have a single persistent symptom , is a challenging situation . evidence - based treatments , such as changes in medication or cognitive - behavioral therapy , may not always be feasible . in this patient , time and economic constraints made it unlikely that the patient would comply with either . hence , an alternative that would be safe , affordable , and with some evidence of effectiveness was required . on account of the rarity of functional hallucinations however , a single case report suggests that carbamazepine may be helpful . in psychiatry , a cochrane review suggests that it may be useful in reducing aggression and tardive dyskinesia , but has little effect on other symptoms . however , one trial has suggested that adjunctive divalproex reduces positive symptoms , and a case series found the addition of valproic acid useful in difficult - to - treat schizophrenia patients . the effect of adjunctive valproate in schizophrenia is probably not due to the elevation of risperidone levels . valproate acts primarily through gamma - amino butyric acid ( gaba)-ergic mechanisms , which may modulate the actions of dopamine . alternately , it may act through an epigenetic mechanism involving the demethylation of relevant sections of the gaba - related genes . finally , given the paroxysmal , event - triggered nature of the patient 's hallucinations , it is possible that valproate 's ability to block sodium channels in a use - dependent fashion , may have contributed to symptom amelioration , as carbamazepine did in the earlier case . although it can not be recommended in all patients , this case suggests that valproate may be beneficial in selected cases , particularly as an add - on to antipsychotics .

functional hallucinations are a rare phenomenon , wherein hallucinations are triggered by a stimulus in the same modality , and co - occur with it . although hallucinations in schizophrenia are normally treated using antipsychotics , not all patients respond to them . the following is the report of a patient with paranoid schizophrenia who experienced persistent functional hallucinations , triggered by the sound of machines in his factory , in the absence of other psychotic symptoms . these occurred despite adequate doses of risperidone , which had controlled his other symptoms . the addition of sodium valproate , titrated up to 1700 mg / day based on response and tolerability , resulted in a marked improvement in this phenomenon and enabled him to return to work . the implications and possible mechanisms of the patient 's response are discussed .

INTRODUCTION CASE REPORT DISCUSSION

PMC2712956

integrins are heterodimeric membrane receptors that mediate cell adhesion to ecm or to another cell ( hynes , 2002 ) . ligand binding provides transmembrane mechanical links to transmit forces from extracellular contacts to intracellular structures ( e.g. , the cytoskeleton ) and signals for a wide variety of cellular processes . for example , binding of integrin 51 to fibronectin ( fn ) plays an important role in fibroblast spreading and motility ( akiyama et al . , 1989 ) , t cell migration ( shimizu et al . , 1990 ) , osteoblastic and myogenic proliferation , and differentiation ( garcia et al . , 1999 ) . integrins are often expressed on cells in an inactive , low affinity state with slow on rate and/or fast off rate for ligand binding , but they can be activated to high affinity states with fast on rates and/or slow off rates ( hynes , 2002 ; luo et al . , 2007 ) . activation can also be triggered by divalent cations and by binding of activating mabs to cell surface or purified integrins ( humphries , 2000 ) . affinity regulation in integrins is thought to be allosteric ( hynes , 2002 ; luo et al . , 2007 ) . the overall shape of the integrin molecule is that of a large head region ( the headpiece ) supported on two long legs . in the low affinity state , integrin legs were observed to have bent knees , which were straightened upon activation ( xiong et al . , 2001 ; takagi et al . , therefore , it seems reasonable to speculate that tensile force applied via a bound ligand may induce unbending of the knees , thereby converting the integrin from a low affinity state with short bond lifetimes to a high affinity state with long bond lifetimes ( chigaev et al . , 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) . indeed , recent studies have provided experimental support for force - enhanced integrin function ( astrof et al . , 2006 ; woolf et al . , 2007 ; friedland et al . , 2009 ) . also , steered molecular dynamics simulations have suggested how force activation of integrin might occur ( jin et al . , 2004 ; puklin - faucher et al . , 2006 ) . the counterintuitive behavior in which force prolongs bond lifetimes is called catch bonds , which is in contrast to the ordinary slip bond behavior where force shortens bond lifetimes ( dembo et al . , 1988 ) . catch bonds have been demonstrated in interactions between selectins and ligands ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , actin and myosin ( guo and guilford , 2006 ) , fimh receptor and mannose ( yakovenko et al . , 2008 ) , and glycoprotein ib ( gpib ) and von willebrand factor ( vwf ; however , loading rate - dependent rupture force measurements with atomic force microscopy ( afm ) force - ramp experiments and dynamic force spectroscopy analysis have not revealed catch bonds for integrin ligand interactions ( li et al . , 2004 ) . using afm force - clamp experiments , we measured lifetimes of single fn51 bonds at forces as low as 4 pn . catch bonds were observed in < 30 pn . changing divalent cations altered lifetimes in the same force range and binding of a mab that reports the extended conformation of 51 , but the catch bonds remained . truncating the 51 leg regions further prolonged bond lifetimes and abolished the response of the lifetime versus force curve to divalent cations ; nevertheless , catch bonds were still observed , showing that leg extension is not required for catch bonds . two activating mabs that bind the headpiece shift catch bonds to a lower force range , indicating that force - induced activation of the headpiece is involved in catch bond formation . thus , catch bond formation appears to involve force - assisted activation of the headpiece but not integrin extension . using afm ( fig . 1 a ) , we measured interactions between an fn fragment ( fniii710 [ fn fragment encompassing the 710th type iii repeats ] ) and a recombinant 51 consisting of either the full extracellular portion ( 51-fc ) or only the headpiece ( truncated 51 [ tr51]-fc ) fused with fc ( coe et al . , 2001 ; we have shown previously that the recombinant proteins closely mimic the function and conformational regulation of the native integrin ( coe et al . , 2001 ; mould et al . , 2002 , 2003a , b , 2005 ) . to avoid uncontrolled adsorption of integrin via different domains , which could produce variable results and restrict conformational changes , tr51-fc was captured by fab of an anti - fc mab ( gg-7 ) preadsorbed on a polystyrene petri dish ( fig . the petri dish was driven by a piezoelectric translator ( pzt ) to contact fniii710 adsorbed on a cantilever tip ( fig . 1 d ) of adhesion was detected upon retraction of the pzt , which was then clamped at a desired force for lifetime measurement ( fig . a laser is focused on the back of cantilever end and bounced onto a photodiode to measure force on the tip that bends the cantilever . a petri dish is mounted on a pzt with an integrated capacitive sensor to allow for distance control with subnanometer precision . molecules depicted represent a composite of several adsorbed or captured on the cantilever tip or the petri dish . extended and bent 51-fc , depicted as heterodimers of an ( light blue ) and a ( red ) subunit fused to an fc ( yellow ) , and tr51-fc , consisting of the propeller ( teal ) and thigh ( dark green ) domains of the subunit as well as the a ( pink ) , hybrid ( dark blue ) , and plexin / semaphorin / integrin ( tan ) domains of the subunit fused to an fc , were captured by an anti - fc mab ( gg-7 ) fab ( blue ) preadsorbed on the petri dish . in some experiments , these were replaced by an anti - fn mab ( hfn7.1 ; brown ) captured by an anti mouse fc antibody ( orange ) preadsorbed on the petri dish . fniii710 ( purple ) was adsorbed on the cantilever tip . in some control experiments , the petri dish was moved up by the pzt to contact the cantilever tip ( blue trace ) , immediately retracted to a small distance ( green trace ) from the tip to reduce nonspecific adhesion , held at this distance for 0.5 s to allow for bond formation ( brown trace ) , and retracted away from the tip to detect adhesion ( red trace ) . the trace illustrates a contact cycle without binding where the retraction curve returned to zero force upon petri dish retraction . the color codes are the same as those in c , which illustrates a contact cycle with binding and lifetime measurement . once the pulling force reached a preset value ( indicated ) , a feedback loop was triggered to keep the cantilever deflection at the set point . the lifetime at that force ( indicated ) was measured until bond failure , signified by the springing back of the cantilever to the level of zero mean force . the bending configurations of the cantilever are depicted with colors matching the corresponding colors of the traces in c and d. experiments were performed in 1 mm ca plus 1 mm mg ( ca / mg ) , 2 mm mg plus 2 mm egta ( mg / egta ) , or 2 mm mn in the absence or presence of blocking or activating mabs or a competing peptide . at the same gg-7coating concentrations , binding of 51-fc ( fig . 2 c ) to fniii710 became progressively more frequent when the cation condition was changed from ca / mg to mg / egta and to mn . this was corroborated by flow cytometry data showing that mg / egta and mn increased the staining of 51-fc coated beads by a mab that reports unbending of the knees ( 9eg7 ; fig . binding was specific , as it was abolished by addition to the solution of mab hfn7.1 , which blocked the integrin - binding site in fniii710 or an rgd - containing peptide cyclo(-grgdsp ) , which competed with the rgd loop in fniii710 for 51 binding ( fig . ( < 20% ) , a requirement for most adhesion events to be mediated by single bonds , progressively lower gg-7coating concentrations were used for experiments in ca / mg , mg / egta , and mn to compensate for the progressively higher binding affinities of tr51-fc ( fig . binding was abrogated when the cantilever tips coated with fniii710 were switched to those coated with bsa or when the petri dishes functionalized with integrins were switched to those that were not functionalized ( fig . frequencies of adhesion between 10 g / ml fniii710 adsorbed on cantilever tips and 10 g / ml 51-fc ( a ) or tr51-fc ( c ) captured by 15 or 100 g / ml gg-7 fab precoated on petri dishes in buffer containing the indicated cations without or with cyclo(-grgdsp ) or hfn7.1 in solution were enumerated in 100 tests for each spot and presented as mean sem of 35 spots . frequencies ( measured with the same protocol as in a and c ) of adhesion between 10 g / ml fniii710 coated on cantilever tips and 10 g / ml 51-fc ( b ) or tr51-fc ( d ) captured by gg-7 fab preadsorbed on petri dishes with indicated concentrations ( closed bars ) are compared with those between bsa - coated tips and tr51-fc functionalized petri dishes ( open bars ) and with those between fniii710-coated tips and gg-7coated petri dishes without incubating with tr51-fc ( hatched bars ) in the indicated cation conditions . mechanical regulation of fn51 dissociation was quantified by the force dependence of mean lifetimes of mostly single fniii71051 bonds measured in each of the three cation conditions ( fig . 3 , a c , circles ) . as force increased , lifetime first decreased to a minimum , then increased to a maximum , and decreased again , exhibiting a triphasic transition from slip bonds to catch bonds and then to slip bonds again . the first slip bond regimen was most clearly observed in ca / mg ( fig . the much less frequent nonspecific binding between bsa - coated cantilevers and polystyrene petri dishes functionalized with 51-fc contributed negligibly to these lifetime versus force curves , as most of these events were ruptured at forces < 20 pn ( fig . s2 a ) , and those that survived the ramping had very short lifetimes ( fig . ( a c ) plots of lifetime versus force of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) . ( d ) a qualitatively similar plot ( triangles ) of m51 reconstituted into lipid bilayer dissociating from fniii710-coated cantilever tips in mg / egta confirmed the catch bond observation . also plotted in a c is the lifetime versus force curve of fc dissociating from gg-7 ( squares ) . for b and c , the black and gray curves overlap at forces > 30 pn , indicating that measured lifetimes beyond 30 pn were caused by fc also shown in c is a lifetime versus force plot ( diamonds ) of 51-fc functionalized petri dish dissociating from bsa - coated cantilever tips measured in mn . ( e ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and 51-fc or between 51-fc and gg-7 . ( f ) schematic of the molecular arrangement for experiments that measured the capturing strength of the fc error bars indicate mean sem . to control for potential artifacts of the chimeric integrin that fuses the extracellular portions of the 5 and 1 chains with a human igg fc region ( coe et al . , 2001 s2 a ) of fniii710 to membrane 51 ( m51 ) purified from human smooth muscle cells reconstituted in glass - supported lipid bilayers in mg / egta . although the lifetimes were shorter , the force - dependent curve of m51 exhibited the same triphasic pattern ( fig . thus , catch bonds were observed at forces ranging from 1030 pn for fn interacting with 51-fc and m51 . because 51-fc was captured by preadsorbed gg-7 , rupture of the molecular complex might result from dissociation of the fn51 or fc gg-7 bond ( fig . we neglect potential detachment of fniii710 from the cantilever tip or of gg-7 from the petri dish because physioadsorption of proteins is generally much stronger than specific protein protein interactions ( rief et al . , 1997 ) . to determine the rupture loci , we overlaid the force - dependent lifetimes of directly adsorbed 51-fc interacting with gg-7 ( fig . 3 f ) on each panel of fig . 3 ( a c , squares ) . slip bonds were observed over the entire force range tested . the mean lifetimes at forces < 25 pn were much longer than those of fniii710 interacting with captured 51-fc , indicating that the observed catch bonds were characteristic of the fn51 bond rather than the fc however , at forces > 30 pn , the black circle curves in fig . 3 ( b and c ) became indistinguishable from the gray square curve , suggesting that the second slip bonds of the former curves result from dissociation of the fc gg-7 bond rather than the fn51 bond . because the dissociation rate of two bonds in series equals to the sum of the two individual dissociation rates , the lifetimes of these two bonds in series would have been significantly shorter than either bond measured separately if lifetime of the fn51-fc bond were comparable with the fc the fact that the lifetime versus force curves of the fn51-fc gg-7 and 51-fc gg-7 bonds were indistinguishable shows that the fn51 bond lifetimes in mg / egta or mn were substantially longer than the fc they might continue to behave as catch bonds at forces > 30 pn before ( and if ) it transitioned to slip bonds . however , the real differences of fn51 bond lifetimes between ca / mg and mg / egta or mn conditions may be much greater than those apparent in fig . 3 ( a c ) . for comparison , we also measured force - dependent lifetimes of 51 and fniii710 bound to their respective blocking mabs , p1d6 and hfn7.1 ( fig . 4 , schematics ) , which were also specific to the antigen antibody interactions ( fig . s2 , c and d ) . similar to the fc gg-7 interaction and consistent with other antibody antigen interactions characterized previously ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , slip bonds were observed in both cases ( fig . 4 ) as the mean lifetimes decreased monotonically with increasing force in the same range where the triphasic transitions between slip and catch bonds were observed for 51 interacting with its physiological ligand ( fig . 4 , compare a with b ) , revealing different interaction characteristics of the different antibody lifetimes of antibody antigen bonds . ( a and b ) plots of lifetime ( mean sem ) versus force of interactions between 51-fc functionalized petri dish and p1d6-coated cantilever tips ( a ) and between hfn7.1 captured by anti a large percentage of 51-fc appeared to exist in a bent conformation in ca / mg , but more of the integrin became extended in mg / egta and mn ( fig . yet in all three cation conditions , lifetimes at low forces ( < 10 pn ) were similarly short ( < 2 s ) . as force increased from 1030 pn , catch bonds were observed , but lifetimes were prolonged less by force in ca / mg ( fig . these data indicate that catch bonds may not result from a force - induced unbending of the integrin . to obtain more definitive evidence 3 ( a c ) using tr51-fc that contains only a five - domain headpiece of the integrin fused with fc ( coe et al . , 2001 ; mould et al . , 2002 ) . as anticipated , catch bonds were still observed , confirming that the integrin legs and the unbending conformational change were not required for the fn51 catch bonds ( fig . 5 , a c ) . interestingly , in the 1025-pn range , truncating the integrin leg regions enabled force to prolong lifetimes to a greater extent than the integrin with legs ( fig . 5 , the lifetime versus force curves in the catch bond regimen were quite similar for all three cation conditions ( compare fig . 5 , a c ) however , similar to the fniii71051 case , the force where the fniii710tr51 catch bonds might transition to slip bonds could not be determined , for the mean lifetimes of the fniii710tr51-fc gg-7 bond at a force > 25 pn in all three cation conditions ( fig . 5 , a 3 ) , indicating that these represented lifetimes of the fc gg-7 bond rather than the fniii710tr51-fc bond . a c ) plots of lifetime ( mean sem ) versus force of tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) . data from fig . the lifetime versus force curves of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares . ( d ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and tr51-fc or between tr51-fc and gg-7 . to explore the structural mechanism of the observed catch bonds , we measured fniii71051-fc bond lifetimes in mn in the presence of ts2/16 ( fig . 6 a ) or 12g10 ( fig . 6 b ) . 6 d , schematic ) to further activate integrins by shifting or stabilizing the position of the 1 helix known to be critical for integrin activation ( mould et al . both mabs left shifted the catch bond region , but ts2/16 yielded longer lifetimes at low forces ( fig . interestingly , ts2/16 also left shifted the fniii710tr51-fc catch bond region but did not prolong lifetimes as much as it did for the fniii71051-fc case ( fig . these data emphasize the importance of the a domain 1 helix and link the fniii710tr51 catch bonds to 51 activation . ( a c ) lifetime ( mean sem ) versus force plots ( circles ) of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( a ) or 12g10 ( b ) or tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( c ) . for comparison , data from fig . 3 c are replotted in a and b , and some of the data from fig . 5 c are replotted in c , where the lifetime versus force curves of tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares . ( d ) schematic of the molecular arrangement indicating binding sites for ts2/16 and 12g10 . catch bonds represent unusual , counterintuitive behaviors that have recently been observed in selectin ligand ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , actin myosin ( guo and guilford , 2006 ) , fimh mannose ( yakovenko et al . , 2008 ) , and gpibvwf ( yago et al . , 2008 ) interactions . based on the integrin structures and their conformational change models , it has been speculated that integrin ligand interactions may also behave as catch bonds ( chigaev et al . , 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ligand catch bonds ( zhang et al . , 2002 , 2004 ; li et al . , 2003 ) . in the previous work , force - ramp experiments were used to measure ramp rate - dependent rupture forces , which were analyzed by dynamic force spectroscopy , assuming that dissociation occurs along a single pathway and off rate increases exponentially with increasing force as modeled by bell ( 1978 ) , which precluded catch bonds . using afm force - clamp experiments , we observed triphasic force - dependent bond lifetimes that deviate from the bell model , demonstrating fn51 catch bonds ( figs . 3 , 5 , and 6 ) . it is known that integrin - mediated adhesion can be strengthened by integrin clustering or binding of multiple integrins cooperatively , which can be induced by force ( galbraith et al . , however , this is unlikely the mechanism for the observed catch bonds for the following reasons . first , ( tr)51-fc molecules were captured by gg-7 fab , which was unlikely to adsorb on petri dishes as clusters . nor was the fniii710 likely to adsorb on afm tips as clusters . without preclustering , without active cellular mechanisms to cluster the molecules , and without lateral mobility , it is unlikely that integrins bind in clusters in our experiments . second , using the same afm setup in the control experiments , we observed only slip bonds for the 51-fc gg-7 interaction was monomeric ( because fab was used ; fig . 3 f ) , 3 a , schematic ) , and fniii710hfn7.1anti mouse fc interaction might be multimeric ( because whole hfn7.1 and anti mouse fc were used ; fig . 3 b , schematic ) . third , we examined the correlation of ( or the lack thereof ) bond lifetime with molecular stiffness ( fig . we have previously shown that the stiffness of multiple bonds is multiple times of that of a single bond ( sarangapani , 2005 ) , which predicts a positive correlation between lifetime and stiffness if the longer lifetimes were caused by greater bond multiplicity . however , such correlation was not observed ( fig . s3 , a and b ) . to the contrary , the mean stiffness values for molecular complexes that had lifetimes clustered around 0.3 , 2.8 , and 10 these data ruled out higher multiplicity of bonds as the cause for longer lifetimes at high forces than the lifetimes at low forces in the catch bond regime . finally , we have previously shown that formation of dimeric bonds can not generate catch bonds if the corresponding monomeric interaction is a slip bond . the effects of dimeric bonds are to shift the lifetime versus force curve of the monomeric bond rightward toward doubling the force and upward toward doubling the lifetime ( marshall et al . , 2003 ) . our flow cytometry data suggest that more 51-fc became extended in mg / egta and mn than in ca / mg ( fig . s1 ) , which is consistent with previous flow cytometric ( humphries , 2000 ) , crystallographic ( xiong et al . , 2001 ) , and electron microscopic ( takagi et al . , 2002 ; zhu et al . , the affinity of fn51 binding was low in ca / mg but high in mg / egta or mn ( mould et al . , 1995 ) , which was manifested as different adhesion frequencies ( fig . 2 , a and c ) . at low forces ( < 10 pn ) , fn51 bonds dissociated rapidly ( lifetimes < 2 s ) in all three cation conditions ( fig . c ) , suggesting that mg / egta or mn increased the on rate for association but did not significantly impact the off rate for dissociation . these are consistent with our previous kinetic measurements of integrin l2 interacting from intercellular adhesion molecule 1 under these cations , which showed fast force - free off rates and a much greater responsiveness to cation conditions of on rates than off rates ( zhang et al . , 2005 ) . in previous tension - free studies , mn has often been considered to be able to activate integrin to a high affinity state for ligand binding ( mould et al . , 1995 ; takagi et al . , 2002 ) , which corresponds to the bond state at low forces in our experiment . then , force is able to further strengthen the bond , inducing new bond states previously not identified ( friedland et al . , 2009 ) . it has been suggested that force applied to a bent integrin may straighten it to an extended integrin , thereby giving rise to catch bonds ( chigaev et al . 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) . however , catch bonds were observed not only for fn interacting with 51 ( fig . c ) in three different cation conditions but also for fn interacting with tr51 that lack the leg regions ( fig . 5 , a c ) , which indicates that force - induced unbending is not a required conformational change for fn51 catch bonds . an essential feature of integrin activation is a swing of the subunit hybrid domain away from the subunit ( takagi et al . , 2002 ; , it has been proposed that , without a lateral force to separate the integrin legs , pulling on an extended integrin would prevent the hybrid domain from swinging outwards , thereby stabilizing the inactive conformation of the headpiece ( zhu et al . , 2008 ) . however , in our experiments , no lateral force was applied to separate the integrin legs , which were restrained . yet , pulling on 51 via a bound fn prolonged bond lifetimes at low forces , indicating that force activates rather than deactivates 51 . it is possible that , at least in our system , the bond strengthening caused by pulling on the 1 helix connection may outweigh the effects of force on hybrid domain movement . similar to the recombinant 51-fc fusion protein , native 51 purified from cell membrane and reconstituted into glass - supported lipid bilayers also formed catch bonds with fn in the same force range , although the apparent lifetimes were shorter ( fig . for the majority of the adhesion events , the shorter lifetimes may not be caused by extraction of 51 from the lipid bilayer because the running adhesion frequency remained stable in a large number of repeated contacts ( fig . s4 b ) , even when the same cantilever tip was used to contact the same lipid bilayer location , indicating that neither the tip nor the bilayer lost functionality over time ( chesla et al . , 1998 ) . however , it is possible that some very long lifetimes ( > 10 s ) could be cut short by extrapolating of integrin from the bilayer . in addition , more 51-fc might be in an extended conformation than m51 in the same cation condition , resulting in longer lifetimes for the catch bonds of fn with 51-fc than with m51 ( mould et al . , 2005 ) . as discussed in the preceding paragraph , in the bent conformation , although catch bonds were observed for both interactions of fn with 51-fc and m51 , the causes of their lifetime differences remain unclear and require further studies . binding of ts2/16 or 12g10 left shifted the fn tr51-fc catch bond region by prolonging lifetimes at low forces ( fig . both mabs bind at or near to the 1 helix of the a domain ( fig . 6 d ; mould et al . , 2002 ) , suggesting that this region could be important in regulating the catch bond behavior . in addition to the rgd - binding site at the a domain of the 1 subunit , the propeller of the 5 subunit may bind the synergy site at the fniii9 domain to strengthen the fn51 interaction ( garcia et al . , 2002 ; mould et al . , 2003b ; friedland et al . , 2009 ) . it has recently been proposed that the fn51 bond can be switched from a relaxed to the tensioned states in response to mechanical force through engaging the synergy site in fn ( friedland et al . , 2009 ) . multiple sites have also been proposed for ligand binding to integrin headpieces ( hynes , 2002 ; liddington and ginsberg , 2002 ; friedland et al . thus , fn51 catch bonds could also involve force - induced binding of multiple sites that strengthen the molecular complex . interestingly , the natural log ( number of measurements with a lifetime > t ) versus t plots for the fn51-fc bond exhibited multiple line segments ( fig . c ) , which are in contrast to the more linear plots of the 51-fc . the negative slope of a line reflects the off rate of the subpopulation of dissociation events associated with that line ( marshall et al . , 2003 ) . multiple line segments suggest multiple states of single fn51-fc bonds rather than multiple fn51-fc bonds because 51-fc were captured by gg-7 fab randomly and sparsely adsorbed and because the lifetimes were measured from single rupture events . similar multiple - bond states were observed for fimh mannose catch bonds ( thomas et al . , 2006 ) . these are different from the selectin ligand catch bonds that show single lines in such plots ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) instead of the aforementioned allosteric , multisite , and multistate models , a sliding rebinding model was proposed for selectin ligand ( lou et al . , 2006 ; lou and zhu , 2007 ) and gpibvwf ( yago et al . , 2008 ) these mechanisms are not mutually exclusive and may work together to give rise to integrin ligand catch bonds . they also transduce signals bidirectionally from inside out and from outside in across the cell membrane . such a dual role makes integrins prime candidates for force - sensing molecules in mechanotransduction ( schwartz and desimone , 2008 ) . catch bonds provide a physical mechanism for force sensing if different bond lifetimes correspond to different activation states that transduce distinct signals . the ability of integrin ligand bonds to be strengthened by force may be of great importance not only for leukocyte trafficking , which occurs under hydrodynamic forces , but also for the migration of many other cell types , which involves cyclic adhesion and detachment between the cell and the ecm . catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces at different times and locations when and where different adhesion strengths are desired . recombinant 51-fc and tr51-fc chimeras were generated by chol761h cells that were transiently transfected with cdna constructs encoding the human 5 ( fitzgerald et al . , 1987 ) and 1 ( coe et al . , 2001 ) subunits or tr5 and tr1 subunits ( coe et al . , 2001 ) . fniii710 with a biotin tag at the n terminus was produced using standard recombinant dna techniques ( petrie et al . , 2006 ) . anti-51blocking ( p1d6 ) and activating ( ts2/16 ) mabs were obtained from millipore , reporting mab ( 9eg7 ) was obtained from bd , and another activating mab ( 12g10 ) was obtained from abcam . the anti - fn mab ( hfn7.1 ) was obtained from developmental studies hybridoma bank . the anti human fc - capturing mab ( gg-7 ) was obtained from sigma - aldrich . the afm is a modification of a previously described system ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) it consists of a pzt on which a petri dish is directly mounted ( fig . 1 a ) . the pzt has a capacitive feedback control that gives subnanometer position resolution . a laser ( oz optics ) focused on the end of the cantilever ( tm microscopes ) is deflected onto a photodiode ( hamamatsu photonics ) to measure cantilever deflection , which is converted to force using the cantilever spring constant . spring constant ( 312 pn / nm ) for each cantilever was calibrated in situ using thermal fluctuation analysis ( hutter and bechhoefer , 1993 ) . a personal computer with a data acquisition board ( national instruments ) was used to control movements of the pzt and to collect signals from the photodiode . labview ( national instruments ) was used as the interface between the user and the data acquisition board . to measure the interaction of 51-fc or tr51-fc with fniii710 ( fig 4 a , schematic ) , cantilever tips were incubated with 1020 g / ml fniii710 or p1d6 overnight at 4c ( fig . 1 b ) . after rinsing with tbs ( 25 mm tris - hcl and 150 mm nacl , ph 7.4 ) , the cantilevers were incubated for 15 min at room temperature in tbs containing 1% bsa to block nonspecific adhesion . gg-7 was cleaved into fab and fc fragments by using the fab preparation kit following the manufacturer 's instructions ( thermo fisher scientific ) . 2 ) was adsorbed on a small spot on a petri dish by overnight incubation at 4c . to capture tr51-fc , the gg-7precoated petri dish was rinsed three times with tbs and incubated with 10 g / ml tr51-fc at the desired cation condition ( ca / mg , mg / egta , or mn ) for 30 min . the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . in some experiments , 10 g / ml hfn7.1 , 1 mg / ml cyclo(-grgdsp ) ( anaspec , inc . ) , or 10 g / ml ts2/16 or 12g10 ( fig . 6 d ) were added to the buffer . to measure the interaction of fniii710 with hfn7.1 ( fig . 4 b , schematic ) or 51-fc with gg-7 fab ( fig . 3 f ) , fniii710 or 51-fc was adsorbed on cantilevers and treated as described in the previous paragraph . 10 g / ml goat anti mouse fc polyclonal antibody or 20 g / ml fragmented gg-7 was adsorbed on a labeled spot on a petri dish overnight at 4c . to capture hfn7.1 , the petri dish was rinsed three times with tbs and incubated with 10 g / ml hfn7.1 for 30 min . the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . to measure the interaction of fniii710 with m51 , cantilevers were coated with streptavidin at 50 g / ml overnight at 4c and further functionalized by incubation with 10 l of 1 g / ml fniii710 for 15 min at room temperature . ( 1993 ) . in brief , vesicles were formed by hydrating a dried lipid film of 1,2-dimyristoyl - sn - glycero-3-phosphocholine and 1,2-dimyristoyl - sn - glycero-3-[phospho - rac-(1-glycerol ) ] ( avanti polar lipids , inc . ) in a 50:50 ratio with 0.1% triton x-100 ( thermo fisher scientific ) in tbs and mixed with m51 , resulting in a final concentration of 0.27 mg / ml ( 0.4 mm ) of lipid and 0.1 mg / ml of integrin . triton x-100 was removed by adsorption to biobeads sm-2 ( bio - rad laboratories , inc . ) at 37c for 4 h. the resulting lipid vesicle solution was stored under argon at 4c and used within several months . coverslips of 40 mm in diameter ( bioptechs ) were cleaned with a mixture of 70% 12 n sulfuric acid and 30% hydrogen peroxide by volume at 100c for 45 min , rinsed extensively with deionized water , and dried completely under an argon stream . the cleaned coverslip , which was used immediately , was placed in a petri dish , and a 4-l drop of m51-incorporated lipid vesicle solution was placed on the coverslip surface . after 20 min of incubation under a damp paper towel , the petri dish was filled with 5 ml tbs with 2 mm mg , 2 mm egta , and 1% bsa . the m51 bilayers that formed had low molecular densities to ensure their infrequent binding to the fniii710-coated cantilever tips , as required for measuring single bonds . the afm force - clamp experiments were performed by repeatedly bringing the petri dish in contact with the cantilever tip , then immediately retracting a small distance ( 05 nm ) , holding at that distance for 0.5 s to allow bond formation , and retracting again at a speed of 200 nm / s . by preventing the cantilever tip from compressing the petri dish during the time for molecular association , the nonspecific binding was greatly suppressed ( fig . 2 ; and fig . s2 , b the presence of adhesion was detected from the force - scan curves ( fig . 1 , c and d ) . a feedback system was used to clamp the force at a desired level to enable measurement of bond lifetime at constant force . approximately 50 lifetimes were measured in the full - force range ( 570 pn ) using a cantilever and a petri dish in each experiment . gg-7 interaction in 1 mm ca / mg condition , 13 experiments were performed to acquire 846 lifetimes at forces ranging from 570 pn . these were segregated into 12 force bins , each of which spanned 56 pn and contained at least 50 lifetimes . plots of natural log ( number of events with a lifetime > t ) versus t were exemplified for the fn51-fc 125 g ( 50 l ) streptavidin - coated magnetic beads ( thermo fisher scientific ) were incubated with gg-7 biotinylated using biotintag micro biotinylation kit ( sigma - aldrich ) in 1 ml pbs at a concentration of 5 g / ml for 30 min . 40 l beads were washed three times in tbs , incubated with 20 g / ml 51-fc in 200 ml tbs with desired cations for 30 min , again washed three times in tbs with appropriate cations , and incubated with 9eg7 at 10 g / ml for 30 min . 9eg7 was preconjugated with allophycocyanin using the lightning - link apc - xl conjugation kit following the manufacturer 's instructions ( innova bioscience ) . after washing three times with tbs containing the appropriate cations , s1 shows flow cytometry analysis of the expression of 9eg7 epitope by 51-fc in different cation conditions . fig .

binding of integrins to ligands provides anchorage and signals for the cell , making them prime candidates for mechanosensing molecules . how force regulates integrin ligand dissociation is unclear . we used atomic force microscopy to measure the force - dependent lifetimes of single bonds between a fibronectin fragment and an integrin 51-fc fusion protein or membrane 51 . force prolonged bond lifetimes in the 1030-pn range , a counterintuitive behavior called catch bonds . changing cations from ca2+/mg2 + to mg2+/egta and to mn2 + caused longer lifetime in the same 1030-pn catch bond region . a truncated 51 construct containing the headpiece but not the legs formed longer - lived catch bonds that were not affected by cation changes at forces < 30 pn . binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range . thus , catch bond formation appears to involve force - assisted activation of the headpiece but not integrin extension .

Introduction Results Discussion Materials and methods Online supplemental material

PMC3471022

skeletal muscle tissues are responsible for the provision of postural control , the coordination of excitation - contraction - relaxation cycles for voluntary movements , the integration of key metabolic and biochemical pathways , and the regulation of heat homeostasis . under normal physiological conditions , hence , supramolecular protein complexes with specialized functions , structures , and connections represent a major biochemical feature of muscle fibres . an excellent example of a large protein assembly present in skeletal muscle is the dystrophin - glycoprotein complex of the sarcolemma [ 15 ] . the crucial importance of the dystrophin - associated protein complex is exemplified by the pathophysiological fact that primary genetic abnormalities in the dystrophin gene result in progressive muscle wasting diseases , such as duchenne or becker muscular dystrophy [ 68 ] . in normal muscle , the dystrophin - glycoprotein complex provides a trans - sarcolemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin . the subsarcolemmal dystrophin matrix and the molecular connection between the basal lamina structure and the muscle interior is believed to prevent damage to the muscle surface from potential membrane - distorting forces during contraction - relaxation cycles . in x - linked muscular dystrophy , dystrophin deficiency results in a drastic reduction of sarcolemmal glycoproteins that triggers a loss of plasmalemmal integrity . muscle fibres are more susceptible to contraction - induced injury and their lateral transmission of force is impaired . cycles of sarcolemmal microrupturing and natural membrane repair mechanisms appear to cause the introduction of ca - leak channels that in turn elevate cytosolic ca - levels and disturb ca - fluxes through the sarcoplasmic reticulum in dystrophic fibres [ 1416 ] . interestingly , a recent study on the therapeutic effect of upregulating the intramuscular heat shock protein hsp72 to ameliorate the dystrophic phenotype revealed that the serca - type ca - atpase is dysfunctional in severely dystrophic muscle . these findings strongly indicate that impaired ca - homeostasis plays a key role in x - linked muscular dystrophy . however , it is not well understood how many molecular and cellular factors are involved in the overall process leading to the highly complex pathology of dystrophinopathy . thus , in order to determine the hierarchy of secondary pathobiochemical effects that render a dystrophic muscle more susceptible to necrosis , it is crucial to elucidate global alterations due to the disintegration of the dystrophin - glycoprotein complex . mass - spectrometry - based proteomics suggests itself as a suitable analytical tool for such large - scale and high - throughput approaches to study the effects of dystrophin deficiency . in contrast to hypothesis - based and targeted bioresearch , proteomics can be considered an unbiased and technology - driven approach for the comprehensive cataloging of entire protein complements [ 1921 ] . skeletal muscle proteomics in particular is concerned with the global identification and detailed cataloguing of the protein constituents of voluntary contractile fibres in health and disease [ 2224 ] . in the long term , comparative proteomics promises to be instrumental for the establishment of comprehensive biomarker signatures of myogenesis , muscle repair mechanisms , physiological adaptations and pathological changes , as well as the natural aging process . most gel electrophoresis - based proteomic studies use high - resolution two - dimensional gel electrophoresis in combination with advanced mass spectrometric analysis for the unequivocal identification of muscle proteins of interest [ 2729 ] . in the case of x - linked muscular dystrophy , a variety of mass spectrometric investigations have attempted to determine proteome - wide changes in dystrophin - deficient muscle tissue in order to establish a dystrophy - specific biomarker signature [ 30 , 31 ] . large - scale proteomic profiling studies have included investigations of serum [ 32 , 33 ] , cardiac muscle [ 34 , 35 ] , and various skeletal muscle tissues [ 16 , 3643 ] from the mdx mouse model of duchenne muscular dystrophy , as well as a proteomic analysis of dystrophic grmd dog skeletal muscle . although the findings from individual studies do not agree on the exact number and extent of protein alterations within the dystrophic muscle proteome , all investigations concur that dystrophin - deficient fibres exhibit a generally perturbed protein expression pattern . previous gel electrophoresis - based proteomic profiling studies of dystrophic samples have focused on the cytosolic fraction from 1 , 3 , and 6 months old hind limb muscle covering a pi range of 47 and using coomassie and silver - staining methods [ 36 , 37 ] , crude extracts from gastrocnemius muscle from 9 weeks old hind limb tissue covering a pi range of 310 and using stains - all labeling , preparations from 6 weeks old gastrocnemius muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , crude extracts from 9 weeks old diaphragm tissue covering a pi range of 310 and using hot coomassie staining , crude extracts from 9 weeks old diaphragm muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , crude extracts from 10 weeks old antisense oligomer - treated diaphragm tissue covering a pi range of 310 and using 2d - dige labeling , crude extracts from 9 weeks old extraocular muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , and crude extracts from aged diaphragm muscle covering a pi range of 310 and using fluorescence rubps labeling . in analogy to the above - outlined proteomic studies , this report has focused on aged tibialis anterior muscle from dystrophic mdx mice . the tibialis anterior is one of the most active lower leg muscles , which exhibits a relatively high degree of resistance to fatigue during periods of intense running , making it an interesting contractile system to study with respect to secondary effects of dystrophinopathy . in addition , previous experimental gene therapy studies have focused on mdx tibialis anterior muscle . labeling of proteins with fluorescent dyes has been extensively applied in proteomic investigations and we have used here fluorescent rubps staining for a comparative proteomic survey of dystrophic leg muscle from 8 weeks , 12 months , and 22 months old mdx mice . the densitometric analysis of two - dimensional gels , covering a pi range of 310 , in combination with mass spectrometry identified significant age - related changes in carbonic anhydrase , aldolase , electron transferring flavoprotein , pyruvate kinase , myosin , tropomyosin , and the small heat shock protein hsp27 in dystrophic mdx tibialis anterior muscle . materials and electrophoresis - grade chemicals for the proteomic analysis of muscle proteins were purchased from amersham biosciences / ge healthcare , little chalfont , buckinghamshire , uk . for protein digestion , sequencing grade - modified trypsin was obtained from promega ( madison , wi , usa ) . chemiluminescence substrate and primary antibodies were obtained from vision biosystems novocastra , newcastle upon tyne , uk ( mab ncl - b - dg to the dystrophin - associated glycoprotein -dystroglycan ) and abcam , cambridge , uk ( ab54913 to the ca3 isoform of carbonic anhydrase ; and ab12351 to the small heat shock protein hsp27 ) . all other chemicals used were of analytical grade and purchased from sigma chemical company , dorset , uk . the mdx mouse is an established model system of x - linked muscular dystrophy and widely used in basic research and for the evaluation of novel therapeutic options to treat diseases of progressive skeletal muscle wasting . the mdx mouse is a naturally occurring mutant that is missing the dp427 protein isoform of the membrane cytoskeletal protein dystrophin due to a point mutation in the dmd gene . in analogy to the etiology of patients suffering from duchenne muscular dystrophy , deficiency in full - length dystrophin results in a drastic reduction of all dystrophin - associated glycoproteins in mdx skeletal muscle , making it a suitable animal model for studying secondary pathobiochemical changes due to dystrophin deficiency . dystrophic tibialis anterior muscle from 8 weeks , 12 months , and 22 months old mdx mice and normal tissues from age - matched c57 mice were obtained from the bioresource unit of the university of bonn . mice were kept under standard conditions and all procedures were performed in accordance with german guidelines on the use of animals for scientific experiments . animals were sacrificed by cervical dislocation and muscle tissues quickly removed and quick - frozen in liquid nitrogen . for the mass spectrometry - based proteomic survey of aged mdx skeletal muscle tissue , tibialis anterior specimens were shipped to ireland on dry ice and stored at 80c prior to usage . in order to obtain muscle protein extracts , 4 dystrophic muscle specimens from each age group were pulverized by grinding tissue pieces in liquid nitrogen using a mortar and pestle . ground muscle powder was solubilized in lysis buffer with the ratio of 100 mg wet weight to 1 ml lysis buffer ( 7 m urea , 2 m thiourea , 4% chaps , 2% ipg buffer ph 310 , 2% ( w / v ) dtt ) . to prevent excess protein degradation , following gentle rocking for 30 minutes , suspensions were centrifuged at 4c for 20 min at 20,000 g and the protein concentration determined . for the separation of muscle proteins , standard two - dimensional gel electrophoresis was carried out by previously optimized methodology using first dimension isoelectric focusing with ph 310 strips , and second dimension slab gel electrophoresis with 500 g protein per gel . twelve slab gels were run in parallel at 0.5 w / gel for 60 min and then 15 w / gel until the blue dye front had disappeared from the bottom of the gel . postelectrophoretic staining for the total protein profile was performed with the fluorescent dye ruthenium ii tris bathophenanthroline disulfonate ( rubps ) . as described previously by rabilloud and colleagues , a stock solution of rubps dye was prepared . following washing twice for 5 min , gels were stained for 6 hours in 20% ( v / v ) ethanol containing 200 nm of ruthenium chelate . gels were re - equilibrated twice for 10 min in distilled water prior to imaging . fluorescently labelled proteins from aged mdx tibialis anterior muscle were visualised using a typhoon trio variable mode imager . gel analysis was performed with progenesis 2d analysis software and protein spots with significantly altered expression levels were identified by mass spectrometry . protein identification was performed with 2d protein spots from coomassie - stained pick gels , following counter - staining of rubps - labelled analytical gels . excised protein spots were treated by standardized in - gel tryptic digestion for the generation of representative peptide mixtures . excision , washing , destaining , and treatment with sequencing - grade trypsin were performed by a previously optimized method . further recovery was achieved by adding 30% acetonitrile/0.2% trifluoroacetic acid to the gel plugs for 10 min at 37c with gentle agitation . samples were dried through vacuum centrifugation and concentrated peptide fractions were then suspended in mass spectrometry - grade distilled water and 0.1% formic acid , spun down through spin filters and added to lc - ms vials for identification by ion trap lc - ms analysis . the mass spectrometric analysis of peptides was carried out with a model 6340 ion trap lc / ms apparatus from agilent technologies ( santa clara , ca , usa ) . separation of peptides was performed with a nanoflow aglient 1200 series system equipped with a zorbax 300sb c18 analytical reversed phase column using hplc - chip technology . mobile phases used were a : 0.1% formic acid , b : 50% acetonitrile and 0.1% formic acid . samples were loaded into the enrichment part of the chip at a capillary flow rate set to 4 l / min with a mix of solvent a and solvent b at a ratio of 19 : 1 . tryptic digests were eluted with a linear gradient of 5% to 70% solvent b over 6 min , 70% to 100% solvent b over 1 min , 100% to 5% over 1 min . a 5 min post - time of solvent a was used to remove any potential carry over . the capillary voltage was set to 2000 v. the flow and temperature of the drying gas were 4l / min and 300c , respectively . database searches were carried out with mascot ms / ms ion search ( matrix science , london , uk ; ncbi database , release 20100212 ) . all searches used mus musculus as taxonomic category and the following parameters : ( 1 ) two missed cleavages by trypsin , ( 2 ) mass tolerance of precursor ions 2.5 da and product ions 0.7 da , ( 3 ) carboxymethylated cysteins fixed modification , ( 4 ) oxidation of methionine as variable modifiaction , ( 5 ) percentage coverage was set at over 10% , and ( 6 ) at least 2 matched distinct peptides . all pi - values and molecular masses of identified proteins were compared to the relative position of their corresponding 2d spots on analytical slab gels . one - dimensional immunoblotting was employed to verify key findings from the proteomic profiling of aged mdx tibialis anterior muscle . gel electrophoretic separation and transfer was carried out with a mini - protean ii electrophoresis and transfer system from biorad laboratories ( hemel - hempstead , herts , uk ) . muscle proteins were transferred to nitrocellulose for 70 minutes at 100 v and at 4c . blocking of membranes was achieved with a milk protein solution ( 5% ( w / v ) fat - free milk powder in 0.9% ( w / v ) nacl , 50 mm sodium phosphate , ph 7.4 ) for 1 hour . nitrocellulose sheets were washed and then incubated for 1 hour with secondary peroxidase - conjugated antibodies , diluted in blocking solution . immunodecorated bands were visualized using chemiluminescence substrate ( roche diagnostics , mannheim , germany ) . prior to the proteomic analysis of differently aged dystrophic tibialis anterior muscle preparations , the protein complement from extracts of normal muscle samples was gel electrophoretically separated and key proteins identified by mass spectrometry . this procedure established a select number of reliable landmark protein spots of a typical proteomic muscle map for control purposes . figure 1 shows a representative fluorescent rubps - labelled gel with the electrophoretically separated protein spot pattern of normal mouse tibialis anterior muscle . major 2d protein spots were treated by in - gel digestion and the most abundant constituent of this area of the gel identified by mass spectrometry . table 1 lists the names of identified muscle marker proteins , their international accession number , pi - values , their relative molecular masses , number of matched peptide sequences , percentage sequence coverage , and mascot scores . identified proteins ranged in molecular mass from 17.1 kda ( spot 22 , myoglobin ) to 70.7 kda ( spot 2 , unknown protein ) , and covered a pi - range from pi 4.6 ( spot 20 , myosin light chain mlc3 ) to pi 8.7 ( spot 15 , fast troponin subunit tni ) . spots 1 to 22 represent major muscle - associated protein species with apparent molecular mass to isoelectric point ratios of 57 kda / pi 5.2 , 71 kda / pi 5.8 , 59 kda / pi 6.7 , 47 kda / pi 6.7 , 43 kda / pi 6.6 , 40 kda / pi 5.8 , 33 kda / pi 4.7 , 33 kda / pi 4.7 , 37 kda / pi 6.2 , 40 kda / pi 8.3 , 36 kda / pi 8.4 , 30 kda / pi 6.9 , 23 kda / pi 5.6 , 23 kda / pi 5.6 , 22 kda / pi 8.7 , 23 kda / pi 5.7 , 21 kda / pi 5.0 , 19 kda / pi 4.8 , 19 kda / pi 4.8 , 19 kda / pi 4.6 , 12 kda / pi 5.0 , and 17 kda / pi 7.1 , respectively ( figure 1 ) . electrospray ionization mass spectrometry identified these marker proteins as isoforms of mitochondrial atp synthase , pyruvate kinase , enolase , creatine kinase , actin , tropomyosin , malate dehydrogenase , aldolase , glyceraldehyde-3-phosphate dehydrogenase , carbonic anhydrase , triosephosphate isomerase , troponin , adenylate kinase , parvalbumin , myoglobin , and various myosin light chains ( table 1 ) . following the optimization and initial mass spectrometric identification of muscle marker proteins in normal mouse tibialis anterior muscle , fluorescence high - resolution two - dimensional gel electrophoresis was employed to detect potential differences in aging - related protein expression patterns in mdx tibialis anterior muscle . figure 2 summarizes analytical gels with 4 biological repeats of 8 weeks , 12 months , and 22 months old total mdx muscle extracts . panels ta mdx 1 to 4 , ta mdx 5 to 8 and ta mdx 9 to 12 represent 8 weeks , 12 months , and 22 months old muscle preparations , respectively . since the overall 2d spot patterns of normal versus dystrophic tibialis anterior muscle were relatively comparable , a detailed denitometric analysis was carried out in order to evaluate potential differences in individual protein species . densitometric scanning was performed with a typhoon trio variable imager and progenesis 2d analysis software was used to establish differential expression patterns during muscle aging . the detailed proteomic survey of dystrophic tibialis anterior revealed distinct age - related changes in 8 muscle protein species between 8 weeks and 22 months old total mdx muscle preparations . a representative fluorescent 2d master gel of mdx tibialis anterior muscle is shown in figure 3 . as compared to a recent study on senescent mdx diaphragm muscle , which showed drastic age - dependent changes in 11 proteins in this severely necrotic tissue , the more mildly affected mdx tibialis anterior muscle showed less pronounced proteome - wide changes during the aging process . this finding agrees with the differing pathology of mdx leg muscle versus mdx diaphragm muscle . skeletal muscle proteins that exhibited significant alterations in expression levels are marked by circles and are numbered 1 to 8 in the 2d gel representing the urea - soluble proteome from mdx tibialis anterior muscle . besides listing the names of identified proteins , their accession number , pi - values , their relative molecular masses , the number of matched peptide sequences , percentage sequence coverage , and ms / ms scores , this table also shows the fold change of individual proteins affected in dystrophin - deficient mdx tibialis anterior muscle during aging . proteins species with a changed concentration in mdx tibialis anterior muscle ranged in molecular mass from 23 kda ( heat shock protein hsp27 ) to 224 kda ( myosin 3 ) and covered a pi - range from pi 4.7 ( tropomyosin ) to pi 8.5 ( electron transferring flavoprotein ) . an increased abundance was shown for the ca3 isoform of carbonic anhydrase ( spots 1 and 4 ) , the glycolytic enzyme aldolase ( spot 2 ) , and electron transferring flavoprotein ( spot 3 ) . the key cytosolic enzyme pyruvate kinase ( spot 5 ) , myosin 3 ( spot 6 ) , tropomyosin ( spot 7 ) , and the molecular chaperone hsp27 ( spot 8) were found to be decreased in mdx tissue . following the mass spectrometric establishment of age - related changes in the urea - soluble mdx tibialis anterior muscle proteome , immunoblotting was used to investigate the concentration of the two most extensively changed new markers ca3 and hsp27 in normal versus dystrophic preparations . antibodies to the dystrophin - associated glycoprotein -dystroglycan ( -dg ) , which forms the main trans - sarcolemmal linker between the extracellular matrix and the cortical actin cytoskeleton in the fibre periphery , were employed to verify the dystrophic status of mdx tissue samples during aging . figure 4(a ) illustrates the drastic reduction of -dg in both 8 weeks and 22 months old mdx tibialis anterior muscle , which is characteristic of dystrophinopathy . equal loading of lanes was ensured by silver staining of gel electrophoretically separated protein preparations ( not shown ) . immunoblotting of young versus old muscle samples with antibodies to the ca3 isoform of carbonic anhydrase ( figure 4(b ) ) and the molecular chaperone hsp27 ( figure 4(c ) ) showed an increased abundance of the metabolic enzyme and a decreased concentration of the small heat shock protein in dystrophin - deficient muscle . thus , both the fibre type - specific protein ca3 and the stress protein hsp27 represent suitable candidate biomarkers of the dystrophic phenotype . duchenne muscular dystrophy is one of the most crippling neuromuscular disorders of childhood , therefore warranting detailed large - scale studies into the establishment of comprehensive biomarker signatures of dystrophinopathy [ 30 , 31 ] . in dystrophinopathy , the almost complete absence of the dp427 isoform of the membrane cytoskeletal protein dystrophin causes a drastic reduction of a large number of surface glycoproteins that in turn triggers a loss of sarcolemmal integrity . the mdx mouse is a widely used model system for the evaluation of novel treatment options to counter - act the symptoms of x - linked muscular dystrophy and basic biomedical research promises to provide the basis of evidence for the development of novel treatment regimes , such as stem cell therapy , myoblast transfer , or exon skipping therapy . previous proteomic studies have established a considerable number of novel biomarkers of secondary changes in dystrophin - deficient organisms . besides the cataloging of generally perturbed protein expression patterns , individual proteomic surveys of mdx muscles of differing subtype and age have demonstrated a drastically altered abundance of adenylate kinase isoform ak1 , the luminal ca - binding protein calsequestrin of the terminal cisternae [ 16 , 41 ] , the cytosolic ca - buffering element regucalcin , mitochondrial isocitrate dehydrogenase , and the muscle - specific molecular chaperone cvhsp [ 40 , 41 ] . a recent aging study of the severely dystrophic mdx diaphragm has demonstrated a drastic increase in the extracellular matrix proteins collagen and dermatopontin , the molecular chaperone b - crystallin , and the intermediate filament protein vimentin , suggesting increased accumulation of connective tissue , an enhanced cellular stress response and compensatory stabilization of the weakened membrane cytoskeleton in severely dystrophic muscle tissue . in the present report , proteomic profiling showed that during the natural aging of the moderately dystrophic tibialis anterior muscle a number of key skeletal muscle proteins change in abundance . we have studied aged mdx muscle , because dystrophic mouse muscle tissue progressively deteriorates with age and thus more closely resembles the neuromuscular pathology seen in duchenne patients . the age - related pathogenesis of mdx muscle is characterized by a drastic loss of myofibres and concomitant replacement by connective tissue [ 6264 ] , progressive motor weakness , the presence of branched fibres that trigger mechanical weakening of the muscle periphery , a reduced life span and increased susceptibility to spontaneous rhabdomyosarcoma , a decline in regenerative potential and alterations in the crucial mtor signaling pathway , and impaired functional and structural recovery after injury . hence , senescent mdx muscle represents a suitable dystrophic phenotype for determining potential global changes in the protein complement during aging . this report has summarized the findings of a comparative proteomic analysis of mildly affected mdx tibialis anterior muscle from 8 weeks versus 22 months old mice . the identification of aldolase , pyruvate kinase , carbonic anhydrase , tropomyosin , myosin , electron transferring flavoprotein and small heat shock protein hsp27 as new indicators of progressive muscular dystrophy might be useful for the establishment of a more comprehensive biomarker signature of dystrophinopathy . the protein with the highest age - related increase was identified as carbonic anhydrase isoform ca3 . in general , carbonic anhydrases catalyze the reversible hydration of co2 and are widely distributed throughout the body . skeletal muscles express several isoforms of this crucial metabolic enzyme in a fibre - type - specific manner . the predominant ca3 isoform is mostly present in the cytosolic fraction of type i and iia fibers . interestingly , metabolic adaptations , altered neuromuscular activity patterns , stretch - induced hypertrophy , and disuse atrophy greatly influence the expression of muscle carbonic anhydrases [ 7173 ] . the higher concentration of the ca3 isoform of carbonic anhydrase in aged mdx muscle , as shown here by mass spectrometry - based proteomics , could be an indication of an increased demand for efficient co2 removal during mdx fibre aging . on the other hand , since the ca3 isoform is predominantly located in slower - twitching fibre populations , its altered density could also be due to age - related fibre - type shifting in the mdx tibialis anterior muscle . this would agree with the findings of a recent proteomic survey of middle aged versus aged vastus lateralis muscle , which revealed increased levels of ca3 in senescent human skeletal muscle . the greatest reduction in a muscle - associated protein during aging of the mdx tibialis anterior was shown to be the small heat shock protein hsp27 . this indicates a potentially blunted cellular stress response in dystrophic tibialis anterior fibres and demonstrates that marked differences exist with respect to expression levels of small heat shock proteins in moderately affected hind limb muscles versus severely dystrophic diaphragm muscle in the mdx model of dystrophinopathy [ 40 , 41 ] . while changes in elements of the contractile apparatus suggest downstream effects of dystrophin deficiency on myosin and tropomyosin organization , altered expression levels in electron transferring flavoprotein and glycolytic enzymes indicate perturbed mdx muscle metabolism . the beta - polypeptide chain of the electron transferring flavoprotein is involved in mitochondrial fatty acid and amino acid catabolism and the enzymes aldolase and pyruvate kinase catalyze the reversible break - down of fructose-1,6-biphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate and the critical oxidoreduction - phosphorylation step that converts adp and phosphoenolpyruvate to atp and pyruvate , respectively . alterations in glycolytic enzymes and mitochondrial proteins in mdx tibialis anterior muscle indicate altered flux rates through key metabolic pathway . with respect to the glycolytic pathway , the activity of four muscle proteins is central to its regulation on the enzymatic level , i.e. the metabolic flux through hexokinase , phosphofructokinase , glycogen phosphorylase , and pyruvate kinase , whereby metabolic silencing of muscle glycolysis is probably mediated by the inactivation of pyruvate kinase . this central role of pyruvate kinase in muscle metabolism makes its changed abundance in the mdx tibialis anterior muscle a crucial finding . pyruvate kinase was previously shown to be a suitable biomarker of the general aging process in skeletal muscle tissues [ 77 , 78 ] . interestingly , the change of metabolic enzymes in aged mdx tibialis anterior muscle , such as pyruvate kinase and aldolase , agrees with the proteomic analysis of golden retriever muscular dystrophy . in the grmd dog model of dystrophinopathy , targets of the transcriptional control factor of energy metabolism pgc-1 , that is , various glycolytic and oxidative enzymes , were found to be reduced . in conclusion , the comparative proteomic survey of dystrophic tibialis anterior muscle from 8 weeks versus 22 months old mdx mice has revealed altered expression levels in a number of critical proteins during skeletal muscle aging . however , the degree of concentration changes was less pronounced in the moderately dystrophic tibialis anterior muscle as compared to the recently analyzed aged mdx diaphragm . these differing proteomic findings agree with the pathophysiological concept that the aged mdx diaphragm muscle is more severely affected as compared to moderately necrotic mdx hind limb muscle . in the long - term , the proteomic identification of new biomarkers of dystrophinopathy might be useful for the establishment of a comprehensive and muscle subtype - specific signature of duchenne muscular dystrophy . this would be useful for improving diagnostic procedures , monitor disease progression , identify novel therapeutic targets and aid in the evaluation of novel treatments , such as exon - skipping therapy or stem cell therapy .

x - linked muscular dystrophy is a highly progressive disease of childhood and characterized by primary genetic abnormalities in the dystrophin gene . senescent mdx specimens were used for a large - scale survey of potential age - related alterations in the dystrophic phenotype , because the established mdx animal model of dystrophinopathy exhibits progressive deterioration of muscle tissue with age . since the mdx tibialis anterior muscle is a frequently used model system in muscular dystrophy research , we employed this particular muscle to determine global changes in the dystrophic skeletal muscle proteome . the comparison of mdx mice aged 8 weeks versus 22 months by mass - spectrometry - based proteomics revealed altered expression levels in 8 distinct protein species . increased levels were shown for carbonic anhydrase , aldolase , and electron transferring flavoprotein , while the expressions of pyruvate kinase , myosin , tropomyosin , and the small heat shock protein hsp27 were found to be reduced in aged muscle . immunoblotting confirmed age - dependent changes in the density of key muscle proteins in mdx muscle . thus , segmental necrosis in mdx tibialis anterior muscle appears to trigger age - related protein perturbations due to dystrophin deficiency . the identification of novel indicators of progressive muscular dystrophy might be useful for the establishment of a muscle subtype - specific biomarker signature of dystrophinopathy .

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

PMC3388456

physical activity ( pa ) has been identified as a hallmark contributor to individuals ' quality of life , most notably among women . indeed , several research endeavours have converged on trying to understand the principal determinants of pa , and in particular , those factors that can be associated with individuals ' volitional control of this specific behaviour . one factor that has been of mounting interest to behavioural scientists is that of affect and/or mood , which is defined as the quality of a subjective feeling state consisting of elements of valence ( i.e. , good / bad ) and activation ( i.e. , high / low ) . notably , studies have revealed associations between engaging in an acute bout of pa and immediate increases in positive affect . in turn , basic affective reactions that are tied to a moderate intensity pa session ( i.e. , increases in positive affect ) have been shown to predict futures bouts of pa six and twelve months later . indeed , repeated positive affective experiences associated with pa may sustain pa motivation over time and this can facilitate long - term pa participation [ 6 , 7 ] . it follows then that studying positive affect in the context of specific pa sessions is an outcome worthy of investigation in its own right , and this within broader attempts to understand how to optimally predict sustainable patterns of pa behaviour for successful weight management and greater well - being . however , experts have highlighted that not all individuals who participate in pa achieve more positive affective states and ensuing increments in general well - being . indeed , results have not been entirely consistent as some participants have witnessed no change or a worsening of their affect with exercise . this may have long - term implications in terms of sustaining adequate amounts of pa as well as levels of well - being . this has led to researchers ' attempts to isolate the conditions under which the specific affective benefits of exercise might be maximized or conversely , thwarted . in particular , one line of research has focused on the intensity of pa and its relationship with exercise - related affect . meta - analytic findings have revealed that the effect of acute pa on positive - activated affect may be stronger at low - to - moderate intensities [ 4 , 10 ] . in past studies , threshold of intensity for the affective benefits of exercise , yet there is insufficient empirical evidence for an inverted u shape dose - response relationship . from a more person - centered view , several researchers now argue that preferred / self - selected intensity might lead to stronger benefits in positive affect than prescribed pa intensities [ 12 , 13 ] . overall , the evidence is inconclusive as there appears to be significant inter - individual variability regarding the benchmark intensity level that is most conducive to increments in positive affect . another line of work that has specifically targeted such individual - based differences has also addressed a call for more theoretical research on possible psychological mechanisms underlying the pa - positive affect relationship [ 14 , 15 ] . in particular , it has been suggested and recently emphasized that individuals ' motivational styles toward pa may supply a missing and understudied link . self - determination theory ( sdt ) , one recognized and well - supported motivational theory , distinguishes between two general types of motivation : self - determined motivation and controlled motivation ( or regulation ) . deci and ryan also classified motivation into several types of behavioural regulations that fall within these two broader categories . overall , self - determined motivation is rooted in feelings of satisfaction , choice and volition . it is characterized as enjoying an activity for its own sake ( i.e. , intrinsic motivation ) and/or assigning it value and personal importance ( i.e. , identified regulation ) . controlled or non - self - determined motivation can be defined as engaging in an activity , such as exercise , so as to avoid self - inflicted shame or guilt , or to gain a personal reward , namely pride ( introjected regulation ) . controlled motivation can also be characterized by being motivated according to external demands ( e.g. , to obtain a reward ; external regulation ) . self - determination theory also postulates another type of motivation , amotivation , which was deemed less relevant in the present study with active participants as it is defined as lacking the intention to act . self - determination , namely , higher levels of intrinsic and identified styles of motivation , has been associated with healthy intentions to engage in health - promoting behaviours such as pa . in women , identified regulation has also stood out as a predictor of the intensity of pa engagement . higher levels of these self - determined regulations have also been positively associated with desirable psychological variables including pa enjoyment and post - pa positive affect , as well as a battery of well - being indicators such as greater self - esteem and lower depression and state anxiety levels . on the other hand and under the more controlling classifications , indeed research has revealed an association between introjected motivation for pa and maladaptive outcomes that include poorer life satisfaction and exercise - dependence symptoms such as strenuous pa . similarly higher scores on external motivation have been linked to self - esteem issues and higher levels of negative affect . although motivation toward pa and the perceived intensity of pa have seemingly been construed as independent influences , it could be that they exert an interaction effect on pa - induced affective changes . such an interplay would be consistent with the expert - noted complexity of underlying mechanisms of the pa - affect ( and well - being ) relationship as well as new evidence regarding the relevance of motivational styles in predicting changes in affect following pa at self - selected intensities [ 15 , 16 ] . especially with introjected regulation , which theory proposes should not be directly linked to well - being ( and affect ) , perceived intensity may exert a particular contributing influence . while other research , albeit scant , has underscored the possible interplay between self - selected pa intensity and psychological factors such as personality and self - efficacy , to our knowledge no empirical research has properly entertained the interaction with motivation . indeed , an interplay between perceived intensity ( self - selected ) and motivation styles in predicting changes in affect with acute pa could reveal a sustainable mechanism for long - term pa and well - being that lies within individuals ' volitional regulation and that also capitalizes on the physical properties of exercise . therefore , the purpose of this controlled laboratory study was exploratory and was to examine whether there was an interaction between sdt 's motivational regulations to engage in a running activity and ratings of perceived exertion ( rpe ; self - selected intensity ) in predicting pre - to post - pa ( i.e. , running ) changes in positive affect . the three types of motivation were targeted given evidence cited above as well as recently revealed associations between motivation types and intensity preferences . lastly , this study was conducted with active women given that the link between pa and affect may be particularly evident in active individuals experienced with pa [ 30 , 31 ] as well as among women [ 32 , 33 ] . sample to highlight the above underlying theoretical relationships ; in addition , this would contribute to an important need to better understand how to develop successful of pa intervention strategies for women . fourty - one healthy and active women ( i.e. , > 20 minutes of moderate to vigorous pa three times per week ) with an average age of 40.98 ( sd = 4.93 ) participated in this study . the godin leisure time exercise questionnaire , a common and validated self - report measure of pa levels , was administered as a screening measure and the mean score for this sample was high at 59.71 . confounding influences in the experimental protocol , it was necessary that participants self - report running as their preferred or most frequent exercise modality . in their leisure time , the women reported running at a mean rpe of 13.37 ( sd = 1.22 ) . they also gave an average of 8.60 km for a usual run and/or 49.37 minutes per run . the women in this sample were well - educated ( 82.9% with a university degree or higher ) and all were employed outside of the home . the positive affect negative affect schedule ( panas ) , comprised of 20 adjective words ( 10 positive , 10 negative ) , was used to assess participants ' affect . according to the circumplex model , affect can be characterized by the dimensions of valence ( positive , negative ) as well as activation ( low , high ) and experts maintain that the panas focuses on the high activation component of affect . for each adjective , participants rated their response from ( 1 ) not at all to ( 5 ) extremely using the stem indicate to what extent you feel this way right now , that is , at the present moment . examples of adjectives include : excited ( positive ) and upset ( negative ) . given the focus on positive affect in the present investigation , only these adjectives were summed and analyzed . in the present study , the cronbach alpha for the positive subscale pre- and post - running were .88 and .91 , respectively . the situational motivation scale ( sims ) was employed to measure situational motivation to run . the sims is comprised of 16 items across four subscales that assess the different behavioural regulations ( intrinsic motivation , identified , external , and amotivation ) . introjected regulation was excluded from the original sims in order to have more succinct instrument for research purposes . in the current study , an enhanced version of the sims with four validated items tapping this type of regulation was used [ 40 , 41 ] . items measuring introjection in the enhanced situational motivation scale ( sims ) : ( 1 ) because i would feel bad not doing it ; ( 2 ) because i would feel guilty not to do it ; ( 3 ) because i want to avoid feeling guilty ; ( 4 ) because i would regret not doing it . using a 7-point likert scale from ( 1 ) corresponds not at all to ( 7 ) corresponds exactly , participants respond to stem why are you currently [ about to run ] , for items that included because i want to avoid feeling guilty ( introjection ) and because i am doing it for my own good ( identified ) . average score were calculated for intrinsic motivation , introjected regulation , and identified regulation . as expected from a sample of active participants , scores on external regulation and amotivation were low and variance levels were negligible , which contributed to low internal consistency values . the cronbach alphas of relevant subscales were acceptable to good with values of .65 , .85 , and .85 for identified regulation , introjected regulation and intrinsic motivation respectively . the rating of perceived exertion ( rpe ) scale was administered in the minutes immediately following the run to assess the perceived intensity / exertion of the running activity . more specifically , and similar to previous studies we employed what some experts have coined session rpe , whereby participants were instructed to rate the overall intensity of the full running session [ 43 , 44 ] . this was expected to minimize the influence of momentary fluctuations in how participants ' perceived their exertion , which could contaminate the accuracy of the overall evaluation . numerical values from 6 to 20 make up the [ session ] rpe scale which is also anchored at every odd integer with a brief descriptor ( i.e. , 7 = very , very light ; 13 = somewhat hard ; 19 = very , very hard ) . the validity and reliability of the rpe are well established given its frequent usage in studies of pa and mood . this study was approved by the ethics review board of the university of ottawa and was part of a larger project . , participants provided written informed consent and promptly responded to the sims and the panas prior to the running activity . participants were taken to a private exercise room equipped with a treadmill , a desk , and a chair and were explained the running protocol . in order to mimic a self - paced run and yet easily log the pace of the run to ensure that at least a moderate - intensity run was being met , the treadmill control panel was physically detached from the treadmill running belt . this allowed the researcher to easily adjust settings in response to any and all demands from the participant . the participants completed a 2 - 3 minute warm - up walk at an average speed of 5.17 km / hour . the researcher remained in proximity in order to speed up or slow down the belt as frequently as necessary , the details and timing of which were duly noted . by weighting any change in pace by the ratio of elapsed time at that pace , an average running pace was computed . there was minimal conversation and eye contact between participant and researcher and the treadmill was maintained at a grade of zero . participants ran for a 30-minute duration in order to stay consistent with previous inquiries on acute exercise and affective states ( e.g. , bartholomew et al . , 2005 ) . afterwards , the belt was slowed to the initial walking speed for a 2-minute cool - down and participants provided the session rpe for the 30-minute running component . all data were entered into spss version 18.0 ; sums , means , and standard deviations were calculated . initial data - screening procedures were conducted according to procedures outlined in tabashnick and fidell . namely , assessments were conducted for data entry errors , missing data , outliers , normality and the basic assumptions of regression analyses . descriptive statistics and reliability analyses were calculated for affect and motivation variables . a repeated - measures t - test compared pre - run and post - run levels of positive affect as a preliminary examination of whether the run had a significant influence on participants ' affect . a standardized residual change score for positive affect was calculated for each participant in order to adequately account for participants ' initial affect scores . namely , a predictor score was computed by regressing post - run affect on pre - run affect and then subtracting this from the observed scores . the residual change score served as the outcome variable in three separate hierarchical multiple - regression models that were employed to test the interaction between rpe and situational motivation for running . specifically , separate product terms for rpe and the three motivational regulations were created using standardized scores and each term was added as the last step in their respective regression models . during the experimental running session , participants ran at an average pace of 9.66 km / hour and provided a mean rpe of 12.79 . this value reflects a moderate to high intensity that was similar to the usual rpe achieved by the women outside of the laboratory . all assumptions regarding normality , linearity , and homoscedasticity were met and there was no evidence of collinearity . positive affect increased significantly from pre- to post - run [ t(40 ) = 4.83 , p < .001 ] . see table 1 for descriptive statistics . results of the hierarchical regression analyses showed no significant interaction between intrinsic motivation and rpe on residual change scores in positive affect [ fchange ( 1,37 ) = .23 , p = .63 ] nor between identified regulation and rpe [ fchange ( 1,37 ) = 1.82 , p = .19 ] . however , there was a significant interaction effect of rpe and introjection [ fchange(1,37 ) = 4.20 , = .30p < .05 ] which explained an additional 9% of variance in the change in positive affect from the variables alone . as displayed in figure 1 , when participants reported low introjection , the influence of perceived running intensity on the change in positive affect was considerable , with higher rpe being associated with a greater increase in affect from pre- to post - run . that is , when participants reported high introjected regulation , the change in positive affect was elevated and fluctuated very little with rising rpe values , and even showed a trend toward diminishing slightly . the results of this experimental study revealed a significant interaction between rpe ( i.e. , intensity ) and introjected regulation but not between rpe and intrinsic or identified styles of motivation in predicting changes in positive affect from pre- to post - running . to our knowledge , this is the first study to have examined this interplay , which builds on previous deliberations by ekkekakis and lind regarding a potentially complex causal chain linking intensity , pleasure from exercise , and adherence , among other factors . in particular , our investigation offers an important adjunct to a study by duncan and colleagues that revealed associations between sdt 's motivational regulations and pa intensity . specifically , we considered the interaction between these variables in explaining a factor that is being increasingly recognized not only as an important consequence of pa but also as a viable determinant of future participation , namely positive affect . our results attest to recent suggestion that a person 's motivational style for pa should be considered when attempting to maximize the affective gains of aerobic pa , especially at a self - selected intensity . the strengths of this investigation include the self - paced nature of the running activity , a situational measure of motivation for running , and the use of a well - controlled laboratory environment . researchers have observed that up to very high exertion levels , there is a basic positive linear association between exercise intensity and affect . while the basic linear relationship ( rpe - positive affect ) in this study was in fact positive for low and moderate levels introjection , it deviated markedly for those high in introjection , such that affective changes were fairly stable regardless of perceived intensity . while introjection has been associated with engaging in vigorous exercise , our results imply that this relationship may have little bearing on the acute mood changes that are experienced through pa , at least among avid female runners . this interplay may even be worrisome given a downward trend in positive affect as the rpe increased among highly introjected runners . this could suggest that active females that are high in introjection achieve some form of an immediate feel - good or relief effect from an activity such as running that serves to prevent or relieve feelings of guilt , thereby materializing irrespective of perceived intensity . this was evidenced in our study by greater overall gains in affect for those high in introjection , which may be akin to what sabiston and colleagues referred to as the reparative properties of motivation from guilt . on the other hand , among runners lower in introjected regulation , changes in positive affect were associated with greater variability in rpe which could indicate a greater appreciation of the sensations and physical properties of pa . lind et al . remarked that individuals usually choose to exercise at a pace that improves or maintains their mood . our findings show that this may be more applicable for exercisers with lower introjection and who are less driven by internal pressures to exercise , thereby freeing them to experience the pa session more fully . therefore , in - task affective states and sensation , which were not assessed in our study , could be a source of discrepancy between persons high and low in introjection . future studies should consider open - ended probes during acute pa sessions in order to ascertain the pertinent sources of affective changes between individuals that differ in motivational style . despite its effect on positive affect in our study , as well its influence on sustaining high intensity pa , introjection has been associated with several negative psychological consequences that were not assessed in our acute exercise study ( e.g. , lower self - worth and life satisfaction ) . thus , cautious interpretation of our results is warranted as immediate improvements in positive affect may not necessarily translate into benefits in general well - being . this could be disconcerting if we consider that individuals with lower levels of well - being are generally less likely to engage in pa , thus initiating a questionable cycle of regular pa maintenance and compounding issues related to leading a healthy and active lifestyle . on a different note , the results showing nonsignificant interactions between more self - determined motivational styles and rpe may be of theoretical significance . similar to what was discussed above with respect to runners with lower levels of introjection , our results hint that those with higher levels of intrinsic and/or identified regulation could have less contingencies attached to their exercise engagement . in this regard , burton and colleagues found that being intrinsically motivated positively predicted well - being independently of the level of performance . moreover , they found that fostering intrinsic motivation may diminish certain contingencies between one 's perceived performance and their well - being . this is consistent with sdt principles and research revealing that despite variability in perceived competence for an activity , self - determined individuals show greater interest , pleasure , and confidence which is exhibited through greater well - being . thus the relationship between self - determined regulations and well - being is more likely of a direct nature , as indicated in the present study whereby perceived intensity , which could be view as an indicator of their performance , did not significantly shape post - pa changes in positive affect among the women higher on self - determined regulations . other authors have also noted a direct relationship between identified and intrinsic motivation and post - pa affect as an indicator well - being . in addition , and from a psychometric perspective , ceiling effects could be partly to blame for nonsignificant findings given the high means and low variances for these variables , especially identified regulation . with respect other possible limitations of this study , there are variant opinions in the literature regarding the optimal time point(s ) at which to assess rpe . we opted to assess session rpe immediately post - pa which is common practice among researchers [ 50 , 56 ] . however , some authors such as singh et al . argue that post - pa rpe can vary significantly in the few minutes following exercise ( e.g. , between 510 minutes ) and that evaluations of rpe taken 1530 minutes after pa are more stable and valid indicators of participants ' perceived intensity . still , other experts gravitate away from session rpe altogether claiming that repetitive rpe measurements at regular intervals during a pa session provide a more representative measure of intensity [ 58 , 59 ] . in future studies , researchers will need to address concerns over the optimal time point(s ) for rpe measurement(s ) in studying exercise - related affect and they may wish to supplement such inquiries with alternative and objective measures of intensity . similarly , researchers might wish to consider the use of research designs that capitalize on longitudinal and in - task effects of motivation and intensity on indicators of well - being . in addition , future studies will need to make use of larger samples and draw from groups of participants that are more diverse in terms of activity levels as this may alleviate some of the psychometric issues regarding the assessment of certain motivational styles . the use of only active , healthy - weight women could also be considered a drawback of the present study in terms of generalizability and it would be worthwhile to test the given interactions in overweight or obese individuals who may experience pa differently . researchers have already shown that autonomous regulations are associated with long - term weight management as well as indicators of well - being in obese populations [ 23 , 61 ] and it would be worthwhile to examine the interplay with pa intensity in order to develop optimal interventions strategies for these individuals . yet it is also interesting to note that some studies have actually shown that affective experiences and pleasure from exercise might not significantly differ between normal weight and overweight women , at least at self - selected intensities [ 29 , 49 ] . in addition , other experts mention that it is actually fruitful to study an active population as much can be learned regarding the determinants of successful pa engagement and associated consequences and this could then be targeted among the insufficiently active . moreover , the purpose of this study was to explore a theory - based interaction mechanism and therefore it was advantageous to select a sample that could maximize the postulated relationships . in sum , while women higher in introjection showed the greatest change in positive affect post - running , further reasoning as to why this increase was not particularly sensitive to the self - selected intensity of the run ( rpe ) , as well as the long - term impact of this relationship on well - being and pa maintenance , is left to future inquiries .

there is evidence that affective experiences surrounding physical activity can contribute to the proper self - regulation of an active lifestyle . motivation toward physical activity , as portrayed by self - determination theory , has been linked to positive affect , as has the intensity of physical activity , especially of a preferred nature . the purpose of this experimental study was to examine the interaction between situational motivation and intensity [ i.e. , ratings of perceived exertion ( rpe ) ] in predicting changes in positive affect following an acute bout of preferred physical activity , namely , running . fourty - one female runners engaged in a 30-minute self - paced treadmill run in a laboratory context . situational motivation for running , pre- and post - running positive affect , and rpe were assessed via validated self - report questionnaires . hierarchical regression analyses revealed a significant interaction effect between rpe and introjection ( p < .05 ) but not between rpe and identified regulation or intrinsic motivation . at low levels of introjection , the influence of rpe on the change in positive affect was considerable , with higher rpe ratings being associated with greater increases in positive affect . the implications of the findings in light of sdt principles as well as the potential contingencies between the regulations and rpe in predicting positive affect among women are discussed .

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

PMC3429803

ingestion of some amino acids has presumable roles in performance improvement in athletes.[13 ] among them , -alanine supplementation has been suggested to improve performance during high - intensity exercises . on the other hand , it has been shown that large amounts of h are produced in the muscles during high - intensity exercise and result in ph reduction . there are many cellular ph buffers defend against exercise - induced acidosis , which include phosphocreatine , inorganic phosphates and histidine - containing dipeptides . carnosine ( -alanyl - l - histidine ) is the main histidine - containing dipeptide in humans . additionally , hill et al . and harris et al . , showed that 28 days of -alanine supplementation increased intramuscular levels of carnosine by nearly 60% . antioxidant function , muscle contractility regulation , and ph buffering , are the possible physiological roles of carnosine in skeletal muscle . thus , the possible role of carnosine could be prevention of skeletal muscle acidity in improving exercise performance . prolonged exercise can result in oxidative stress and muscle fatigue , which may be prevented by carnosine due to its antioxidative properties . on the other hand , -alanine administration could increase carnosine content of skeletal muscles by 4080%.[1719 ] increase of carnosine concentrations in muscle results in altered buffering capacity,[1819 ] and thus affects performance . furthermore , some studies have shown that carnosine acts as a ca sensitizer for the sarcomeres in muscles[2021 ] and thus could prevent fatigue . however , synthesis of carnosine in muscle is limited by the availability rate of -alanine , which can be overcome by -alanine supplementation . although it has been estimated that carnosine is responsible for nearly 10% of the total buffering capacity in human muscle there are few studies on -alanine supplementation and its possible effects on endurance exercise ; therefore , the purpose of this study is to assess the effects of -alanine administration on vo2 max , time to exhaustion and lactate concentrations in male physical education students . these students were fit ( bmi<25 ) and active ( physical activity2 hr / d ) , but not involved in professional sports . participants age , weight and height were 21.10.7 years , 71.88.8 kg and 1787 cm , respectively , for -alanine ( n=20 ) group and 21.91.5 years , 74.98.3 kg and 1805 cm for placebo group ( n=19 ) , respectively ( ns ) . before initiating the study , all participants were informed of all procedures of the study and signed an informed consent . none of the participants had ingested -alanine , or any other nutritional supplements , for a minimum of 3 months before the initiation of the study . participants were asked to abstain from exercise 24 h before trial initiation and to maintain their current physical activity and dietary patterns . after pre - testing , the participants were randomly assigned to one of the two groups : a ) -alanine ( 2 g / day ) , b ) placebo ( 2 g dextrose per day).the supplements had the same appearance , and ingested four times per day for 42 consecutive days before post - testing . all participants completed all experiments , and there were no complaints of side effects of the supplements . participants were supplemented orally for 6 weeks with either -alanine ( ajinomoto , usa , inc ) or placebo ( dextrose ) . the study was approved by the ethics committee ( esfahan sport medicine association , iran ) . supplements were provided in capsules of 400 mg and were administered each day as five divided single doses , with at least 2 h in between ingestions . venous blood samples were obtained from all participants between 5:00 and 6:00 p.m , after intensive endurance exercising , at the baseline and after intervention . all measurements were done before the start of the supplementation ( pre ) and after the intervention ( post ) . prior to and following the supplementation protocol , participants performed a continuous graded exercise test ( gxt ) on an electronically braked cycle ergometer ( lode , the netherlands ) to determine vo2 max and time to exhaustion ( tte ) . for each gxt , the primary power output was set at 30 w and elevated 30 w every 2 min until the participant could not maintain the required power output at a pedaling rate of 70 rpm due to fatigue . plasma samples were obtained for the determination of plasma lactate and glucose concentrations immediately prior to each gxt and 2 min post - exercise . glucose and lactate were analyzed using ysi auto - analyzer ( yellow springs , oh ) . statistical analyses were conducted using the statistical program for the social sciences ( spss version 13 , inc , chicago , il ) computer software package . paired t test was used to analyze before and after test data for each group differences . table 1 shows the mean sd values of exercise performance indices for the pre - and post - supplementation . supplementation with -alanine demonstrated a significant increase in vo2 max ( p<0.05 ) . on the other hand , tte and lactate concentrations decreased after 6 weeks of supplementation with -alanine ( p<0.05 ) . the placebo group showed a significant increase in lactate concentrations ( p<0.05 ) , but a non significant increase in tte . comparison of exercise performance indices , pre - and post - supplementation ( mean sd ) the post - exercise concentrations of plasma lactate were significantly higher ( p<0.05 ) than baseline in two groups . however , the post - exercise concentrations of lactate were significantly lower ( p<0.05 ) during the alanine supplementation compared to the placebo group . dietary intake before each trial was similar for energy and macronutrients [ table 2 ] . the findings of our study suggest that supplementation with -alanine may improve the endurance exercise performance as measured by the vo2 max , tte and plasma lactate concentrations . , showed that supplementation with creatine + -alanine resulted in significant increases ( p<0.01 ) in muscle carnosine content . the increased muscle carnosine content was accompanied with an improvement in vo2 max and tte in response to a maximal graded exercise test performed on a cycle ergometer . another study demonstrated that supplementation with both -alanine and creatine improved cycling performance ( tte ) . they concluded that this was due to h buffering by carnosine during this transitional period . our data demonstrate that the significant improvements in the performance indices with -alanine supplementation were due to ph reduction . the improvement in tte seen in the placebo group participants might be due to the encouragement provided by our staff and also the participants psychological status . also , the findings of the present study might have been influenced by the fluctuations in the skeletal muscle response to oral supplementation with -alanine . our data suggest that supplementation with -alanine may delay the onset of fatigue and thus improve performance during incremental cycle exercise in men . the glucose concentrations did not change significantly in our study , due to different individual response and insufficient dose or duration . the participants in this study , however , were male physical education students rather than untrained participants . however , according to the hypothesis , -alanine supplementation would prevent the drop in intracellular ph during high - intensity contractions and result in less circulating acidosis finally due to elevation of myocellular carnosine content . furthermore , several studies have shown the importance of ph regulation on performance during endurance exercise , by a pre - exercise alkalosis intervention . this suggests that the difference between groups is related to the presumable enhancement of muscle carnosine content . future studies should examine muscle carnosine levels along with vo2 max and plasma lactate concentration during strenuous exercise with variable quantities of -alanine supplementation also , further investigations are necessary to determine the effects of -alanine supplementation during more prolonged and submaximal exercise . it can be concluded from this study that -alanine administration can reduce acidosis during high - intensity exercise and thus can improve exercise performance in endurance athletes . also it is found that six weeks of supplementation with -alanine at the mentioned prescribed dose did not result in significant changes in glucose concentrations .

objectives : supplementation with -alanine has been proposed to improve performance in some exercises such as cycling and running . also , it has been demonstrated that great deals of proton ions are produced in the skeletal muscles during exercise that result in acidosis , whereas -alanine may reduce this effect . therefore , the aim of this study is to assess the effects of alanine supplementation on vo2 max , time to exhaustion and lactate concentrations in physical education male students.methods:thirty-nine male physical education students volunteered for this study . participants were supplemented orally for 6 week with either -alanine ( 5 * 400 mg / d ) or placebo ( 5 * 400 mg dextrose / d ) , randomly . vo2 max and time to exhaustion ( tte ) with a continuous graded exercise test ( gxt ) on an electronically braked cycle ergometer ; and serum lactate and glucose concentrations were measured before and after supplementation.results:supplementation with -alanine showed a significant increase in vo2 max ( p<0.05 ) and a significant decrease in tte and lactate concentrations ( p<0.05 ) . a significant elevation in lactate concentrations and a non significant increase in tte were observed in placebo group . plasma glucose concentrations did not change significantly in two groups after intervention.conclusion:it can be concluded that -alanine supplementation can reduce lactate concentrations during exercise and thus can improve exercise performance in endurance athletes .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC5034370

during the late 1970s and early 1980s , the lack of commercial development for drugs that treat rare diseases became an important political issue in the us . these so - called orphan drugs were largely neglected by the pharmaceutical industry because they represented small markets that were unlikely to be profitable . the lobbying and public awareness efforts of a number of rare disease patient organizations in the us eventually culminated in the passage of the orphan drug act of 1983 ( oda ) , which provides industry with support and incentives to develop orphan drugs , defined as drugs intended for use in treating a condition that affects less than 200,000 persons in the us . the substantial increase in the development of drugs for rare diseases over the past three decades is often directly attributed to the passage of the oda , and the act is widely considered to be a success . since the oda was passed in 1983 , more than 400 orphan drugs have been developed and marketed in the us , which suggests that the incentives are having an effect . moreover , the last 1015 years have been the most successful period of development for orphan drugs . according to the fda , nearly 200 orphan drugs enter development each year and approximately one third of new drugs approved by the fda are for the treatment of rare diseases . a concurrent trend that is contributing to the shift toward niche market development is recent advances in new genomic technologies that are now making the completion of the human genome project the international effort to map the entire human genomelaid the foundation for the development of new health care technologies and therapies , including genetic tests to assist in the diagnosis and prevention of disease and drug therapies that are tailored to the genetic characteristics of individual patients. pharmacogenomics , the study of the influence that genetic factors have on drug response , has emerged from genomics - related research and the development of new diagnostic approaches based on biomarkers . there is increasing interest in the pharmaceutical sector toward pairing pharmaceutical products with diagnostic tests that can stratify broader disease categories into rarer disease genotypes . significantly , a growing number of products in clinical development now rely on a clinical biomarker , which suggests the mounting importance of pharmacogenomic - based drug development . advances in pharmacogenomic research and increasing industry interest in personalized medicine have important implications for the way that orphan drug policies are interpreted and applied . in the us , the office of orphan products development ( oopd ) has indicated that pharmacogenomic products are treated the same as any other orphan drug submissions . however , concerns have been raised regarding orphan drug legislation generally and the impact of pharmacogenomics in this context in particular . some express concern that orphan drug incentives are not adequately targeted to the diseases with greatest unmet medical needs and that the oda has favored the development of treatments for diseases that can , through omics data and technologies , be recast as rare , or belong to the larger , more lucrative therapeutic class of oncology products. moreover , there are concerns that the oda does not adequately distinguish between true orphan drugs and trojan applicants that seek to co - opt the benefits for drugs that should not qualify as orphans. however , in 2013 , the fda made a number of important amendments to the oda regulations that purport to address many of the challenges raised by the evolving drug development environment , including advances in pharmacogenomics . the oda has served as a model for legislation in a number of other jurisdictions , including europe , japan , and australia . although the legal framework implemented in these countries is similar , the definition of an orphan disease , the criteria which must be satisfied to obtain a designation of orphan status , the incentives provided to encourage the development of orphan drugs and the authorization process for orphan drugs , varies from jurisdiction to jurisdiction . although for years the canadian government denied the need for the country to develop its own orphan drug policy , the government has recently reversed its policy and in december 2012 , released a draft orphan drug policy for discussion . the release of the 2013 amendments to the oda regulations provides an opportunity to examine what lessons canada can learn from the fda 's experience in drafting its own orphan drug policy , or indeed , whether a canadian orphan drug policy is even necessary given the increasingly lucrative nature of niche markets . this article considers whether orphan drug legislation can be drafted in a way that will maximize benefits and minimize concerns relating to the impact of pharmacogenomics on orphan drug research and development . after reviewing the issues that may arise at the intersection of orphan drug policies and pharmacogenomics , this article will discuss the potential impact of pharmacogenomics at two critical points : orphan designation and approval of the drug product . at each of these points , the relevant aspects of current us orphan drug legislation are examined , focusing on the extent to which recent amendments may address concerns that have been raised previously . this analysis will then provide the foundation for a critical review and recommendations regarding the proposed new canadian orphan drug framework . in recent years , many concerns have been raised about the potential impact of pharmacogenomics and new genomic technologies on our understanding of how disease categories are delineated , and subsequently , how the concept of rare disease should be defined for the purposes of orphan drug policies . it is estimated that over 80% of rare diseases are genetically - based , so it makes sense that pharmacogenomics could play an important role in the discovery and development of new treatments for rare disease . a 2013 report by thomson reuters suggests that the tremendous growth in orphan drug development over the past decade coincides with the increasing focus on personalized medicine , and that orphan disease markets will propel the evolution of [ personalized ] medicine. as argued by haffner and colleagues , [ i]n an environment in which medicine is increasingly adapted to the needs of patients , the incentives of the orphan drug act could arguably take on even greater importance. the obama administration 's announcement in january 2015 of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}215 million investment to support personalized approaches to medicine that take into account individual differences in people 's genes , environments , and lifestyleis yet another indication of the burgeoning importance of this area of research and development . many pharmacogenomic drugs have already qualified for orphan drug status under the oda , although at present these drugs represent only a small fraction of the total number of products that have received orphan drug designation . in certain cases , there may be a legitimate need to incentivize research into pharmacogenomic treatments that may only be effective in a particular subset of patients with a particular genetic biomarker . the nuffield council on bioethics , for example , highlights that stratifying more common diseases into rarer disease genotypes may result in some patient subsets being so small that developing specific medicines targeting these groups may not be financially viable for drug developers . in such cases , orphan drug policies may provide the necessary incentives to encourage pharmaceutical companies to develop medicines for these narrow populations , perhaps even allowing drugs that would have otherwise failed to be targeted to a smaller subpopulation in which the drug is more likely to be safe and/or effective . on the other hand , [ t]he nature of pharmacogenomics drugs may allow some pharmaceutical companies to game the system and abuse the oda 's built - in incentives for drug development. the oda provides incentives to manufacturers at two stages . first , in the development phase , sponsors can apply for an orphan drug designation , which gives them access to a variety of support and incentive measures , including a tax credit of 50% for the costs of clinical research , access to the oodp 's clinical research grants program , a waiver of fda user fees , and development and regulatory assistance . second , if the drug is then approved , the manufacturer is granted a seven - year period of market exclusivity for the orphan indication(s ) for which the drug is approved . this means that the fda will not approve another drug for the same indication(s ) during the exclusivity period , unless the holder of the exclusive license consents or can not supply sufficient quantities of the drug , or the other product is shown to be clinically superior ( and therefore not the the exclusivity applies only to the approved orphan indication(s ) and does not prevent the same drug from being designated or approved for a different use . pharmacogenomics can impact the way that the oda operates at a number of points in the product lifecycle . first , the use of pharmacogenomics to identify patient subsets can impact the way that disease categories are defined , and thus the size of the target population for the purposes of orphan designation . as the science advances , the nomenclature and classification of disease is becoming increasingly complex and consequently , the number of potential orphan diseases appears to be on the rise . herder argues that new insights from genomics and epigenomics are rendering the boundary between common and rare disease increasingly mutable , potentially exploding the scope of legislated definitions of orphan disease. however , orphan designation is only the first step toward market approval for an orphan drug product , and many orphan designated drugs never reach the market or are not ultimately approved for the orphan indication . second , pharmacogenomics can have an impact on the way that orphan exclusive approval is granted within the rare disease or condition , or orphan subset , for which the designation was given . pharmacogenomics can contribute to the narrowness of approved indications because stratification of the disease may increase the specificity with which the approved indication is defined , and may lead to multiple , narrow approvals within a single orphan designated disease or subset . repurposing efforts that is , the discovery of new useful activity in an older clinically used drugallowing some drugs to achieve multiple orphan drug designations and approved indications . in these cases , orphan designation and exclusive approval may add to the profitability of a drug that has already been approved and widely marketed for other uses . the oda thus provides an incentive for sponsors to invest in studying potential new uses of a drug for rare diseases . one concern that has long plagued the oda is the potential for drug developers to exploit the benefits of the act by artificially subdividing diseases to create subgroups of patients that fall under the orphan drug prevalence threshold a practice referred to as salami slicing. a classic example of salami slicing is the drug epogen ( epoetin alpha ) , which received an orphan designation from the fda in 1986 for the treatment of anemia associated with end - stage renal disease . after the drug was approved by the fda in 1989 , the drug became widely prescribed for a wide variety of patients with anemia , not only anemia caused by end - stage renal failure . consequently , through off - label use prescribing a drug for indications not formally approved by drug regulators in patients without end - stage renal disease , epogen became a blockbuster drug and generated billions of dollars in revenue for its manufacturer . loughnot argues that pharmacogenomics could potentially take salami slicing to a new level by allowing drug developers to genetically subdivide diseases that affect a large portion of the population into groups small enough to qualify for orphan drug status. the potential to stratify broader disease categories based on biomarker status significantly increases the potential number of orphan subsets that may be defined for the purpose of orphan designation . the fda acknowledges that what is considered a distinct disease or condition may change over time as scientific understanding evolves , which would affect prevalence determinations. maher and haffner point out that [ w]hether such increasingly precise disease descriptions constitute separate diseases would normally appear to be an academic exercise , unless , as is now more often the case , a specific therapy targeting a specific mutation is developed. that is , the very development of a new treatment can play an integral role in shaping the classification of a new rare disease or condition , or orphan subset . the potential for salami slicing is not a new concern , having been previously considered in earlier revisions of the us orphan drug legislation . in discussing the criteria for orphan drug designation , the 1991 notice of proposed rulemaking stated that a subset of a common disease or condition would qualify for designation only if the subset is medically plausible and that arbitrary however , the 1992 regulations offered little guidance on the meaning of this rather ambiguous phrase , providing only that the concept of medically plausible is interpreted flexibly depending on the specific facts of each case . moreover , a request to further define the term arbitrary in this context was rejected on the basis that every fda decision on arbitrariness would necessarily be highly fact dependent. according to herder , until recently , the addition in 1992 of the requirement that the disease in question be considered medically plausible was the only relevant constraint that has been adopted by the fda to distinguish between rare diseases that have long been identified as such and those which have been reclassified as rare by virtue of new scientific insights. the question of orphan subsets was addressed again , in more detail , in the most recent regulatory amendments . in the 2011 notice of proposed rulemaking [ b]ecause the term medically plausible has not been further clarified through regulations or guidance , it has been misinterpreted to mean any medically recognizable or any clinically distinguishable subset of persons with a particular disease or condition , and that inappropriate application of the medically plausible concept could result in artificially narrow subsets . although it can certainly be beneficial for manufacturers to develop more effective treatments for some subsets of common diseases and conditions , it could be said that this departs from the original intent of the oda to target very rare diseases that currently lack any effective treatments . indeed , the fda believes such an interpretation would frustrate the intent of the oda and divert resources away from research and development of true orphan drugs by allowing a non - rare disease or condition to be artificially subdivided into smaller groups for establishing subsets that are under the prevalence limit for designation. the fda therefore proposed to amend the regulation to remove the term medically plausible and instead provide a description of how an appropriate subset may be identified for the purpose of orphan drug designation. the final rule adopted in 2013 added a definition of orphan subset to clarify that an appropriate subset may exist where use of the drug in a subset of persons with a non - rare disease or condition may be appropriate but use of the drug outside of that subset ( in the remaining persons with the non - rare disease or condition ) would be inappropriate owing to some property(ies ) of the drug. the fda insists that the 2013 amendments are consistent with the agency 's longstanding approach to identifying some may continue to question how effectively the fda can distinguish between true and artificial subsets of disease , particularly since evaluation of orphan subsets is complex and is often based on uncertain evidence . may be difficult given the potential information asymmetries between the fda and the companies it regulates. if there were significant concerns about the fda 's previous approach , the fact that it maintains that the new language is consistent with this approach may undermine confidence in the potential for the new language to better distinguish between real and however , an analysis of the revised wording , along with some examples of subsets that have been accepted or rejected by the fda , suggests that these concerns may have been mitigated somewhat . in the 2013 final rule , although the fda accepted that biomarker - based and other targeted treatments could be used to define subsets , it expressly rejected the proposition that an orphan subset can exist whenever there is a basis for using the drug in the subset of interest , regardless of whether the drug can also be used in the remaining persons with the disease or condition. at the core of the analysis is consideration of the property or properties of the drug that preclude its use in the remaining persons with the non - rare disease or condition , outside of the orphan subset [ emphasis added]. according to the stated approach of the fda , sponsors have to not only demonstrate why this subset should be targeted for an orphan drug treatment , but also why the drug can not also be used outside the subset . the 2013 amendments provide specific guidance as to what factors may or may not inform whether an appropriate orphan subset exists . this is likely due to the fact that that much of the new wave of concern around salami slicing has arisen from the impact of new genomic technologies on the classification of rare diseases and conditions . first , the 2013 final rule provides that where a drug 's mechanism of action suggests that the drug would not have significant activity outside of a subset of patients with a particular type of tumor or biomarker , this may establish an orphan subset . second , the final rule indicates that where previous clinical experience with the drug indicates that the drug does not demonstrate significant activity in a particular subset of patients , this may inform whether an acceptable orphan subset exists . pharmacogenomic research can assist in the identification of patient subsets that are more likely to respond to drug therapy . third , the final rule lists the drug 's toxicity profile as a relevant factor in defining orphan subsets . for example , patients with a particular non - rare disease or condition who are refractory to , or intolerant of , other less toxic drugs could be a subset for the purposes of a more toxic drug , whereas other patients with the same disease or condition would not be appropriate candidates for that drug . pharmacogenomic research is often used to identify those patients who may be at a higher risk of adverse drug reactions due to their genetic profile . the fda may grant multiple orphan designations for a particular disease or orphan subset indeed , this has become common practice . however , it is worth noting that the fda 's acceptance of an orphan subset for one drug does not mean that the same subset will be accepted for subsequent applicants . the fda states that the prevalence estimate may be narrowed owing to one or more properties of the drug that allow for the existence of an orphan subset [ emphasis added]. that is , although a particular orphan subset may be designated for a given drug product , this same orphan subset may be rejected for another drug because the appropriateness of the subset is evaluated separately based on the specifics of each drug . similarly , the factors that are not sufficient to define an orphan subset are informative in the pharmacogenomic context . the 2013 final rule also notes that clinical trial eligibility , a sponsor 's plans to study the drug only for a select indication , the particular grade or stage of a disease , or a low likelihood of use in a broader population are not sufficient in themselves to establish an orphan subset . with pharmacogenomics , biomarkers can be used to prospectively select patient subpopulations a strategy known as enrichment of the study population that are more likely to respond to a given drug therapy so that the treatment effect is more likely to be detected . in such cases , later - stage studies may only be conducted in patient groups with a particular biomarker status . according to the fda , restricting clinical trial eligibility to biomarker - positive patients , or only choosing to study the drug in a particular patient subset , is not sufficient to establish an orphan subset . again , the sponsor is required to show not just that the drug is more effective or less likely to cause adverse effects within the subset population , but also that the difference between the subset and the larger group is sufficient to prevent the drug from being a viable treatment option for patients in the larger group . as such , this approach does show some potential for limiting what could be seen as abuse , particularly since the sponsor bears the burden of showing that drug is inappropriate for use outside of the orphan subset . loughnot suggested in 2005 that the increased precision that pharmacogenomics brings to pharmacology might eventually help provide the fda with the ability to define medically plausible. ultimately , as the science advances , pharmacogenomics will likely increase not only the number of potential rare diseases and orphan subsets , but also the precision with which these diseases and subsets may be delineated . as noted by haffner and colleagues , advances in genomics and proteomics have led to increasingly precise disease definitions. as advances in pharmacogenomics increase the ability of researchers to measure non - response , this may raise the bar for sponsors who are trying to establish an orphan subset . maher and haffner note that a clearer understanding of drug non - response is often revealed when response is stratified. consequently , as the lines between response and non - response are more clearly defined , orphan subsets will hopefully become less prone to manipulation . salami slicing would primarily arise in circumstances where the prevalence of the broader disease category is over 200,000 cases in the us , but the prevalence of the medically plausible subtype is less than 200,000 cases . there are a few cases where a pharmacogenomic - based biomarker was likely a determinative factor in bringing an orphan subset below the prevalence threshold . for example , although non - small cell lung cancer accounts for 85% of the approximately 400,000 cases of lung cancer in the us thus placing the disease well above the orphan designation threshold in 2010 , xalkori ( crizotinib ) received an orphan designation for the treatment of alk - positive , met - positive , or ros - positive non - small cell lung cancer. in this case , the biomarker status of the non - small cell lung cancer ( ie alk - positive , met - positive , or ros - positive ) appears to have been the factor that brought the indication under the prevalence threshold to qualify for orphan drug status . as another example , in 2011 , there were some 960,000 americans living with melanoma , but in 2010 , zelboraf ( vemurafenib ) received an orphan designation for the treatment of patients with iib to stage iv melanoma positive for the braf ( v600 ) mutation. if , as the 2013 amendments suggest , the stage of disease alone is usually not sufficient to define an appropriate orphan subset ( see below ) , then the biomarker selection for the braf(v600 ) mutation appears to be important to bringing the target population below the 200,000 patient prevalence threshold . in clarifying their longstanding approach to eligibility for orphan subsets , the guidance provided by the fda in the 2013 amendments may help to decipher the reasons behind their decisions to deny orphan drug designations to some pharmacogenomic drugs in the past . for example , before receiving approval for the breast cancer ( specifically , her2-positive metastatic breast cancer ) drug herceptin ( trastuzumab ) in september 1998 , its manufacturer , genentech , applied for orphan drug status with the fda , but the designation was denied . at the time , there was an estimated 165,000 metastatic breast cancer patient in the us , of whom approximately 30% , or 49,500 people , had her2 overexpressing tumors well below the 200,000 cut - off for orphan drug designation . however , the fda denied herceptin orphan drug status . while the exact reason for the denial was unclear , the oopd indicated that the most common reasons for refusal is disagreement between drug sponsors and regulatory authorities over how the target population is defined . in 2002 , shah suggested that herceptin was most likely not approved for orphan designation because the size of the population of her2 overexpressers was underestimated , as it is overexpressed in cancers other than that of the breast. in particular , it was clear from the success of clinical trials that herceptin could potentially also be used in the treatment of a range of other possible cancers including bladder , pancreatic , ovarian , colorectal , and prostate . however , it seems unlikely that the possibility of treating multiple types of cancer with herceptin was in fact the reason for the denial since in the 2013 final rule , the fda explicitly states that [ a ] drug that shows promise in multiple , different rare diseases or conditions may be eligible for multiple designations , one for each disease or condition , because fda considers the prevalence within each disease or condition. rather , it is more likely that the reason for the denial was that the stage of the disease ( metastatic , or stage iv breast cancer ) was not an acceptable subset to limit the target population since fda currently considers stage i breast cancer to be the same disease or condition as stage iv breast cancer when evaluating orphan drug designation requests for products that treat breast cancer. since breast cancer was estimated to affect nearly three million women in the us in 2011 , the sponsor presumably failed to demonstrate that the drug would not be effective in a broader subset of patients ( ie in her2-positive breast cancer in other stages ) . in most cases , stratification based on genetic biomarkers does not appear to be necessary to bring the target population below the 200,000 patient threshold ; pharmacogenomic data is often used to stratify diseases that are already rare enough to be eligible for orphan designation . indeed , greenbaum notes that the vast majority of pharmacogenomics drugs fall within the literal definition of an orphan drug. that is , most orphan designations that have been granted for pharmacogenomic drug products are for diseases where the broader disease category already falls below the 200,000 person threshold , such as chronic myelogenous leukemia , pancreatic cancer , or acute lymphoblastic leukemia . moreover , biomarker status is only one of many different factors that can be used to subdivide disease categories . most rare diseases and orphan subsets granted orphan designation are already subdivided based on a wide range of factors such as chronic or acute state , age of the target population ( eg adult vs. pediatric ) , or the underlying cause of disease , just to name a few . only once a larger body of examples is available to examine will it be possible to fully assess to what extent the oda remains open to abuse through salami slicing. the clarification provided in the 2013 final rule does seem to reduce the potential for abuse , and experience to date seem to suggest that instances in which subsets are artificially created to bring products below the orphan drug prevalence threshold will be fairly rare ; in most cases , subdivision based on biomarkers may be entirely legitimate . while this is an issue that should continue to be monitored , other jurisdictions , like canada , can learn from the fda 's recent efforts to define the orphan subset concept in a way that minimizes the potential for abuse . concerns about the potential misappropriation of orphan designation through salami slicing may be tempered by the fact that obtaining an orphan designation is only the first step toward market approval . while many of the benefits under the oda accrue as soon as an orphan designation is received ( namely assistance in clinical trials , the 50% tax credit for clinical trial costs , and access to federal grants ) , these benefits will ultimately be of little value if they do not lead to market authorization for the indication ( or a subset thereof ) for which the orphan designation was granted . the seven - year market exclusivity , also known as orphan exclusive approval , is arguably the most important incentive for drug developers seeking orphan designation . the bar for obtaining orphan designation is significantly lower than that for obtaining market approval and only a small fraction of orphan designated drugs ever reach the us market . as noted by maher and haffner , [ e]valuation of [ a request to consider a subset of a prevalent disease for orphan designation ] frequently rests on both incomplete knowledge of disease etiology and uncertain therapeutic mechanism of action , and is both difficult and complex. for example , as of 2012 , there had been 2661 successful orphan product designations granted by the fda , which had led to 408 approved orphan products ( representing about 15% of orphan drug designations ) . as with other orphan drugs , only a portion of pharmacogenomic drugs that receive an orphan designation are approved for the us market , though the proportion of designated pharmacogenomic - based drugs ultimately approved appears to be somewhat higher than for other classes of drugs . once a drug that has been granted orphan designation is approved for a rare disease or condition , the market exclusivity provisions in the oda prevent the fda from approving such drug for such disease or condition for a period of seven years from the date of approval , unless the holder of the first approval consents or can not supply sufficient quantities of the drug . the regulations specify that once a designated drug receives exclusive approval , no approval will be given to a subsequent sponsor of the same drug for the same use or indication for 7 years unless one of the exceptions applies . same drug is defined by regulation to mean a drug that contains the same active moiety or principal molecular features as a previously approved drug and is intended for the same use . same drug if it can be shown to be clinically superior to the first drug. the regulations define a clinically superior drug as one that is shown to provide a significant therapeutic advantage over and above that provided by an approved drug , through greater safety , efficacy , or other major contribution to patient care. a sponsor can obtain an orphan designation for a previously approved drug for the same rare disease or condition if it can present a plausible hypothesis that its drug may be clinically superior to the first drug , and then can receive its own exclusive orphan drug approval if it can demonstrate this clinical superiority . the plausible hypothesis of clinical superiority standard for orphan designation is easier to establish than the is achieved through liberally granting designation based on a plausible hypothesis of clinical superiority , allowing drugs to benefit from development incentives that flow from designation. maher and haffner note that unlike the fda market approval review , the orphan designation review takes place at an earlier stage of product development , sometimes even prior to any clinical studies having been performed. it is also worth noting that demonstration of clinical superiority at the approval stage need not be on the same basis as the hypothesis presented at the designation stage . the current approach to requiring proof of clinical superiority at the approval stage was called into question by the september 2014 decision of the us district court for the district of columbia in the case of depomed inc . the case arose from a 2012 complaint launched by the pharmaceutical firm depomed challenging the fda 's decision to deny orphan drug exclusivity for the drug gralise . other drugs with the same active ingredient ( gabapentin ) had previously been approved and marketed for the same indication ( post - herpetic neuralgia ) . having provided a plausible hypothesis of clinical superiority over these earlier products , gralise was granted orphan drug designation . however , fda refused to grant exclusive approval because the sponsor had not proved the clinical superiority of its product . none of the previously approved products had received orphan drug designation , however , so depomed argued that the clinical superiority requirement should not apply . looking at the relevant statutory provisions , the district court found that a plain - language reading of the oda mandates the fda to recognize exclusivity for any drug that the fda has designated [ as an orphan drug ] and granted marketing approval. it was therefore not open to the fda to impose the additional requirement of proving clinical superiority as a condition of granting exclusivity . as a result , the fda was ordered by the district court to grant orphan drug exclusivity for gralise without requiring proof of clinical superiority or imposing any additional conditions on depomed. it is important to note that the district court 's decision expressly acknowledges that fda can still impose conditions for orphan drug designation because it has been granted the authority in the oda to make regulations on this issue . therefore , the fda can still use clinical superiority to determine whether a drug for which orphan drug designation is sought is the same drug as one previously designated and approved . in the court 's view , this should allay fda 's concerns that removing the clinical superiority requirement for exclusive approvals could lead to sponsors evergreening or obtaining serial exclusivity for their products ( an issue discussed in more detail in the next section ) , contrary to the policy goals of the oda . a sponsor can only obtain orphan drug exclusivity for a product that has been designated as an orphan drug , and the fda can deny this designation to the sponsor of a drug that is the same as ( ie not clinically superior to ) a previously approved drug . following this decision , in december 2014 , the fda issued a clarification of policy in which the agency stated that the district court decision was limited to the specific case of gralise and that as such , the agency will continue to apply its existing regulations which require the sponsor of a designated drug that is the same as a previously approved drug to demonstrate that its drug is clinically superior to that drug upon approval in order for the subsequently approved drug to be eligible for orphan drug exclusivity. this seems to be a fairly aggressive position , considering that the district court 's decision questioned the fda 's authority to impose these conditions on exclusivity , and has led to considerable speculation about its implications . the issue is unlikely to be definitively resolved unless and until these issues are relitigated in further court challenges or appeals . while considerable uncertainty remains , the specific implications for the issues discussed in this article may be limited , given that fda 's authority to set conditions for orphan drug designation remains undisturbed , and the specific factual context of the depomed case where the same drug had been previously approved but not designated as an orphan drug are quite unusual . the pharmaceutical sector is fiercely competitive and brand name drug companies are always seeking ways to squeeze more profits out of drug products , particularly those that are approaching patent expiry or that are no longer under patent protection . a common tactic is to seek new periods of patent protection or market exclusivity by discovering new uses or new target populations for existing drug products a strategy often referred to as repurposing. the national institutes of health in the us have described repurposing as a key initiative to fight against stagnation in drug development. in particular , repurposing is an important strategy in the development of therapies for rare diseases : the sheer number of unmet medical needs to be found among orphan and rare disease suggests that drug repurposing among existing clinically used drugs may be a major solution to this societal medical need. in addition , drug developers may seek to reformulate or improve upon existing drug products in order to qualify for a new period of market exclusivity . it is worth noting that once the patent and market exclusivity periods have expired on a pharmaceutical product , anyone may seek an orphan designation and orphan exclusive approval for that product . a common means of extending the lifecycle of a patented drug product is through drug reformulation where a drug company modifies the characteristics of an existing drug product enough to qualify for a new patent or period of data exclusivity . pharmaceutical companies are often accused of evergreening their products by making trivial and needless modifications to patented medicines in order to extend the term of patent protection or exclusivity . the fda has acknowledged that one of the potential concerns with allowing new periods of orphan exclusivity for an already approved drug is that it could permit inappropriate evergreening of exclusive approval periods by allowing a sponsor to apply for a new designation ( and then exclusive approval ) near the end of a previous exclusivity period . as noted by the fda evergreening would allow orphan exclusivity to be extended indefinitely for the same drug for the same use without any meaningful benefit to patients , a result at odds with the seven - year exclusivity period provided by the statute. however , reformulation is not considered to be inappropriate if the sponsor can demonstrate clinical superiority ; a sponsor that improves its own previously approved drug can be eligible for a new exclusivity period if clinical superiority is shown . as the fda notes , the requirement of clinical superiority is intended to encourage the development of potentially safer and more effective orphan drugs rather than encouraging minor modifications to already approved drugs that confer no meaningful benefit to patients. indeed , if the intent is to create incentives to improve treatment options , then arguably it should n't matter who develops the clinically superior alternative , whether the original sponsor or a competitor . in this way , the oda may incentivize research into improved versions or new applications of existing drug products for a rare disease or condition . it is important to note that the scope of the market exclusivity is determined by the approved indications , not by the orphan designation . the fda generally grants orphan - drug designation for use of a drug in all patients with a rare disease or condition and expects sponsors to seek approval on this basis , but sometimes the approval will be narrower if the data submitted only supports use in a subset of patients or indications . in the 2013 amendments , the fda set out to clarify the scope of market exclusivity by replacing the term subset [ of uses] with select indication(s ) or use(s). the regulations now provide that if orphan exclusive approval is limited to only particular indication(s ) or uses(s ) within the rare disease or condition for which the drug was designated , fda may later approve the drug for additional indication(s ) or uses(s ) within the rare disease or condition not protected by the exclusive approval. that is , the sponsor may obtain multiple periods of seven - year market exclusivity for each approved indication or use that falls within the orphan designation . each new period of market exclusivity will begin to run from the date of approval for the new ( ie not previously approved ) indication or use thus staggering the market exclusivity based on the approval date of each new orphan indication . loughnot expresses concern that pharmacogenomics could contribute to evergreening tactics by help[ing ] identify patients who are susceptible to adverse drug reactions , allowing a sponsor to create significantly better clinical trial results without altering a drug at all by including only patients who are less likely to have adverse reactions . enriching clinical trial populations with patients who are most likely to benefit from the drug under study has become common practice in the development of pharmacogenomic products , as well as in other areas of drug development . as noted above , the 2013 amendments make clear that for the purposes of orphan designation , clinical trial eligibility and the decision to only study the drug in a particular patient population are insufficient to establish an acceptable orphan subset . subsequently , at the approval stage , if the issue is the sponsor attempting to evergreen an existing orphan exclusive approval on its own product , simply retargeting a drug at a narrower patient population within an already approved orphan indication would likely not be sufficient to obtain a new period of market exclusivity . an already approved drug could be targeted toward an unapproved indication within the orphan designation , since the oda clearly permits multiple approvals within a designation . however it is common for an orphan designated drug to be ultimately approved for a narrower indication than was set out in the designation . for example , the pharmacogenomic - based drug xalkori ( crizotinib ) initially received orphan designation for the treatment of alk - positive , met - positive , or ros - positive ( three different types of biomarkers ) non - small cell lung cancer but has so far only received fda approval for treatment of alk - positive non - small cell lung cancer . a broader orphan designation widens the potential scope of orphan exclusive approvals that may be obtained under a single designation ; if the scope of the orphan designation is too narrow , additional designation will likely need to be obtained . as noted above in the context of orphan designation , pharmacogenomics may both multiply the number of potential rare diseases and orphan subsets that may be designated and increase the precision with which these diseases and subsets may be defined ; [ w]ith therapies becoming increasingly guided by a more complete understanding of both genomics and proteomics , the number of potential orphan diseases should be expected to increase. similarly , at the approval stage , pharmacogenomics may narrow the scope of approved indications within a designated rare disease or subset since the approval may specify increasingly precise conditions of use or target populations . accordingly , pharmacogenomics may increase the trend toward having multiple orphan approvals within a single orphan designation since pharmacogenomics increases the stratification of disease and the specificity with which disease subtypes and/or subpopulations may be defined . nonetheless , as discussed in the next section , the potential for off - label prescribing may erode the distinction between approved and unapproved indications within an orphan designation . through subgroup analyses , pharmacogenomics can assist in drug repurposing efforts by identifying new targets for treatment or pinpointing patient subpopulations in which an existing drug may be more effective . as greenbaum notes , [ n]ew technological tools are now being used to determine if there are additional targets of current drugs on the market , or so called off - targets. such repurposing is an example of retrospective pharmacogenomic drug development , where sponsors can use data generated in previous clinical trials to identify potential new indications . in addition , pharmacogenomic research may be able to rescue drugs that may have failed all comer clinical trials by defining a more appropriate patient population and conducting enriched clinical trials ; traditional randomized clinical trials may mask treatment efficacy by including participants for whom the drug has poor efficacy . further , pharmacogenomics enables genetic profiling of subpopulations at an increased risk of adverse drug events . the fda , for example , has acknowledged that if new science enables us to determine that the adverse events are restricted to a small , identifiable segment of the population , public health could be improved by making the drug available to others who could benefit without undue risk. consequently , pharmacogenomics may even allow drugs that have been withdrawn from the market due to rare but serious adverse events to be reintroduced for a specific subpopulation under more restricted terms of authorization . a previously approved drug may receive an orphan designation for an unapproved use , regardless of whether the previous approval was for a rare or non - rare disease or condition . even where a drug was previously approved for a common indication , it can receive orphan designation for a different indication that qualifies as an orphan disease , and manufacturers can be granted market exclusivity for an off - patent drug for orphan indications . the support and incentives provided under the oda may result in previously discarded treatments being revived or in new orphan applications for already successful drug products . haffner and colleagues note that orphan exclusive approval under the oda is important for the development of older drugs namely , drugs that are no longer covered by patent protection . sponsors may be able to maximize the sales potential for existing drugs by obtaining new orphan designations . the fda has explicitly stated that [ a ] drug that shows promise in multiple , different rare diseases or conditions may be eligible for multiple designations , one for each disease or condition , because fda considers the prevalence within each disease or condition. gleevec ( imatinib ) , for example , has received seven separate orphan designations and orphan exclusive approvals . according to a report by thomson reuters , about 15% of orphan drugs analyzed in one study had subsequent launches for additional rare diseases . the fda openly encourages drug developers to pursue orphan indications for drugs that have already been approved for more common conditions . the oopd has created a database of products that have received both orphan status designation for a rare disease and a market authorization for the treatment of more common diseases . this database offers sponsors a useful tool for finding special opportunities to develop niche therapies that are already well - advanced through development and thus represent a far easier lift to drug developers than beginning with an untested new therapy compound. even drugs that have achieved blockbuster sales in broad patient markets may be eligible for orphan designation where that same drug can also be used to treat an orphan condition . indeed , the fda has granted orphan designation to over 100 drugs with existing approvals for more common diseases , including to some highly successful blockbuster drugs such as prozac ( fluoxetine ) , viagra ( sildenafil citrate ) , and neurontin ( gabapentin ) . however , of these only gabapentin has received market authorization for an orphan indication . it is problematic when incentives are used in situations where they are not intended or needed which could be the case where orphan drug policies intended to encourage sponsors to develop products that would not otherwise be financially viable are used simply to enhance the profitability of products that would already viable without any incentives . in these situations , concerns of abuse or exploitation may be raised . the oda does not consider the previous profitability of the drug in determining whether a new orphan application should receive an orphan designation , and subsequently , orphan exclusive approval . rather , the oda is aimed at encouraging the development of orphan treatments that would otherwise not be developed . even if a drug had already achieved blockbuster sales for another indication , this does not necessarily translate into making it financially viable to pursue new orphan indications for the drug product since this additional research and development can entail significant cost . thus , the incentive to investigate and test the drug 's potential for a particular indication could be necessary and useful , even if the drug is already profitable . therefore , on balance , it is legitimate to allow orphan designations for drugs previously approved for other indications , including common diseases . it is quite fair to say that [ f]rom the perspective of the patient with a rare disease , whether a drug is also effective in treating a more prevalent disorder is irrelevant. from this perspective , anything that could encourage a sponsor to identify and test the drug as a potential therapy for the patient 's condition might be beneficial . finally , as noted above , the potential for off - label prescribing may erode the value of the orphan drug exclusivity : although manufacturers are prohibited from marketing drugs for off - label uses , physicians are free to prescribe drugs off - label , potentially allowing generic drugs to be prescribed for indications that are protected by orphan exclusivity . typically , generic drugs must have the same labeling as the innovative drug product to which they are compared in their application for market approval . however , where the innovator has patent or exclusivity protection for a particular use or condition , if the generic copies these protected elements in the innovator 's labeling , they risk an infringement action . however , the generic may seek permission from the fda to carve out the protected language , which would theoretically limit the generic 's market to conditions or uses that are not covered by patent or exclusivity rights . while a detailed discussion of the carve - out policy is beyond the scope of this article , it is worth noting that these provisions could potentially undermine an innovator 's incentive to pursue repurposed orphan applications for existing drug products . as noted by mahn , innovative drug companies are concerned that the carve out rule , coupled with the practice of prescribing and substituting generic drugs off label , threatens their ability to recover the large investments needed to discover new uses or to improve the safety or efficacy profiles for old drugs. in the past , suggestions that canada consider a us - style orphan drug framework had been rejected by health canada on the basis that sufficient flexibility and incentives already existed in the canadian legislation . in a 1997 policy statement , health canada cited several mechanisms which could be applied to orphan drugs , including tax incentives , fee reductions for drugs with small market potential , and access to unapproved drugs through the special access program . health canada concluded that these mechanisms were sufficient because approximately 60% of us - approved orphan drugs were available in canada . the number of persons with rare diseases in canada may not be sufficient to support substantial clinical trial research and development in the area of orphan drugs. currently , canadians may access orphan drugs through the health canada 's special access program , by participating in clinical trials , or where the drug has been approved through the regular drug approval process . [ t]here has not been significant pressure from industry or special interest groups in canada to develop an orphan drug policy. recently , however , there have been renewed calls for an orphan drug policy in canada . the canadian organization for rare disorders , for example , has argued that without an orphan drug policy , manufacturers have no motivation to seek market approval for orphan drugs in canada and consequently , canadians with rare disorders run the risk of being among the last in the developed countries to gain access to new medicines , if at all. biotecanada , the national association representing the biotechnology industry , also strongly supports the development of a canadian orphan drug framework , stating that the initiative will help canada to compete in attracting investment to nurture [ orphan drug ] products into the marketplace , and see new canadian solutions for unmet medical needs developed in canada. orphan drug policies have also been framed as a means to promote personalized medicine : a decade ago , in a report to the canadian government , the external advisory committee on smart regulation noted that [ w]ith the recent developments in pharmacogenomics , and the resulting ability to target treatments for sub - groups of the population , it may be timely to consider implementing a legislative framework to facilitate access to these drugs. according to the canadian organization for rare disorders , health canada 's proposed orphan drug framework would be the first step towards canada taking a leadership position in personalized medicine. despite its earlier position that there was no need for an orphan drug policy in canada , in october 2012 , the federal government announced its plans to develop an orphan drug framework for the designation , authorization and monitoring of orphan drugs . soon after in december 2012 , health canada released an initial draft discussion document for a canadian orphan drug framework , which proposed a regulatory framework for orphan drugs , including provisions relating to orphan drug designation , scientific and clinical protocol advice , special market authorization for orphan drugs , and post - market assessment and management . the criteria for orphan designation outlined in the proposed canadian framework mirror those of the european legislation , with the exception that they do not consider the economic viability of the drug . in particular , the canadian framework adopts the same prevalence criteria as the european union legislation in the definition of an orphan drug : a drug that is intended for the diagnosis , treatment , mitigation or prevention of a life - threatening , seriously debilitating , or serious and chronic disease or condition affecting not more than five in 10 thousand persons in canada. as is the case in other jurisdictions , health canada 's proposed legislation contains a series of incentives including priority review for marketing authorization , fee reductions for small to medium enterprises , and scientific and clinical protocol advice . currently , the canadian orphan drug framework is still in the draft discussion phase , and health canada has provided only a high - level description of the elements of the proposed framework . as such , it is too early to assess how pharmacogenomics products will be handled under the canadian orphan drug policy . as the proposed framework is developed and implemented , canada can learn from the us experience with the oda and the debate surrounding how rare diseases are categorized and understood in order to maximize the benefits and minimize the potential for abuse of an orphan drug regime . in particular , health canada will have to consider how orphan subsets will be defined for the purposes of orphan designation and the terms and conditions for the approval and market exclusivity for orphan drugs . under the draft orphan drug framework , to qualify for orphan designation , the drug must either not currently be authorized for the canadian market , or if already approved , must provide a potentially substantial benefit for the patient distinguishable from the existing therapy. the proposed framework also permits a sponsor to submit an application for orphan designation on the basis of a designation from a recognized country if the proposed orphan drug and indication in canada is the same to that under which the foreign designation was issued . as in the us , a single drug may be eligible for multiple orphan designations for different rare diseases . while the draft canadian framework contains many of the same types of incentives that are currently offered under the us system , there are some important incentives that are not included in the draft framework or that are offered under more restricted terms . perhaps most significantly , the oda offers a tax credit for 50% of the cost of clinical trials , whereas the draft canadian orphan drug framework does not currently propose any type of specific tax credit for orphan designated drugs . in addition , under the us system , drugs that have been granted an orphan designation are exempt from the application fee that sponsors must normally pay to the fda when making a regulatory submission ; these application fees can be over two million dollars per product depending on the type of application . in contrast , although few details are provided at this stage , the canadian draft framework proposes only a fee reduction rather than a fee exemption , and further this reduction is aimed at small and medium enterprises , whereas the us exemption is available to any applicant . as a result , the financial incentives available upon orphan designation under the proposed canadian orphan drug framework are arguably less enticing than those offered under the oda and consequently , may be less likely to encourage the creation of artificial subsets . finally , as noted above , concerns about the potential misappropriation of orphan designation through salami slicing should be tempered by the fact that designation is only the first step toward market approval , and only a fraction of orphan drugs will ultimately be approved . under the canadian draft framework , the government will be required to maintain and make available to the public a list of orphan drug designations. this is in line with the orphan drug product designation database maintained by the fda and the register of designated orphan medicinal products maintained by the european medicines agency ( ema ) . however , it is worth noting that while the fda does not make public any information about the drug products that have been denied orphan designation , in europe , the ema also maintains a register of drug products that have been refused orphan drug designation , which includes links to various documents giving the basis for the decision . making public information on which sponsors have applied for and been refused orphan designation could potentially add a level of public scrutiny to the activities of pharmaceutical companies that apply for orphan drug designation , hopefully discouraging companies from making questionable applications for designation this type of transparency measure is important for ensuring the accountability and responsiveness of the orphan drug regime , and health canada should aim to make public information about both successful and unsuccessful applications for orphan designation . as the orphan drug framework develops , health canada must establish criteria for defining acceptable orphan subsets , particularly given the controversy that had arisen over this subject south of the border . although designation will ultimately be assessed on a case - by - case basis , the long running uncertainty in the us around the definition of a medically plausible subset , and the subsequent need for clarifications under the 2013 amendments demonstrate the need for regulatory authorities to provide more detailed guidelines around what factors may and may not inform the existence of an acceptable orphan subset particularly in light of advances in new genomic technologies and evolution in our understanding of disease categories . the us approach of requiring the sponsor to demonstrate why patients outside of the orphan subset are not good candidates for the drug appears to be a good approach particularly as it puts the onus on the sponsor to justify their framing of the target population . further , given that the current canadian draft framework proposes to allow a sponsor to submit an application on the basis of designation of a recognized country , the policies adopted by the fda in respect of orphan subsets may very well lead to a corresponding orphan designation in canada if the us orphan designation is used as the basis for the application . at the approval stage , the canadian orphan drug framework does not propose any new category of exclusivity for orphan drug products . these provide eight years of exclusivity for an innovative drug , defined as a drug that contains a medicinal ingredient not previously approved in a drug and that is not a variation of a previously approved medicinal ingredient. the exclusivity operates by preventing a competitor from obtaining market approval for another drug based on a direct or indirect comparison with the innovative drug . an additional six months of exclusivity is provided for innovative drugs that have been the subject of studies relating to use of the drug in pediatric populations . similar provisions for new chemical entity and pediatric exclusivity exist in the us but are distinct from orphan drug exclusivity . by proposing linkage to the innovative drug and pediatric exclusivity periods , health canada 's draft orphan drug framework appears to indicate that up to an eight - and - a - half year exclusivity period would be available for orphan drugs . this period is longer than the seven - year period of market exclusivity granted for orphan drugs in the us , though shorter than the ten years provided by the european orphan drug legislation . however , there is an important distinction between how the existing data exclusivity offered by health canada operates in comparison with the orphan exclusivity offered under the us orphan drug regime : in the us , drugs that have been previously approved are still eligible for the seven - year orphan exclusivity for each new approved orphan indication , whereas under the proposed canadian system , a previously approved drug would not be eligible for a new period of exclusivity if the sponsor subsequently sought approval for an orphan indication . that is , the sponsor would not be granted a fresh period of eight - year exclusivity for a new orphan indication if the drug has already been approved for another indication because the exclusivity is only available for innovative drugs . at most , perhaps , an extension ( similar to the six - month pediatric extension ) might lengthen a previously granted period of exclusivity . unless there are plans to alter the existing exclusivity to allow an orphan drug to receive multiple periods of exclusivity , the draft canadian orphan drug framework is unlikely to provide much incentive for pharmaceutical companies to pursue research into new orphan applications for existing drugs that have already been approved , or that are no longer under patent protection . while only granting a single period of data exclusivity for each drug product has the advantage of preventing abuse of the orphan drug incentives through evergreening strategies , the disadvantage is that this approach will do little to encourage the repurposing of existing drug products for orphan indications , or the development of clinically superior reformulations of existing treatments for orphan diseases . health canada will need to do more than simply apply the existing eight- or eight - and - a - half - year exclusivity to the orphan drug context if it wants to provide these incentives . rather , as demonstrated by the controversy around the assessment of clinical superiority under the us orphan drug policy , health canada will have to clearly articulate the circumstances under which orphan drug products will be eligible for market exclusivity . as the canadian framework takes shape , the orphan exclusivity provisions should encourage the development of alternative treatments that offer genuine and significant benefits for patients by setting strict standards for what constitutes a clinically superior product that entitles a sponsor to a new exclusive approval for what would otherwise be the same drug. the difficulty is that it is currently unclear how the canadian exclusive approval would work , and therefore , how the issue would play out under the new framework . greater uncertainties may exist for orphan drugs given the complexities of the diseases , the small and vulnerable populations and the treatment environment itself , and that consequently , greater abilities to plan for and resolve those uncertainties are needed once the drug is on the market. accordingly , the draft canadian orphan drug framework includes expanded transparency measures , including the publication of the key information on when an application for market authorization for an orphan drug has denied . health canada notes that [ t]he publication of negative decisions and the basis for these decisions is important for orphan drugs in circumstances when an existing marketed re - purposed drug could continue to be used off - label. unfortunately , the fda does not currently make publicly accessible , in their online orphan drug database or otherwise , any specific information about when an application for market authorization for an orphan - designated drug has been unsuccessful . given the rapid evolution of the field of pharmacogenomics , its impact on orphan drug policies will need to be continually reassessed to ensure that policies remain responsive to the current drug development paradigm . the recent revision of the oda regulations in 2013 is encouraging because the fda has at least demonstrated that it is alive to the concerns raised by the increasing detail with which potential orphan subsets can be defined . although questions remain about whether the amendments will be sufficient to prevent abuse of the provisions , it appears likely that some of the concerns have been mitigated . the us experience can provide lessons for other jurisdictions , including canada as it develops its new orphan drug policy . a central dilemma in addressing the issue of salami slicing in orphan designation artificial subdivision of disease categories an assessment that must be made with reference to the purposes of the orphan legislation . however , it is important to note that this does not necessarily imply that developing treatments for artificial groups that do n't qualify as orphan subsets is not valuable . as noted by herder , [ i]n the abstract , it is not obvious that orphan disease policy should prioritize diseases that have long been understood as rare over diseases like breast cancer that may soon be subdivided into multiple rare diseases since either may be in need of new and improved treatment options . rather , the more important question is whether the objectives of the oda are fulfilled by incentivizing development of such treatments . in pharmaceutical development , there is a general desire to encourage the development of more and better treatment options for patients with rare diseases . while this is a worthy goal , we should bear in mind that the more specific objective of orphan drug legislation is to target incentives toward the development of drugs that would not otherwise be developed due to a lack of financial viability . some argue that orphan drug legislation is overinclusive because it provides incentives where they are not really needed . the marked increase in applications for orphan designations in recent years has led to suggestions that the oda risks becoming a victim of its own prodigious success , and tends to confirm suspicions that the orphan drug market is one that the pharmaceutical industry now views as potentially lucrative . there are orphan drugs that are actually very profitable due to the very high prices that can be charged for rare disease therapies or to extensive off - label use for more common conditions both of which are in themselves sources of significant controversy . the outstanding question is whether it is better to err on the side of offering orphan incentives more liberally , even if this leaves the policy open to abuses such as salami slicing , or to be more restrictive in granting incentives but risk of dissuading the development of some orphan drugs , such as those that target biomarker - based orphan subsets . another area that should be of interest to canada as it develops its orphan drug framework is the potential to encourage the use of pharmacogenomics research to repurpose drugs for use in treating rare disorders . in the us , the possibility of a new period of orphan drug exclusivity provides an incentive for manufacturers to invest in repurposing , and there is even talk of adding a new type of extension to exclusivity or patent terms to further stimulate this activity . as discussed above , the exclusivity proposed in the canadian draft framework for orphan drugs would need to be modified to provide an effective incentive for repurposing approved drugs for new orphan indications . finally , the impact of pharmacogenomics on orphan drug development makes it all the more important to ensure that orphan drug policies be implemented with a high level of transparency and coordination . in introducing the draft orphan drug framework , the canadian government stated that a key focus of this new approach will be on international information - sharing and collaboration for the development and regulation of orphan drugs. specifically , the proposed canadian orphan drug regulations would allow health canada to operationally align and participate in well - established activities of the us and the european union including designation , scientific / protocol advice and pre- and post - market information sharing. transparency is also addressed through plans to publish the basis for decisions on marketing authorization . as the science of drug development becomes more and more complex , measures to minimize abuse of incentives and to enhance the safety of off - label use by sharing information among regulatory agencies , manufacturers , and the public are essential . this work was funded by the government of canada through genome canada and the ontario genomics institute ( ogi-064 : enhanced care for rare genetic diseases in canada ) , and through a second genome canada grant ( ethical and legal issues of cancer initiating stem cell research ) .

advances in pharmacogenomic research and increasing industry interest in personalized medicine have important implications for the way that orphan drug policies are interpreted and applied . concerns have been raised about the potential impact of pharmacogenomics and new genomic technologies on our understanding of how disease categories are delineated , and subsequently , how the concept of rare disease should be defined for the purposes of orphan drug policies . this article considers whether orphan drug legislation can be drafted in a way that will maximize benefits and minimize concerns relating to the impact of pharmacogenomics on orphan drug research and development . after reviewing the issues that may arise at the intersection of orphan drug policies and pharmacogenomics , this article will discuss the potential impact of pharmacogenomics at two critical points : orphan designation and approval of the drug product . at each of these points , the relevant aspects of current us orphan drug legislation are examined , focusing on the extent to which recent amendments may address concerns that have been raised previously . this analysis will then provide the foundation for a critical review and recommendations regarding the proposed new canadian orphan drug framework .

INTRODUCTION PHARMACOGENOMICS AND ORPHAN DESIGNATION PHARMACOGENOMICS AND ORPHAN DRUG APPROVAL Proposals for a Canadian Orphan Drug Framework CONCLUSION FUNDING CONFLICT OF INTEREST

PMC2279163

zinc is an essential element in the nutrition of animals , human beings , and plants [ 1 , 2 ] . zinc plays an important role in replications , gene expressions , and the metabolism of nucleic acids and different proteins there are many methods for zinc determination such as spectrophotometry [ 35 ] , atomic absorption spectrometry [ 6 , 7 ] , flame atomic absorption spectroscopy , graphite furnace atomic absorption spectrometry [ 9 , 10 ] , inductively coupled plasma atomic emission spectroscopy , and inductively coupled plasma mass spectrometry . but some are expensive , some are time - consuming , some are troublesome , and others are toxic . preconcentration technique [ 1316 ] can improve the detection limit as well as the selectivity of the method . flow injection analysis ( fia ) [ 1719 ] can improve analytical procedures such as high analytical throughput , precision , accuracy , low reagents , and sample consumption . therefore , a new method was proposed for online preconcentration and spectrophotometric determination of low - concentration zn ( ii ) in water . the proposed method is based on the fact that zn reacts with pan to produce zn ( ii)-pan complex formation in the ph 9.3 buffer solution . to avoid the extraction procedure , it has been found that the pan reacts with zinc to form a water - soluble complex at the presence of tween-80 . a model zj - la automatic metallic element analyzer developed and produced successfully by professor xinshen zhang of our own laboratory was used . the analyzer has the functions of flow injection analysis ( fia ) , ion chromatography , and automatic sample injection . the function of automatic reference fia was applied to determine zn ( ii ) . two analytical pumps ( shanghai huxi analytical instrument plant , shanghai , china ) of fia were used to deliver solutions . one was used to deliver elution solution and reagent solution while the other delivered sample solution . polytetrafluoroethylene ( ptfe ) tubing of 0.5 mm in internal diameter was used as the channels for all solutions to circulate . ultraviolet and visible spectra were obtained using a spectrumlab s54 ( lengguang technology co. , ltd , shanghai , china ) . data acquisition and processing were performed with hw-2000 chromatography software ( qianpu software co. , ltd , shanghai , china ) running under windows xp . all reagents used including 1-(2-pyridineazo)-2-naphthol ( pan ) ( tianjin ruijinte chemical reagent plant , tianjin , china ) , tween-80 , sodium tetraborate , o - phenanthroline , ddtc , and sodium hexametaphosphate ( chengdu fang zhou chemical reagent plant , chengdu , china ) , oxalic acid dihydrate , lithium hydroxide monohydrate , ammonium thiocyanate , and citric acid ( beijing donghua chemical reagent plant , beijing , china ) were of analytical grade and all solutions were prepared with deionized water . zinc stock solution : zinc stock standard solutions at a concentration of 1000 mg l were obtained from china environmental monitoring terminus , beijing , china . working solutions were prepared by suitable dilution of the stock solution . mixed chromogenic reagent solution ( r ) : 10 ml of 4 10 mol l pan ethanol solution , 20 ml of 8% tween-80 , 20 ml of ph 9.3 of sodium tetraborate suffer solution , 1.5 ml of 10% sodium hexametaphosphate , 1 ml of 0.1% ddtc , and 2 ml of o - phenanthroline were mixed and the mixture solution was diluted to 100 ml . elution solution : a total of 1.26 g of oxalic acid dihydrate , 1.576 g of citric acid monohydrate , and 1.15 g of lithium hydroxide monohydrate were dissolved in 1000 ml of water . the preconcentration column developed by our laboratory was filled with macroporous spherical resin with 4-(2-pyridylazo ) resorcinol ( par ) functional group . the par functional group can form chelate complex with determined metal ion in the preconcentration column and the chelate complex can be eluted by the eluent solution of oxalic acid - citric acid - lithium hydroxide . the filling granularity of resin was 80100 m . the preconcentration column length was 40 mm and its internal diameter was 5 mm . by filling the preconcentration column , mix resin and deionized water into pasty matter and drop slowly into column with burette . after sedimentating for a moment , resin was dropped slowly until the preconcentration column became full . then , cover strainer , cover on the column , and wash a few minutes with deionized water for further work . the sample solution was injected into preconcentration column to preconcentrate with a six - way injection valve . when the instrument was put into analyzing position , the concentrated zn ( ii ) was eluted by eluent solution , pushed to the three - way cock , and mixed with chromogenic reagent solution in the reaction coil . the zn ( ii)-pan complex was then formed and carried into the flow cell . the absorbance intensity of zn ( ii)-pan complex was determined at 560 nm by detector and transformed to a signal of the peak which was recorded by hw-2000 software on a pc . in the flow injection analysis , the absorbance of complex is affected by ph of the reaction medium . the effect of ph on the zn ( ii)-pan complex was tested in the ph range 411 . the test results showed that the maximum peak height could be obtained when ph range was between 8 and 10 . the effect of pan concentration on the peak height were tested from 5 10 mol l to 1.2 10 the results showed that the peak height rose when concentration of pan changed from 5 10 mol l to 4 10 mol l , and the peak height declined when concentration of pan changed from 4 10 mol l to 1.2 10 mol l. therefore , 4 10 mol l pan was selected for further work . according to the reaction kinetics , the reaction rate will inevitably increase when increasing concentrations of the reagent . however , the reaction rate will not increase when concentrations of the reagent reaches a certain value . therefore , when pan consumption reaches a certain value , the concentration of pan which reacts with zinc should be certain because zinc concentration is fixed in the sample . thereby , the absorbency will not increase when increasing pan concentration . on the contrary , the background color ( baseline noises ) will increase because the free state pan in the system will increase . thus , tween-80 was selected as reagent which could increase solubility of the system . the test results in figure 3 showed that the peak height increased when concentration of tween-80 changed from 1.0% to 1.6% and that the peak height did not increase even when concentration of tween-80 changed from 1.6% to 2.2% . therefore , 1.6% tween-80 was selected as the optimal concentration for the further work . in the fia , the flow rate could affect the sensitivity of the proposed method . in the study , the flow rate was adjusted by the interior diameter of pump pipe . therefore , the effect of the flow rate of reagent solution on the peak height was studied from 0.10 ml min to 0.72 ml min . the test results showed that the best flow rate could be obtained when the flow rate of reagent ( r ) was set at 0.23 ml min . in the flow injection analysis , the reaction time is adjusted by the length of reaction coil . the study results in figure 4 showed that 3 m of the reaction coil length was the optimum and further increase of reaction coil length did not increase peak height . length of preconcentration column and filling granularity could affect the effect of preconcentration . in the study , filling granularity of 80 100 m was selected and length of concentration column from 20 mm to 100 mm was tested . the results showed that the shorter the length of concentration column , the higher the sensitivity . however , if the length of concentration column was too short , the concentration of preconcentration zn ( ii ) in samples was not adequate , which lowered the sensitivity . the highest sensitivity was obtained when the length of concentration column was 40 mm . the results in figure 5 showed that the preconcentration time was longer , the zinc concentration of the preconcentration column would be higher which causes the analysis sensitivity to be higher but the sampling frequency to be lower . taken a comprehensive consideration of analysis sensitivity and sampling frequency , preconcentration time of 5 minutes was decided for further work . too high - preconcentration rate would increase analysis sensitivity but augment pressure of preconcentration column which would cause leakage or pump pipes rupture . too low - preconcentration rate would reduce analysis sensitivity and sampling frequency but augment the preconcentration time . taken a comprehensive consideration of analysis sensitivity and sampling frequency , preconcentration rate of 1.5 min l was chosen for further work . if eluent concentration was too low , zn ( ii ) in preconcentration column would elute incompletely . if eluent concentration was too high , it would waste reagent . in the study , nitric acid and oxalic acid - citric acid - lithium hydroxide were tested as eluents . the experimental results showed the sensitivity of the eluent of oxalic acid - citric acid - lithium hydroxide was higher than that of nitric acid . thus , oxalic acid - citric acid - lithium hydroxide was selected as eluent solution and its concentrations were 0.01 mol l , the sensitivity was greatly affected by flow rate of eluent . if flow rate was too high , the sensitivity augmented and baseline noises increased . if flow rate was too slow , zn ( ii ) in concentration column would elute incompletely , which caused sensitivity decline . the result showed that flow rate of 0.5 ml min of eluent was optimal for further work . the interference of some possibly coexisting foreign metallic ions was examined to make sure the proposed method can be applied to determine zn ( ii ) in water . the tolerance limit was defined as the concentration of added solutions causing less than 5% relative error during the determination of 10 g l of zn ( ii ) . no interference was observed from a great deal of k , na , nh , mg , ca , cr , cr , al , co , hg , as , and so forth . however , the metallic ions of ni , cd , mn , pb , cu , and fe interfered . the interference was eliminated ( 100 g l of each ion of ni , cd , mn , pb , cu , and fe ) by adding 1.5 ml of 10% sodium hexametaphosphate , 1 ml of 0.1% ddtc , and 2 ml of o - phenanthroline as appropriate masking reagents into mixed chromogenic reagent solution . thus , the results showed that the proposed method had good selectivity . to assess the reproducibility and accuracy of the proposed method , under the selected conditions above the linearity was tested by a series of working standard solutions of zn ( ii ) ranging from 0.5 g l to 400 g l. test showed that the peak height versus zn ( ii ) concentration was linear within the range of 2.0 g l to 360 g l. the curve was h = 0.427c + 3.638 ( h : peak height ; c : concentration of zn ( ii ) ( g l ) ) and the correlation coefficient obtained was 0.9995 and the detection limit ( 3 multiples of baseline noises ) was 0.42 g l. the test of precision was conducted by eight injections of 5.0 g l and 50 g l of zn ( ii ) standard solution , respectively . the results showed that the relative standard deviation ( rsd ) was calculated as 3.55% and 2.14% , respectively . the proposed method was applied to determine zn ( ii ) in water samples using the zj - la automatic metallic element analyzer . the analysis results of samples were shown in table 1 . to prove the accuracy of the method , a test of recovery would be conducted by accurately adding known concentration of zn ( ii ) into sample whose original zn ( ii ) had been determined . the test results in table 1 showed that the recovery was between 97.1% and 104.8% and analytical results of the proposed method were satisfactory . therefore , the proposed method was suitable for determination of trace amounts of zn ( ii ) in water . the proposed method was significant with regard to the development of a simple , reliable , and sensitive flow injection method for online preconcentration and determination of trace amounts of zinc ( ii ) . high sensitivity , selectivity , broad determination range ( 2.0 g l to 360 g l ) of zn ( ii ) , low - detection limit ( 0.42 g l ) , and fully automated analysis were just some of the advantages of the proposed method . especially , compared with other existing methods of zinc preconcentration , eluent concentration is even low to mmol l. the detection limit of the proposed method can be further improved by increasing preconcentration time . the model zj - la automatic metallic elements analyzer employed in this study was cheap , small , and easy to move . the proposed method has been successfully applied to determine zinc ( ii ) in water . the method is suitable for determination of zn ( ii ) in both water samples and other samples for its good selectivity and the above mentioned advantages .

a simple , sensitive , reliable and flexible flow injection spectrophotometric method is proposed for on - line preconcentration and determination of trace amounts of zinc in water . at the presence of tween-80 in ph 9.3 buffer solutions , the shade of color of zn ( ii)-pan complex is in a linear relation to the zinc amount at the point of the maximum absorption peak of 560 nm . the optimal experimental conditions , including reaction conditions and preconcentration conditions , had been obtained . the linear range of the proposed method was between 2.0 and 360 g l1 and the detection limit was 0.42 g l1 . the relative standard deviation was 3.55% and 2.14% for 5.0 g l1 and 50 g l1 of zinc standard solution ( n = 8) . the method had been successfully applied to zinc determination in water samples and the analytical results were satisfactory .

1. INTRODUCTION 2. EXPERIMENT 3. RESULTS AND DISCUSSION 4. CONCLUSION

PMC2734265

in the last few decades , computer simulation has become an important tool to investigate various phenomena in cardiac biology , including studies of single ion channel properties , action potentials of the myocyte [ 3 , 5 ] , dynamics of action potential propagation in tissue , subcellular calcium dynamics , etc . in spite of the advancement of computational technology , the simulation of action potential waves in three - dimensional ( 3d ) cardiac tissue with a realistic geometry is still considered as a large - scale simulation . general - purpose computing on gpus ( gpgpu ) is a recently emerging technology [ 1 , 4 , 8 ] , which uses gpus , instead of cpus , to compute large simulations in parallel . . each gpu may contain 128240 stream processors whereas today s cpus contain 2 , 4 , or 8 cores . in this paper , we demonstrate that the gpu is about 30~40 times faster than the cpu , enabling it to perform whole heart electrophysiology simulations within practical time . in this study , we chose the simulation of the propagation of the action potential in cardiac tissue , which is modeled as the propagation of a wave in an excitable medium . therefore , this technique can be applied to a number of phenomena in physics , chemistry , and biology . the first was a 2d homogeneous sheet , and the second was an anatomic rabbit ventricular model with fiber rotation , that is , an anisotropy that varies from point to point in the heart . the gpu simulation was performed with a single nvidia geforce 8800 gt 1 gb graphic random - access memory ( ram ) and an nvidia geforce 9800 gx2 1 gb graphic ram . these graphic cards were installed into a system with a dual - core 2.0 ghz amd opteron processor and 4 gb error correction code ( ecc ) ram . the cpu simulation was performed with an 8-node high performance - computing ( hpc ) cluster . each node has two dual - core 2.0 ghz amd opteron processors ( i.e. , 4 cores in each node ) and 4 gb ecc ram . all 2d simulations , and all 3d simulations with one gpu , were performed with the nvidia geforce 8800 gt . 3d simulations with two or four gpus were performed with the nvidia geforce 9800 gx2 . because these gpus support only single precision , all floating - point calculations were done using single precision across both gpu and cpu simulations . when the gpu kernel code is executed , it is similar to a cpu based parallel implementation accomplished through a series of threads , with each thread running independently in parallel . similar to a cpu implementation , it was necessary to synchronize all threads after each ordinary differential equation ( ode ) or partial differential equation ( pde ) kernel execution . we can then thread these intra - gpu as they control the processing within a single gpu . in addition to having to manage threads intra - gpu , it was also necessary to have inter - gpu threads to control each gpu . for instance , the nvidia geforce 9800 gx2 graphics card has two gpus on one card . in order to utilize each gpu as with a cpu cluster with distributed memory , it is also necessary to manage the distributed gpu memory . however , unlike a cpu where data can be moved from one cpu to another , gpus can and must communicate with the cpu memory , that is , data is transferred from one gpu to the other gpu via the main ram ; gpu1ramgpu2 . the cardiac tissue was modeled using the following partial differential equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{\partial v}{\partial t } } = - { \frac{i}{{c_{m } } } } + \nabla \cdot d\nabla v , $ $ \end{document}where v is the transmembrane voltage , i is the total ionic current , cm is the transmembrane capacitance , and d is the diffusion tensor . we solved this reaction - diffusion equation with the forward euler method , using the technique of operator splitting . the time step was adaptively varied between 0.01 and 0.1 ms and the space step was 0.015 cm . details of the modeling of cardiac tissue are described in our previous study . for each time step , the ode part was solved once and the pde part was solved four times for the 2d simulation and six times for the 3d simulation ( fig . 1sample gpu code to solve the reaction - diffusion equation in 2d tissue . at each time step , the ode part is called once and the pde part is called four times . the ode kernel code and the pde kernel code are in the appendix sample gpu code to solve the reaction - diffusion equation in 2d tissue . at each time step , the ode part is called once and the pde part is called four times . the ode kernel code and the pde kernel code are in the appendix to test the gpu code , we induced spiral waves in 2d and 3d tissue using cross - field stimulation , that is , two successive perpendicular rectilinear wave fronts . in each case 2).fig . 2action potential propagation in 2d tissue and in the anatomic heart model . the spiral wave was induced by cross - field stimulation . c spiral wave breakup in 2d tissue . g electrocardiogram from the anatomic heart simulation f action potential propagation in 2d tissue and in the anatomic heart model . the spiral wave was induced by cross - field stimulation . c spiral wave breakup in 2d tissue . g electrocardiogram from the anatomic heart simulation f for the 2d tissue simulations , the benchmark protocol involved pacing the tissue from the corner for 3 s of simulated time at a pacing cycle length of 150 ms . tissue size was varied from 100 100 ( 1.5 cm 1.5 cm ) to 800 800 ( 12 cm 12 cm ) . for the 3d tissue simulations , the benchmark protocol consisted of pacing the whole heart from the apex for 3 s of simulated time , at a pacing cycle length of 150 ms . finally , we investigated where the computational we split the program into three parts , the ode calculation , the pde calculation , and the data transfer . in order to measure the data transfer time , the ode calculation and the pde calculation were skipped , and the total time elapsed was then assigned to data transfer . then , skipping the ode calculation , we could measure the time for the pde calculation plus the data transfer . the time for the pde calculation was then estimated by subtracting the data transfer time from the ( pde + data transfer ) time . the time for the ode calculation was obtained by subtracting the ( pde + data transfer ) time from the time for the whole simulation . when tissue is homogeneous , parallel computation is very efficient . to compute 1 s of simulated time in 100 100 tissue , a single gpu took 8.2 s , whereas the cpu took 201 s. for larger tissue ( 800 800 ) the gpu took 283 s , while the cpu took 13,113 s. this is because as the tissue size becomes larger , the boundary / non - boundary ratio becomes smaller and the parallel computation becomes more efficient . in these cases , 3comparison between gpu and cpu . a time to compute 3 s of simulation time in 2d tissue . righty - axis is acceleration ( i.e. , computation time with cpu / computation time with gpu ) . b time to simulate the whole heart for 1 s of the simulation time . c computation ratio of the ode , the pde , and the data transfer for each simulation comparison between gpu and cpu . a time to compute 3 s of simulation time in 2d tissue . righty - axis is acceleration ( i.e. , computation time with cpu / computation time with gpu ) . b time to simulate the whole heart for 1 s of the simulation time . c computation ratio of the ode , the pde , and the data transfer for each simulation to simulate the anatomic rabbit ventricular model for 1 s , the hpc cluster with 32 cpus ( 8 nodes ) took 45 min . on the other hand , one gpu took about 72 min and two and four gpus took about 43 and 27 min respectively for the same simulation ( fig . on the other hand , the bottleneck of the computation with gpus is mainly in the pde part ( fig . we demonstrate that gpus are substantially faster than cpus in the simulation of action potential propagation in cardiac tissue . a single gpu simulation of the whole heart is only 1.6 times slower than the simulation in an hpc cluster , and two or four gpus are even faster than the hpc cluster , making the gpu a new tool for cardiac simulations . however , like parallel cpu implementations , management of threads and memory must be well thought out if maximum performance is to be achieved .

in this technical note we show the promise of using graphic processing units ( gpus ) to accelerate simulations of electrical wave propagation in cardiac tissue , one of the more demanding computational problems in cardiology . we have found that the computational speed of two - dimensional ( 2d ) tissue simulations with a single commercially available gpu is about 30 times faster than with a single 2.0 ghz advanced micro devices ( amd ) opteron processor . we have also simulated wave conduction in the three - dimensional ( 3d ) anatomic heart with gpus where we found the computational speed with a single gpu is 1.6 times slower than with a 32-central processing unit ( cpu ) opteron cluster . however , a cluster with two or four gpus is faster than the cpu - based cluster . these results demonstrate that a commodity personal computer is able to perform a whole heart simulation of electrical wave conduction within times that enable the investigators to interact more easily with their simulations .

Introduction Methods Results Conclusions

PMC4182423

in vertebrates , melatonin and its signaling through g - protein - coupled receptors ( gpcrs ) play a fundamental role in the circadian modulation of physiology and behavior . melatonin is a highly diffusible hormone secreted at night by the pineal organ , which in nonmammalian vertebrates is directly light sensitive ( lamb , 2013 ) . as a hormone of darkness , melatonin levels change according to circadian , lunar , and seasonal cycles ( reiter , 1993 ) . a conserved function of melatonin in vertebrates , from fish to mammals , is the regulation of sleep ( dollins et al . , 1994 ; zhdanova et al . , 2001 ) . in mammals , this occurs through direct modulation of neuronal excitability in the suprachiasmatic nucleus , the brain circadian pacemaker ( jiang et al . , 1995 ) , and in the thalamus , where sleep is initiated ( ochoa - sanchez et al . , 2011 ) . melatonin is one of the oldest biologically active molecules in nature , present in a nearly ubiquitous range of organisms , including bacteria and plants . its rhythmic production has been reported not only in vertebrates , but also in various protostomes , in cnidarians , and in dinoflagellates ( balzer and hardeland , 1991 ; hardeland and poeggeler , 2003 ; roopin and levy , 2012 ) . the ubiquitous presence of melatonin ( hardeland and poeggeler , 2003 ) has been linked to its chemical properties , which make it one of the most powerful radical scavengers known in nature ( tan et al . , 2007 ) . beyond that , the presence of melatonin receptors in all animal lineages , except sponges ( feuda et al . , 2012 ) , indicates that melatonin acquired an additional signaling function early in animal evolution . it has been speculated that the role of melatonin as the hormone of darkness has evolved directly from its antioxidant properties : in a system with constant melatonin synthesis , the reduction of melatonin levels during the day due to its light - dependent oxidation would make it a suitable signal for darkness ( hardeland et al . , 1995 ) . chemical indicator of darkness for the circadian regulation of some physiological process and/or behavior . however , the nature of such melatonin - controlled behavior has so far remained elusive , as the role of melatonin signaling has been poorly investigated outside vertebrates . in few cases , it has been demonstrated that melatonin has modulatory ( often inhibitory ) effects on locomotion ( anctil et al . , 1991 ; bentkowski et al . , 2010 ; tanaka et al . , 2007 ; tilden et al . , 2003 ) ; yet it is not clear whether these effects are linked to circadian rhythms and how they relate to the ancient role of melatonin signaling in animals . to broaden our perspective on the function of melatonin signaling in metazoans , we investigated its possible role in the day / night control of zooplankton locomotion . primary larvae forming most of the zooplankton are part of the life cycle in the majority of animal phyla . they swim using locomotor cilia , which are either distributed over the entire body surface or concentrated in specialized ciliary bands . in the ocean , almost all of these larvae show a pronounced rhythmic behavior , known as diel vertical migration ( dvm ) , which generally consists of an upward swimming phase at dusk and a downward displacement phase throughout the night and/or at dawn ( alldredge and king , 1980 ; forward , 1988 ; rudjakov , 1970 ) . the control of vertical migration in the oceans has been linked to the origin of animal circadian rhythms ( gehring and rosbash , 2003 ; pittendrigh , 1993 ) , as a mechanism to escape damaging uv irradiation during the daytime ( calkins and thordardottir , 1980 ; rhode et al . , 2001 ) . we thus considered it an attractive hypothesis that melatonin signaling may play a role in the diurnal control of ciliary swimming . to elucidate the possible interplay between ambient light detection , melatonin signaling , and circadian larval swimming activity in an invertebrate zooplankton larva , we chose the annelid platynereis dumerilii , a marine animal model amenable to molecular and behavioral studies , which has conserved vertebrate - type ciliary photoreceptors ( arendt et al . , 2004 ; jkely et al . , as an entry point , we found that the most specific marker of melatonin synthesis , the gene hydroxyindole - o - methyltransferase ( hiomt ) ( nowak et al . , 1993 ) , is conserved in several bilaterian genomes ( figure s1 available online ) . therefore , in order to identify the platynereis melatonin - producing cells , we performed whole - mount in situ hybridization ( wmish ) with probes detecting transcripts of platynereis hiomt and of marker genes for phototransduction and circadian clock ( figure 1 ) . expression was detected in the episphere , where the larval brain differentiates ( figures 1a and 1b ) , starting at 34 hr postfertilization ( hpf ) in dorsomedial cells ( figures 1d1f ) and covering the extended ciliary photoreceptor region at 48 hpf ( dashed circles in figures 1g1j ) . this area comprises the previously described ciliary photoreceptors expressing c - opsin1 ( arendt et al . , 2004 ) . in vertebrate ciliary photoreceptors , phototransduction involves the g - alpha subunit gt , a member of the gi family , and nonselective cationic cyclic nucleotide - gated ( cng ) channels , which lead to an off response ( hyperpolarization ) in the presence of light . the same phototransduction components were expressed specifically in the platynereis dorsal brain ( figures s2a s2c , 1e , 1h , and 1k ) . intriguingly , markers of the circadian clock , like bmal ( arendt et al . , 2004 ) , vrille , and l - cry , the ortholog of the drosophila light - sensitive cryptochrome , were expressed in the same region ( figures s2d , 1f , and 1i ; see also zantke et al . , 2013 ) . coexpression of these genes was apparent from the similar position of stained cells adjacent to the photoreceptor cilia ( arrows in figure 1 ) and confirmed with the profiling by image registration ( primr ) technique ( tomer et al . , 2010 ) , which revealed coexpression of platynereis hiomt , l - cry , cnga , and c - opsin1 in a few cells of the dorsal brain , including the ciliary photoreceptors ( blue in figure 1c ) . interestingly , the expression of melatonin synthesis , phototransduction , and clock markers was broader than the highly restricted c - opsin1 expression ( magenta in figure 1c ) and overlapped with that of other opsin family representatives , such as neuropsin ( figure s2e ) and peropsin ( figure 1j ; compare extended red coexpression region in summary figure 1l ) . our data suggest that photosensitivity , circadian entrainment , and melatonin release are directly coupled , at the cellular level , in the platynereis dorsal brain , just as in pineal photoreceptors . the first experimental evidence for such coupling was obtained from a quantification of gene expression dynamics during the light - dark cycle . we measured simultaneously the expression levels of clock genes , neuropeptides , opsins , and genes of the melatonin synthesis pathway using a high - throughput quantitative pcr ( qpcr ) screen ( figures 2 and s3 ) . to this aim , platynereis larvae were sampled between 45 and 75 hpf ( with the night phase between 56 and 64 hpf ) . the circadian system is already active and entrainable at these larval stages : the expression levels of clock genes ( such as clock , period , and tr - cry ; zantke et al . , 2013 ) oscillated throughout the light - dark cycle , and in sibling larvae raised in an inverted light - dark cycle ( shifted by 12 hr ) , the phase of clock gene expression was likewise shifted by 12 hr ( figure 2b ) . clustering analysis of gene expression changes over our entire data set retrieved two major groups : genes with expression levels constantly increasing throughout development ( reflecting the steady increase in the number of differentiated cells at these stages ) and genes upregulated specifically during the night ( figures 2a , 2c , and s3 ) . the first group included neuronal differentiation markers , such as synaptotagmin ( syt ) and prohormone convertase 2 ( phc2 ) , several neuropeptides ( conzelmann et al . , 2011 ) , and rhabdomeric opsins expressed in the eyes ( r - ops1 and r - ops3 ; randel et al . , 2013 ) . the second group included several genes expressed in the hiomt+ cells , such as peropsin , neuropsin , and cng channel subunits , indicating a night - dependent change of light sensitivity . moreover , all genes involved in the melatonin synthesis and degradation pathway ( tph , ddc , mao , aanat , and hiomt ) were upregulated at night , whereas those exclusively involved in serotonin signaling ( vmat , sert ) were not ( figures 2c and 2d ) . these findings suggested that melatonin itself is produced and released during the night , as reported in other invertebrate species ( hardeland and poeggeler , 2003 ) , and that melatonin release is directly controlled by light and/or by the circadian clock . to identify and characterize a possible effect of nocturnal melatonin release on larval behavior , we assayed larval ciliary swimming . in platynereis , the larval brain controls ciliary locomotion by direct innervation of the prototroch , an equatorial girdle of cells equipped with multiple motile cilia ( figures 1a and 1b ) . changes in the ciliary beating frequency ( cbf ) and in ciliary closure time ( i.e. , length and frequency of ciliary arrests ) differentially affect swimming speed , depth , and direction ( conzelmann et al . thus , we assayed cbf and ciliary arrests at 52 hpf , comparing larvae in their midday and midnight ( zt8 and zt20 ; zt , zeitgeber time ; figure 3a ) . although cbf did not differ significantly between day and night ( figure 3b ) , we found a significant increase in overall ciliary closure time at night ( figure 3c ) . treatment at night with the melatonin receptor antagonist luzindole dramatically decreased closure time ( figure 3d ) , indicating that the nocturnal increase in ciliary closure time depends on melatonin signaling . consistently , daytime treatment with 100 m melatonin was sufficient to almost double ciliary closure time ( figure 3e ) . finally , we found that absence of light during daytime was not sufficient by itself to increase ciliary closure time ( figure 3f ) . our data are thus consistent with the possibility that the circadian clock contributes to the regulation of the melatonin rhythm ( as in vertebrates ; falcn et al . , 2010 ) . next , we reasoned that the ciliary closure time depends on two parameters : the frequency and the length of the closure events . therefore , we dissected how these parameters change during day and night . at night , the frequency of ciliary arrests is significantly higher compared to daytime ( figure 3 g ) . moreover , the bimodal distribution of ciliary closure lengths ( figure 3h ) indicated the existence of two distinct kinds of arrests , the short and long closures ( with an average length of 0.2 and 2.2 s , respectively ; figure 3h ) . the day - night transition involves a significant increase of the proportion of long closures ( figures 3i and 3i ) . the melatonin receptor antagonist luzindole applied at night recapitulated the diurnal condition : the overall frequency of arrests was reduced ( figure 3j ) and the long closures were suppressed ( figures 3k and k ) . conversely , treatment with melatonin during the day increased the proportion of long closures to night levels ( figures 3l and 3l ) . taken together , our data indicate the existence of two alternative behavioral states during day and night , which correlate with the day - night differences in gene expression profile . melatonin signaling is necessary and sufficient to increase ciliary closure frequency and length , establishing the nocturnal behavioral state . to dissect the mechanism of ciliary arrest ciliomotor neurons driving ciliary arrests and to test whether they are responsive to melatonin signaling . given that the prototroch cells express the acetylcholine receptor 9/10 ( jkely et al . , 2008 ) , the best candidates were the early developing cholinergic neurons previously identified in the larval brain ( jkely et al . , 2008 ) . consistent with this , application of acetylcholine ( ach ) significantly increased ciliary closure time , whereas the acetylcholine receptor antagonist mecamylamine was able to suppress the ach effect and reduce ciliary arrests ( figure 4a ) . notably , mecamylamine specifically suppressed the long closures that are regulated by melatonin signaling ( figure 4b ) . acetylcholinesterase staining confirmed direct innervation of the prototroch by cholinergic axons ( figure 4c ) . in particular , the ventral cholinergic neurons , some of which directly innervate the prototroch ( cfr mn3l / mn3r in randel et al . , 2014 ) , qualified as candidate melatonin - responsive neurons , as indicated by the coexpression of the platynereis melatonin receptor ( mtnr ; figure s2 ) and the cholinergic marker chat ( figure 4d ) . to test whether melatonin signaling modulates neuronal activity , we performed two - photon imaging on larvae expressing the genetically encoded calcium indicator gcamp6s . to facilitate the identification of the ventral cholinergic neurons , we took advantage of the fact that these cells are located at the interface between the two halves of the larval brain developing from the ab and cd blastomeres ( figure s4 ) . therefore , we expressed gcamp6s and h2b - rfp in the cd lineage by single - blastomere injections ( figure 5a ) . in control conditions , we detected sparse , arrhythmic small - amplitude calcium transients in these neurons and along their projections . unexpectedly , the application of melatonin elicited enhanced neuronal activity in the most ventral cell of the cholinergic cluster . this heightened neuronal activity also revealed a direct contralateral projection to the prototroch cells through the ventral cerebral commissure ( figure 5b ) . melatonin robustly induced a sustained rhythmic activity pattern , characterized by the appearance of gcamp6s peaks of regular amplitude and frequency ( mean frequency : 0.43 hz ; figure 5c ; movie s1 ) . the melatonin - induced calcium peaks were readily suppressed by subsequent addition of the melatonin receptor antagonist luzindole ( figure 5c ) . moreover , we detected this activity pattern specifically in the prototroch - projecting ciliomotor neuron and never in any of the neighboring cd cells ( figure 5d ) . to test whether the melatonin - induced activity recapitulates the nocturnal endogenous activity , we performed the same experiments at night . in untreated larvae , we found spontaneous rhythmic calcium peaks reminiscent of the melatonin - induced activity ( mean frequency : 0.31 hz ; figure 5e ) . these peaks were suppressed by application of luzindole ( figure 5e ) , indicating that at night this rhythmic activity is induced by endogenous melatonin release . analysis of gcamp6s peak frequency and temporal distribution confirmed that daytime administration of melatonin mimics the spontaneous nocturnal activity ( figures 5f and 5 g ) . notably , the rhythmic activity of the cholinergic neuron proved insensitive to mecamylamine treatment ( figure s5 ) , indicating that the effect of cholinergic agonists and antagonists on ciliary arrests occurs downstream of these neurons ( i.e. , at the cholinergic ciliomotor synapses on the prototroch cells ) . to better understand the neurophysiological processes that underlie the observed rhythmic firing and the nocturnal change in ciliary arrests , we performed intracellular sharp electrode recordings from prototroch cells . ciliary arrests coincided with electrical activity , as reported previously from extracellular field potential recordings ( conzelmann et al . , 2011 ) ; however , intracellular recordings with simultaneous imaging of ciliary beating revealed that these arrests coincide with depolarizations that arise from an intrinsic electrical excitability of the prototroch cell ( figure 6a ) . consistent with excitatory cholinergic transmission , application of acetylcholine induced prototroch depolarization , and this effect was suppressed by concurrent mecamylamine application ( figure s6 ) . application of melatonin caused a significant increase in prototroch spike frequency ( figures 6a6c ) , as well as a decrease in the distribution of interspike intervals ( figure 6d ) , reflecting the sustained electrical activity that would be expected given the effect melatonin has on the length of ciliary arrests . intracellular recordings also revealed the presence of excitatory postsynaptic potentials ( epsps ) . in agreement with the calcium imaging data , melatonin changed prototroch epsps from single sparse events to regular periodic bursts , each characterized by a duration of 0.25 0.08 s ( figure 6e ) . the effect of melatonin was reversible with application of the melatonin receptor antagonist luzindole ( figure 6e ) . moreover , application of mecamylamine after melatonin treatment completely eliminated excitatory synaptic transmission , indicating that this activity was exclusively cholinergic ( figure 6e ) . these results indicate that each nocturnal peak of gcamp6s fluorescence corresponds to a burst of action potentials in the presynaptic cholinergic cells , increasing the likelihood of prototroch spiking . to further assess the effect of melatonin - induced bursting on synaptic transmission this revealed a significant increase of the mean epsp amplitude , indicating that the number of synaptic vesicles released per action potential more than doubles during the melatonin - induced bursting ( figures 6f and 6 g ) . at the same time , the mean resting membrane potential of the prototroch cells ( which do not express mtnr ) was not significantly altered in presence of melatonin ( from 70.45 to 70.39 mv , n = 7 , ns ) . therefore , we conclude that at night melatonin signaling directly controls the excitability of presynaptic cholinergic neurons , that it induces rhythmic bursting and potentiates synaptic transmission at the ciliomotor - prototroch cells synapses , and that this augmented release of acetylcholine enhances the frequency and duration of ciliary arrests . our combination of expression profiling , neuronal activity imaging , and intracellular recordings in a zooplankton model reveals a role of melatonin signaling in the circadian control of ciliary swimming . in platynereis , melatonin is necessary and sufficient to establish a nocturnal behavioral state characterized by enhanced ciliary arrests . we can distill the underlying circuit architecture into two major components ( red and blue in figure 7 ) . the first is the sensory - neuromodulatory component , constituted by melatonin - releasing , ambient light - detecting photoreceptors that harbor a circadian clock . the second is the effector component , represented by the cholinergic ciliomotor neurons , which are direct targets of nocturnal melatonin signaling and respond by rhythmic bursting . this system represents a minimalistic example of integration of sensory - neuromodulatory and motor circuits in animal nervous systems . comparative evidence indicates that at least parts of these circuits are of more widespread occurrence in animals and may thus be evolutionary ancient . the shared key feature of the dorsal brain neurons described here is the coexpression of hiomt with markers of opsin - based phototransduction and the circadian clock . the circadian clock is already entrained in early larvae , and several genes expressed in these cells , including hiomt itself , are upregulated at night , suggesting that ambient light detection and the circadian clock control melatonin synthesis . a coupling of these three processes in the same cells is also observed in the pineal photoreceptors of lower vertebrates ( lamb , 2013 ) , and intriguingly , the platynereis ciliary photoreceptors ( previously compared to pineal and retinal photoreceptors ; arendt et al . , 2004 ) are part of the clock - controlled , melatonin - releasing system . the expression of multiple opsins in nonvisual photoreceptors of the most anterior ( or apical ) part of the larval nervous system has been documented in other protostomes and in deuterostomes ( ooka et al . , 2010 ; passamaneck et al . , a shared feature of this so - defined larval apical nervous system ( specified by conserved developmental patterning mechanisms ; marlow et al . , 2014 ) is the presence of multiple sensory cells that project into a neurosecretory neuropil and release hormones and neuromodulators ( tosches and arendt , 2013 ) . the expression of different opsins in subsets of hiomt+ cells in platynereis hints at a sophisticated responsiveness of the apical nervous system to light . opsins responding to different wavelengths would allow dissecting the spectral composition of light at different depths or during dusk and dawn and may also contribute to moonlight detection for the regulation of circalunar rhythms , which in adult worms has been attributed to the brain region harboring the ciliary photoreceptors ( zantke et al . , 2013 ) . the upregulation of peropsin , neuropsin , and cng channels during the dark phase might then prepare the larvae to sense moonlight . the expanding array of functional tools available for platynereis ( zantke et al . , 2014 ) will allow testing these hypotheses and unraveling the contributions of individual opsins and neuropeptides to the annelid ambient light - dependent behaviors . on the effector side , we identify a pair of cholinergic ciliomotor neurons as regulators of ciliary arrests . these neurons belong to a larger cholinergic system that innervates larval ciliary bands , as revealed by acetylcholinesterase staining ; within this system , they are the only neurons expressing the melatonin receptor and responding to melatonin treatments . cholinergic innervation of ciliary bands has been observed in most protostome and deuterostome larvae , and a general requirement of cholinergic transmission for ciliary arrests has been also documented ( lacalli and gilmour , 1990 ; lacalli et al . , 1990 ) , indicating that the cholinergic ciliomotor system described here is of broader relevance for understanding zooplankton neurobiology . in platynereis larvae , the melatonin - sensitive cholinergic system regulates distinct , long - lasting diurnal and nocturnal behavioral states . at the level of the ciliomotor neurons , this regulation involves the switch from sparse firing to rhythmic bursting . this finding is especially interesting as it may shed light on the evolutionary origin of circadian behavioral states in animals . circadian behavioral states , such as sleep - wake cycles , are widespread in the animal kingdom , indicating that the day - night regulation of activity rhythms is crucial for homeostasis and survival ( allada and siegel , 2008 ) . a defining and conserved feature of sleep , which makes it distinct from rest , is the disconnection of the brain from the sensory environment and hence the increase of arousal threshold . platynereis larvae can respond to a variety of sensory cues , which would ultimately affect ciliary arrests through the same ciliomotor system . for example , ciliomotor neurons are innervated by interneurons of the larval visual system , indicating that they respond to visual stimuli , together with other motoneurons ( randel et al . , 2014 ) . but could any sensory cue be relayed in presence of rhythmic bursting ? if not , the nocturnal rhythmic burst firing might be a mechanism that filters incoming sensory information and reduces responsiveness to sensory stimuli . in a similar way , in mammals , the switch of thalamic relay neurons to rhythmic burst firing is responsible for filtering sensory information during sleep ( mccormick and feeser , 1990 ) and can be induced directly by melatonin ( ochoa - sanchez et al . , 2011 ) . it will be interesting to determine whether these functional analogies correspond to conserved molecular mechanisms , for instance , to conserved targets downstream of melatonin signaling . promising candidates are shaker voltage - gated potassium channels , key determinants of neuronal excitability , which are regulated by melatonin ( yang et al . , 2011 ) and are necessary for sleep in flies and mammals ( cirelli et al . , 2005 ; douglas et al . , 2007 ) the most prominent behavior associated with day - night cycles is dvm ( see introduction ) . we propose that the day - night changes in ciliary closure time evoked by melatonin signaling in platynereis correspond to different phases of dvm in the field . in platynereis , a long ciliary closure time correlates with a low vertical position ( conzelmann et al . , 2011 ) ; therefore , the nocturnal behavior would lead to the slow descent of larvae in the water column . in line with this conclusion , field studies have shown that planktonic annelids reach their highest position in the water column at dusk and then sink throughout the whole night ( alldredge and king , 1980 ) . other zooplankton species show different temporal dvm regimes that often involve muscular rather than ciliary swimming , suggesting a more complex regulation of locomotor activities downstream of melatonin signaling as an adaptation to variable ecological constraints ( lampert , 1989 ) . interestingly , it has been proposed that circadian rhythms and ambient light detection evolved in the context of dvm , an advantageous behavior facilitating escape from light - induced oxidative stress ( gehring and rosbash , 2003 ; pittendrigh , 1993 ) . light - sensitive cryptochromes , present in all basal metazoan phyla , including sponges ( rivera et al . , 2012 ) , might have represented the first link between ambient light detection and clock entrainment . we now extend this reasoning with the idea that melatonin signaling has been added to this system as an efficient paracrine signal , ensuring better coordination of the organismal response and whole - body physiology and becoming a perfect chemical indicator of darkness after its original radical scavenger role . because melatonin receptors and opsins arose from the same duplication of an ancestral g - protein - coupled receptor ( feuda et al . , 2012 ) , we further hypothesize that melatonin signaling and opsin - based phototransduction evolved in the same dvm context . as photopigment , opsin outperformed cryptochrome by increased signaling speed , signal amplification , coupling with different transduction cascades and an evolvable spectral tuning that , after repeated duplications , allowed perception of colors at different depths and times of the day ( nilsson , 2013 ) . this way the full system involving opsin - based ambient light detection , circadian clock , and melatonin signaling became a key regulator of circadian behavior and persisted in the dorsal brain of annelids and in the pineal and retinal photoreceptors of vertebrates . platynereis genes were either obtained from expressed sequence tag libraries or cloned with rapid amplification of cdna ends ( race ) pcrs and/or rt - pcr using gene specific primers ( pcr primers are listed in the extended experimental procedures ) . in situ hybridizations and acetylcholinesterase stainings ( ache ) were performed and imaged following established protocols , as detailed in the extended experimental procedures . platynereis larvae were fertilized at zt8 ( midday ) and raised in an 18c incubator , under a 16l:8d cycle . nine groups were sampled at different time points , as indicated in figures 2 and s3 . at night , animals were collected under dim red light . for the experiments with the inverted cycle conditions , immediately after fertilization , sibling larvae were raised under an inverted light cycle ( 12 hr shift ) . cdna was synthesized from 100 ng of total rna ; 1.25 l of each cdna was used as a template for a preamplification reaction ( preamp ) of target genes . diluted preamps were loaded on a 48.48 fluidigm biomark dynamic array chip ( spurgeon et al . , 2008 ) . the chip was run following manufacturer s instructions ( see the extended experimental procedures for further details on the protocol and the list of primers used ) . fluidigm data were analyzed with the bioconductor htqpcr package ( dvinge and bertone , 2009 ) . the genes cdc5 and rps9 were used to obtain the normalized dcq values , and for each time point the two dcqs from the two preamps were averaged . expression relative to the first time point ( 45 hpf ) was calculated as 2 . the expression values of all the genes ( except the housekeeping genes ) were used for clustering analysis . clustering was performed with the average method using the euclidean distance . for clustering , expression values were scaled to the same range in order to compare similar trends regardless of the amplitude of expression changes . the following drugs were used : l - cis - diltiazem ( enzo life sciences ) in natural sea water ( nsw ) , luzindole ( tocris ) , stock in dmso , mecamylamine ( sigma ) , stock in 95% etoh or nsw , and melatonin ( sigma ) and acetylcholine ( sigma ) solutions freshly prepared in nsw . drugs were diluted to their final concentration in nsw ; corresponding concentrations of vehicle were used as controls . ciliary beating and arrests were imaged as described ( conzelmann et al . , 2011 ) , with a dmk 21bf04 camera ( the imaging source ) and a frame rate of 60 or 15 frames / s , a 750-nm - long pass filter was always interposed between the light source and larvae . behavioral data were analyzed in r. all the experiments were repeated at least twice with larvae from different embryonic batches . platynereis cd blastomeres were injected with gcamp6s ( chen et al . , 2013 ) and h2b - rfp mrnas ( final concentration of 250 g/l each ) . injected larvae at the 4852 hpf stage were mounted in 3% low - melting agarose on glass - bottom culture dishes ( mattek ) and maintained in nsw . imaging was performed using a zeiss lsm780 microscope , with a 32-ch gaasp detector and a two - photon light source ( chamaleon , coherent ) set at 910 nm . two - photon imaging was chosen to avoid any interference of light with behavioral responses . movies ( 248 250 pixels ) were acquired under a 40 oil - immersion objective and with a temporal resolution between 8.26 and 16 hz . responses to drugs were imaged 520 min after application to the bath and without any change of imaging settings . gcamp6s movies were analyzed using fiji and r , as described in the extended experimental procedures . the same region of interest ( roi ) was used to quantify fluorescence before and after drug application . sharp electrode recordings with simultaneous high - speed imaging were performed on 4060 hpf platynereis larvae . holding pipettes were made from borosilicate glass ( science products ) with an outer diameter ( od ) of 1 mm and were fire polished to minimize damage to the larvae . recording electrodes were made from pipettes with an od of 1.5 mm , filled with 3 m kcl , and showed resistances between 1525 m. to facilitate electrode placement , larvae were digested with 46.7 g / ml of proteinase k for 1015 min . signals were acquired at 20 khz and analyzed using clampfit 10.3 ( molecular devices ) . input resistances of prototroch cells were monitored by delivering small hyperpolarizing currents via the recording electrode , and only prototroch cells that displayed resting potentials between 65 to 80 mv and input resistances between 1025 m were used for analysis . simultaneous high - speed ( 20 hz ) imaging of ciliary beating was performed on an andor neo s - cmos camera and analyzed using fiji . larvae were raised in natural seawater ( nsw ) , at 18c under a 16 hr light - 8 hr dark cycle.sources of platynereis genes and phylogenetic analysisgenesnew platynereis genes were cloned either directly , with rt - pcr and gene specific primers , or first with race pcrs using a mixed stages cdna library ( from 24hpf , 39hpf , 48hpf , 56hpf , 72hpf and 5 dpf larvae , invitrogen generacer kit ) . pcr primers were designed on the basis of available transcriptome and est data , and for amplification we used either the hotstart taq polymerase ( qiagen ) or the phusion polymerase ( new england biolabs ) . pcr fragments were cloned in the pcrii - topo vector ( invitrogen ) and verified by sequencing.the following race primers were used to isolate the hiomt cdna sequence : ttggagcataaatgattgggctgatgg , aaggtggcgatattccttccctgaacg , atgcctccaccagggttgattttctca , gctcagcaggtagaggtcggcttca , tgatgaagaagtccccgggcaaa , tgcatgagagatacagggccttcgaca , agtgctgtcgatttaggagctggcaca.the following pcr primers were used to amplify cdnas and produce probes for in situ hybridization : gene nameforward primerreverse primeraanattggtgtaggtatagcacaagtcacatctttttatgttgaaattgagtttggatttcngaacttggattgcagggtacttggagcaaacattatgaccatggcgttccaactgacngbcagtctgcagatgcagcgaagacaaattgtgagcttcagggtagtcactcatgggchgactccagcgacgtcagtcctatccatgtttgattggtggactgcacattttcggiacaaatctttaagacaggatggagggttcgtaacaacactgtacchiomtcaggggaaggacggaaagcacaattaccaccaaaactttgtgcacatatcacctcamaoaggagcaggcataagtggtctctcagccacagtcagaaagcacacactggcaganeuropsinttcccgggatcctgttacccagttagggagggctctgaccgactcttgttgtgaperopsintggtgatgtgccactgctagtcagcatgcaaactgcataaaccagagggacacctgvmattactcggacgacaaacaaaggagcagagcagccatcttgaaacgcacacataagvrilleggacaccaccaagtgtgccactccactgttttcgcttgtggcggtgttactin order to design qpcr primers and/or produce probes for in situ hybridization , the sequences for the following genes were obtained from previous publications : ddc ( raible et al . , 2005 ) , fmrf , phc2 , tph and vtn ( tessmar - raible et al . , 2007 ) , chat ( denes et al . , 2007 ) , fvri ( jkely et al . , 2008 ) , dlamide , flamide , fvamide , yfamide , ryamide and wldamide ( conzelmann et al . , 2011 ) , r - opsin4 ( randel et al . , 2013 ) , tr - cry and clock ( zantke et al . , 2013 ) and npy-4 ( conzelmann et al . , 2013).phylogenetic analysessequence data were retrieved from the jgi genome portal webserver , ncbi and ensembl . multiple sequence alignments of protein sequences were generated with muscle ( http://www.ebi.ac.uk/tools/msa/muscle/ ) and with webprank ( lytynoja and goldman , 2008 ) , inspected and corrected in jalview ( waterhouse et al . phylogenetic trees with the maximum likelihood method were computed with phyml 3.0 ( guindon et al . , 2010 ) , with 100x bootstrap . phylogenetic trees were plotted with figtree.imaging of gene expression patternsin situ hybridizationswhole mount in situ hybridizations and tubulin counterstaining were performed as described previously ( arendt et al . , 2001 ) , with few modifications . for hiomt in situs , probe binding specificity was improved by carrying hybridization and post - hybridization washes at 62c and by using a lower ssc concentration for the last post - hybridization washes ( 0.1x ssc instead of 0.2x ssc ) . in situ hybridizations were normally counterstained with an anti - acetylated tubulin antibody ( sigma , t7451 ) , which labels the axonal scaffold and the ciliary bands . for early developmental stages , larvae were acetylated after the proteinase k digestion with the following protocol : 5 min in 1% triethanolamine , 5 min in 1% triethanolamine 3 l / ml acetic anhydride , 5 min in 1% triethanolamine 6 acetylated larvae were counterstained with the anti - tyrosinated tubulin antibody ( sigma t9028).imagingwe used a zeiss axio imager.m1 microscope for light microscopy and a leica tcs spe and a leica tsc sp8 for confocal microscopy . confocal stacks of expression patterns were acquired with reflection microscopy ( jkely and arendt , 2007 ) , using a 40x oil immersion objective . contrast and brightness were adjusted equally throughout the images.image registrationprofiling by image registration ( primr ) was performed as described in tomer et al . average expression patterns was performed using built - in functions in fiji and imaris.acetylcholinesterase staining and imagingacetylcholinesterase ( ache ) staining was performed according to denes et al . ( 2007 ) and karnovsky and roots ( 1964 ) , with some modifications . before fixation , larvae were digested for 1 min with 46.7 g / ml of proteinase k in nsw . larvae were then fixed in ice - cold etoh for 2 min or in 4% pfa for 10 min , and stained for 3 - 4 hr at room temperature in 65 mm phosphate buffer ph 6 , 0.5mg / ml ach iodide , 5 mm sodium citrate , 3 mm copper sulfate and 0.5 mm potassium ferricyanide . staining was stopped with 50% etoh , then larvae were dehydrated with an etoh series and transferred in 87% glycerol for imaging . reflection microscopy was used for confocal imaging of the ache signal.qrt-pcr screen with fluidigm dynamic arrayssample preparationeight batches of platynereis larvae were fertilized at the same time at zt8 ( midday ) . they were subsequently pooled , separated again into nine groups and raised in a 18c incubator , under a 16 hr light - 8 hr dark cycle . the nine groups were sampled at different time points between 45hpf and 75hpf , and frozen in liquid nitrogen . the time points were selected as follows ( see also figure 2a ) : 1 ) 45hpf zt5 ; 2 ) 48hpf zt8 ; 3 ) 54hpf zt14 ; 4 ) 57hpf zt17 ; 5 ) 60hpf zt20 ; 6 ) 63.5hpf zt23.5 ; 7 ) 67.5hpf zt3.5 ; 8) 72.5hpf zt8.5 ; 9 ) 75hpf zt11 . for the nighttime samples , animals were collected under dim red light . for the experiments with the inverted cycle conditions , immediately after fertilization sibling larvae were transferred into a different 18c incubator with inverted light cycle conditions ( 12 hr shift ) , and then sampled at the same developmental stages . total rna was extracted from frozen samples using trizol ( peqlab ) and phenol - chloroform , and rna quality was assessed with bioanalyzer ( agilent technologies).reverse transcription , preamplification of cdna , and qrt - pcrfor each sample , 100ng of total rna were reversed transcribed with superscriptiii first - strand synthesis supermix for qrt - pcr ( invitrogen ) , in a 10 l reaction with oligo - d(t ) and random hexameres . 1.25 l of each cdna ( corresponding to 12.5ng of initial rna ) were used as template for a preamplification reaction ( preamp ) with the abi preamp mastermix kit ( pn4391128 ) . for each sample , preamps were run in duplicate.each preamp consisted in the specific target amplification ( sta ) of all the 48 target genes . the gene specific primers were pooled to a final concentration of 50 nm each , and preamplification was carried out for 14 cycles . after preamp , sta primers were removed with exonuclease i ( neb ) , and the cdnas were diluted 10 times . conventional qpcr was used to confirm the linearity of the preamp reactions.to quantify gene expression levels , we used a high - throughput microfluidics 48.48 fluidigm biomark dynamic array chips were used to measure the expression of 48 target genes , following the manufacturer instructions . the inlets were loaded with 2.25 l of the diluted preamp cdnas or with gene specific primers , to an inlet concentration of 5 m ( corresponding to a 500 nm concentration in the final reaction ) . serial dilutions of the templates were included to control the linearity of the amplification . the fluidigm real time pcr analysis software was used to set an optimal global threshold for all the target genes and obtain cq values . these results were confirmed with independent qpcr experiments on a subset of target genes.data analysisthe fluidigm data were analyzed with the bioconductor htqpcr package ( dvinge and bertone , 2009 ) . the genes cdc5 and rps9 , previously shown as reliable normalization genes ( dray et al . , 2010 ) , were used to obtain the normalized dcq values , and for each time point the two dcqs from the two preamps were averaged . the expression values for all the genes ( except the housekeeping genes ) were used for clustering analysis . clustering was performed with the average method using the euclidean distance . for clustering , expression values were scaled in order to compare similar trends regardless of the amplitude of expression changes . we obtained comparable results for other two biological replicates assayed independently.qpcr primersqpcr primers were designed to target , whenever possible , sequences without snps , and to span exon - exon junctions ( as assessed from internal transcriptomic and genomic resources ) . the efficiency of all the primer pairs was determined experimentally using as template a dilution series of cdna ; only primers with efficiency between 90% and 110% were used . the selected primers have an average tm of 60c and an average amplicon size of 91.4bp . all the primers used for qpcr are listed in the following table ( gene name , forward primer , reverse primer):gene nameforward qpcr primerreverse qpcr primercdc5cctattgacatggacgaagatgttccctgtgtgttcgcaagrps9cgccagagagttgctgactactccaatacggaccagacgaanatccaaacatcatggcactgacagtggcagcttcatcacttgddcatgttgttccgaccgatgacgaaagcacatgtcggagttggchaaagccctcctctacttcacctctcggacaatcaccatctchiomtacatgtgggacaccttcatcaggtggcgatattccttccmaoatctgggaggtcgataatgggctgacttgcattgaacctcmtnratcaatcccttcagcattacagcgacagaggcccaaattatcsertcaacgagaccaacgtcaaaccccaagcctcttgctttatctphccctcaagaagctgaatctgatttcgagagttggggtctcvmatgatggttatcagccggattcgaatgggatacaagccaagcbmalcagcatgccataaaaagaaacgcaggatttcaaataaccagcaaaaclockgaggttcccgaatattcaaatcagtcagagagatcggttcgtagl - cryagaagcccttcaacaagtgggcgttcaatcatgagacgagtr - cryatgggacaagaatccagaggcatgacgagcaagatgatggvrilleactggatgaagagacgcaagttagctcacgacgaaggttgperiodtcctgcagcacatgaagaagtggcaccaaggagttctatgc - ops1tctgcaagtggtatggcttcaaacttccaagcctcaggacneuropsatttcgccgagaggtgatagtcgccatggttgcattagperopscgaggtatgctttggacaaggaagcagctcatggttatggr - ops1accctatgccgtcgttggaggcgagcagagggtggatr - ops3tcattcactgggtcatagggtggacggagttcgtaatgagr - ops4aaagccatccagcactcaccaatagccagatgggttgagbsxcccagaaagagtggaattggtgcggtgaatgtgactgttcnotaaagtcaggagggatggttccctaaccctctttgctttgcdlamidegcattgccttacagaagcagtgagaagacactgacgaccagflamidecacacttaaaggaggcaagcgagcctacattggcactttgfmrfattcgggaaacgggaaagcttcccaaacctcatgaaccfvamidectcactaggtgggcaaaatgtcatcttcgtcatcctctcgfvricaaagaccgacttcaccaagcatactgtcacaacggactggyfamidecaatggtgaactgcaagaccggatatctcttctcgctgtcgnpy-4gcaagaggacaatagcagagtggtggatatgttccatggcttcpdfaggatgggatcaagcaagacagacaacctgcatgacgaacryamidectctcattctcgctttagcccctcgcttgtcatcttcatcvtncagtgtttcggacccaacattaacgtgttaatgtagcatcctattgawldamideaaacgccgtcctaactcaaggtggcagtttgtttgctgtgcngattgctggtcacagaaagaggaatcagtgggaaggatggaccngatgccgtccatagcagtaaaccaaagcgcactacaaagctgcngbagtcctgcctaagatcctgaagaagagctgcactttgctgagphc2caacggagaacacaactggacatatccgaagaggtggttgasytaaagaaaaggtgcagcctgatcatagatggcgaacaccagbehavioral experimentsanimalsfor all the behavioral experiments , animals were raised in an 18c incubator , under a 16 hr light - 8 hr dark cycle . all experiments were typically performed between 51 and 55hpf . for the midday conditions ( zt8 ) , fertilizations were set up at zt4 , whereas for midnight conditions ( zt20 ) fertilizations were set up at zt16 . therefore the night experiments were specifically performed in the second half of the night phase.pharmacologythe following drugs were used : l - cis - diltiazem ( enzo life sciences ) , 10 mm stock in nsw ; luzindole ( tocris ) , 100 mm stock in dmso ; mecamylamine ( sigma ) , 10 mm stock in 95% etoh or freshly dissolved in nsw ; melatonin ( sigma ) and ach ( sigma ) solutions were freshly prepared in nsw . the drugs were diluted to the final concentration in nsw ; corresponding concentrations of vehicle were used as controls.imagingciliary beating and ciliary arrests were imaged as described ( conzelmann et al . , 2011 ) , with a zeiss axiophot microscope equipped with a dmk 21bf04 camera ( the imaging source ) . movies were acquired with a frame rate of 60 or 15 frames / s , respectively . a 750 nm long pass filter for the nighttime measurements , larvae were quickly mounted under a dim red light and imaging was performed in complete darkness . for the ach experiments ( figure 4 ) , each larva was imaged after acute treatment with ach and/or mecamylamine ( within 1 min after drug application ) . for the melatonin and luzindole experiments , larvae were incubated with the drugs for at least 20 min before mounting and imaging.data analysisbehavioral data were analyzed in r ( r core team , 2012 ) . all the experiments were repeated at least twice with larvae from different embryonic batches . for the analysis of closure length distribution , the log10 transformed data set was used in the histograms of figure 3 , and the data are normalized according to the number of larvae imaged in each condition.two-photon calcium imaging with gcamp6smicroinjection of zygotesplatynereis zygotes were injected using a zeiss axiovert 40c inverted microscope , equipped with a joystick micromanipulator ( narishige ) and a microinjector ( femtojet , eppendorf ) . injection needles were pooled from glass capillaries ( borosilicate thin wall with filament , 1.0 mm outer diameter , 0.78 mm inner diameter , harvard apparatus ) using a sutter needle pooler . embryos were accommodated on an agarose stage , customized for platynereis injections.gcamp6s ( chen et al . , 2013 ) was kindly provided by the genie project , janelia farm research campus , howard hughes medical institute ( addgene plasmid 40753 ) . the gcamp6s coding sequence was subcloned in the pcs2 + vector , and mrna was in vitro transcribed with the sp6 mmessage mmachine high yield capped rna transcription kit ( ambion ) . gcamp6s mrna was injected in the cd blastomere to a final concentration of 250 g/l . h2b - rfp mrna was co - injected at the same concentration in order to label the cd lineage.two-photon imagingfor calcium imaging , 48 - 52hpf larvae were mounted on glass bottom culture dishes ( mattek ) in 3% low melting agarose , and maintained in natural seawater . imaging was performed using a zeiss lsm780 microscope , with a 32-ch gaasp detector and a two - photon light source ( chamaleon , coherent ) running at 910 nm . movies ( 248x250 pixels ) were acquired under a 40x oil - immersion objective , for at least one minute and with a temporal resolution ranging from 8.26hz to 16hz . drugs were applied directly to the seawater , and responses were imaged 20 min after application ( or 5 min after application for the mecamylamine experiment in figure s5 ) . for the calcium imaging experiments during the night , larvae were mounted under dim red light and imaged in complete darkness.image analysisgcamp6s movies were analyzed using fiji and r. the cholinergic ciliomotor neuron was identified on the basis of its position within the cd domain and the presence of a contralateral projection . rois ( region of interest ) were manually selected around the cell body and/or the axon of the cholinergic ciliomotor neuron . in each experiment , the same roi was used to compare gcamp6s fluorescence before and after the drug treatments . for the calculation of normalized f / f0 data with a global baseline , f0 was set as the average fluorescence of the 11-frames interval centered on the minimum fluorescence value . for the calculation of normalized f / f0 data with the moving average approach , a sliding window of 0.8 s was used to calculate f0 . for the analysis of peak frequency and peak - to - peak intervals , peaks were extracted from the imaging data with custom - written algorithms.electrophysiologysharp electrode recordings along with simultaneous high - speed imaging were performed on 4060 hpf platynereis larvae . holding pipettes were made from borosilicate glass ( science products gmbh , hofheim ) with an outer diameter ( o.d . ) of 1 mm and were fire polished to minimize damage to the larvae . of 1.5 mm , filled with 3 m kcl , and showed resistances between 15 25 m. to facilitate electrode placement , larvae were digested with 46.7 g / ml of proteinase k for 10 - 15 min.electrophysiological recordings were performed on a multiclamp 700a amplifier . signals were acquired at 20 khz and analyzed using clampfit 10.3 ( molecular devices , union city , ca ) . input resistances of prototroch cells were monitored by delivering small hyperpolarizing currents via the recording electrode and only prototroch cells which displayed resting potentials between 65 to 80 mv and input resistances between 10 25 m were used for analysis . simultaneous high - speed ( 20 hz ) imaging was performed on an andor neo s - cmos camera and analyzed using fiji . larvae were raised in natural seawater ( nsw ) , at 18c under a 16 hr light - 8 hr dark cycle . new platynereis genes were cloned either directly , with rt - pcr and gene specific primers , or first with race pcrs using a mixed stages cdna library ( from 24hpf , 39hpf , 48hpf , 56hpf , 72hpf and 5 dpf larvae , invitrogen generacer kit ) . pcr primers were designed on the basis of available transcriptome and est data , and for amplification we used either the hotstart taq polymerase ( qiagen ) or the phusion polymerase ( new england biolabs ) . pcr fragments were cloned in the pcrii - topo vector ( invitrogen ) and verified by sequencing . the following race primers were used to isolate the hiomt cdna sequence : ttggagcataaatgattgggctgatgg , aaggtggcgatattccttccctgaacg , atgcctccaccagggttgattttctca , gctcagcaggtagaggtcggcttca , tgatgaagaagtccccgggcaaa , tgcatgagagatacagggccttcgaca , agtgctgtcgatttaggagctggcaca . the following pcr primers were used to amplify cdnas and produce probes for in situ hybridization : gene nameforward primerreverse primeraanattggtgtaggtatagcacaagtcacatctttttatgttgaaattgagtttggatttcngaacttggattgcagggtacttggagcaaacattatgaccatggcgttccaactgacngbcagtctgcagatgcagcgaagacaaattgtgagcttcagggtagtcactcatgggchgactccagcgacgtcagtcctatccatgtttgattggtggactgcacattttcggiacaaatctttaagacaggatggagggttcgtaacaacactgtacchiomtcaggggaaggacggaaagcacaattaccaccaaaactttgtgcacatatcacctcamaoaggagcaggcataagtggtctctcagccacagtcagaaagcacacactggcaganeuropsinttcccgggatcctgttacccagttagggagggctctgaccgactcttgttgtgaperopsintggtgatgtgccactgctagtcagcatgcaaactgcataaaccagagggacacctgvmattactcggacgacaaacaaaggagcagagcagccatcttgaaacgcacacataagvrilleggacaccaccaagtgtgccactccactgttttcgcttgtggcggtgttact in order to design qpcr primers and/or produce probes for in situ hybridization , the sequences for the following genes were obtained from previous publications : ddc ( raible et al . , 2005 ) , fmrf , phc2 , tph and vtn ( tessmar - raible et al . , 2007 ) , chat ( denes et al . , 2007 ) , fvri ( jkely et al . , 2008 ) , dlamide , flamide , fvamide , yfamide , ryamide and wldamide ( conzelmann et al . , 2011 ) , r - opsin4 ( randel et al . , 2013 ) , tr - cry and clock ( zantke et al . , 2013 ) and npy-4 ( conzelmann et al . , 2013 ) . multiple sequence alignments of protein sequences were generated with muscle ( http://www.ebi.ac.uk/tools/msa/muscle/ ) and with webprank ( lytynoja and goldman , 2008 ) , inspected and corrected in jalview ( waterhouse et al . , 2009 ) , and trimmed with gblocks ( castresana , 2000 ) . phylogenetic trees with the maximum likelihood method were computed with phyml 3.0 ( guindon et al . , 2010 ) , with 100x bootstrap . whole mount in situ hybridizations and tubulin counterstaining were performed as described previously ( arendt et al . , 2001 ) , with few modifications . for hiomt in situs , probe binding specificity was improved by carrying hybridization and post - hybridization washes at 62c and by using a lower ssc concentration for the last post - hybridization washes ( 0.1x ssc instead of 0.2x ssc ) . in situ hybridizations were normally counterstained with an anti - acetylated tubulin antibody ( sigma , t7451 ) , which labels the axonal scaffold and the ciliary bands . for early developmental stages , larvae were acetylated after the proteinase k digestion with the following protocol : 5 min in 1% triethanolamine , 5 min in 1% triethanolamine 3 l / ml acetic anhydride , 5 min in 1% triethanolamine 6 acetylated larvae were counterstained with the anti - tyrosinated tubulin antibody ( sigma t9028 ) . we used a zeiss axio imager.m1 microscope for light microscopy and a leica tcs spe and a leica tsc sp8 for confocal microscopy . confocal stacks of expression patterns were acquired with reflection microscopy ( jkely and arendt , 2007 ) , using a 40x oil immersion objective . profiling by image registration ( primr ) was performed as described in tomer et al . ( 2007 ) and karnovsky and roots ( 1964 ) , with some modifications . before fixation , larvae were digested for 1 min with 46.7 g / ml of proteinase k in nsw . larvae were then fixed in ice - cold etoh for 2 min or in 4% pfa for 10 min , and stained for 3 - 4 hr at room temperature in 65 mm phosphate buffer ph 6 , 0.5mg / ml ach iodide , 5 mm sodium citrate , 3 mm copper sulfate and 0.5 mm potassium ferricyanide . staining was stopped with 50% etoh , then larvae were dehydrated with an etoh series and transferred in 87% glycerol for imaging . eight batches of platynereis larvae were fertilized at the same time at zt8 ( midday ) . they were subsequently pooled , separated again into nine groups and raised in a 18c incubator , under a 16 hr light - 8 hr dark cycle . the nine groups were sampled at different time points between 45hpf and 75hpf , and frozen in liquid nitrogen . the time points were selected as follows ( see also figure 2a ) : 1 ) 45hpf zt5 ; 2 ) 48hpf zt8 ; 3 ) 54hpf zt14 ; 4 ) 57hpf zt17 ; 5 ) 60hpf zt20 ; 6 ) 63.5hpf zt23.5 ; 7 ) 67.5hpf zt3.5 ; 8) 72.5hpf zt8.5 ; 9 ) 75hpf zt11 . for the nighttime samples , animals were collected under dim red light . for the experiments with the inverted cycle conditions , immediately after fertilization sibling larvae were transferred into a different 18c incubator with inverted light cycle conditions ( 12 hr shift ) , and then sampled at the same developmental stages . total rna was extracted from frozen samples using trizol ( peqlab ) and phenol - chloroform , and rna quality was assessed with bioanalyzer ( agilent technologies ) . for each sample , 100ng of total rna were reversed transcribed with superscriptiii first - strand synthesis supermix for qrt - pcr ( invitrogen ) , in a 10 l reaction with oligo - d(t ) and random hexameres . 1.25 l of each cdna ( corresponding to 12.5ng of initial rna ) were used as template for a preamplification reaction ( preamp ) with the abi preamp mastermix kit ( pn4391128 ) . for each sample each preamp consisted in the specific target amplification ( sta ) of all the 48 target genes . the gene specific primers were pooled to a final concentration of 50 nm each , and preamplification was carried out for 14 cycles . after preamp , sta primers were removed with exonuclease i ( neb ) , and the cdnas were diluted 10 times . conventional qpcr was used to confirm the linearity of the preamp reactions . to quantify gene expression levels , we used a high - throughput microfluidics qpcr system from fluidigm ( spurgeon et al . , 2008 ) . 48.48 fluidigm biomark dynamic array chips were used to measure the expression of 48 target genes , following the manufacturer instructions . the inlets were loaded with 2.25 l of the diluted preamp cdnas or with gene specific primers , to an inlet concentration of 5 m ( corresponding to a 500 nm concentration in the final reaction ) . the fluidigm real time pcr analysis software was used to set an optimal global threshold for all the target genes and obtain cq values . the fluidigm data were analyzed with the bioconductor htqpcr package ( dvinge and bertone , 2009 ) . the genes cdc5 and rps9 , previously shown as reliable normalization genes ( dray et al . , 2010 ) , were used to obtain the normalized dcq values , and for each time point the two dcqs from the two preamps were averaged . the expression values for all the genes ( except the housekeeping genes ) were used for clustering analysis . clustering was performed with the average method using the euclidean distance . for clustering , expression values were scaled in order to compare similar trends regardless of the amplitude of expression changes . qpcr primers were designed to target , whenever possible , sequences without snps , and to span exon - exon junctions ( as assessed from internal transcriptomic and genomic resources ) . the efficiency of all the primer pairs was determined experimentally using as template a dilution series of cdna ; only primers with efficiency between 90% and 110% were used . the selected primers have an average tm of 60c and an average amplicon size of 91.4bp . all the primers used for qpcr are listed in the following table ( gene name , forward primer , reverse primer):gene nameforward qpcr primerreverse qpcr primercdc5cctattgacatggacgaagatgttccctgtgtgttcgcaagrps9cgccagagagttgctgactactccaatacggaccagacgaanatccaaacatcatggcactgacagtggcagcttcatcacttgddcatgttgttccgaccgatgacgaaagcacatgtcggagttggchaaagccctcctctacttcacctctcggacaatcaccatctchiomtacatgtgggacaccttcatcaggtggcgatattccttccmaoatctgggaggtcgataatgggctgacttgcattgaacctcmtnratcaatcccttcagcattacagcgacagaggcccaaattatcsertcaacgagaccaacgtcaaaccccaagcctcttgctttatctphccctcaagaagctgaatctgatttcgagagttggggtctcvmatgatggttatcagccggattcgaatgggatacaagccaagcbmalcagcatgccataaaaagaaacgcaggatttcaaataaccagcaaaaclockgaggttcccgaatattcaaatcagtcagagagatcggttcgtagl - cryagaagcccttcaacaagtgggcgttcaatcatgagacgagtr - cryatgggacaagaatccagaggcatgacgagcaagatgatggvrilleactggatgaagagacgcaagttagctcacgacgaaggttgperiodtcctgcagcacatgaagaagtggcaccaaggagttctatgc - ops1tctgcaagtggtatggcttcaaacttccaagcctcaggacneuropsatttcgccgagaggtgatagtcgccatggttgcattagperopscgaggtatgctttggacaaggaagcagctcatggttatggr - ops1accctatgccgtcgttggaggcgagcagagggtggatr - ops3tcattcactgggtcatagggtggacggagttcgtaatgagr - ops4aaagccatccagcactcaccaatagccagatgggttgagbsxcccagaaagagtggaattggtgcggtgaatgtgactgttcnotaaagtcaggagggatggttccctaaccctctttgctttgcdlamidegcattgccttacagaagcagtgagaagacactgacgaccagflamidecacacttaaaggaggcaagcgagcctacattggcactttgfmrfattcgggaaacgggaaagcttcccaaacctcatgaaccfvamidectcactaggtgggcaaaatgtcatcttcgtcatcctctcgfvricaaagaccgacttcaccaagcatactgtcacaacggactggyfamidecaatggtgaactgcaagaccggatatctcttctcgctgtcgnpy-4gcaagaggacaatagcagagtggtggatatgttccatggcttcpdfaggatgggatcaagcaagacagacaacctgcatgacgaacryamidectctcattctcgctttagcccctcgcttgtcatcttcatcvtncagtgtttcggacccaacattaacgtgttaatgtagcatcctattgawldamideaaacgccgtcctaactcaaggtggcagtttgtttgctgtgcngattgctggtcacagaaagaggaatcagtgggaaggatggaccngatgccgtccatagcagtaaaccaaagcgcactacaaagctgcngbagtcctgcctaagatcctgaagaagagctgcactttgctgagphc2caacggagaacacaactggacatatccgaagaggtggttgasytaaagaaaaggtgcagcctgatcatagatggcgaacaccag for all the behavioral experiments , animals were raised in an 18c incubator , under a 16 hr light - 8 hr dark cycle . all experiments were typically performed between 51 and 55hpf . for the midday conditions ( zt8 ) , fertilizations were set up at zt4 , whereas for midnight conditions ( zt20 ) fertilizations were set up at zt16 . therefore the night experiments were specifically performed in the second half of the night phase . the following drugs were used : l - cis - diltiazem ( enzo life sciences ) , 10 mm stock in nsw ; luzindole ( tocris ) , 100 mm stock in dmso ; mecamylamine ( sigma ) , 10 mm stock in 95% etoh or freshly dissolved in nsw ; melatonin ( sigma ) and ach ( sigma ) solutions were freshly prepared in nsw . the drugs were diluted to the final concentration in nsw ; corresponding concentrations of vehicle were used as controls . ciliary beating and ciliary arrests were imaged as described ( conzelmann et al . , 2011 ) , with a zeiss axiophot microscope equipped with a dmk 21bf04 camera ( the imaging source ) . movies were acquired with a frame rate of 60 or 15 frames / s , respectively . a 750 nm long pass filter was always interposed between the light source and larvae . ciliary closure time was measured from 1 min long movies . for the nighttime measurements , larvae were quickly mounted under a dim red light and imaging was performed in complete darkness . for the ach experiments ( figure 4 ) , each larva was imaged after acute treatment with ach and/or mecamylamine ( within 1 min after drug application ) . for the melatonin and luzindole experiments , larvae were incubated with the drugs for at least 20 min before mounting and imaging . all the experiments were repeated at least twice with larvae from different embryonic batches . for the analysis of closure length distribution , the log10 transformed data set was used in the histograms of figure 3 , and the data are normalized according to the number of larvae imaged in each condition . platynereis zygotes were injected using a zeiss axiovert 40c inverted microscope , equipped with a joystick micromanipulator ( narishige ) and a microinjector ( femtojet , eppendorf ) . injection needles were pooled from glass capillaries ( borosilicate thin wall with filament , 1.0 mm outer diameter , 0.78 mm inner diameter , harvard apparatus ) using a sutter needle pooler . gcamp6s ( chen et al . , 2013 ) was kindly provided by the genie project , janelia farm research campus , howard hughes medical institute ( addgene plasmid 40753 ) . the gcamp6s coding sequence was subcloned in the pcs2 + vector , and mrna was in vitro transcribed with the sp6 mmessage mmachine high yield capped rna transcription kit ( ambion ) . gcamp6s mrna was injected in the cd blastomere to a final concentration of 250 g/l . h2b - rfp mrna was co - injected at the same concentration in order to label the cd lineage . for calcium imaging , 48 - 52hpf larvae were mounted on glass bottom culture dishes ( mattek ) in 3% low melting agarose , and maintained in natural seawater . imaging was performed using a zeiss lsm780 microscope , with a 32-ch gaasp detector and a two - photon light source ( chamaleon , coherent ) running at 910 nm . movies ( 248x250 pixels ) were acquired under a 40x oil - immersion objective , for at least one minute and with a temporal resolution ranging from 8.26hz to 16hz . drugs were applied directly to the seawater , and responses were imaged 20 min after application ( or 5 min after application for the mecamylamine experiment in figure s5 ) . for the calcium imaging experiments during the night gcamp6s movies were analyzed using fiji and r. the cholinergic ciliomotor neuron was identified on the basis of its position within the cd domain and the presence of a contralateral projection . rois ( region of interest ) were manually selected around the cell body and/or the axon of the cholinergic ciliomotor neuron . in each experiment , the same roi was used to compare gcamp6s fluorescence before and after the drug treatments . for the calculation of normalized f / f0 data with a global baseline , f0 was set as the average fluorescence of the 11-frames interval centered on the minimum fluorescence value . for the calculation of normalized f / f0 data with the moving average approach , a sliding window of 0.8 s was used to calculate f0 . for the analysis of peak frequency and peak - to - peak intervals sharp electrode recordings along with simultaneous high - speed imaging were performed on 4060 hpf platynereis larvae . holding pipettes were made from borosilicate glass ( science products gmbh , hofheim ) with an outer diameter ( o.d . ) of 1 mm and were fire polished to minimize damage to the larvae . of 1.5 mm , filled with 3 m kcl , and showed resistances between 15 25 m. to facilitate electrode placement , larvae were digested with 46.7 g / ml of proteinase k for 10 - 15 min . signals were acquired at 20 khz and analyzed using clampfit 10.3 ( molecular devices , union city , ca ) . input resistances of prototroch cells were monitored by delivering small hyperpolarizing currents via the recording electrode and only prototroch cells which displayed resting potentials between 65 to 80 mv and input resistances between 10 25 m were used for analysis . simultaneous high - speed ( 20 hz ) imaging was performed on an andor neo s - cmos camera and analyzed using fiji .

summarymelatonin , the hormone of darkness , is a key regulator of vertebrate circadian physiology and behavior . despite its ubiquitous presence in metazoa , the function of melatonin signaling outside vertebrates is poorly understood . here , we investigate the effect of melatonin signaling on circadian swimming behavior in a zooplankton model , the marine annelid platynereis dumerilii . we find that melatonin is produced in brain photoreceptors with a vertebrate - type opsin - based phototransduction cascade and a light - entrained clock . melatonin released at night induces rhythmic burst firing of cholinergic neurons that innervate locomotor - ciliated cells . this establishes a nocturnal behavioral state by modulating the length and the frequency of ciliary arrests . based on our findings , we propose that melatonin signaling plays a role in the circadian control of ciliary swimming to adjust the vertical position of zooplankton in response to ambient light .

Introduction Results Discussion Experimental Procedures

PMC4327201

o objetivo deste estudo foi avaliar , in vitro , a qualidade do preenchimento de canais radiculares curvos com a pasta de hidrxido de clcio [ ca(oh)2 ] ultracal ( ultradent ) , associada ou no a cones de guta - percha contendo hidrxido de clcio ( calcium hydroxide plus points , roeko ) . cento e vinte razes de dentes humanos extrados , aleatoriamente distribudas em 3 faixas de curvatura ( leve - 0 a 14 ; moderada - 15 a 29 ; severa > 30 ) foram utilizadas . aps o preparo qumico - mecnico , as razes foram divididas em 4 grupos ( n=30 ) de acordo com o mtodo de aplicao da medicao intracanal : grupo 1 aplicao da pasta de ca(oh)2 com espiral de lentulo ; grupo 2 aplicao da pasta de ca(oh)2 com espiral de lentulo , seguida da introduo de um cone de ca(oh)2 ; grupo 3 aplicao da pasta de ca(oh)2 com a ponta navitip ( fornecida com o sistema ultracal ) ; grupo 4 aplicao da pasta de ca(oh)2 com a ponta navitip seguida da introduo de um cone de ca(oh)2 . as razes foram diafanizadas e avaliadas quanto qualidade do preenchimento do tero apical dos canais por um examinador calibrado . os espcimes foram examinados em lupa estereoscpica e classificadas por escores de 1 a 4 ( desde preenchimento inadequado at preenchimento total dos canais ) . os resultados foram comparados atravs da anlise de varincia e teste post hoc de duncan com nvel de significncia de 5% . no se observaram diferenas estatisticamente significantes ( p>0.05 ) entre os graus de curvatura dos grupos 1 , 3 e 4 quanto ao preenchimento dos canais . os grupos que associaram pasta e cones de ca(oh)2 ( 2 e 4 ) apresentaram melhor preenchimento apical em relao aos demais grupos , porm essa diferena foi estatisticamente significante ( p<0,001 ) somente para as razes com curvatura severa . de acordo com os resultados deste estudo , o grau de curvatura no influenciou na qualidade do preenchimento . as tcnicas que utilizaram cones de guta percha contendo ca(oh)2 promoveram um melhor preenchimento the disinfection of the root canal system is one of the most important aspects that account for the success of endodontic therapy . the use of a calcium hydroxide [ ca(oh)2]-based intracanal dressing between sessions has a major role in decreasing the microbial population within the root canals6 . for optimal effect , ca(oh)2 should be in intimate contact with the dentinal walls , along the whole canal extension5,6,7 . nevertheless , three - dimensional filling is not easily obtained , especially in curved canals , in which failures occur mostly in the apical third4,15,17,18 . the similarity between ca(oh)2 and conventional gutta - percha points facilitates their placement into root canals up to the working length2,11,13,14 . there are no studies in the literature that associate the use of ca(oh)2 paste and points . therefore , this in vitro study evaluated the quality of ca(oh)2 paste filling , associated or not with ca(oh)2-containing gutta - percha points in curved root canals . the research project was submitted to review by research ethics committee of lutheran university of brazil and the designed methodology was approved ( process # 2004 - 138h ) . one hundred and twenty roots were selected from extracted human first molars and single - rooted teeth . buccolingual and mesiodistal radiographs were taken to confirm absence of anomalies of form and size , full development of roots and presence of a single canal per root . maxillary first molars with more than one canal in the mesiobuccal root were included in the study , but only the main canal was used . the length of the roots was standardized between 14 and 18 mm by sectioning the crowns at the cementoenamel junction . the roots were classified according to their degree of curvature as mild ( 0 to 14 ) , moderate ( 15 to 29 ) and severe ( above 30 ) , as proposed by fontanella , et al.8 ( 2004 ) . forty roots were selected for each of the three ranges of curvature degree . a size 10 k - type file was introduced into the canal until the tip of the instrument was visualized at the apical foramen . working length ( wl ) was calculated by subtracting 1 mm from this measurement . before shaping , the cervical third was prepared with sizes 1 , 2 and 3 laxxess instruments at 650 rpm . the middle and apical thirds were shaped according to the step - back technique up to a size 50 k - type file , the size 30 k - type file being the memory file . at each change of instrument , the canals were alternately irrigated with 2 ml of 1% sodium hypochlorite and 2 ml of 17% trisodium edta . , south jordan , ut , usa ) and size 30 ca(oh)2-containing gutta - percha points ( calcium hydroxide plus points ; roeko , langenau , germany ) , which were delivered to root canals following different protocols . prior to the placement of intracanal dressings , the sample received a stratified randomization to guarantee uniform distribution of the different curvature degrees in each group of treatment . group 1 : a size 25 lentulo spiral ( maillefer instruments sa , ballaigues , switzerland ) carrying small portions of ca(oh)2 paste was placed into the canals at 2 mm from the working length and powered at low speed until paste reflow was observed . petrpolis , rj , brazil ) was used at canal entrance to condense the intracanal dressing towards the root apex . group 2 : root canals were filled in the same way as described for group 1 . however , after the paste was condensed , a size 30 ca(oh)2-containing gutta - percha point was introduced into the canal up to the working length . group 3 : root canals were filled using a navitip tip ( supplied with ultracal kit ) . the navitip tip was attached to ultracal syringe and introduced into the canals up to 2 mm from the working length . the syringe embolus was then pushed and the syringe was pulled backwards slowly , until paste reflow was observed . ltda ) was used at canal entrance to condense the intracanal dressing towards the apex . group 4 : root canals were filled in the same way as described for group 3 . however , after the paste was condensed , a size 30 ca(oh)2-containing gutta - percha point was introduced into the canal up to the working length . both areas were sealed with conventional glass ionomer cement ( ketac - fill plus , espe , seefeld , germany ) , light - cured composite resin ( heraeus- kulzer , hanau , germany ) and araldite resin handled according to the manufacturers ' instructions . the teeth went through a clearing process3,10 by immersion in 5% nitric acid ( qumica delaware , porto alegre , rs , brazil ) for 72 h ( the solution being changed every 24 h ) , washing in running water for 4 h and successive dehydration in 80% alcohol for 12 h , 90% alcohol for 1 h and 99% alcohol for 3 h ( changed every hour ) . the teeth were then placed in methyl salicylate ( qumica delaware ltda . , porto alegre , rs , brazil ) , which rendered them transparent . the cleared roots were examined under a stereomicroscope ( gsz , zeiss , germany ) at 16x magnification and the quality of canal filling was assessed . a calibrated experienced examiner , blinded to curvature degrees and groups of treatment , examined all surfaces of the apical third of each root and attributed scores to the specimens according to the following ranking scale : score 1 : inadequate root canal filling 0 - 50% of intracanal space was filled ( figure 1 ) ; score 2 : root canal filling with great failures 51 - 70% of intracanal space was filled ( figure 2 ) ; score 3 : root canal filling with minor failures 71 - 85% of intracanal space was filled ( figure 3 ) ; score 4 : complete root canal filling 86 - 100% of intracanal space was filled ( figure 4 ) . intra - examiner reliability was assessed by intraclass correlation coefficient ( icc ) after reexamination of 15 specimens . data referring to the quality of filling obtained with the different techniques , as a function of curvature degree , were described as means and standard deviations . analysis of variance compared the results of the groups at curvature levels and duncan 's post hoc test identified the differences . data were assessed using spss software , version 11 ( statistical package for the social sciences , adobe systems inc . , san jose , ca , usa ) and sigma plot ( sigma - aldrich corp . during clearing procedures , 5 specimens were lost because of infiltration of liquids used in the process . means and standard deviations of the scores attributed to each group are shown table 1 . regarding the degree of curvature , it was observed that in groups 1 ( lentulo ) , 3 ( navitip ) and 4 ( navitip + point ) there were no statistically significant differences ( p>0.05 ) among the curvature ranges . in group 2 ( lentulo + point ) there was bordering statistical significance ( p=0.05 ) for severe curvature in comparison to the other curvature degrees . regarding the filling techniques , the groups that associated the use of ca(oh)2 paste and points ( 2 and 4 ) showed better filling of the apical third than the groups that received ca(oh)2 paste alone as intracanal dressing ( 1 and 3 ) . however , this difference was statistically significant ( p<0.001 ) only for the roots with severe curvature . this study evaluated the quality of intracanal dressing placement in root canals filled with ultracal ca(oh)2 paste . this paste has a new injectable application system , navitip , which was developed for use in curved canals . lentulo spirals , which are still the most indicated instruments for this purpose , were also used in comparison to the manufacturer - supplied tips4,5,12,15,17,18 . the hypothesis that the placement of ca(oh)2 points into canals filled with ca(oh)2 paste would produce an embolus effect and improve apical filling quality the roots used in this study were classified in each of the curvature ranges not only by their angulation . the initial position of curvature was also considered because both parameters determine the severity of root curvature1,11 . apical foramen was provisionally sealed with wax prior to application of intracanal dressing to prevent air escape from the canal . foramen patency could produce a falsely adequate filling that would not reflect the clinical situation18 . the findings of this study indicate that , under the investigated conditions , the quality of root canal filling was not influenced by curvature degree in the groups filled using lentulo spiral , navitip and navitip + point . there were no statistically significant differences ( p>0.05 ) among the three curvature ranges ( mild , moderate and severe ) for these groups . these results are not consistent with those of torres and luft17 ( 2003 ) , who reported better root filling quality in teeth with mild curvature than in severely curved roots , regardless of the technique used . this discrepancy of results may stem from the variability of root anatomy . far more than curvature , the complexity of the root canal systems , with its numerous ramifications , is a variable difficult to control and may either facilitate or impair the filling of the canals . bordering statistical significance ( p=0.05 ) was observed in group 2 ( lentulo + point ) when degree of curvature was compared . in this case , the action of the ca(oh)2 point seemed to be beneficial for filling severely curved canals but did not affect the quality of filling in the other curvature ranges . regarding the filling technique , groups 2 and 4 presented higher scores than the other groups in mild and moderate curvature ranges , but this difference was not significant statistically ( p>0.05 ) . the superiority of the techniques employing ca(oh)2 points was established in roots with severe curvature . in this range , groups 2 and 4 presented better root canal filling ( p<0.001 ) than the other groups in which ca(oh)2 paste alone was used . the results of this study are in agreement with those of estrela , et al.5 ( 2002 ) , who reported an adequate root canal filling when a ca(oh)2 paste prepared with an aqueous vehicle was used as an intracanal dressing . in the present study , in all groups , ca(oh)2 paste was condensed at canal entrance towards the apex using a paper point of great diameter . in the groups where ca(oh)2 points were used , condensation of the intracanal dressing occurred in apical third . these findings suggest that the physical embolus action of the ca(oh)2 point resulted in better filling quality , pushing the paste into an intimate contact with root canal walls . therefore , a similar effect may be expected if conventional gutta - percha points are used in the same way . the filling obtained with use of ultracal paste alone corroborates the findings of previous studies . this system seems to be inadequate for use in curved canals because the diameter of the tip supplied with the kit ( 0.76 mm ) is not compatible with that of severely curved root canals . according to staehle , et al.16 ( 1997 ) , needles with diameters greater than 0.6 mm can be employed only for straight canals enlarged apically at least up to a size 50 file , which did not occur in the present study . in view of these findings , it seems clear that the quality of curved root canal filling with an aqueous calcium hydroxide paste is considerably improved when ca(oh)2 points are also used . the association of paste and points seemed to make the difference , mainly in canals with more accentuated curvatures . according to the methodology proposed and based on the results of this study , the following conclusions may be drawn : all techniques were unable to fill root canal apical third completely.when lentulo spiral , navitip tip and navitip + ca(oh)2 points were employed , the curvature range did not influence the quality of apical third filling.when lentulo spiral was associated with ca(oh)2 point , canals with severe curvature presented better filling than those with mild to moderate curvatures.the techniques that associated ca(oh)2 paste and point ( lentulo + point and navitip + point ) yielded better filling quality at root canal apical third in roots with severe curvature . when lentulo spiral , navitip tip and navitip + ca(oh)2 points were employed , the curvature range did not influence the quality of apical third filling . when lentulo spiral was associated with ca(oh)2 point , canals with severe curvature presented better filling than those with mild to moderate curvatures . the techniques that associated ca(oh)2 paste and point ( lentulo + point and navitip + point ) yielded better filling quality at root canal apical third in roots with severe curvature .

purposethe aim of this study was to evaluate , in vitro , the quality of calcium hydroxide [ ca(oh)2 ] paste filling ( ultracal , ultradent ) associated or not with ca(oh)2-containing gutta - percha points ( calcium hydroxide plus points , roeko ) in curved root canals.material and methodsone hundred and twenty roots of extracted human teeth , randomly divided into three curvature ranges ( mild - 0 to 14 ; moderate - 15 to 29 ; severe - > 30 ) were used . after chemomechanical preparation , the roots were assigned to 4 groups ( n=30 ) , according to the technique of intracanal dressing placement : group 1 - ca(oh)2 paste was applied with a lentulo spiral ; group 2 - ca(oh)2 paste was applied with a lentulo spiral and a ca(oh)2 point was inserted into the canal ; group 3 - ca(oh)2 paste was applied with a navitip tip ( supplied with ultracal system ) ; group 4 - ca(oh)2 paste was applied with a navitip tip and a ca(oh)2 point was inserted into the canal . the roots were cleared and the quality of apical third filling was assessed by a calibrated experienced examiner . the specimens were examined under stereomicroscopy and scored 1 to 4 ( i.e. , from inadequate to complete root canal filling ) . the results were analyzed statistically by anova and duncan 's post hoc test at 5% significance level.resultsthere were no statistically significant differences ( p>0.05 ) among the curvature degrees in groups 1 , 3 and 4 . severely curved roots in group 2 presented bordering significance ( p=0.05 ) . the groups that associated the use of ca(oh)2 paste and points ( 2 and 4 ) showed better apical filling than the other groups , but this difference was statistically significant ( p<0.001 ) only for roots with severe curvature.conclusionaccording to the results of this study , the curvature degree did not influence the quality of filling . the techniques that used ca(oh)2-containing gutta - percha points yielded better filling of the apical third in roots with severe curvature .

Objetivo. Material e Mtodos. Resultados. Concluso. INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC4677204

type 2 diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia in association with insulin resistance , impaired relative and/or absolute insulin production , and altered glucagon secretion [ 1 , 2 ] . at the onset of diabetes two main processes are involved in its pathogenesis : progressive decline in pancreatic islets function and reduced insulin sensitivity in peripheral tissues . in particular , insulin resistance ( ir ) occurs when insulin effect on muscle and fat tissues glucose uptake is defective and is not capable of inhibiting endogenous glucose production by the liver . because skeletal muscle is responsible for 70%80% of total insulin - stimulated glucose uptake , skeletal muscle ir is a major determinant of type 2 diabetes . interactions between genetic and environmental factors , overnutrition , and sedentary behavior promote the progression and pathogenesis of ir . in particular , the modifications that occurred in the global food system during the past 3 - 4 decades have created an obesogenic environment contributing to the increase of the obesity epidemic and consequent ir incidence increase . unhealthy diet and physical inactivity are considered among leading causes of the same diseases characterized by ir . currently , alleviating this condition is still one of the key strategies to treat [ 2 , 6 ] . metf , a widely prescribed drug in type 2 diabetes , is being increasingly considered for treatment and prevention of sedentariness damages , as well as for the extension of healthy lifespan . recent data showed that long - term diet supplementation with metf extends healthy lifespan in c. elegans and in middle - aged male mice [ 8 , 9 ] . in addition , our group demonstrated how acute metf treatment may induce the generation of neohypertrophic myotubes , by using an in vitro model of satellite cells ( c2c12 cells line ) . regular practice of physical exercise plays a very important role in maintaining a good state of health and physical well - being . in particular , recent publications showed the active function of exercise in the reduction and counteraction of the mechanisms underlying muscle atrophy and degeneration related to the onset of peripheral ir [ 12 , 13 ] . in exercising muscle , increased energy metabolism and atp production is obtained by an increased glucose utilization . one of the most relevant metabolic effects of exercise is the enhancement of insulin action . many factors may contribute to increasing insulin sensitivity induced by exercise : a reduction in fat mass , an increase in muscle mass , and the increase of membrane - bond glucose transporters ( glut4 ) in muscle cells . the effects of physical exercise may have relevant implications in the prevention and treatment of metabolic diseases . in fact , by increasing insulin sensitivity , physical activity can reduce the risk of pathological conditions such as type 2 diabetes and metabolic syndrome . understanding the complex mechanisms that regulate insulin response and the onset of peripheral insulin resistance represents a primary goal in the treatment of diabetes and obesity complications , particularly by targeting skeletal muscle . given the growing prevalence of the disease and the conditions of relative sarcopenia related to it , new therapeutic interventions are able not only to reduce the loss of skeletal muscle mass but also to stimulate muscle regeneration while preserving the physiology of viable muscle satellite cells become necessary . in order to determine if metformin could relieve the sedentariness damages , we studied metf effects in sedentary adult young mice , focusing our attention on metf ability to maintain mouse physical performance during submaximal incremental test . mouse c2c12 myoblasts were purchased from the european collection of animal cell cultures ( ecacc ) . reagents were purchased from sigma chemical co. ( saint louis , mo , usa ) . primary antibodies against akt ( c-20 ) , camkii ( m-176 ) , calnexin ( h-70 ) , erk1 ( k-23 ) , erk2 ( c-14 ) , gapdh ( fl-335 ) , myod ( c-20 ) myogenin ( d-10 ) , myhc ( h-300 ) , myf5 ( c-20 ) , n - cadherin ( h-63 ) , p70s6 ( c-18 ) , sod2 ( fl-222 ) , perk1/2 ( e-4 - 4 ) pp70s6 ( sc-7984 ) , peroxidase - conjugated secondary antibodies for western blot analysis , and rhodamine - conjugated antibodies for immunofluorescence analysis were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . primary antibodies phospho - akt ( ser473 ) ( d9e ) xp and phospho - ampk alpha ( thr172 ) ( 40h9 ) were purchased from cell signaling technology ( danvers , ma , usa ) . male c57bl/6 mice ( n = 10 ) , purchased from charles river laboratories ( boston , ma , usa ) , were used for the study at 12 weeks of age . all animals were kept on a 12 h/12 h light / dark cycle with unlimited access to standard rodent chow food and water . mice were divided into two paired groups : one treated with metf and the other not treated ( contr ) . blood glucose was measured in blood collected from the tip of the tail with a portable glucose measuring device ( bayer , basel , switzerland ) . metf ( sigma chemical co. , saint louis , mo , usa ) was added to the drinking water at dose of 250 mg / kg body weight per day , for 60 days . our pilot study confirmed that c57bl/6 mice consumed 7 ml water per day ; metf addition did not influence water consumption . water and metf were changed daily and the dose adjusted to weight gain each week ( figures 1(a ) and 1(b ) ) . muscular performance exercise was evaluated by a submaximal incremental test prior to and upon completion of the study . briefly , mice were placed in adapted treadmill ( columbus instruments ) for 5 min at a 0 incline , and then treadmill speed was increased according to the scheme shown in figure 1(a ) with 1.5-min intervals at a 15 incline ( figure 1(a ) ) . animals run until exhaustion , which is defined as remaining on the shocker plate for more than 5 seconds . at the end of experiment , mice were sacrificed and tissues were harvested , frozen in liquid nitrogen , and stored at 80c for further analyses . this study was conducted in compliance with approved institutional animal care of the university of milan . c2c12 cells were maintained at 37c in humidified 5% co2 atmosphere in a growth medium ( gm ) containing dmem ( dulbecco modified eagle medium ) supplemented with 20% ( v / v ) fetal bovine serum ( fbs ) , 1% penicillin streptomycin , and 1% l - glutamine up to 70% confluence . cell differentiation was initiated by placing 70% confluent cell cultures in differentiation medium ( dm ) , containing dmem supplemented with 1% horse serum ( hs ) , antibiotics , and 1% l - glutamine . in our in vitro differentiation model , early myotubes appeared 2448 hours ( h ) after serum starvation and neomyotubes formation was completed after 72 h . proliferating cells , myoblasts during differentiation process , and neomyotubes were treated with 400 m metf . in the control cells metf was not added to medium . figure 2 explains experimental study design in each phase of the protocol , with cell confluence percentage , treatments start time , and duration . to study metf action on c2c12 myoblast proliferation , we performed growth curve assay as described . briefly , c2c12 myoblasts were plated in 60 mm 15 mm culture dishes at 40% confluence and grown in gm with or without metf and in dm . medium was changed every 24 h and the experiment lasted until control cells achieved 70% of confluence ( 3 days ) . every day , the cells were trypsinized , stained with trypan blue , and were counted using a hemocytometer and the average values for each single day were used to plot a growth curve . cell viability was calculated by dividing the nonstained viable cell count by the total cell count . protein extracts , performed as described elsewhere , were obtained from mouse tissues or cell cultures by using the following lysis buffer containing : 50 mm tris / hcl , ph 7.4 , 150 mm nacl , 1% triton x-100 , 1 mm sodium orthovanadate ( na3vo4 ) , 1 mm edta , 1 mm pmsf , 1 mg / ml aprotinin , 1 mg / ml leupeptin , and 1 mg / ml pepstatin . aliquots of 30 g supernatant proteins , quantified using bradford method , were resolved on sds - page gel and transferred onto nitrocellulose membrane ( protran , whatman schleicher & schuell ) . the membranes were incubated with specific primary antibodies and then with hrp conjugated anti - species - specific secondary antibodies . to confirm equal protein loading per sample , we used antibody anti - calnexin or anti - gadph . quantitative measurement of immunoreactive bands intensities , visualized by an enhanced chemiluminescence method ( amersham pharmacia biotech , piscataway , nj , usa ) , was performed by densitometric analysis using the scion image software ( scion corporation , frederick , md , usa ) . data were then converted into fold - changes ( fc ) of the controls . for tissues analysis , after that , slides were washed in pbs and incubated for 1 hour at room temperature with 10% horse serum in pbs with 0.05% triton x-100 to block nonspecific binding sites , while c2c12 cells , fixed and permeabilized as described , were blocked with pbs containing 1% bovine serum albumin . slides or cells were then immunostained with specific antibodies rhodamine - conjugated and nuclei revealed with dapi staining . cells were observed using nikon eclipse 50i microscopy and images were captured using nis - elements d 4.00 software ( nikon instruments europe bv , netherlands ) . data were displayed and analyzed using adobe photoshop cs4 . live c2c12 cells were examined and images were acquired by phase contrast microscopy using the same microscope and digital system described above . previous data [ 8 , 9 ] suggested the capacity of metf to extend lifespan in the nematode c. elegans and in middle - aged male c57bl/6 mice , improving healthspan mice . we aimed to investigate the possible role of metf in the prevention of sedentariness induced damages . to achieve this scope , 12-week - old male c57bl/6 mice were chosen , maintained in a condition of total absence of exercise , and treated for 60 days with metf , added in water at the dose of 250 mg / kg body weight per day ( figure 1(a ) ) . we did not observe a significant modification in body weight between mice randomized to receive metf and those that did not receive metf ( figure 1(b ) ) . a weight reduction was observed in both groups , comparing the beginning and the end of the experiment . in contrast , we did not observe significant effect in glycemia levels ( figure 1(b ) ) . metf effect on muscle performance evaluation revealed that metf treatment increased speed , time , work , and estimated maximal oxygen consumption ( vo2max ) of acute submaximal incremental exercise in the metf group as compared to their baseline values ( figure 1(c ) ) . overall , muscular performance capacity was therefore increased in the metf group with respect to the pretreatment condition , despite aging and despite the lack of previous exercise training performed whatsoever . the akt / mtor pathway , crucial regulator of skeletal muscle mass , is upregulated during hypertrophy and downregulated during muscle atrophy [ 23 , 24 ] . in gastrocnemius muscle of metf treated animals , akt activation improved compared with controls ( figure 1(d ) ) . in addition , a trend of an akt activation was detected in quadriceps femoris muscle in metf treated mice ( figure 1(d ) ) . liver erks activation is associated with oxidative stress and metabolic dysfunctions , main features of obesity and diabetes . interestingly , erks activation was significantly decreased in liver of metf treated mice ( figure 1(e ) ) . immunofluorescence analysis ( figure 1(f ) ) showed how metf positively modulates ca / calmodulin dependent protein kinase ( camkii ) protein levels in liver tissue with respect to controls , suggesting an important role of metf in cellular calcium homeostasis . all together , our in vivo data indicated that metf could ameliorate not only age induced damage but also sedentariness induced injury . we also tested metf action on skeletal muscle proliferation , differentiation , and hypertrophic process , using a c2c12 cell lines ( figure 2 ) . c2c12 murine immortalized cell line provides a good in vitro model for the study of the major steps of myoblasts proliferation and differentiation [ 27 , 28 ] . during c2c12 myogenic phenotype achievement , myogenic regulator factors ( mrfs ) are expressed in a defined sequence ; myod and myf5 are primarily expressed , while myogenin expression is only induced upon muscle differentiation [ 27 , 28 ] . 400 m metf did not alter c2c12 proliferative potential and did not induce cytotoxic effects , as shown in figure 3(a ) and confirmed in phase contrast images in figure 3(b ) . metf led to a significant rise in myod content , similarly to dm , compared with control ( figure 3(b ) ) . immunofluorescence analysis during proliferation phase revealed that metf increased the protein expression of two key markers of early differentiation : myf5 and myod , suggesting an important role in differentiation induction and promotion of the myoblast commitment to myotube ( figure 3(d ) ) . to confirm this , phalloidin assay ( figure 3(e ) ) showed that the cells lost their characteristic circular shape , typical of the active proliferation phase , to achieve a new elongated morphology . we analyzed metf action on differentiation ( figure 2 ) , from the first phase of differentiation induction ( 24 h ) , middle phase ( 48 h ) , and to the end of the process ( 72 h ) , when all fusion - competent myocytes can form multinucleated myotubes . as shown in figure 4(b ) , metf treated cells were characterized by a significant rise in principal marker of myotube maturation , myosin heavy chain ( myhc ) protein levels with respect to control cells . the similar metf positive action was observed for n - cadherin , a central cytoskeletal protein involved in cytoskeletal rearrangement , required to the fusion of myoblast in new myotubes . to corroborate our results , which indicated the active role of metf in differentiation progression metf enhanced myogenin protein content and , in particular , its expression peak at 48 h ( figure 4(b ) ) . protein level of superoxide dismutase ( sod2 ) , an enzyme that efficiently converts superoxide to the less reactive hydrogen peroxide , was significantly increased in metf cells compared to control cells ( figure 4(b ) ) . this result suggests that metf could counteract the damage caused by a sedentary lifestyle by strengthening the antioxidants mitochondrial functions . finally , we investigated metf action on the principal signaling cascades involved in skeletal muscle formation : erks and p70s6 kinase pathways . metf enhanced differentiation process through erks activation , while it decreased p70s6 kinase pathway ( figure 4(b ) ) . after 48 h from differentiation induction , immunofluorescence analysis revealed that camkii protein expression was increased in neoformed myotubes treated with metf ( figure 4(d ) ) . based on our previous data on effect of short time metf stimulation on the hypertrophic process , we studied the effect of long time metf treatment on neoformed myotubes ( figure 4(c ) ) . 24 h of metf stimuli significantly increased myhc protein levels ( figure 4(e ) ) . as observed in all differentiation phases , metf effects were mediated by erks signaling pathways activation ( figure 4(e ) ) . also in immunofluorescence images , we observed an important increase in myhc and n - cadherin protein content after metf treatment on neoformed myotubes . furthermore , metf treatment caused important morphological changes in terms of morphological parameters ( myotubes length and diameter as shown in figure 4(f ) ) . the effects of physical exercise have relevant implications in the prevention and treatment of damage induced by obesogenic environment characterized by a positive balance between energy intake and energy expenditure . moreover , physical activity represents a primary goal in the treatment of diabetes and obesity complications , in particular in skeletal muscle system . to corroborate the fundamental role of physical activity , the world health organization has identified physical inactivity as the fourth - leading risk factor for global mortality . we investigated the potential effects of in vivo treatment with metf , currently candidate drug for lifespan extension , on the prevention of sedentariness induced damages . the study of a protocol of exercise training in mice ( figure 1(c ) ) indicated that metf could have positive effects on muscular performance . our results , obtained from studying a model of sedentary healthy mice , confirm the positive metf action on skeletal muscle function previously obtained in older mice models . several recent works suggest how this biguanide drug could be used in the prevention of aging induced damages [ 8 , 9 , 31 ] . in this perspective akt signaling is central in the regulation of muscle function and , in particular , akt inactivation is associated with muscle damages induced by aging [ 23 , 33 ] . we observed that metf increased the activation of akt in gastrocnemius and quadriceps femoris muscles ( figure 1(d ) ) , suggesting a hypothetical novel use of this drug not only in aging - related conditions , but also in sedentary - related damaged muscle conditions . precisely , for the first time , our work showed how the beneficial effects of metf occur not only in groups already characterized by pathological conditions ( e.g. , obesity , diabetes , and aging - related disorders ) but also in healthy sedentary populations . an additional positive action was observed in metf treated mouse liver ; metf deactivates erk and promotes camkii signaling . erk activation is crucial in favoring the development of several liver dysfunctions , such as liver fibrosis and hepatocellular cancer , while camkii pathways activation is fundamental to preserve liver functions . from these data , it is reasonable to conclude that metf supplementation could be utilized to keep liver healthy . we demonstrated in healthy humans that constant aerobic physical exercise is the clue to avoid lipid steatosis . to further clarify metf cellular mechanisms underlying the effects obtained in the mouse model , we studied metf action using an in vitro model of myoblasts , c2c12 cell line [ 27 , 28 ] . this cell line represents the gold standard of immortalized cells to study not only myogenesis but also the hypertrophy process and its use has allowed us to investigate metf action on cellular pathways involved in muscle training , characteristic process of healthy subject . first , we observed that metf did not modify c2c12 proliferation rate and viability ( figure 3(a ) ) , confirming the possible use of this biguanide drug without side effects on skeletal muscle . those results have important implications since several tumors are associated with sarcopenia and cachexia , which might benefit from metf treatment . after 24 h of metf stimuli on neomyotubes , we observed an increment in morphological parameters ( figure 4(f ) ) , similar to our previous work where on acute metf treatment was administered . our data indicate that metf should not be considered not only a drug capable of inactivating the cellular mechanisms related to muscle injury , but also a drug capable of activating cellular processes related to muscle strengthening and hypertrophy . we speculate that this metf capacity to enhance myotubes formation and hypertrophy could represent a possible explanation of data obtained , in vivo , in mice . finally , our results obtained during differentiation phases showed that sod2 protein content is increased after metf stimuli ( figure 4(b ) ) . the intracellular enzymes of the sod family act as a primary line of defense to cope with the deleterious effects of ros , thereby contributing to an overall decrease in oxidative damage . lower sod activity is associated with sedentary lifestyle , characterized by insulin resistance , suggesting that reduced capacities of antioxidant enzymes lead to increased oxidative stress in diabetes and obesity . in conclusion , our study reports several novel findings regarding the use of metf in a condition of absence of physical activity and specifically : ( 1 ) it improves mice physical aerobic performance ; ( 2 ) it ameliorates myotubes formation , regulating the principal molecular mediators of skeletal muscle hypertrophy and atrophy ; ( 3 ) it prevents oxidative stress damage , modulating erk and sod signaling . the relevance of our results resides in a potential use of metf ( or drugs with similar biological proprieties ) to counteract the damages consequent to sedentariness either directly ( acting on molecular targets involved in stress condition ) or indirectly , by enhancing the known beneficial physical activity effects . in this framework , additional research is necessary , also in humans , to test combined therapeutical use of metf , exercise , and diet to prevent damages of sedentariness .

metformin ( metf ) , historical antihyperglycemic drug , is a likely candidate for lifespan extension , treatment and prevention of sedentariness damages , insulin resistance , and obesity . skeletal muscle is a highly adaptable tissue , capable of hypertrophy response to resistance training and of regeneration after damage . aims of this work were to investigate metf ability to prevent sedentariness damage and to enhance skeletal muscle function . sedentary 12-week - old c57bl/6 mice were treated with metf ( 250 mg / kg per day , in drinking water ) for 60 days . metf role on skeletal muscle differentiation was studied in vitro using murine c2c12 myoblasts . muscular performance evaluation revealed that metf enhanced mice physical performance ( estimated vo2max ) . biochemical analyses of hepatic and muscular tissues indicated that in liver metf increased ampk and camkii signaling . in contrast , metf inactivated erks , the principal kinases involved in hepatic stress . in skeletal muscle , metf activated akt , key kinase in skeletal muscle mass maintenance . in in vitro studies , metf did not modify the c2c12 proliferation capacity , while it positively influenced the differentiation process and myotube maturation . in conclusion , our novel results suggest that metf has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages .

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

PMC3205804

the recovery of exfoliated cells from biological fluids is a non - invasive technology which is in high demand in the field of translational research as well as during long - term experiments designed to minimize the sacrifice of long - lived or precious animals . exfoliated epithelial cells can be used as surrogate for tissue biopsies in predicting changes in gene expression , dna methylation , dna damage , protein expression , and accumulation of dietary components [ 1 , 2 ] . exfoliation has also been described as an active biochemical process linked to the homeostasis of gut epithelium [ 36 ] . it is believed that epithelial cells , loosing contact with companion cells ( like fibroblasts ) as well as extracellular matrix , enter anoikis . recent in vitro models are opening new avenues to conceptualize the exfoliation of gut epithelia in order to explain this highly context - dependent phenomenon . loss of extracellular matrix contact induces autophagy in normal epithelial cells , and autophagy promotes the survival of detached cells during both anoikis and lumen formation in 3d epithelial cell culture [ 8 , 9 ] . under these assumptions , exfoliation may be understood as a natural process to remove external cells from the luminal surface of an epithelium . consequently , exfoliation may have a physiological role by allowing the formation of a lumen , preserving the epithelium 's architecture , and , we can surmise , by providing sufficient flexibility to preserve the physical integrity of epithelia and allow its growth . in three - dimensional epithelial cell cultures , both autophagy and apoptosis are observed during lumen formation [ 8 , 9 ] . by loosing contact with the original mucosa , indeed , quiescent exfoliated epithelial cells without signs of apoptosis can be recovered under specific clinical conditions in gastric fluid aspirates or by suction from breast glands [ 11 , 12 ] or extensive rinsing at the end of routine colonoscopy . many exfoliated quiescent epithelial cells can be cultured suggesting that detachment - induced autophagy contributes to the viability of these cells . however , the survival of quiescent epithelial cells outside the tissue structure is highly variable . human mammary epithelial cells die after 2448 hours of detachment ; certain epithelial cells , notably rat intestinal epithelial cells , perish within a few hours following substratum detachment [ 9 , 14 ] . this paper presents current understanding of exfoliation along with the influence of methodology on the isolation of exfoliated gut epithelial cell phenotypes and , finally , speculates on the balance between anoikis and apoptosis to explain the survival of epithelial gut cells in the environment . exfoliation can be understood as a natural process to preserve tissue architecture . following that first point of view , exfoliation is a loss of cellular material retaining the basic cytological features of typical cells ( plasma membrane , cytoplasm , and nucleus ) . exfoliated epithelial cells can be obtained from a wide range of mucosae whose line body passages and cavities communicating directly or indirectly with the exterior like mammary glands , oral , bronchial , urothelial , or gastrointestinal epithelia . epithelia can be classified as simple cylindrical cell monolayers like colon or pseudostratified like urothelium . according to histology , epithelia are organized in functional units containing different cellular compartments ( stem , proliferative , mature , or functional and senescent ) as shown in figure 1 . these functional units are always at the interface with the environment . at a given time point , a mucosal epithelium is supposed to loose different categories of cells by different mechanisms of exfoliation . however , the cell turnover of these epithelial cells is driven by a delicate balance between cellular loss and proliferation . proliferation is running on two cellular compartments , the proliferative cells capable of rapid mitosis to amplify tissue regeneration and the stem cells which are giving rise to all phenotypes by asymmetric mitosis . the speed of mitosis in proliferative compartment is dependent on cellular loss at the top of the structure and on tightly regulated cell migration along the functional units . cell migration in the small and large bowels of mice shows a strong circadian rhythm , with cell velocity maximal at 9 a.m. and minimal at 5 p.m . other rhythms which could be controlled by circadian clocks have been observed in the intestine like cellular proliferation [ 20 , 21 ] or apoptosis . cell proliferation is also believed to be under the control of clockwork not only in hepatocytes but also along the rat 's gut . seasonal rhythms of proliferation have been described in adult rats [ 25 , 26 ] . circadian as well as seasonal rhythms in cell proliferation seem clearly relevant to the recovery of exfoliated quiescent cells retaining specific and functional biomarkers . however , there is a second point of view where exfoliation is a loss of cells in the environment due to external mechanical forces like brushing or friction . such forces are deeply altering the epithelium architecture but allow to yield rapidly high amounts of epithelial cells retaining phenotypes and physiological status as close as possible to the mucosal cells remaining in the epithelium . manual exfoliation has been reported with brushing or scraping technique on oral epithelium of cheek or tongue , cervical , or rectal swabbings , airway epithelial cells in sputum and buccal mucosal cells obtained by rinsing the mouth or chewing - gum ( betel chewers ) , esophageal cells , mammary by nipple aspirate , ductal lavage klein et al . , , breast milk , or bladder urothelial cells present in urine samples [ 32 , 33 ] . manual exfoliation has also been proposed as a way to recover intact , normal epithelial cells on tissue biopsies made on colon resection [ 34 , 35 ] . some device has also been designed to recover surface exfoliated cells of human rectal mucosa by a minimal invasive scraping . the technique partially purifies the cell preparation by taking advantage of the cell 's inherent biology . epithelial cells remained in small groups or sheets , detached from any stromal elements that may have been scraped off . the problem is that in most clinical situation , there is no direct access to the mucosa and the technique simply can not be used . consequently , we may wonder whether some useful biological information can be recorded from relatively low numbers of cells , isolated as a mixture of cellular phenotypes with different physiological states ? magnetic beads and antibodies are well - known systems to recover low numbers of epithelial cells in biological fluids or to recover highly purified epithelial phenotypes . antibodies against human cell surface antigens like anti - hep , or antiepithelial surface marker from an original antibody described by moldenhauer et al . , or anti - ber - ep4 [ 34 , 35 ] from an original antibody described by sheibani et al . labeled with paramagnetic particles are used to capture and to purify epithelial cells . viability of recovered cells by this exfoliation / enrichment method as well as by other similar techniques is on average between 90 and 100% by the trypan blue exclusion assay . however , in our experience , shieving is necessary to perform immunocapture . with samples containing sheets of 5 to 30 cells , shieving or percoll gradients in addition , microbial contaminations are not easily removed by such density gradient methods because microbes are tightly associated with cells . even with the manual exfoliation technique , the exfoliated cell populations may contain other cell types , most notably lymphocytes and plasma cells . it should be underlined that the problem of cross - contaminations by other sources of exfoliated epithelial cells like breast cells from the milk with gastric cells of lactating infants or exfoliated cells from manipulators is particularly difficult to ward off calling for the development of biomarkers of tissue origin which can guarantee both the cellular origin and the affordability of testing . the next section discusses recent works in 3d mammary reconstruction and the functioning of acini which have shed new light on the capacity of surface epithelial cells to survive outside their epithelium . laboratory rodent models are also discussed as they open the possibility to induce exfoliation by nutritional manipulations . primary cells are inoculated as single - cell suspensions or small clumps of cellular aggregates . these cells have also lost contact with the tissue architecture ( companion fibroblasts , epithelial cell neighbors , and with the extracellular matrix ) , as well as with the nervous regulation or the blood nutriments . from tissue cultures , we know that a molecule of nutriment has to be within 50 nm away from a single cell to be accessible . so even if some cells are exfoliated in a nutritious matrix ( milk for instance ) , they may have to trigger a survival mechanism . some set of genes are progressively turned - down like clock genes , but in this particular situation , they can be reinduced under specific stimulation in culture . over the years , the conditions of culture have been adapted to mimic the tissue architecture by creating three - dimensional ( 3d ) environment . recent works have shown that autophagy can be observed during lumen formation in 3d cell cultures in vitro . the mcf-10a cells are a nontransformed human mammary epithelial cell line , which can form spherical structures ( called acini ) in which a layer of polarized epithelial cells surrounds a hollow lumen , mimicking the glandular epithelium in vivo [ 8 , 9 ] . the lesson we can learn from this 3d reconstruction of mammary gland is that epithelial cells are able to flexibly leave or reenter an epithelium . the property is useful for tissular growth as well as to heal rapidly microlesion in the epithelium cell lining . . their biochemical state should be close to the state of cells having lost contact with the 3d reconstructed gland . in the next section , we present recent works in laboratory rodents which have shown that this capacity of an epithelial cell to adapt to changing environmental conditions is highly context dependent . laboratory rodent models have been developed to study the inducing effect of nutrient intake on the exfoliation of epithelial cells in the digestive lumen . on adult rats fastened for 24 hours and refed for one hour , the feeding intake induces exfoliation of quiescent parietal cells at the top of the gastric gland through an unknown exfoliation factor . under these conditions , stem cells located in the neck region of gastric glands are believed to be recruited actively to repopulate the surface of the adult rat stomach . on lactating rat pups , we obtained similar results by fastening the pups for 5 hours and allowed them to be reunited with their mother for one hour before sacrifice . by contrast , on adult laboratory mice , fatty acids ( like palmitate ) are inducers of intectin , a protein implicated in the exfoliation of apoptotic cells at the top of the villus of the small intestine within an hour after meal . these models of nutritional manipulations to induce exfoliation on small intestine of mice or gastric mucosa of rats indicate that the mechanism of exfoliation is highly context dependent , but they also open the possibility to develop in vivo studies of anoikis and autophagy in relation with the functioning of peripheral circadian clocks . the disponibility of laboratory rodent models is of paramount importance to develop in vivo studies on anoikis and its connection with molecular circadian clocks to evaluate the stability of chronobiological molecular information in exfoliated cells . the proof that exfoliation of quiescent cells is following a circadian rhythm is still missing , probably because the set of physiological parameters leading to the induction of active exfoliation is difficult to handle and the interpretation of data obtained from manual exfoliation is also highly context - sensitive . in conclusion , exfoliation is a broad term recovering many different biological or experimental situations but as illustrated by the next section , progress in the understanding of the delicate balance between autophagy and apoptosis will help scientists to design new bioassays tailored for specific clinical situations . exfoliated cancerous cells of epithelial origin have been the first to be used to help design noninvasive screening assay of cancerous patients [ 52 , 53 ] . the relatively high loss of cancerous epithelial cells by patients as well as the stability of molecular information ( genetic alterations related to colon cancer , for instance ) have helped to establish the methodology . recently , chapkin et al . have developed and patented a transcriptomic approach to explore exfoliation in stools of infants as well as in adults . the weak point of this approach is that the morphological information of the cell population is lost during the extraction process of mrna , and there is no possibility to check for the exact cellular origin of these molecules . exfoliation in stools is still highly debated ; some visual proof of typical intestinal cells have been published [ 4 , 48 ] , but in my experience if whole crypt material or typical colonocytes can be found , most of the time the criticism of loktionov that these cells can not be distinguished from epithelial cells of the anal zone is correct . the proof of similarity between exfoliated epithelial cells with the ones remaining in the mucosa will be probably easier to perform on gastric epithelium following the seminal work of aoyama et al . . however , the detection of proteins and structural elements will remain possible only in a limited number of clinical situations narrowing the possibility of using exfoliated epithelial cells as indicator of good health . however , a better understanding of the key factors allowing the cellular survival outside the tissue architecture will open new avenues to derive useful screening assays from clinical material . the detachment of epithelial cells from the tissue architecture triggers both pro- and antiapoptotic signals , such as nuclear factor kappa - b and inhibitor of apoptosis protein family members ; these antiapoptotic mechanisms presumably delay the onset of apoptosis and allow cells to survive [ 5456 ] . the balance between these signals and the duration of detachment determine further fate of these cells . antiapoptotic signals presumably delay the onset of anoikis , allowing cells to survive provided that they can reestablish extracellular matrix contact in a timely manner . in cells having lost contact with tissular structure , autophagy corresponds to the recycling of cellular material as well as to the cell capacity to mobilize reserves during periods of starvation . autophagy is a biochemical pathway allowing survival during fasting period which can be stopped at the organism level to prevent self - digestion . there are three main forms of autophagy : microautophagy , macroautophagy , and chaperone - mediated autophagy [ 5759 ] . in macroautophagy , a portion of the cytosol or organelles are sequestered in a double - membrane - bound vesicle , the autophagosome ( figures 2(a ) and 2(b ) ) . a core molecule in autophagy regulation is the kinase mammalian target of rapamycin ( mtor ) . by sensing signals that monitor nutrient levels , mtor can trigger protein translation by specific phosphorylation of the ribosomal protein s6 kinase ( ps6k ) . recent works on the molecular pathway regulating microtubule - associated protein light chain 3b ( lc3b ) and autophagy support the idea that regulation of autophagy is interconnected with regulation of apoptosis . lc3b may regulate the extrinsic apoptosis pathway in the lung through direct interactions with caveolin-1 and fas . implication of beclin-2 modifying factor and the antiapoptotic proteins of the bcl-2 family in the anoikis process have been proposed to play a central role in the survival of human intestinal epithelial cells ; this work partly explains , at the molecular level , the low survival rate of exfoliated epithelial intestinal cells . however , autophagy has been demonstrated to occur in vivo in the surface epithelial cells of neonatal small intestine of piglets . , the reports of nair et al . and chandel et al . are indicating that high amount of living colonocytes can be recovered from stools ( 5 102 10 cells / g of stool ) . from a physiological point of view in infants as well as in adults , some epithelial cells may survive in a state of macroautophagy close to the surface of the epithelium up to finding their way back in the cellular lining . a device can then easily remove such cells ( i.e. , exfoliate these cells ) by mechanical forces . in theory , the physiological status and the genetic profile of these epithelial cells should be close to the ones at the surface of the mucosa . at the intracellular level , autophagosomes are connected to mtor and clock pathways , and if needed to apoptotic pathways . autophagy can be described in amino - acid - free situations ( figure 2(a ) ) as well as in glucose - free situations ( figure 2(b ) ) . the balance is specially relevant in protein kinetics in preterm infants where amino acids are provided by intravenous solutions . the relationship between autophagy and circadian rhythms has been proposed , but the molecular link between these two phenomena is not yet known . cells have to process diverse signals such as temperature , ph , and nutrient concentrations in order to maintain a normal physiology . in vivo , cells are believed to use clock genes to organize and adapt cellular metabolisms and coordinate three - dimensional macromolecular organization ( their phenotype ) in a noisy molecular environment ( molecular signals criss - crossing in and between cells and irrelevant chemical messages ) . exfoliated epithelial cells in anoikis can be seen as a way to obtain chronobiological information dating back to the time of cells leaving the top of the functional units . signals encoded in the amplitude domain are predominantly based on concentrations of signaling molecules , a parameter difficult to measure on exfoliated cells in anoikis without proper normalization related to the single cell level on highly purified cellular phenotypes . in addition , some highly labile biochemical modifications like phosphorylation are probably lost during the storage of biological fluids and in the isolation process or can be made irrelevant to the pathophysiology of the mucosa due to the turnover of the signal by the cellular machinery . significant improvements in the quality of cellular extracts may be achieved by using extraction buffers suitable to preserve clock gene products . the biological information encoded in the frequency domain of an oscillatory signal can be transmitted as concatenated signals with multiple biologically significant signals to gene behind the regulatory sequence within the promoter . on transgenic mammals , oscillations can be measured by recording rhythms of light emissions by cells in which the promoter of some clock gene is linked to a luciferase reporter . on human or nontransgenic mammals , oscillations per se can not be measured in absence of spectrofluorimetric methods applicable on freshly recovered living cells , but indirect evidence of gene - circuit activation may be recovered . long - lasting or resilient information may be accessible either through ( 1 ) the machinery of transcription at the site of fixation on the dna of the cells or ( 2 ) by the histone code as these epigenetics modifications are believed to be acquired with a stability related to the original tissue ( and to the time of day ) . the chronobiological information that we can extract from exfoliated epithelial cells depends on the techniques used to isolate cellular material or the manipulation of physiological parameters ( figure 1 ) and on the affinity of interactions of clock molecular components with stable molecules like dna or the persistence of the physiological effects that they are inducing . we may speculate that quiescent cells like epithelial gastric cells are retaining fully functional clocks , that is , consistent information with their time at exfoliation and subsequent cell survival out of the organism . the induction of gastric cell exfoliation by nutrient cycle developed in rats that we have adapted on lactating rat pups can be used in the future to address questions about the stability of clock information during anoikis . exfoliated epithelial cells can be followed by microscopic examination from the initial step of loss of contact at the mouth of the gastric gland to the recovery of cells in the stomach lumen . in addition , the model is clearly relevant to clinical situation in which patients are equipped with nasogastric tubing . however , there may be tissue - specific differences in the molecular composition of the circadian clock , and clock components that have subtle effects on the central clock function may play a more prominent role in the regulation of peripheral clocks . have used spontaneously immortalized mouse embryo fibroblasts to explore the main clock components ( proteins and mrna ) suggesting that peripheral clocks in cultured cells may be similar in composition and regulation as central clock , but all these components are not always present in cells depending on their tissular origin . the most striking example is the apparent redundancy of clock with its homolog npas2 , which are largely equivalent molecules with strict structural differences [ 72 , 73 ] . three main physiological pathways have been described associated to the regulation of autophagy : akt ( energy sensing ) , egr - r ( growth factor sensing ) , and bcl-2 ( stress - related programmed - cell death ) . according to gan et al . clock components are probably also altered during this process , but there are no data on the relation between autophagy and clock regulation . by contrast , explants of tissue isolated from transgenic rat for period1 gene is giving some chronobiological information about the chaotic expression of this gene under the drastic external conditions of explantation . recording of luminescence emitted in vitro by the explants maintained in classic tissue culture conditions ( 37c , 5% co2 ) has clearly shown that the chaotic light emissions by the transgene system stands up to 1214 hours , thereafter the rhythms of light emissions by liver explants are organized according to an oscillatory model reminiscent of period1 's in vivo oscillations ( as of stokkan et al . , , the phase of the peak has been recorded during the first subjective day in culture i.e. , between 12 and 36 hours ) . to avoid such chaotic evolution with the loss of chronobiological information , experimenters are using mechanical punches of mucosae which are directly snap frozen in liquid nitrogen . this strategy is a reliable but invasive solution to study clock gene expression in time series . otherwise , tissue biopsies can be explanted in culture to derive primary cells or cell lines and record clocks functioning just like with exfoliated cells . however , cell lines have lost contact with body 's network , and their clock systems are probably quickly reorganized to tune up with their new in vitro environment [ 76 , 77 ] . in the future , the use of transgenic mice for autophagic gene circuitry will also help to appreciate the exfoliation status and the molecular link between clocks and autophagy . the development of non - invasive methods is crucial to allow easy sampling of human populations in nutritional / clinical intervention studies . exfoliated epithelial cells could be extremely useful to deal with subtle environmental influences during development causing persistent changes in epigenetic regulation . they represent an alternative to fibroblasts which can be easier to collect on adults , and they putatively are giving molecular information from inner tissues difficult to access . the problem in the recovery of exfoliated cells is to relate the cell phenotypes and their physiological status with the intact tissue structure of the donor . in addition , the degradation of biological information depends on the bioactive compounds present in the biological fluids which can be heavily loaded with enzymes like digestive fluids . a specific application explored in our laboratory is to develop studies in exfoliation in order to improve nursing care in preterm infants and to prevent the onset of such a syndrome during adult life . tracking exfoliated epithelial cells can be used to follow the renewal of the gastric epithelium in order to monitor nutritional or pharmacological practices . recently , we have shown that circadian clock genes were disregulated following an episode of perinatal denutrition . the isolation of exfoliated epithelial cells from pups or infants suffering from perinatal denutrition at the onset of the problem or later in adult life may help to know whether the histone acetyltransferase 's activity of clock can be used to explore the stability of epigenetic profile in exfoliated epithelial cells . however , there is a lack of biomarkers to study exfoliated epithelial cells and the role of clock genes , if any , in autophagy . among many unsolved questions which can be listed , we can wonder what is the biological information retained , altered , or lost during anoikis ? future works may focus on the mtor signaling pathway which has been found downregulated in detached epithelial cells and , in adipocytes , linked to diurnal gene expression and metabolic regulation . recent data on the molecular biology of clock components indicate that central and peripheral clocks differ in their coupling with the different categories of synchronizers as well as in their output on rodent models [ 81 , 82 ] as well as on human data . a better understanding of exfoliation may be useful not only to translational research but also to tissue reconstruction of mucosa .

the recovery of exfoliated cells from biological fluids is a noninvasive technology which is in high demand in the field of translational research . exfoliated epithelial cells can be isolated from several body fluids ( i.e. , breast milk , urines , and digestives fluids ) as a cellular mixture ( senescent , apoptotic , proliferative , or quiescent cells ) . the most intriguing are quiescent cells which can be used to derive primary cultures indicating that some phenotypes retain clonogenic potentials . such exfoliated cells are believed to enter rapidly in anoikis after exfoliation . anoikis can be considered as an autophagic state promoting epithelial cell survival after a timely loss of contact with extracellular matrix and cell neighbors . this paper presents current understanding of exfoliation along with the influence of methodology on the type of gastrointestinal epithelial cells isolated and , finally , speculates on the balance between anoikis and apoptosis to explain the survival of gastrointestinal epithelial cells in the environment .

1. Introduction 2. Exfoliation of Epithelial Cells: A Source of Reliable Biological Information on the Mucosa Physiology? 3. In Vitro and Animal Model to Study Exfoliation 4. Stability of Biological Information in Exfoliated Epithelial Cells 5. Perspectives

PMC4427746

healthy horses : eleven foals ( 7 males and 4 females under one year old ) and an yearling female thoroughbred were used for evaluation of the development stages of the gastric mucosae of healthy horses . details of the program for endoscopic examination are summarized in table 1table 1.endoscopic examination of the healthy horsesm : male , f : female . the number of horses is shown in parentheses , eleven foals ( 7 males and 4 females under one year old ) and an yearling female thoroughbred were used for evaluation of the development stage of the gastric mucosa of healthy horses .. m : male , f : female . the number of horses is shown in parentheses , eleven foals ( 7 males and 4 females under one year old ) and an yearling female thoroughbred were used for evaluation of the development stage of the gastric mucosa of healthy horses . affected foals : in the clinical field , it has been empirically demonstrated that foals suffering from gastric ulcer frequently exhibit depression , dysorexia or diarrhea [ 1 , 3 , 11 ] . fifty - six foals having these symptoms were used for examination of gastric ulcer by endoscope , as shown in table 2table 2.endoscopic examination of the affected horsesage ( days)number of examinations at each age ( days)malefemaletotal4305383160981761901091991120718121198224total332356 . we devised a grading system for the gastric ulcers of the foals according to andrews criteria and calculated the distribution of the lesions . the grading system was as follows : a score of 1 indicates that the mucosa is intact , but associated with areas of hyperemia with erosion ( fig . 1.a grading system for the gastric ulcers of foals ( modified andrews criteria , 1999 ) . ( a ) score 1 : the mucosa is intact , but associated with areas of hyperemia with erosion ( arrows ) . ( b ) score 2 : small , single or multifocal superficial ulcers with hyperemia ( arrows ) . ( c ) score 3 : large , single multifocal or extensive superficial ulcers with hyperemia ( arrows ) . ( d ) score 4 : extensive lesions with areas of apparent deep ulceration ( arrow ) . ) . a score of 2 indicates a small , single or multifocal superficial ulcer with hyperemia ( fig . a score of 3 indicates a large , single , multifocal or extensive superficial ulcer with hyperemia ( fig . finally , a score of 4 indicates extensive lesions with areas of apparent deep ulceration ( fig . 1d ) . a grading system for the gastric ulcers of foals ( modified andrews criteria , 1999 ) . ( a ) score 1 : the mucosa is intact , but associated with areas of hyperemia with erosion ( arrows ) ( b ) score 2 : small , single or multifocal superficial ulcers with hyperemia ( arrows ) . ( c ) score 3 : large , single multifocal or extensive superficial ulcers with hyperemia ( arrows ) . ( d ) score 4 : extensive lesions with areas of apparent deep ulceration ( arrow ) . endoscopic examination : the endoscope ( vq-8303a , olympus , tokyo , japan ) used in the present study has an effective length of 3,000 mm , a tip diameter of 10.6 mm , a suction unit and an image recording apparatus . the endoscopic procedure was performed according to previous reports [ 10 , 13 ] . for the purpose of sedation and pain relief , a combined solution of xylazine ( 0.5 mg / kg , iv , nippon zenyaku kogyo co. , ltd . , koriyama , japan ) and butorphanol ( 0.01 to 0.02 mg / kg , iv , bristol - myers squibb co. , tokyo , japan ) was used before endoscopic examination . the endoscope was inserted via the nasal cavity of foals and reached the stomach cavity expanded by air from a biopsy channel . endoscopic examination was performed on 4 regions of the squamous mucosa in the lesser curvature ( lc - s ) , squamous mucosa in the greater curvature ( gc - s ) , glandular mucosa in the greater curvature ( gc - g ) and pylorus ( p ) . statistical analysis of the mean scores was performed with the t - test . healthy horses : at the 6th day after birth , the margo plicatus was clear , and serrated ridges were not noticeable . the squamous mucosa ( lc - s and gc - s ) wall was very thin , and part of the spleen attached to the outer wall of the stomach was observed through the thin gastric mucosa . the gc - g exhibited a thin membrane - like appearance with a clear color . the margo plicatus became clear at 14 days old , and the formation of serrated ridges was not observed at that time . the squamous mucosa wall thickened somewhat , had a translucent luster and appeared to be wet . the squamous mucosa wall thickened further , and the clarity of the stomach wall was lost . the gc - g was wet and had a surface structure containing irregularities that were finely granular . at 28 days old , a serrated ridge was evident at the boundary of the squamous mucosa and glandular part . the gc - g also thickened , and the granular structure of the mucosal surface showed a more pronounced pink color . throughout the period from 6 to 28 days old , epithelial desquamation although epithelial desquamation of the lc - s was noted until 60 days old , it was still observed in the gc - s at 90 days old ( fig . old , the squamous mucosa wall had an increased thickness , and the serrated ridges had grown , appearing similar to those observed in the squamous mucosa of a yearling horse . after 30 days , the gm thickened and showed a reddish color . the thickened squamous mucosa wall of the yearling lacked epithelial desquamation , and the surface of the mucous membrane showed dryness . the mucosa of the gastric fundus and pylorus exhibited a shiny and smoothly granular structure . during the period of endoscopic examination , there were no abnormalities in the gastric mucosa in any of the foals . affected foals : the number of foals having a gastric ulcer during the 198 days after birth is summarized for each 30-day period in fig . the incidence ( 30.4% ) of gastric ulcer sharply increases between 31 and 60 days old . the highest incidence ( 33.9% ) of gastric ulcer is between 61 and 90 days old . the incidence ( 14.3% ) of gastric ulcer sharply declines at 91 days old or older .. the minimum age of the foals having a gastric ulcer was 4 days old . the incidence of gastric ulcer sharply increased by 30.4% after 31 days old . the highest incidence ( 33.9% ) of gastric ulcer after 91 days old , the incidence of gastric ulcer sharply declined ( 14.3% ) . the incidence ( 30.4% ) of gastric ulcer sharply increases between 31 and 60 days old . the highest incidence ( 33.9% ) of gastric ulcer is between 61 and 90 days old . the incidence ( 14.3% ) of gastric ulcer sharply declines at 91 days old or older . the age ( days old ) at the time of endoscopic examination and distribution of gastric ulcers in 56 foals are shown in fig . 3.fig . gastric ulcers occurred more frequently in the squamous mucosa ( lc - s and gc - s ) ( 74.8% ) than in the glandular mucosa ( gc - g and p ) ( 25.2% ) during the period of examination . gastric ulcer occurred more frequently in the squamous mucosa ( lc - s and gc - s ) ( 74.8% ) than the glandular mucosa ( gc - g and p ) ( 25.2% ) during the period of examination . in the group of foal at up to 30 days old , gastric ulcer was frequently observed in the gc - s . in the group of foal at 31 to 60 days old , the ulcers increased in the lc - s in a manner similar to that in the gc - s . in the group of foals at 61 to 90 days old , the incidence of gastric ulcer sharply increased by more in the gc - s than in the lc - s . gastric ulcers formed predominantly in the gc - s between 4 and 90 days old and had a peak incidence between 61 and 90 days old . in the group of foals at 91 to 120 days old , the incidence of ulcers was reduced , and they were found predominantly in the squamous mucosa ( lc - s and gc - s ) . at over 120 days , there were no differences observed in the incidence of ulcer in the 4 parts of the gastric mucosa . gastric ulcers occurred more frequently in the squamous mucosa ( lc - s and gc - s ) ( 74.8% ) than in the glandular mucosa ( gc - g and p ) ( 25.2% ) during the period of examination . the age ( days old ) at the time of endoscopic examination , and the grading scores of gastric ulcers in 56 foals are shown in fig . 4fig . 4.the average score of gastric ulcers in all parts of the gastric mucosa . in the group of foals at up to 30 days old , the mean sd grading score of ulcers for each mucosal site was 0.53 0.24 ( lc - s , 0.13 0.13 ; gc - s , 0.75 0.16 ; gc - g , 0.88 0.40 ; p , 0.38 0.26 ) . in the group of foals at 31 to 60 days old , the mean sd grading score for each mucosal site was 0.53 0.21 ( lc - s , 0.24 0.14 ; gc - s , 0.71 0.22 ; gc - g , 0.69 0.27 ; p , 0.50 0.20 ) . in the group of foals at 61 to 120 days old , the mean sd grading score in the gc - s was between 1.26 0.25 and 1.13 0.55 . the mean sd grading score in the gc - g was between 2.00 0.38 and 1.50 0.60 . in the group of foals at more than 120 days old , the mean sd grading score for each mucosa was 1.19 0.68 . the grade of gastric ulcer shows a tendency to increase with age in days .. the average score for gastric ulcers in each of the parts showed a tendency to increase over time . in the group of foals at up to 30 days old , the mean sd grading score for ulcers at each mucosal site was 0.53 0.24 ( lc - s , 0.13 0.13 ; gc - s , 0.75 0.16 ; gc - g , 0.88 0.40 ; p , 0.38 0.26 ) . in the group of foals at 31 to 60 days old , the mean sd grading score for each mucosal site was 0.53 0.21 ( lc - s , 0.24 0.14 ; gc - s , 0.71 0.22 ; gc - g , 0.69 0.27 ; p , 0.50 0.20 ) . in the group of foals at 61 to 120 days old , the mean sd grading score in the gc - s was between 1.26 0.25 and 1.13 0.55 . the mean sd grading score in the gc - g was between 2.00 0.38 and 1.50 0.60 . in the group of foals at more than 120 days old , the mean sd grading score in each mucosa was 1.19 0.68 . the average score of gastric ulcers in all parts of the gastric mucosa . in the group of foals at up to 30 days old , the mean sd grading score of ulcers for each mucosal site was 0.53 0.24 ( lc - s , 0.13 0.13 ; gc - s , 0.75 0.16 ; gc - g , 0.88 0.40 ; p , 0.38 0.26 ) . in the group of foals at 31 to 60 days old , the mean sd grading score for each mucosal site was 0.53 0.21 ( lc - s , 0.24 0.14 ; gc - s , 0.71 0.22 ; gc - g , 0.69 0.27 ; p , 0.50 0.20 ) . in the group of foals at 61 to 120 days old , the mean sd grading score in the gc - s was between 1.26 0.25 and 1.13 0.55 . the mean sd grading score in the gc - g was between 2.00 0.38 and 1.50 0.60 . in the group of foals at more than 120 days old , the mean sd grading score for each mucosa was 1.19 0.68 . healthy horses : a few reports have described the development process of the gastric mucosa of neonatal foals using an endoscope [ 9,10,11 , 13,14,15 ] . however , long and continuous observations of the mucosal development of the stomach for 119 days after birth have not been reported as far as we know . in this report , endoscopic observation revealed the development process of the gastric mucosa of the foal , during the 4 months following birth . immediately after birth , part of the spleen attached to the outside stomach wall and after 28 days of development , the stomach wall was thick enough to prevent observation of the spleen through the wall . the appearance of the mucosal surface was very similar to that of a yearling horse . during examination , formation of epithelial desquamation on the mucosal surface was characteristically observed in the squamous mucosa ( lc - s and gc - s ) . histologically , the stratum corneum of the squamous mucosa forms beginning in the late fetal stage , and the stratum corneum grows slowly thicker after birth . the keratinized epithelium of the stratum corneum detaches from the mucosal surface gradually , and this is called epithelial desquamation . epithelial desquamation is considered a physiological phenomenon and is characteristic of the development process of the gastric mucosa [ 11 , 15 ] . in our study , endoscopic examination revealed the physiological development process of the gastric mucosa during the 4 months following birth . earlier studies reported that epithelial desquamation of the squamous mucosa disappears at around 40 days old . however , epithelial desquamation was observed in the lc - s in our foals until 60 days old and in the gc - s until 90 days old . this finding suggests that there is a difference in the development of the gastric mucosa by region and that desquamation can continue over a long term , a term longer than studies have previously reported . affected foals : as reported in western countries , gastric ulcers of foals in japan ( hidaka district ) occur between 30 and 90 days old , but the reason for this is still unclear . a deficiency in passive immunity is considered to be one of the internal factors that contribute to gastric ulcers that occur frequently in this period [ 7 , 8 ] . many severe diseases , such as pneumonia ( e.g , rhodococcus equi ) , enteritis ( e.g , rotavirus ) [ 5 , 6 , 22 ] , infectious arthritis and osteomyelitis occur frequently in foals with immunodeficiency . on the other hand , changes in feed and rearing environment can be considered as external factors . in the hidaka district , the weaning of foals usually begins at around 100 days old and ends at around 180 days . in the healthy foals , the mother s milk and secretion of by the foal prompts the proliferation of epithelial cells and maturation of the gastric mucosa in preparation for weaning . while living with the mother , the growing foal may frequently eat the concentrated feed given to the mare . the decreases in gastric juice ph , intake of the mother s milk and saliva secretion caused by this surreptitious eating are considered to disturb the development of the gastric mucosa before controlled weaning . our study revealed that maturation of the gastric mucosa of the foal occurs up to at least 90 days old . before weaning , surreptitious ingestion of the mother s feed therefore , the deficiency of passive immunity and failure of feeding management can be considered important factors in the occurrence of gastric ulcers in foals before weaning . the increased gastric ulcer score along with age in the affected foals suggests that these factors may contribute to formation of severe lesions before weaning of the foals . on the other hand , ulcers in the weaning period after 120 days old showed a high score in the present study . weaning of foals may be involved in weakening the immune system and weight loss . the gastric ulcers were found to have occurred mostly in the squamous mucosa ( lc - s and gc - s ) throughout the examination period . the results suggest that gastric ulcers may occur given the specific background of the development process of the mucous membrane and structure of the stomach . the mucosal surface of the gm is protected by mucus produced by the surface mucus cells , and secretion of digestive juice is not active in the lactation period . the membrane of the squamous mucosa ( lc - s and gc - s ) lacks surface mucus cells and is protected solely by the action of saliva . the mother s milk in the lactation period contributes to protect the mucosal membrane and to the development of the gastric mucosal epithelium . therefore , anatomical and functional differences of the stomach , which are horse specific , may cause the differences in sites of lesions and scores of gastric ulcers caused by the various internal and external factors . in other words , when the causes of gastric ulcer overlap , the squamous mucosa is the most affected region , and the lesion may occur most frequently in the gc - s , in which development of the mucosa continues until around 90 days old . the process of development of the gastric mucosa of healthy foals during the 119 days from the 6th to the 125th day after birth was revealed in this study . characteristic changes were seen in the squamous mucosa ( lc - s and gc - s ) , and the maturation of the squamous mucosa ( lc - s ) was more rapid than that in gc - s . the epithelial desquamation of the squamous mucosa ( lc - s and gc - s ) indicates the process of development of the squamous mucosa membrane and is considered to be a normal physiological phenomenon . gastric ulcers formed predominantly in the squamous mucosa ( especially in the gc - s ) due to the immature mucosa of the foals before the weaning period , and the peak incidence was between 61 and 90 days .

to contribute to early diagnosis and treatment of gastric ulcer of foals , we examined the gastric mucosa of healthy and affected foals using an endoscope . in healthy foals , the characteristic changes in the development of the squamous mucosa were seen mainly in the squamous mucosa , and maturation of the squamous mucosa in the greater curvature ( gc - s ) occurred more slowly than that of the squamous mucosa in the lesser curvature ( lc - s ) . epithelial desquamation in the lc - s and gc - s was observed between 6 and 90 days but was not observed in the lc - s at about 60 days , whereas it was observed in the gc - s until 90 days old . these findings suggest that there is a difference in the development of the gastric mucosa by region and that desquamation continues over a term longer than studies have reported in the past . in the affected foals , the minimum age at which gastric ulcer was observed was 4 days old . gastric ulcers formed predominantly in the squamous mucosa ( lc - s and gc - s ) of foals with an immature mucosa before the weaning period , and the peak incidence occurred between 61 and 90 days old . the differences in the ulceration sites were considered to depend on the difference in the development ( maturation ) stage of the squamous mucosa . the grading score of the gastric ulcer increased with the growth of the affected foals . the gastric ulcer might be enhanced greatly by stress in the weaning period .

MATERIALS AND METHODS RESULTS DISCUSSION

PMC3692048

micrornas ( mirnas ) are small non - coding rnas ( 2123 nt in length ) that post - transcriptionally regulate gene expression by blocking translation or inducing degradation of the targeted mrna ( 1 ) . since their first identification in caenorhabditis elegans in 1993 ( 2 ) , the number of annotated mirnas and mirna - related publications increase in a super linear rate , clearly depicting their central position in the rna revolution ( 3 ) . in silico mirna target identification is a crucial step in most mirna experiments , as the mirna interactome has not yet been adequately mapped , even for the most studied model organisms . early mirna - related research efforts have highlighted the necessity of computational analyses in order to assist the experimental identification of mirna targets . this has resulted to the development of numerous mirna target prediction algorithms ( 4 ) , which are now considered indispensable for the design of relevant experiments . these algorithms identify in silico mirna targets as candidates for further experimentation or for computational processing , such as target enrichment analyses . predictions of the available computational algorithms can be acquired from relevant interaction databases or web servers ( 4,5 ) . the diana - microt web server v4.0 ( 6 ) is focused on providing in silico predictions of mirna mrna interactions . it is characterized by a user - friendly interface and provides extensive information for predicted mirna target gene interactions such as a global score for each interaction , as well as detailed information for all predicted target sites . each target site can be visualized , and the user can examine its local prediction score , target site conservation and mirna mrna binding structure . the server also provides connectivity to online biological databases and offers links to nomenclature , sequence and protein databases . here , we present diana - microt web server v5.0 , a significantly updated version , which hosts the state - of - the - art target prediction algorithm , diana - microt - cds ( 7 ) . microt - cds is the only algorithm available online , specifically designed to identify mirna targets both in 3 untranslated region ( 3utr ) and in coding sequences ( cds ) . the new server detects mirna targets in mrna sequences of homo sapiens , mus musculus , drosophila melanogaster and c.elegans . furthermore , it has been updated to mirbase v18 ( 8) and ensembl v69 ( 9 ) . specific attention has been paid to the web server interface , which follows the diana design framework , to be instantly familiar to users of previous versions , or other diana tools ( figure 1 ) . on the other hand , online help , informative tooltips and easy - to - use menus the fifth version of the diana - microt web server focuses also on advanced users and laboratories requiring support for sophisticated pipelines . the server provides programmatic access to services of multiple diana algorithms and a complete integration with the taverna workflow management system ( wms ) ( 10 ) , using the in - house developed diana - taverna plug - in . furthermore , a new section of the web interface hosts ready - made advanced workflows that can perform extensive mirna - related analyses on results derived from high - throughput techniques , such as microarrays or next - generation sequencing ( ngs ) . the interface presents information regarding the specified predicted mirna mrna interactions . mirna and gene - related information , as well as the advanced search options have been expanded . links to external databases , graphical representation of the binding sites , as well as mirna - recognition elements ( mres ) conservation and prediction scores are displayed in the relevant sections . the left side of the page is devoted to personal user space , reporting latest searches and bookmarks . the interface presents information regarding the specified predicted mirna mrna interactions . mirna and gene - related information , as well as the advanced search options have been expanded . links to external databases , graphical representation of the binding sites , as well as mirna - recognition elements ( mres ) conservation and prediction scores are displayed in the relevant sections . the left side of the page is devoted to personal user space , reporting latest searches and bookmarks . initial research efforts have unveiled that mirnas regulated gene expression through their binding on the 3utr of protein - coding genes ( 2 ) . however , accumulated experimental evidence has revealed that mirna - binding sites within coding sequences are also functional in controlling gene expression ( 11 ) . the new algorithm microt - cds ( 7 ) can identify mirna targets in 3utr , as well as in cds regions . further details on the microt - cds algorithm and the utilized training sets can be found in the relevant publication by reczko et al . diana - microt - cds provides increased accuracy and the highest sensitivity at any level of specificity over other available state - of - the - art implementations , when tested against pulsed stable isotope labeling by amino acids in cell culture ( psilac ) proteomics data sets ( 12 ) . the selection of diana - microt - cds as its core algorithm renders the new web server the only available online resource capable of incorporating mirna targets in 3utr as well as in cds . the server is compatible with the new mirna nomenclature ( 3p/5p ) introduced in mirbase v18 , as well as with previous mirna naming conventions . it currently supports 7.3 10 h.sapiens , 3.5 10m.musculus , 4.4 10 d.melanogaster and 2.5 10c.elegans interactions between 3876 mirnas and 64 750 protein - coding genes . gene ( 9 ) and mirna ( 13 ) expression annotation has been incorporated into the web server , enabling the user to perform advanced result filtering based on tissue expression . furthermore , users can also restrict predictions between uploaded lists of expressed genes and/or mirnas . for example , this feature can be used to identify interactions between a list of repressed ( or overexpressed ) genes and overexpressed ( or repressed ) mirnas , in the case of a differential expression analysis pipeline . moreover , the web server hosts an updated version of the kegg database providing a relevant search module based on kegg pathway descriptions ( 14 ) . a redesigned optional user space has also been implemented , which provides personalized features and facilitates the interconnectivity between the web server and the available diana software and databases ( figure 1 ) . as high - throughput data have become the new backbone of biological research the new diana - microt web server aims to facilitate users , not having access to extensive computational infrastructures and support , to perform ready - to - use sophisticated pipelines . diana - microt web server v5.0 hosts numerous integrated analyses in the form of ready - made advanced pipelines , covering a wide range of inquiries regarding predicted or validated mirna gene interactions and their impact on metabolic and signalling pathways . these pipelines can be used to analyse user data derived from small scale and high - throughput experiments directly from the diana - microt web server interface , without the necessity to install or implement any kind of software . for instance , one of the available workflows ( figure 2 ) can analyse mrna and mirna expression data ( expression and fold change ) . the workflow performs enrichment analysis of experimentally validated targets derived from diana - tarbase v6.0 ( 3 ) or / and predicted interactions from microt - cds . this step is considered crucial to identify mirnas that significantly regulate the differentially expressed genes . interactions between user - defined mirna and gene sets are in silico identified in 3utr and cds regions using diana - microt - cds . a subsequent mirna target enrichment analysis identifies mirnas controlling significantly the sets of differentially expressed genes . the pipeline is automatically repeated for different prediction thresholds ( from more sensitive , to more stringent ) . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds . in the last step of the pipeline , mirna - targeted pathway analysis is implemented with diana - mirpath v2 . interactions between user - defined mirna and gene sets are in silico identified in 3utr and cds regions using diana - microt - cds . a subsequent mirna target enrichment analysis identifies mirnas controlling significantly the sets of differentially expressed genes . the pipeline is automatically repeated for different prediction thresholds ( from more sensitive , to more stringent ) . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds . in the last step of the pipeline , mirna - targeted pathway analysis is implemented with diana - mirpath v2 . the prediction score threshold can significantly affect the analysis steps that follow . in the case of predicted interactions , the pipeline can be optimized by automatic repetitions of different prediction thresholds ( from sensitive to more stringent ) , to minimize the effect of the selected settings to the result . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds ( 15,16 ) . in the last step of the pipeline , the identified mirnas are subjected to a functional analysis , where pathways controlled by the combined action of these mirnas are detected using diana - mirpath v2.1 ( 16 ) . other available pipelines can handle mirna and gene lists , to perform the enrichment analysis , or even select the type of used interactions ( predicted or experimentally validated ) . in the latter workflow , the algorithm it initially identifies the number of available interactions in diana - tarbase and diana - microt - cds ( validated versus predicted ) and automatically selects to use validated targets only in the cases of well - annotated mirnas . the new diana - microt web server enables users to perform such analyses directly from the on - line user interface , or create even more extensive pipelines programmatically or by using visual tools ( taverna wms ) . diana - microt web server v5.0 was completely redesigned to provide the necessary building blocks for easily incorporating mirna functional analysis in complex pipelines . the new diana - microt web server aims to facilitate advanced users in creating novel or enhancing existing pipelines with mirna target identification and functional analysis tools . to this end , diana - microt web server v5.0 provides a complete integration with the taverna wms , using our in - house developed diana - taverna plug - in . diana - taverna plug - in enables the user to directly access our target prediction server ( microt - cds ) from the graphic interface of taverna and incorporate advanced mirna analysis functionalities into custom pipelines . furthermore , the plug - in enables the extension of such pipelines through the use of other diana tools and databases , providing access to the most extensive collection of validated mirna targets ( diana - tarbase v6.0 ) and to diana - mirpath v2.1 , a tool designed for the identification of mirna targeted pathways . furthermore , the web server also supports direct programmatic access to all aforementioned utilities in the form of services , to facilitate users having already implemented pipelines using scripting or programming languages . initial research efforts have unveiled that mirnas regulated gene expression through their binding on the 3utr of protein - coding genes ( 2 ) . however , accumulated experimental evidence has revealed that mirna - binding sites within coding sequences are also functional in controlling gene expression ( 11 ) . the new algorithm microt - cds ( 7 ) can identify mirna targets in 3utr , as well as in cds regions . further details on the microt - cds algorithm and the utilized training sets can be found in the relevant publication by reczko et al . diana - microt - cds provides increased accuracy and the highest sensitivity at any level of specificity over other available state - of - the - art implementations , when tested against pulsed stable isotope labeling by amino acids in cell culture ( psilac ) proteomics data sets ( 12 ) . the selection of diana - microt - cds as its core algorithm renders the new web server the only available online resource capable of incorporating mirna targets in 3utr as well as in cds . the server is compatible with the new mirna nomenclature ( 3p/5p ) introduced in mirbase v18 , as well as with previous mirna naming conventions . it currently supports 7.3 10 h.sapiens , 3.5 10m.musculus , 4.4 10 d.melanogaster and 2.5 10c.elegans interactions between 3876 mirnas and 64 750 protein - coding genes . gene ( 9 ) and mirna ( 13 ) expression annotation has been incorporated into the web server , enabling the user to perform advanced result filtering based on tissue expression . furthermore , users can also restrict predictions between uploaded lists of expressed genes and/or mirnas . for example , this feature can be used to identify interactions between a list of repressed ( or overexpressed ) genes and overexpressed ( or repressed ) mirnas , in the case of a differential expression analysis pipeline . moreover , the web server hosts an updated version of the kegg database providing a relevant search module based on kegg pathway descriptions ( 14 ) . a redesigned optional user space has also been implemented , which provides personalized features and facilitates the interconnectivity between the web server and the available diana software and databases ( figure 1 ) . as high - throughput data have become the new backbone of biological research , there is an increasing need to support advanced high - throughput analysis pipelines . the new diana - microt web server aims to facilitate users , not having access to extensive computational infrastructures and support , to perform ready - to - use sophisticated pipelines . diana - microt web server v5.0 hosts numerous integrated analyses in the form of ready - made advanced pipelines , covering a wide range of inquiries regarding predicted or validated mirna gene interactions and their impact on metabolic and signalling pathways . these pipelines can be used to analyse user data derived from small scale and high - throughput experiments directly from the diana - microt web server interface , without the necessity to install or implement any kind of software . for instance , one of the available workflows ( figure 2 ) can analyse mrna and mirna expression data ( expression and fold change ) . the workflow performs enrichment analysis of experimentally validated targets derived from diana - tarbase v6.0 ( 3 ) or / and predicted interactions from microt - cds . this step is considered crucial to identify mirnas that significantly regulate the differentially expressed genes . interactions between user - defined mirna and gene sets are in silico identified in 3utr and cds regions using diana - microt - cds . a subsequent mirna target enrichment analysis identifies mirnas controlling significantly the sets of differentially expressed genes . the pipeline is automatically repeated for different prediction thresholds ( from more sensitive , to more stringent ) . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds . in the last step of the pipeline , interactions between user - defined mirna and gene sets are in silico identified in 3utr and cds regions using diana - microt - cds . a subsequent mirna target enrichment analysis identifies mirnas controlling significantly the sets of differentially expressed genes . the pipeline is automatically repeated for different prediction thresholds ( from more sensitive , to more stringent ) . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds . in the last step of the pipeline , mirna - targeted pathway analysis is implemented with diana - mirpath v2 . the prediction score threshold can significantly affect the analysis steps that follow . in the case of predicted interactions , the pipeline can be optimized by automatic repetitions of different prediction thresholds ( from sensitive to more stringent ) , to minimize the effect of the selected settings to the result . by using meta - analysis statistics , the server combines the p - values from each repetition into a total p - value for each mirna , signifying its effect on the selected genes for all used thresholds ( 15,16 ) . in the last step of the pipeline , the identified mirnas are subjected to a functional analysis , where pathways controlled by the combined action of these mirnas are detected using diana - mirpath v2.1 ( 16 ) . other available pipelines can handle mirna and gene lists , to perform the enrichment analysis , or even select the type of used interactions ( predicted or experimentally validated ) . in the latter workflow , the algorithm it initially identifies the number of available interactions in diana - tarbase and diana - microt - cds ( validated versus predicted ) and automatically selects to use validated targets only in the cases of well - annotated mirnas . the new diana - microt web server enables users to perform such analyses directly from the on - line user interface , or create even more extensive pipelines programmatically or by using visual tools ( taverna wms ) . diana - microt web server v5.0 was completely redesigned to provide the necessary building blocks for easily incorporating mirna functional analysis in complex pipelines . the new diana - microt web server aims to facilitate advanced users in creating novel or enhancing existing pipelines with mirna target identification and functional analysis tools . to this end , diana - microt web server v5.0 provides a complete integration with the taverna wms , using our in - house developed diana - taverna plug - in . diana - taverna plug - in enables the user to directly access our target prediction server ( microt - cds ) from the graphic interface of taverna and incorporate advanced mirna analysis functionalities into custom pipelines . furthermore , the plug - in enables the extension of such pipelines through the use of other diana tools and databases , providing access to the most extensive collection of validated mirna targets ( diana - tarbase v6.0 ) and to diana - mirpath v2.1 , a tool designed for the identification of mirna targeted pathways . furthermore , the web server also supports direct programmatic access to all aforementioned utilities in the form of services , to facilitate users having already implemented pipelines using scripting or programming languages . high - throughput techniques have significantly increased the number of users requiring in - depth analysis of mirna mrna interactions . these techniques provide great numbers of differentially expressed mirnas and genes , which have to be analysed in sophisticated pipelines . the new diana - microt web server aims to further increase the target prediction accuracy and usability of the server interface , while facilitating users aiming to perform complex mirna - related analyses . compared with the previous version , the new web server has received a major upgrade and is currently up to date with key mirna / gene repositories , such as ensembl ( v69 ) and mirbase ( v18 ) . the taverna plug - in and the integration of cutting - edge workflows in the web interface provide the missing link between experimental results and state - of - the - art functional meta - analyses . the project [ 09 syn-13 - 1055 ] mikrorna by the greek general secretariat for research and technology and the project tom which is implemented under the aristeia action of the operational programme education and lifelong learning and is co - funded by the european social fund ( esf ) and national resources . funding for open access charge : projects tom and mikrorna .

micrornas ( mirnas ) are small endogenous rna molecules that regulate gene expression through mrna degradation and/or translation repression , affecting many biological processes . diana - microt web server ( http://www.microrna.gr/webserver ) is dedicated to mirna target prediction / functional analysis , and it is being widely used from the scientific community , since its initial launch in 2009 . diana - microt v5.0 , the new version of the microt server , has been significantly enhanced with an improved target prediction algorithm , diana - microt - cds . it has been updated to incorporate mirbase version 18 and ensembl version 69 . the in silico - predicted mirna gene interactions in homo sapiens , mus musculus , drosophila melanogaster and caenorhabditis elegans exceed 11 million in total . the web server was completely redesigned , to host a series of sophisticated workflows , which can be used directly from the on - line web interface , enabling users without the necessary bioinformatics infrastructure to perform advanced multi - step functional mirna analyses . for instance , one available pipeline performs mirna target prediction using different thresholds and meta - analysis statistics , followed by pathway enrichment analysis . diana - microt web server v5.0 also supports a complete integration with the taverna workflow management system ( wms ) , using the in - house developed diana - taverna plug - in . this plug - in provides ready - to - use modules for mirna target prediction and functional analysis , which can be used to form advanced high - throughput analysis pipelines .

INTRODUCTION MATERIALS AND METHODS Integration of DIANA-microT-CDS Web server update and extension Web server support of advanced pipelines Web server integration with Taverna WMS CONCLUSION FUNDING

PMC2039814

picture archiving and communication systems ( pacs ) and other imaging - related information technology ( it ) systems have grown in popularity over the last decade . initially used within departments as radiology - focused tools with a small number of users , these systems have now grown to become enterprise - class resources for departments outside of radiology . as demand for and reliance on these systems have continued to grow , so has the demand for an always - on infrastructure and system architectures that can support mission - critical availability and uptime.1 meeting these requirements is often a challenge for pacs and radiology it professionals . many system architectures delivered as standard offerings by industry vendors lack redundancy and/or failover capabilities . most pacs teams rely on their clinical users to alert them to problems as they occur . this is a less - than - ideal management approach because it requires that a system fail sufficiently to adversely affect users before the pacs team is notified of the problem . moreover , it places the onus of problem - reporting responsibility on the already time - challenged end - users . users often grow dissatisfied in this role and either ignore the issue or wait hours to report it , delaying the repair time , allowing the problem to affect growing numbers of their end - user colleagues , and , in worst - case scenarios , adversely affecting patient care . with the truly mission - critical nature of these systems , the need to understand possible failure modes and react quickly is high . a much preferred and more straightforward approach is proactive monitoring of system availability.2 system monitoring tools are available to automatically watch over all systems in a department or hospital.3 these tools can remotely monitor the ports of a server for activity and can alert it staff if no activity is detected . digital imaging and communications in medicine ( dicom ) , health level 7 ( hl7 ) , databases , and web services all run on different ports and can be monitored separately . by using these tools , pacs support team members can be automatically notified by pager when a possible problem exists and can work to repair issues more quickly . monitoring tools can also provide useful statistics ( e.g. , uptime reporting to the system service level ) , restart failed services , and interface with ticketing systems to perform issue tracking and trending analysis . implementation of these tools provides the added advantage of having an independent system monitor the availability of a pacs system . this can be quite useful when trying to hold a vendor accountable to an uptime guarantee in a service - level agreement . although commercial packages are available , these are often expensive and lack custom tools to monitor imaging - specific criteria ( such as dicom availability ) . commercial packages may also lack the requisite flexibility to monitor such criteria as message queues for radiology information systems ( ris ) . we report here on the implementation of an open source monitoring tool customized to our departmental needs and on our assessment of its utility in routine clinical imaging operations . we implemented the open source availability monitoring package nagios4 and configured it to be a comprehensive availability monitoring solution in a heterogeneous environment of 68 systems with 108 monitored services . we installed the nagios packages on an hp dl360 server ( hewlett - packard , palo alto , ca , usa ) with dual central processor units and 2 gb ram running the gentoo5 linux operating system . each contact had a name , e - mail address , pager e - mail address , and availability window for paging . these contacts were split into groups , such as pacs_team and ris_team . the host servers were then set up in the system . each host was configured with a name , internet protocol address , interval for monitoring ( 24 7 in most cases , but 8 5 for those up only during business hours ) , and notification options ( down , recovered , warning states ) . the interval functionality was useful for those devices and modalities that were turned off during specific time periods ( such as overnight ) . groups were created from the list of hosts and assembled on the basis of function . some of the groups we formed were pacs_archives , pacs_databases , and ris_servers . one of the most powerful features of nagios was the ability to create customized approaches to search for more than the availability of a single port . nagios uses a series of programs or scripts for monitoring as part of its standard functionality . the exit codes used are 0 ( ok ) , 1 ( warning ) , and 2 ( critical ) . one example of the utility of these codes is in a queue checker we created . in this scenario , a script connects to a database and pulls the value of a specific queue . if the value is > 50 , the program exited with a code of 2 ( critical ) . if it is between 25 and 49 , the program exited with a code of 1 ( warning ) . if the value is < 25 , the program exited normally with an exit code of 0 ( ok ) . one advantage of using an open source application in this role was the relative ease with which codes can be extended to add functionality . our system was customized to monitor criteria beyond system availability , to include port monitoring , queue status , and dicom availability of pacs gateways and subspecialty visualization work stations , and was more broadly used for an overall picture of the health of our information systems . additional plug - ins were created to restart services in the event of a failure , to log a ticket in our open source issue tracking system ( radtracker),6 as well as page our team members in the event of an issue . table 1specific ports and services used by nagios for system monitoringports and servicesicmp or ping for general availabilityport 80 for http web server availabilityport 443 for https secure web server availability ( including ssl certificate expiration)port 104 for dicom ( specifically c - echo)port 53 for domain name service port 21 for file transfer protocol port 25 for simple mail transport protocol port 3306 for mysql database availabilityport 1433 for microsoft sql availabilityport 1521 for oracle database availabilityport 389 for lightweight directory access protocol web services health checker status screen scraping of queue monitoring web pages with scripting to enable a restart of interface via url string specific ports and services used by nagios for system monitoring we installed this system in 2006 and monitored its usefulness over a 6-month period from december 2006 to may 2007 . the system was extremely effective at providing an overall view of the health level of our systems as well as specific data about availability . benefits that were realized as a result of this implementation included ( but were not limited to ) : automated monitoring with web - based access to system management data . the software provided a comprehensive view of the systems in our environment , monitoring 68 servers on a 24 7 basis . in addition , remote radiology sites were monitored , giving the added benefit of measuring wide area network ( wan ) availability to remote locations ( fig . the system was integrated with our inhouse paging system via simple mail transfer protocol to our local e - mail gateway . by integrating the system in this fashion , alerts could be sent directly to staff members pagers via e-mail.integration with problem management systems . the system was integrated with our paging system via direct open database connectivity insertion into our local issue tracking system s mysql database ( fig . 2 ) . this allowed the team to track any specific issue as part of the normal call resolution process and provided a knowledge base in the system for root cause analysis as well as a methodology for repair.service level agreement reporting . the system provided an overall system uptime view with time - frame customization reporting for availability , with detail down to the service level for each specific host or host group ( fig . the system could monitor these interfaces via a screen scrape of the web page and restart those interfaces that allowed for a restart when passed via a uniform resource locator string.health check status of systems via web service . when provided by vendors , the system could also monitor health and other status via web service capabilities.over the 6-month period , the system generated 461 issues in our issue tracking system . a breakdown of these issues by category 3service log entries.table 2breakdown of alerts submitted into ticketing system over 6 monthstype of eventnumber of occurrenceshost availability of ping209interface or queue problems104dicom c - echo51web server failure40web - services - related37lightweight directory access protocol ( ldap ) bind failures8misc . the software provided a comprehensive view of the systems in our environment , monitoring 68 servers on a 24 7 basis . in addition , remote radiology sites were monitored , giving the added benefit of measuring wide area network ( wan ) availability to remote locations ( fig . 1 ) . the system was integrated with our inhouse paging system via simple mail transfer protocol to our local e - mail gateway . by integrating the system in this fashion the system was integrated with our paging system via direct open database connectivity insertion into our local issue tracking system s mysql database ( fig . 2 ) . this allowed the team to track any specific issue as part of the normal call resolution process and provided a knowledge base in the system for root cause analysis as well as a methodology for repair . the system provided an overall system uptime view with time - frame customization reporting for availability , with detail down to the service level for each specific host or host group ( fig . the system could monitor these interfaces via a screen scrape of the web page and restart those interfaces that allowed for a restart when passed via a uniform resource locator string . when provided by vendors , the system could also monitor health and other status via web service capabilities . over the 6-month period , the system generated 461 issues in our issue tracking system . a breakdown of these issues by category these types of failures were either total system failures or network - related , oftentimes indicating a wan link failure . not all notifications were failures , however , because normal network maintenance at our facility was also included . on several occasions , the use of this tool was effective in providing warning notifications to wan vendors of a network failure or performance degradation before the vendor had been alerted to a problem . the second most common failure mode was related to interfaces either dropping or having a high number of messages that exceeded a predetermined threshold for that queue . by keeping interfaces available and within guidelines , we were able to keep the flow of data to a more real - time level . by scripting interface restarts and turning these into an automated process this failure mode was critical , because it effectively monitors the ability of systems to send images via dicom to pacs . this functionality does not normally exist in commercial monitoring packages and was of great importance since we were able to effectively monitor our system s ability to function as a dicom sender or receiver . the nagios plug - in for this feature was created using the dcmtk dicom toolkit7 to both perform the c - echo and determine if the association was completed successfully . in one case , we observed that by simply monitoring the dicom service for a particular host with c - echos at 5-min intervals , we were able to prevent dicom service failures that had previously been a frequent occurrence on the device . other remaining failure modes included web servers , web services , and lightweight directory access protocol bind issues , among others . monitoring for these failures was a critical addition and a great benefit for our group , because traditional availability - only monitoring ( through internet control messaging protocol or ping alone ) would not in most cases have revealed these failure modes as these types of failures most often occur for specific services within servers themselves . in such a scenario , servers would answer a ping and would appear to be available on a network but , in fact , would be experiencing an internal failure that would leave them nonfunctional or in a degraded state and unavailable to users . by capturing these failure modes through our monitoring efforts , we were able to respond immediately to issues that might not be obvious to standard monitoring systems . we found the nagios availability tool to be effective in the proactive support of mission - critical radiology and other clinical imaging systems . support staff were notified in real time with monitoring tool alerts to warning signs of possible system failures . although some pacs and ris vendors offer their own monitoring packages , we found that the addition of an independent monitoring tool that was outside of vendor control was effective and allowed the monitoring of multiple vendors through the same interface . this comprehensive approach to monitoring departmental and enterprise applications across all support environments provided a comprehensive view of system availability . we found that open source monitoring tools such as nagios could provide cost - effective independent monitoring . the benefits of using an open source tool included independent monitoring , agility in adaptation , and an open tool box for customizations . if a new application or system needed to be monitored , custom or prepackaged plug - ins could be utilized without the need of a purchase order or lengthy development turnaround time . the use of nagios as a system alerting and availability tool allowed us to monitor our systems for failures and notify our support staff immediately when problems occurred . the result was enhancement of our continuing efforts to achieve always - on systems and mission - critical delivery of services to our customers .

the goal of all radiology information technology ( it ) support organizations is excellent customer service through the availability of critical clinical information services , such as picture archiving communication systems and radiology information systems . despite these goals , it support personnel often act like firefighters , reacting to each problem , but unable to prevent or predict other problems . proactive support is always more desirable than reactive support . warning signs may exist well before a technical issue becomes system wide or the user is affected . the objective for it support organizations in health care should be to maximize system uptime by using proactive monitoring systems for failures and to automatically detect failures through systems management tools . we report on the implementation of nagios , an open source monitoring tool , as an availability management system in a diagnostic imaging department and on customized applications and protocols specific to radiology needs .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3144622

demonstration of coronary artery pathology in autopsies is vital for the elucidation of sudden death cases related to these lesions and for the development of new treatment approaches . congenital coronary artery anomalies are frequently observed in athletes , representing the second most common cause for athletic field deaths ( in about 20% of cases ) . the incidence of sudden cardiac death was reported to be 57% among 49 cases of anomalous left coronary artery . autopsy was conducted on a thirty - year male who died in a road traffic accident and brought dead to the hospital . heart dissection showed the absence of right coronary artery and the presence of left main trunk with unusual dominant course of the left circumflex coronary artery which was supplying whole of the right heart . however , the left anterior descending artery was normal [ figure 1 ] . cut section of the left circumflex coronary showed focal elevated areas [ figure 2 ] which microscopically showed [ figure 3 ] the features of pathological intimal thickening . the family members informed that he did not had any medical / cardiac signs and symptoms , and was absolutely normal before death . gross photograph of dissected heart showing only the left coronary artery with anterior descending and left circumflex branches gross photograph of dissected left circumflex coronary artery showing focal elevated areas in tunica intima microphotograph of the left coronary artery showing intimal thickening . coronary artery anomalies that entail a risk of sudden death are often associated with complex cardiac malformations but may occasionally be solitary . single coronary artery anomaly is most relevant clinically and associated with sudden cardiac death in young athletes and military personnel . a single coronary artery is an unusual congenital anomaly where only one coronary artery arises from the aortic trunk by a single coronary ostium and supply the entire heart . if a single common ostium is present , the pattern is considered to represent single coronary artery . the separate origin ( absent left main ) of the left anterior descending artery / left circumflex artery ( 30.4% ) and the anomalous origin of the left circumflex artery ( 27.7% ) are the two most common coronary anomalies . the anomaly observed in this case was the absent right coronary artery and an unusual dominant course of the left circumflex artery running along the posterior surface of the heart and supplying whole of the right heart , which is an extremely rare occurrence . to the best of our knowledge such an anomaly has only been reported twice in the english literature . the clinical outcome in patients with an anomalous coronary artery is heterogeneous with manifestations such as angina , syncope , myocardial infarction and sudden cardiac death . these are most common causes for sudden and unexpected death in young individuals , particularly during sporting activities and the greatest incidence of sudden death occurs during heavy physical exertion . the cause of sudden death varies from 25% for anomalous right coronary artery to 57% for anomalous left coronary artery . ischemia is the consequence of anatomical malformations , including the acute angle takeoff of the anomalous vessel , with a narrowed slit - like orifice that collapses in a valve like manner , thereby limiting the blood flow . younger patients ( 30 years old ) are reported to die suddenly or during exercise than older patients , despite their low frequency of significant atherosclerotic coronary artery disease . it was proposed that ischemia may be caused by sporadic spasm of the anomalous coronary artery induced by endothelial injury . hence one should be aware of the incidence of single coronary artery in particular , the rarest single dominant left coronary artery especially in athletes and military personnel , who can be screened and proper measures can be taken . in conclusion , we present a case with the absent right coronary artery and the presence of left main trunk with an unusual dominant course of the left circumflex coronary artery which was supplying whole of the right heart with normal left anterior descending artery . to the best of our knowledge such an anomaly has only been described twice in the english literature . performing autopsies for a better understanding of the coronary artery anomalies associated with sudden death

coronary artery anomalous course is rare , reported incidence is approximately 0.31.3% of patients undergoing coronary angiography and approximately 1% of routine autopsy examinations . a single coronary artery is an unusual congenital anomaly where only one coronary artery arises from the aortic trunk from a single coronary ostium , supplying the entire heart . we describe here a rare case with an unusual dominant left circumflex artery and absent right coronary artery .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSIONS

PMC3296267

magnetic resonance imaging ( mri ) is an important application of compressive sensing ( cs ) [ 14 ] . cs in mri has the potential to significantly improve both the speed of acquisition and quality of mr images , but requires an iterative reconstruction that is more computationally intensive than traditional inverse fourier reconstruction . compressive sensing accelerates mr acquisitions by reducing the amount of data that must be acquired . reconstruction of this partial data set is then accomplished by iteratively constraining the resulting image to be sparse in some domain while enforcing consistency of the measured subset of fourier data . one practical barrier to the routine adoption of cs mri is the delay between acquisition and reconstruction of images . compressed sensing solvers work almost entirely with vector and image arithmetic , making them an excellent candidate for acceleration through using graphics processing units ( gpus ) for parallelization . here we illustrate how gpus can be used to achieve significant increases in speed of cs reconstructions of large mri data sets . high - end video cards can contain hundreds of separate floating - point units , allowing for massively parallel computations at a fraction of the cost of cpu - based supercomputers , as measured on a per gigaflop basis ( one gigaflop is equivalent to one billion floating point operations per second ) . gpu computing works best in single instruction , multiple data ( simd ) situations , such as the solution of large systems of linear equations . the power of gpu computing is already being realized in several advanced medical image reconstruction applications [ 69 ] . the reconstruction platform tested was a high - performance gpu server designed to serve an mri scanner as a dedicated reconstructor . this system contained a six - core , 2.67 ghz xeon x5650 with 12 gb of 1333 mhz ddr3 ram , 32 kb of l1 cache , 256 kb of l2 cache , and 12 mb of l3 cache . the tesla c2050 contains 448 cuda cores arrayed as 14 streaming multiprocessors ( sms ) with 32 cores per sm . it has 3 gb of vram and 64 kb of memory per sm . all sms share 768 kb of l2 cache . the optimization problem we are choosing to explore is the reconstruction of partial mri data sets using cs . mri is proving to be a fertile application for cs because many mr images can be highly compressed by transform coding with little loss of information . conventional mri data is acquired according to the fourier sampling pattern required to satisfy the nyquist criterion while producing an image of a given field of view and spatial resolution . to sample the fourier domain compressively , only a random subset of the full nyquist fourier sampling scheme is acquired . reconstruction of the conventional fully sampled mri data requires simply an inverse fourier transform , ( 1)u = f1b , but the inverse problem becomes underdetermined when part of the fourier data is omitted , so approximate methods must be used . one highly successful method in particular has been to formulate the cs mri reconstruction as a sparse recovery problem , in which an image is found that is consistent with the acquired fourier data while having the sparsest representation in a chosen basis ( e.g. , gradient and wavelet ) . the typical formulation of the reconstruction of a complex image u from a partial fourier data set b is then ( 2)u = arg minx||x||1+||axb||22 , where a is a measurement operator that transforms the sparse representation x to the image domain then performs the subsampled fourier measurement , and the l1 and l2 norms are , respectively , ( 3)||x||1=i|xi|,||x||22=ixixi , where the bar denotes complex conjugation . with the addition of the l1 norm , the problem is more difficult to solve , and iterative techniques such as interior point methods [ 10 , 11 ] , iterative soft thresholding [ 12 , 13 ] , and gradient projection [ 1416 ] are typically employed . the open - source split bregman code of goldstein and osher was chosen as the starting point for the gpu - based cs solver . this solver was chosen for its rapid convergence and lack of array reduction steps , which hinders parallelization . we modified the original code to work with jacket 1.8.0 ( accelereyes , atlanta , ga ) and matlab r2010b ( mathworks , natick , ma ) . algorithm 1 briefly outlines the procedure for running the split bregman reconstruction on the gpu . note that the split bregman algorithm runs for a fixed number of iterations , so there is no variation in run time due to different descent trajectories as with a tolerance - based stopping criterion . furthermore , the choice of image reconstructed has no bearing on the results , since the fixed number of iterations ensure that the same number of operations are performed on any input data set . at the beginning of the reconstruction , the fourier data in main memory must be transferred to the gpu with jacket 's gsingle and gdouble matlab commands . next , temporary storage is allocated on the gpu using jacket 's gzero and gones commands . all subsequent arithmetic operations , including the fourier transform , are carried out on the gpu using function overloading . function overloading simplifies code syntax by enabling a single function to encapsulate different functionality for different types of arguments . the specific behavior is typically chosen by the compiler or at run time . in our case , matlab automatically calls the jacket library if an operation is requested on a matrix that lies in gpu memory , while identical operations on a matrix in main memory are carried out with matlab 's built - in functions . after the last loop iteration on the gpu , the solution is transferred back to main memory with overloaded versions of matlab 's double and single commands ; all temporary storage is automatically freed . the first was a pure matrix multiplication , designed to measure practical peak floating - point performance of the cpu and gpu as realized by jacket 1.8.0 . to remove dependencies on the multithreading performance of matlab r2010b and provide easier comparison of the cs reconstructions the second experiment , and the focus of this work , was a cs mri reconstruction of a t1-weighted breast image subjected to a 50% undersampling in fourier ( spatial frequency ) space . total variation was used as the sparsity constraint and was defined as the sum of the magnitudes of pixels in the gradient image . cs mri reconstructions were performed for powers - of - two image sizes ranging from 32 to 8192 ( up to 4096 only for double precision , due to memory limitations ) . this range covers the range of realistic mr acquisition matrix sizes for 2d scans with allowance for specialty techniques at very low or very high resolutions or future developments in imaging capabilities . the largest matrix sizes can also be indicative of the performance of three - dimensional reconstruction problems . for example , a 256 data set is the same size as a 4096 one . all reconstructions were timed eleven times , with the first iteration discarded and the following ten iterations averaged . jacket uses just - in - time ( jit ) compilation to improve performance of repeated function calls , so the first call to the jacket library is slowed by this compilation step . the cuda driver , upon initial invocation , optimizes the low - level gpu code for the particular hardware being used . these two processes increase code performance across multiple runs but reduce it for the initial function calls . for timing experiments , warmed up with a similar problem before timing the full - scale computation . here we used the simplest approach of discarding the first iteration . in principle , though , the warmup problem can be much smaller in data size as long as it uses the same set of jacket functions needed in the full - size reconstruction . table 1 shows the result of the matrix multiplication experiment with and without cpu multithreading enabled . in terms of cpu performance , matlab r2010b accelerated the cpu - based matrix multiplication by ~6 on this six - core processor using multithreading . using the gpu then yielded an additional factor of ~5 beyond this , with a combined speedup of ~30 over a single cpu core . based on the measured gpu single- and double - precision performance of 650 and 311 gflops , respectively , we can see that matlab r2010b combined with jacket 1.8.0 reached 60% , and 63% of the theoretical maximum single- and double - precision performance , respectively , of the gpu . the results of the cs mr image reconstructions are shown in tables 2 and 3 and figure 2 . the maximum speedup was 27 for a single - precision image of size 2048 . for a typical double - precision mri acquisition matrix of 256 to 512 figure 2 shows the speed advantage of the gpu - based code over using both one and multiple cpu cores . the difference between enabling multithreading or not was large , and should be considered to fairly evaluate the speed improvement of using a gpu . as can be seen in figure 2 , the speedup was less than one for images smaller than about 64 , followed by a rapid gain in speedup factor for images of 2048 and a decline in speedup factor for the largest matrix sizes . despite the performance falloff , the single - precision gpu code was able to reconstruct images up to 8192 before running out of gpu memory since single - precision matrices require half the storage per element of double - precision matrices . two notable features are evident in tables 2 and 3 . first , the gpu reconstruction times are very similar for images below 256 , regardless of numerical precision . this is due to the communication overhead in transferring the data from the cpu memory to the gpu memory . for the smallest images , this cost dominated the computation time and , for images below 64 , even caused the gpu reconstruction to take longer than the cpu reconstruction this suggests that for very small acquisition matrices an efficient gpu reconstruction should combine multiple 2d data sets , such as different slices or echoes , into one reconstruction problem . the second interesting feature of tables 2 and 3 is that gpu reconstruction times for images of size 1024 and smaller were effectively instantaneous . a typical rapid gradient echo sequence may employ a repetition time of 5 ms , so a 50% undersampling of an n fourier matrix would require roughly 2.5 n ms to acquire ( ignoring other acceleration methods for simplicity ) . the smallest image tested here would thus take 40 ms to acquire , while the cs reconstruction would take only 60 ms to complete , even with the severe communication penalty . the largest double - precision image we tested ( 4096 ) would take 15 s to acquire and 10 s to reconstruct . thus our gpu - based platform has the capacity to produce iteratively reconstructed cs - accelerated gradient echo images in real time for some mri applications . the decline in acceleration at 4096 was likely due to memory limitations ( yalamanchili , private communication ) . the nvidia tesla c2050 card is designed such that each streaming multiprocessor ( sm ; comprised of 32 floating point units ) shares a single 64 kb block of local memory . for a double - precision complex matrix multiplication , each matrix element requires 16 bytes of storage , so a maximum of 4096 elements can be stored in the sm 's shared memory . matlab stores arrays in column major order , so a matrix column must fit entirely into the shared memory of the sm in order to minimize memory access overhead . thus memory access patterns will become inefficient above matrix sizes of 2048 for double - precision complex matrix multiplication . we do in fact see a leveling off of performance at 1024 and a dramatic decline in performance above 2048 , which is consistent with this prediction . many alternate methods of accelerating the cs reconstruction on a gpu platform exist , including writing custom low - level code in cuda c or opencl , using free low - level libraries like cula and cufft , using matlab 's built - in gpu library through the parallel computing toolbox ( r2010b later only ) , or using other free high - level matlab - cuda interfaces , such as gpumat . the pace of algorithmic development is accelerating in compressed sensing , as demonstrated in figure 3 , and one advantage of using a high - level interface to the gpu is rapid prototyping and implementation . debugging time is shorter with high - level languages , and coding effort is reduced , allowing the newest algorithms to be implemented quickly while still retaining a majority of the theoretical computational benefit of the gpu . matlab 's built - in gpu library does not support the array indexing needed for the gradient operation , so we could not compare it here . gpumat is free and open source and a viable alternative to jacket in theory , but we were unable to use it with our reconstruction code due to an unresolvable memory access error . jacket is the only high - level software package to support sparse matrix operations on the gpu , which allows classes of compressed sensing algorithms that use sparse matrix operators to be used . also , the overloaded gpu functions in jacket are implemented as mex files , which are precompiled c / c++ functions callable from within matlab ; so writing custom cuda subroutines could only eliminate the function call overhead and not speed up the individual simd operations . we have shown that gpu computation significantly accelerated cs mri reconstruction of all but the smallest of the tested image sizes . the combination of matlab and jacket yields a processing package that is able to realize over half of the theoretical maximum performance of the gpu , while requiring minimal code development . the speedup realized by the gpu for the smallest images was progressively hampered by communication overhead , while the largest images suffered from the limited gpu ram . the optimal image dimensions , however , seem to be serendipitously close to that of high - resolution mri data ; so gpu computing , coupled with the goldstein and osher split bregman algorithm , appears to be a well - suited platform for rapid cs mri reconstruction . future improvements to these methods include algorithm modifications to allow unified reconstruction of multiple two - dimensional or a single three - dimensional fourier data set on the gpu with a single call to the reconstructor , thus reducing the communication penalty . additional cores on the gpu card could allow higher acceleration , since we found that the split bregman algorithm parallelizes extremely well . and finally , more ram onboard the gpu would allow larger data sets to be reconstructed more efficiently .

compressive sensing ( cs ) has been shown to enable dramatic acceleration of mri acquisition in some applications . being an iterative reconstruction technique , cs mri reconstructions can be more time - consuming than traditional inverse fourier reconstruction . we have accelerated our cs mri reconstruction by factors of up to 27 by using a split bregman solver combined with a graphics processing unit ( gpu ) computing platform . the increases in speed we find are similar to those we measure for matrix multiplication on this platform , suggesting that the split bregman methods parallelize efficiently . we demonstrate that the combination of the rapid convergence of the split bregman algorithm and the massively parallel strategy of gpu computing can enable real - time cs reconstruction of even acquisition data matrices of dimension 40962 or more , depending on available gpu vram . reconstruction of two - dimensional data matrices of dimension 10242 and smaller took ~0.3 s or less , showing that this platform also provides very fast iterative reconstruction for small - to - moderate size images .

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

PMC3675286

polymethyl methacrylate ( pmma ) is primarily used for removable partial and complete denture fabrication as a base material . reinforcements such as fibers , glass1 and polyethylene or activation and processing techniques such as injection - molding and microwave2 activation provided new benefits in polymer knowledge . however , this material still has some limitations about mechanical properties such as dimensional stability , color and flexural strength . eventually fracture of acrylic resin denture base is known to be the common clinical problem in prosthodontic practice . causes of such fractures are known to be related to porosity , residual monomer , poor fit of denture base , occlusal imbalance , design and fabrication failures , material choices , stress after long clinical usage and accidents.3,4 besides , in certain circumstances before complete fracture , a crack formation may be propagated bound to different types of stresses that denture base materials are subjected to . it is known that rough surfaces present suitable conditions for microorganism colonisation and/or biofilm formation,5,6 denture base cracks may become to be one of the best sites for microorganism propagation . biofilm is a microbial community that has dense and complex structure and may represent multiple organisms.7 they are often encapsulated within a matrix of exopolymeric material that consists of intricate networks of cells attached to biotic surfaces . they resist antimicrobials and immune cell challenge8 and are deeply embedded into cracks and porosities of dental materials as mentioned before . metallic and non - metallic medical devices like catheters , implants , dental materials are suitable sites for colonization of various types of microorganisms.9 corrosion at the interfacial surfaces of non - metallic materials usually starts with swelling after infiltration of little molecules or microorganisms . interfacial electrochemical process may be activated with the formation of biofilm on metallic and non - metallic surfaces resulting in an increased corrosion of colonized substratum . this development can detoriorate the materials with the presence of biofilm and is termed as biocorrosion.9 three - dimensional structure of biofilm is known to provide a highly complex arrangement of microorganisms.10 several studies regarding the developments and structures of biofilms on different dental materials including denture bases and their effects over oral health have been constituted.8 - 13 however the relationship between the biofilm related biocorrosion and crack and/or fracture formation still remains complicated even undefined . the aim of this study was to investigate the destructive effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms by evaluating the diffusion potential of the microorganisms , fracture propagation and scanning electron microscope ( sem ) images ( calculating biofilm covered surfaces ) on three different denture base materials . three different heat polymerized acrylic resins ( table 1 ) were used to prepare 50 15 4 mm ( type a ; for three point bending ( tpb ) test , n=210 ) , 8 8 1 mm ( type b ; for sem analysis , n=54 ) and 2 2 2 mm ( type c ; for spectrophotometer analysis , n=180 ) samples ( table 2 ) . next , each specimen were deflasked and finished with 320 , 400 and 600-grit silicone carbid papers . to simulate a crack line on the denture base , " v " type notch was carved in the middle of each specimen of impact test groups ( a type ) along with the 15 mm surface by using a milling machine and a milling tool as shown in the fig . all type of specimens were ultrasonically cleaned for 20 minutes and immersed in distilled water for 48 hours at 37 before tests . s. aureus , e. faecalis , p. aeruginosa , e. coli , s. mutans and c. albicans strains were inoculated in trypticase soy broth media and grown to stationary phase overnight . the samples were diluted 1:100 and each diluted bacterial culture ( 200 l ) was inoculated into each well in a fresh 96-well flat - bottom microtiter polystyrene plates , which also contain " c " type acrylic resin samples . plates were incubated for 48 hours at 37 and visualized by staining with 0.5% crystal violet for five minutes after washing with water . the biofilm was quantified in duplicate , after adding 100 l of 95% ethanol and the contents were transferred to new wells of microtiter plate . optical density ( od ) of stained adherent bacteria was determined with a micro elisa auto reader at wavelength of 620 nm spectrophotometrically.14,15 these od values were calculated as : " od - control " and considered as an index of bacteria adhering to acrylic surfaces and forming biofilms . ten specimens of type " a " , 3 specimens of type " b " and 10 specimens of type " c " denture base materials from 3 different brands ( table 1 ) were randomly inoculated into one of ; staphylococcus aureus ( atcc 6538 ) , streptococcus mutans ( atcc 35688 ) , enterococcus faecalis ( atcc 10541 ) , escherichia coli ( atcc 25922 ) , pseudomonas aeruginosa ( atcc 2327 ) or candida albicans ( atcc 18804 ) culture as indicated in table 2 at 0.5 mcfarland scale which corresponds to 10 cfu / ml for 168 hours at 37. a previously described method was modified and performed for biofilm formation.11 in brief , 1 ml aliquot of the bacterial and yeast cultures were introduced into 500 ml of brain heart infusion broth media in conical flasks and prepared denture base materials were inoculated into media . for the maintenance of bacterial and yeast density near the steady - state growth phase , 50% of the media were drained and replaced with the equal amount of a fresh sterile medium every two days and on day seven , the denture base materials were retrieved from the inoculated media for sem examination . the control group was composed of 10 non - contaminated " a " type samples of each denture base material ( 30 total ) and was kept in distilled water at 37 until the tpb test . the contaminated samples of type " a " , were rinsed with pbs and kept in running water for 15 minutes . the impact values of the specimens were measured under 2500 n maximum load ( 1 n preload ) with 1 mm / min cross - head speed with tpb test by using an universal testing machine ( lloyd lrx , west sussex , uk ) . the specimens were then supported on the jigs with a diameter of 3.2 mm with span length 50 mm . the " v notch " was placed face down on the jigs and the load was applied to the centre of the specimens . the data of the measurements were transferred to a personal computer , and the results were recorded . after incubation , each specimen of type " b " was removed and kept in an ultrasonic cleaner for 1 minute and rinsed with pbs to remove non or weak adhered microorganisms . the samples were fixed in 2.5% glutaraldehyde solution for 1h at room temperature , then rinsed with pbs . ethanol solutions with concentrations graded from 75% to 95% were used in 5 steps to dehydrate the specimens . specimens were then dried and placed on stubs to coat with 20 a gold / palladium for sem ( jeol 6400 , jeol corp . digital photographs as tiff files at 5,000 magnification were obtained from three different regions of each sample surface ( fig . the images were transferred to a personal computer to calculate total area and area fractions of biofilms using image j software.16 since cleaning and dehydrating processes were performed accurately , the area aspects apart from denture surface were all accepted as biofilm surface of that microorganism . area fraction of each image was recorded as the data of that sample . descriptive data were expressed as median , maximum , minimum and mean standard deviation . statistical analysis was performed using " pasw 18.0 statistics " and " statistica-7 " statistical software . when normality assumptions were satisfied analysis of variance ( anova ) otherwise the equivalent non - parametric test : kruskal - wallis was used for group comparisons . when significant differences found between groups , we used tukey and dunnett 's test ( after anova ) and dunn 's test ( after kruskall wallis ) for multiple group comparisons . table 3 shows the mean quantitative biofilm values and statistical significances according to microorganism and denture base material . there were significant differences among the adhesion potential of 6 different microorganisms and there were significant differences among their adhesion onto 3 different denture base materials . the highest median value within all the denture base materials was obtained with p. aeruginosa ( 0.095 0.018 ) followed by s. aureus ( 0.085 0.014 ) , c. albicans ( 0.081 0.017 ) , s. mutans ( 0.079 0.010 ) , e. faecalis ( 0.070 0.013 ) and e. coli ( 0.070 0.012 ) . the difference of adhesion between p. aeruginosa and s. aureus was not significant however there were significant differences between p. aeruginosa and other 4 microorganisms . the adhesion potential of the microorganisms over lucitone denture base material was higher than the other materials . the difference was not significant compared with qc-20 but it was significant when compared with ivocap plus . p. aeruginosa exhibited the highest median value on lucitone 550 denture base material surface ( 0.105 0.020 ) while c. albicans exhibited the lowest value on ivocap plus ( 0.067 0.021 ) . sem images revealed the regions of typical biofilm formation of each selected microorganism on denture base materials ( fig . 2 ) . table 4 shows the mean biofilm covered regions ( % ) on denture base materials and statistical significances according to microorganism and denture base material . the type of denture base material did not alter the diffusion potential of the microorganisms significantly . the percentages of biofilm covered areas of denture base materials ranged from 52.57% to 70.96% . s. aureus and p. aeruginosa had significantly higher ( 68.66% ) diffusion potential than the other tested microorganisms , but the difference among them was not significant . e. coli had the least diffusion potential and was significantly different from all the tested groups . table 5 shows the mean and standard deviation values of tpb test and statistical significances according to microorganism and denture base material . biofilm formation of the tested microorganisms decreased the tpb test values compared to the control group and this was statistically significant . lucitone 550 and ivocup plus denture base materials were significantly more resistant than qc-20 when mean tpb test values were consulted . e. coli produced the minimum destructive effect to the base materials when mean tpb test values of materials were evaluated , however this was not significantly different from e. faecalis ( p=.699>.05 ) , s. mutans and c. albicans . this study investigated the effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms on three different denture base materials . all tested microorganisms significantly decreased the tpb test values of the tested denture base materials . after contamination with microorganisms , the tpb test values of denture base materials were decreased significantly compared with the control groups . it may be speculated that the structure of all the denture base materials were decomposed by the biofilm formation and/or biocorrosive activity of microorganisms . several studies have investigated the fracture resistance of denture materials.17 - 20 in this study ivocap plus appears to be the most resistant base material when the peak data of control group and mean values at tpb tests were evaluated . this was compatible with the results from the study of hedzelek and gajdus.21 however , when the percentages of tpb test value reduction were examined , lucitone 550 was the most resistant material . the mean tpb test value of samples exhibited 18% resistance reduction at lucitone 550 samples , 19% at ivocap plus and 24% at qc-20 denture base materials compared with control group after contamination processes of 6 different microorganisms . evaluating the subgroups data of denture base materials and microorganisms of this study , the highest tpb test value reduction was observed in p. aeruginosa - qc group with 36% . even e coli , known to have the least degenerative bioactivity,12 decreased the tpb test values of lucitone 550 by 8% . eventually considering all the mean tpb test values of three denture bases of our study it can be affirmed that microorganisms had biocorrosive activity and deteriorated at least 15% of the initial physical composition of tested denture base materials . however , the difference of mean biofilm covered regions on denture base materials at sem display were not statistically significant the mean quantitative biofilm values of microorganisms on ivocap plus was significantly different from lucitone 550 . this may be due to the dissimilar accumulation of microorganisms on the material surface ( fig . 2 ) that varies according to the production of extracellular polysaccharides which can only be demonstrated using advanced techniques like three - dimensional confocal scanning laser microscopy.13 consequently , the two - dimensional sem display may have inhibited quantitative estimation of this phenomenon . serrano - granger et al.22 claimed that there was no relationship between the microorganism adhesion and acrylic resin type and/or composition . this was particularly compatible with our study ; when biofilm covered regions were evaluated when we observed that microorganisms showed minimal adhesion to ivocap plus . this result was not statistically significant when compared with qc but significant compared with lucitone 550 . this may be attributed to the special fabrication technique of this material ( injection molding ) which may form samples with smoother surface and lesser microporosities . the other reason for minimal adhesion may be the higher residual monomer release after using injection molding technique.23 it is known that the pmma monomer is toxic for living cells.23,24 during the experimental process , with the mentioned destructive effect , pmma monomer may have inhibited the survival and/or adhesion of microorganisms tested on denture base materials . on the contrary , the higher adhesion to lucitone 550 may be attributed to the reinforcing materials that poorly adhere to the polymer matrix forming microporosities that become suitable for microorganism accumulation.25 c. albicans was shown to have higher adhesion potential to denture base materials in most of the studies.10 conversely , in our study c. albicans - ivocap plus group exhibited the least adhesion potential when all the subgroups of quantitative biofilm values were examined . this result was compatible with the literature that p. aeruginosa and s. aureus have higher survival success over the plastic and/or metallic devices used in medicine.26 additionally the mean biofilm covered regions on denture base materials at sem display indicated the highest percentages for p. aeruginosa and s. aureus confirming the results of mean quantitative biofilm values of our study . within the limitations of this study it was shown that microorganisms diffused at least 52% of the denture base surface that can not be neglected in dental practice . however it was a remarkable result that this rate did not exceed 71% ( table 4 ) which may be attributed to the environmental or local conditions of this study or the floral equilibrium of microorganisms in certain circumstance . it can be reported that all the tested microorganisms had destructive effect over the structure and composition of the denture base materials .

purposethe aim of this study was to investigate the destructive effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms.materials and methodsthree different heat polymerized acrylic resins ( ivocap plus , lucitone 550 , qc 20 ) were used to prepare three different types of samples . type " a " samples with " v " type notch was used to measure the fracture strength , " b " type to evaluate the surfaces with scanning electron microscopy and " c " type for quantitative biofilm assay . development and calculation of biofilm covered surfaces on denture base materials were accomplished by sem and quantitative biofilm assay . according to normality assumptions anova or kruskal - wallis was selected for statistical analysis ( =0.05).resultssignificant differences were obtained among the adhesion potential of 6 different microorganisms and there were significant differences among their adhesion onto 3 different denture base materials . compared to the control groups after contamination with the microorganisms , the three point bending test values of denture base materials decreased significantly ( p<.05 ) ; microorganisms diffused at least 52% of the denture base surface . the highest median quantitative biofilm value within all the denture base materials was obtained with p. aeruginosa on lucitone 550 . the type of denture base material did not alter the diffusion potential of the microorganisms significantly ( p>.05).conclusionall the tested microorganisms had destructive effect over the structure and composition of the denture base materials .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC1522195

certain phenyl - substituted hydrocarbons of environmental concern have the potential to disrupt the endocrine system of animals , apparently in association with their estrogenic properties . competition with natural estrogens for the estrogen receptor is a possible mechanism by which such effects could occur . we used comparative molecular field analysis ( comfa ) , a three - dimensional quantitative structure - activity relationship ( qsar ) paradigm , to examine the underlying structural properties of ortho - chlorinated hydroxybiphenyl analogs known to bind to the estrogen receptor . the cross - validated and conventional statistical results indicate a high degree of internal predictability for the molecules included in the training data set . in addition to the phenolic ( a ) ring system , conformational restriction of the overall structure appears to play an important role in estrogen receptor binding affinity . hydrophobic character as assessed using hydropathic interaction fields also contributes in a positive way to binding affinity . the comfa - derived qsars may be useful in examining the estrogenic activity of a wider range of phenyl - substituted hydrocarbons of environmental concern.imagesfigure 1.figure 2 .

Images

PMC3446042

left ventricle non - compaction ( lvnc ) or persistence of spongy myocardium is a rare form of congenital cardiomyopathy which presents with cardiac failure , thromboembolic events , arrhythmia and sudden death . we report a family with two deceased children and two alive offspring diagnosed with left ventricular ( lv ) non - compaction . a 9-year - old boy and a 16-year - old girl of our reported family suddenly died following exercise and emotional provocation . both had a history of convulsion and syncope , with the latter taking anticonvulsant medication . the remaining two offsprings , one boy and one girl were diagnosed as having isolated lvnc . syncope and convulsion can be first manifestations of lvnc and heralding signs for sudden death in patients with lvnc . isolated left ventricular non - compaction ( lvnc ) is one of the rare genetic heart diseases recently known , and characterized by heterogeneous defects in endocardium accompanied by increased trabeculation due to abnormal embryogenesis of the normal myocardium . in this disease , the myocardium is composed of two layers : spongy or non - compacted and normal or compacted ones . the clinical manifestations include symptoms and signs of left ventricular failure ( common ) , those of right ventricle ( rare ) , ventricular arrhythmias , sudden death , mural thrombi and embolic events with cardiac origin . we report a family having the history of two offspring deaths , at the ages of 9 and 16 , with two live ones suffering from lvnc . we reported sudden death of a 9-year old boy with a history of two episodes of unprovoked convulsion and syncope in school and his 16-year old sister due to sudden death while she was getting back home after a feast . a 12year - old boy and a 7-year - old girl were referred to pediatric cardiology clinic for cardiac evaluation following occurrence of sudden death in their siblings . the first pregnancy of the mother ended in abortion at the age of two months of gestation , and the second offspring was a girl who died one week after birth following an operation to repair esophageal atresia . the first victim of suspected lvnc in this family was a nine year old boy with a history of two episodes of unprovoked convulsion and syncope in school . the first attack was in break time with spasm of neck and body , ended uneventfully . the next one was during exercise , with sudden fall but no urinary incontinence , eye deviation or sialorrhea . the electrocardiogram ( ecg ) , electroencephalography and lab tests showed no abnormalities at that time . the family lost their 16-year old daughter due to sudden death while she was getting back home after a feast . she also had experienced two seizure episodes associated with loss of consciousness , one at the age of 9 during exercise at school and the other at the age of 10 on vacation . both episodes had ended uneventfully , and brain ct scan with contrast demonstrated no pathologic finding . the 12-year - old boy had no history of seizure , syncope , easy fatigability , exertional dyspnea or hospitalization . on 2d transthorasic echocardiography , the parasternal short axis and four chamber views demonstrated increased left ventricular ( lv ) wall thickness accompanied by trabeculations and deep recesses that were indicative of lv non - compaction . four chamber and short axis views demonstrating the ratio of non - compacted to normally compacted segments the coronary arteries were normal and no pericardial effusion and intracavitary thrombi were detected . the 7-year - old girl also had no positive history of hospitalization , seizure , easy fatigue . her ecg was normal , however , her 2d echocardiograms were compatible with the diagnosis of lv non - compcation , based on the diagnostic criteria already reported by frischknecht et al , with increased lateral , inferior and apical wall thickness and deep recesses and trabeculations . the coronary arteries were normal and no pericardial effusion and intracavitary thrombi were detected ( fig . parasternal long axis view and m - mode echocardiography showing abnormal trabeculation and spongy pattern of left ventricle the parents were evaluated clinically and by echocardiography . this case report highlights the importance of syncope and seizure as heralding signs for sudden death in two deceased children of a family with two offspring having documented lv non - compaction . although syncope and sudden death have been previously reported in adults with lvnc but to date , as to best of our knowledge , sudden death with prior heralding sign of syncope and seizure has not been reported in children with lv non - compaction . in our reported family , both of the unexpected deaths occurred after previous episodes of uneventful seizure and syncope . considering the common embryologic origins of central nervous system and myocardium , occurrence of syncope and seizure in lvnc could be explained . lv non - compaction or persistence of spongy myocardium , first described in 1984 , is a rare form congenital cardiomyopathy with a prevalence of 0.05 - 0.24/year . the limitation of our report is lack of echocardiographic evidence for confirmation of diagnosis in the two deceased and not timely - referred and timely - diagnosed children . we suggest careful biannual follow up ( ecg , holter monitoring , echocardiography ) and anticoagulation therapy ( aspirin , dose : 3 - 5mg/ kg / daily ) for alive sibling . unexplained seizure and syncope in a child , particularly in a familial pattern , may warrant further echocardiographic evaluation for early diagnosis of left ventricular non - compaction to prevent catastrophic events . although the numbers of our patients are limited , but it seems that seizure and syncope can be considered as warning signs in those patients with lvnc who are at high risk for sudden death .

backgroundleft ventricle non - compaction ( lvnc ) or persistence of spongy myocardium is a rare form of congenital cardiomyopathy which presents with cardiac failure , thromboembolic events , arrhythmia and sudden death.case presentationwe report a family with two deceased children and two alive offspring diagnosed with left ventricular ( lv ) non - compaction . a 9-year - old boy and a 16-year - old girl of our reported family suddenly died following exercise and emotional provocation . both had a history of convulsion and syncope , with the latter taking anticonvulsant medication . following their demise , the other members of the family were evaluated by echocardiography . the remaining two offsprings , one boy and one girl were diagnosed as having isolated lvnc.conclusionsyncope and convulsion can be first manifestations of lvnc and heralding signs for sudden death in patients with lvnc . echocardiography can be helpful for early diagnosis .

Background Case Presentation Conclusion Introduction Case Presentation Discussion Conclusion

PMC4848083

cardiovascular diseases ( cvd ) is the leading cause of death globally , causing nearly 17.3 million deaths in 2008 , with the number expected to increase to 23.3 million by 2030 , . according to world health organization , over 80% of the world s deaths from cvds occur in low and middle income countries , as they have less access to effective and equitable healthcare services which respond to their needs , including early detection service . easy to use diagnostic and screening devices , amenable to field deployment for screening and triaging are important in such scenarios . carotid artery stiffness has been demonstrated to have strong association with increased intima - media thickness and even with severity of plaques in aorta . hence , non - invasive measurements of carotid artery stiffness , that can estimate cardiovascular health , are an attractive option for screening and early detection . however , state of the art systems for measuring arterial stiffness are highly expensive , and have a laborious operating procedure , and are hence not suited for field applications . ultrasound echo tracking systems , such as aloka e - tracking , artlab etc . , require expert sonologists to examine ultrasound data of the artery to perform stiffness measurements . there exists an unmet need for a practical , affordable , easy - to - use technology to non - invasively measure arterial stiffness in an automated manner , which could be used by general medical practitioners and health workers . such a non - expert operable device could overcome the skill barrier and also reduce the time taken for test , thereby making it suitable for large scale cardiovascular screening in addition to diagnosis . to address this need , we have developed artsens ( arterial stiffness evaluation for non - invasive screening ) , a device that overcomes the limitations of present systems effectively , while performing the measurements with accuracy and precision according to clinically accepted standards , . we had previously presented the concept of an image - free system for measurement of arterial stiffness and also demonstrated the accuracy and repeatability of measurements in controlled laboratory settings , . in this paper , we start with a technical overview of artsens device , and its operation , and present a detailed validation of the ability of the device to perform accurate measurements of arterial stiffness in - vivo , in clinical settings . the feasibility of using stiffness measurements performed in sitting posture is also investigated . from the list of major clinically accepted estimates of arterial stiffness enlisted in table 1 , it is evident that evaluation of the arterial stiffness requires an accurate measurement of arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } which is defined as the change in diameter of the artery from its mean position during each pulse , end - diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d})$ \end{document } , systolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{s}= d_{d}+\delta d ) $ \end{document } and the systolic and diastolic blood pressures ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p_{s}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { p}_{d})$ \end{document}.table 1clinically accepted measures of arterial stiffness.measure of arterial stiffnessequationpressure strain elasticity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}=\frac{d_{d } \times \delta p}{\delta d}$ \end{document}arterial compliance , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac= \frac{\pi(d_{s}^{2 } - d_{d}^{2})}{4\delta p}$ \end{document}stiffness index , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta=\frac{{\rm in}({p_{s}}/p_{d})}{(\delta d / d_{d})}$ \end{document } artsens utilizes a single element ultrasound transducer to interrogate the artery and obtain all relevant arterial dimensional measurements in an automated manner . the principle of image - free measurement and system architecture of artsens is presented in section ii . this section explains the hardware designed to excite the transducer , receive echo signals from the artery and digitize the signals , and also provides a detailed description of algorithms used to extract required parameters from the echo signals . section iv presents the results of the study , and illustrates the accuracy of artsens by comparison with state of the art echo tracking system for evaluating carotid artery stiffness . 1 . a single element ultrasound probe , operated in the pulse echo modality is used to investigate arterial dynamics . this probe , placed on the neck over the carotid artery , sends sharp pulses of high frequency ultrasound into the body that are reflected by the artery walls and other structures in the sound propagation path . intelligent signal processing and automated measurement algorithms automatically identify the arterial wall echoes , track wall motion and measure arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } and end - diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{d})$ \end{document } over multiple cycles . these are used to compute various measures of arterial stiffness such as stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) , $ \end{document } pressure strain elasticity(\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } and arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document}. figure 1.system architecture of artsens . the measurement requires no intervention from the operator other than positioning of the probe over the artery as illustrated in fig . the system utilizes a single element 5 mhz ultrasound transducer ( 10 mm diameter , hengxuannanshi , china ) . the ultrasound frequency of 5 mhz was selected based on the conflicting requirements of high resolution while ensuring required depth of penetration which is about 60 mm for measurements on carotid artery considering the attenuation coefficient of soft tissue as 0.7db / cm / mhz . the transducer s narrow half angle beam width of about 1.3 ensures that strong and distinct echoes will be obtained only when the transducer is kept normal to the artery . custom designed probe housings were developed for the single element transducer , to enable easy operation . the white probe was used in the current study . the custom analog front end electronics hardware developed for artsens 5 . the board has a low voltage ( + /15 v , 500 ma ) power supply section for the digital logic and analog front end sections , and a high voltage ( 100 v , 50 ma ) section used for ultrasound transducer excitation . digital pulses generated using a microcontroller are translated to high voltage levels and used to excite the transducer in the pulse echo modality . the reflected echo signals are then passed to an analog signal conditioning section consisting of a high pass filter of cut off frequency 2.56 mhz and a dual stage amplifier with a total gain of 40 db . a transmit receive switch is used to protect the amplifier section from the high voltage transmit pulses . figure 4.single element ultrasound probes designed for artsens . the conditioned analog signals are digitized at the rate of 100 ms / s using a ni usb 5133 high speed digitizer ( national instruments ) . the digitized signals , referred to as frames , are then given to subsequent signal processing algorithms for automated measurement . the digitized signals are transferred in real time into a laptop computer and processed using a signal processing virtual instrument , developed in labview using state machine architecture . the raw ultrasound data frames ( where each frame is defined as the echo obtained for a transducer excitation pulse ) are pre - processed to improve its signal to noise ratio ( snr ) . the frames are filtered using a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 4^{\mathrm { th}}$ \end{document } order zero phase butterworth band pass filter with upper cut off frequency of 8 mhz and lower cut off frequency of 1 mhz and then passed through a time gain compensation ( tgc ) block to compensate for the signal attenuation encountered as it passes through the soft tissue , as seen in fig . 7 . the arterial near ( proximal ) and far ( distal ) walls are identified based on the inherent out of phase motion of the wall echoes , as the artery contracts and relaxes during systole and diastole respectively . the phase of wall motion is detected by locating artery wall echo peaks in the first frame , and then using the next few ( 5 to 10 ) frames to identify the two consecutive artery wall echoes that move opposite to each other . figure 7.(a ) raw echo signal ( frame ) received from the artery ( b ) signal after pre - processing . notice significant improvement in snr , elimination of transducer ringing artefacts and compensation for echo attenuation to improve amplitude of farther echoes . ( a ) raw echo signal ( frame ) received from the artery ( b ) signal after pre - processing . notice significant improvement in snr , elimination of transducer ringing artefacts and compensation for echo attenuation to improve amplitude of farther echoes . once the artery wall locations are identified , the system goes to tracking state where the movements of near wall and far wall echoes are tracked for a few cardiac cycles as they move out of phase w.r.t each other in a quasi - periodic manner . correlation based algorithm is used to find the shift in the echo location from that of the previous frame . arterial distension \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d(t)$ \end{document } is calculated from each frame as the sum of relative near and far wall shifts w.r.t . the carotid artery lumen diameter is calculated as the distance between the intima layers on both walls , corresponding to the trailing edge of the near wall echo and leading edge of the far wall echo . a smoothened hilbert envelope of the region of interest ( roi ) a wall motion negative correlation check running simultaneously along with tracking ensures that tracking is done on near and far walls without fail , . this is ensured by checking that the near and far wall echoes are moving opposite to each other , i.e. , the absolute values of the near and far wall motion patterns have a negative correlation . if the condition is not satisfied , the system goes back to wall identification state . the distension and diameter values as obtained from the tracking stage are recorded and stored in a buffer of size \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b$ \end{document } , calculated as the number of points required to store distension of around two cardiac cycles . once the buffer is filled , the valley locations are found out to obtain distension cycles , from which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d$ \end{document } is calculated . the diameter values corresponding to these valley points in the distension array are identified as end diastolic diameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document } . gaussian error elimination is performed to eliminate any random data which could have been included in the measurement . the arterial stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } , arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } and pressure strain elasticity ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } are computed using valley to peak distension \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d$ \end{document } and end diastolic diameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document } , measured by artsens , and systolic and diastolic pressure ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p_{s } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { p}_{d})$ \end{document } , separately measured and entered by the operator prior to artsens measurement . a snapshot of the various versions of the artsens device that were developed is illustrated in fig . the hardware for data acquisition and subsequent signal processing modules were embedded together in the device , making it easier for the operator to use . figure 8.technology evolution of artsens ( a ) desktop prototype ( b ) tablet version ( c ) rugged version for field deployment ( d ) handheld version under development . technology evolution of artsens ( a ) desktop prototype ( b ) tablet version ( c ) rugged version for field deployment ( d ) handheld version under development . the performance of the signal processing algorithms , the repeatability and reproducibility of the instrument and artsens usability in controlled laboratory settings have already been validated and reported . here we present an extensive validation of artsens in clinical setting . the objectives of this study are the following . ( a)to establish feasibility of artsens measurements in clinical setting on large number of subjects(b)to verify the ability of artsens to provide accurate estimates of stiffness with the subject in sitting posture.(c)to establish the measurement accuracy of artsens in comparison with state of art image based ultrasound echo tracking system.the aloka \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \alpha 10 $ \end{document } prosound e - tracking system was taken as the reference equipment for evaluation of carotid artery stiffness . to establish feasibility of artsens measurements in clinical setting on large number of subjects to verify the ability of artsens to provide accurate estimates of stiffness with the subject in sitting posture . to establish the measurement accuracy of artsens in comparison with state of art image based ultrasound echo tracking system . the procedure of arterial stiffness measurements and the overall study protocol approved by the institutional review board are explained below and are shown in fig . figure 9.artsens validation study protocol ( a ) informed consent ( b ) blood pressure ( c ) blood sampling ( d ) anthropometry ( e ) artsens measurement ( f ) aloka e - tracking measurement . artsens validation study protocol ( a ) informed consent ( b ) blood pressure ( c ) blood sampling ( d ) anthropometry ( e ) artsens measurement ( f ) aloka e - tracking measurement . male and female subjects , above the age of 18 years , with no documented history of cardiovascular or peripheral vascular disease were included . all subjects were informed of the study objectives , backgrounds and protocol before data collection and informed consents were obtained from them . each subject was registered to the study with a unique identifier , and the personal details were filled in . subjects were given a light breakfast and allowed to relax for at least 15 minutes before proceeding with measurements . blood pressure at the brachial artery was measured using a sphygmomanometer in both sitting and supine position . the stiffness of the carotid artery of each subject was evaluated both using artsens , and also using an ultrasound echo tracking system ( aloka \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \alpha 10 $ \end{document } prosound e - tracking system ) . the left carotid artery location was identified by palpation and the ultrasound probe was placed nearly 2.5 cm below the carotid bifurcation . the angle of the probe was adjusted to get strong echoes from the near and far wall of the artery . artsens was configured to give the average of five measurement results where each measurement comprised of five continuous cycles of distension . 10 shows a screenshot of artsens graphical user interface ( gui ) indicating all measurements . screenshot of artsens gui indicating measurements . for measurement using the e - tracking system , 3-lead electrocardiogram ( ecg ) was placed on the subject s body . the near and far walls of the artery were manually identified on the b - mode image and the wall motion was tracked by the aloka \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \alpha 10 $ \end{document } prosound e - tracking system . when a steady waveform was obtained for 45 cycles , image is frozen to acquire the data which were accumulated over the period to start arterial stiffness analysis . in the analysis window , measurements were taken in both sitting and supine postures with corresponding systolic and diastolic pressures entered . 10 shows a screenshot of aloka measurement and post analysis screen . the agreement between the arterial stiffness readings given by artsens to those given by the ultrasound imaging system least square regression models , performing linear fit for stiffness estimates from imaging systems and those from artsens were obtained under the assumption that random error is associated with only artsens results and that the results from imaging system are true values without random errors . further , a bland altman analysis was performed to examine the degree of variation between the two readings . the limits of agreement were defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \pm 2sd$ \end{document } . two artsens measurements were performed on a few ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \text{n}=33 $ \end{document } ) randomly selected subjects . in such cases , the repeatability of measurements performed by artsens was evaluated by computing coefficient of variability ( repeatability ) as the ratio of the standard deviation of the differences between the two artsens measurements to the average of the means . a trend analysis was also performed to investigate the ability of artsens to detect age - related changes in arterial stiffness . artsens was used to measure the carotid artery stiffness of all these subjects . typical time taken for a measurement was less than 5 minutes . this demonstrated the ability of artsens to perform in - vivo measurements of arterial stiffness . in very few cases , the measurement took more time , as it was difficult to position the probe at the correct location to get stable distension waveforms . of the total 125 subjects , a few subjects on whom it was difficult to perform the measurements on the exact same locations due to change in posture or probe dimensions illustrated less repeatability in measurements . such suspected cases of measurement error , in which there was significant variation in the stiffness values recorded by the two instruments ( artsens and aloka e - tracking ) were eliminated as outliers . after eliminating a few suspected cases of data entry error and cases with missing data , a total of 111 subjects were selected for analysis . a summary of the linear regression analysis is provided in table 2 and table 3 . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d , d_{d},d_{s},\beta , ac$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } were found to have a strong positive correlation with the corresponding values obtained from the imaging system , measured in sitting posture , as indicated in table 2 . arterial distension measured using artsens was found to have a correlation coefficient ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r$ \end{document } ) of 0.82 with the value given by the imaging system . the correlation of both end - diastolic diameter and systolic diameter were observed to be 0.5 . this slightly lower values of correlation coefficient are expected in direct comparison of dimensional measurements , as these measurements were performed sequentially , using two different instruments and hence perfect matching of the site of measurement is not possible due to practical limitation of positioning the larger imaging probe on the exact same location as that of the smaller artsens probe . moreover , physiological variations in arterial distension with time , associated with slight changes in the instantaneous blood pressure values , also affect this result.table 2linear regression analysis of arterial stiffness measurements by artsens with those given by imaging system in sitting posture.parameterimaging system in sitting posturerrslopeinterceptcoeff.p value\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta_{d}$ \end{document}0.820.680.703 < 0.0010.076\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document}0.510.260.765<0.0010.364\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{s}$ \end{document}0.490.240.737<0.0010.592\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta$ \end{document}0.910.830.991<0.0010.009\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document}0.940.891.035 < 0.001 2.699\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document}0.770.590.579<c0.0010.112table 3linear regression analysis of arterial stiffness measurements by artsens with those given by imaging system in supine posture.parameterimaging system in supine posturerrslopeinterceptcoeff.p value\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta_{d}$ \end{document}0.810.650.641<0.0010.102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document}0.480.230.753<0.0010.347\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{s}$ \end{document}0.460.220.703<0.0010.724\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta$ \end{document}0.810.660.845<0.0010.592\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document}0.890.790.938<0.0013.402\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document}0.670.450.411<0.0010.253 however , the material property of the vessel wall is not expected to show such significant variations , especially under controlled settings as was achieved in the study . this is also illustrated by the high correlation coefficient value of 0.9 of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } stiffness index value measured by artsens with respect to that given by the imaging system , as shown in fig . the pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured by artsens also shows strong correlation with those obtained using the imaging system , with a correlation coefficient of 0.94 . the correlation of arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } , was lower ( 0.77 ) , owing to its direct dependence on dimensional parameters . figure 11.screenshot of aloka e - tracking ( a ) b - mode indicating cursor locations ( b ) post analysis screen . figure 12.comparison of arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . figure 13.comparison of end - diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{d})$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . figure 14.comparison of systolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{s})$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . figure 15.comparison of stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . screenshot of aloka e - tracking ( a ) b - mode indicating cursor locations ( b ) post analysis screen . comparison of arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . comparison of end - diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{d})$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . comparison of systolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{s})$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . comparison of stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . strong correlation was observed even when the artsens stiffness estimates measured on subjects in sitting posture were compared with those made using the imaging system on subjects in supine posture . it may be observed from table 3 , that the arterial stiffness estimates ( both \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured by artsens showed strong correlation , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r$ \end{document } values of 0.81 and 0.89 respectively . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r$ \end{document } values of arterial distension and end - diastolic diameter were 0.81 and 0.48 respectively . it may be remembered that there is a difference in the arterial pressures , both systolic and diastolic , when the subject moves from sitting to supine posture , and this will cause corresponding differences in the arterial distension and end - diastolic diameter values . however , the strong observed correlation of arterial stiffness estimates validates the use of artsens to evaluate stiffness in the sitting posture . this is relevant , as a supine measurement may not be often possible in field settings , when artsens is deployed for screening . to evaluate the degree of agreement between stiffness measurements provided by artsens and the imaging system , bland - altman plots with limits of agreement defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \pm 2sd$ \end{document } , were created for all the directly measured parameters and the stiffness estimates . figure 16.comparison of pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } ) measurements from artsens with those obtained from imaging system in sitting posture . figure 17.comparison of arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . figure 18.bland altman plot of end diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d})$ \end{document } measured from artsens ( dd\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( dd\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. figure 19.bland altman plot of arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } measured from artsens ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta \text{d}_{\mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta \text{d}_{\mathrm { image(sit)}})$ \end{document}. figure 21.bland altman plot of pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured from artsens ( ep\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( ep\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. figure 22.bland altman plot of arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measured from artsens ( ac\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( ac\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. figure 23.distribution of the differences between beta measurements given by artsens and imaging system in sitting posture . figure 24.distribution of the differences between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } measurements given by artsens and imaging system in sitting posture . figure 25.distribution of the differences between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document } measurements given by artsens and imaging system in sitting posture . comparison of pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } ) measurements from artsens with those obtained from imaging system in sitting posture . comparison of arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measurements from artsens with those obtained from imaging system in sitting posture . bland altman plot of end diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d})$ \end{document } measured from artsens ( dd\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( dd\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. bland altman plot of arterial distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } measured from artsens ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta \text{d}_{\mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta \text{d}_{\mathrm { image(sit)}})$ \end{document}. bland altman plot of pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured from artsens ( ep\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( ep\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. bland altman plot of arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measured from artsens ( ac\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { artsens}})$ \end{document } and imaging system in sitting posture ( ac\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ _ { \mathrm { image(sit)}})$ \end{document}. distribution of the differences between beta measurements given by artsens and imaging system in sitting posture . distribution of the differences between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } measurements given by artsens and imaging system in sitting posture . distribution of the differences between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document } measurements given by artsens and imaging system in sitting posture . it may be observed that there is a high degree of agreement between artsens readings and stiffness values provided by the imaging system . the mean of differences , known as the bias , for directly measured artery dimensional parameters is slightly higher than those reported in literature using image based systems , owing to reasons explained earlier in section iv.c1 . the bias is close to zero for the arterial stiffness results \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p}$ \end{document } , showing that artsens is not expected to underestimate or overestimate the stiffness values . it can be also be inferred from the plots that the bias and the limits of agreement are very low when compared to the absolute values of the parameters measured . the variability in measurements exhibits even distribution in all plots , irrespective of the magnitude of the value measured . it may be noted that the analysis also takes into account the minor differences introduced owing to minor changes in probe location and subject physiology as the measurements are performed sequentially.table 4results of bland altman analysis of arterial stiffness measurements by artsens with those given by imaging system in sitting posture.parameter measuredmean ( bias)standard deviation\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta_{d}$ \end{document } ( mm)0.090.096\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document } ( mm)1.060.800\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta$ \end{document}0.060.976\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{{\rm p}}$ \end{document } ( kpa)0.5413.180\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac ( { \mathrm { mm}}^{2}/{\mathrm { kpa}})$ \end{document}0.310.237 mean difference between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } measurements given by imaging system and artsens was found to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 0.06 \pm 1.04 $ \end{document } , indicating no bias in measurement . bland altman plot indicates the difference to be randomly distributed , with 99% of the measurements within 2sd as shown in fig . the difference between the stiffness index measurements , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } is less than 2 , which is within clinically acceptable limits of variation expected in stiffness for estimation of local arterial stiffness for screening . the noticeable bias in the lumen diameter measurement may be attributed to the fact that the automated algorithm in artsens measures the inner lumen diameter , whereas it is normally difficult to locate lumen interface on ultrasound imaging system and hence the measurement by imaging system is expected to overestimate the diameter . figure 20.bland altman plot of arterial stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } measured from artsens ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { \mathrm { artsens}}$ \end{document } ) and imaging system in sitting posture ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { \mathrm { image(sit)}})$ \end{document}. bland altman plot of arterial stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } measured from artsens ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { \mathrm { artsens}}$ \end{document } ) and imaging system in sitting posture ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { \mathrm { image(sit)}})$ \end{document}. it may be observed that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document } measured by artsens express slightly higher bias and limits of agreement when compared to the absolute value of measurement . also , as already mentioned , the correlation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document } between the two methods was slightly lower when compared to other stiffness readings . hence it may be concluded that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac$ \end{document } is not the best measure of arterial stiffness provided by artsens . the stiffness index , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta , $ \end{document } and the elastic modulus \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { e}_{p } $ \end{document } , are the best estimates of stiffness provided by artsens . two artsens measurements were performed on a few randomly selected subjects ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \text{n}=33 $ \end{document } ) . the repeatability and reproducibility of artsens in a controlled environment has already been reported . the repeatability of the arterial stiffness index value ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } , pressure strain elasticity ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } , arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } , distension ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta d)$ \end{document } and end diastolic diameter ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { d}_{d})$ \end{document } were analysed to investigate the performance of the device in field . the coefficient of variability ( repeatability ) computed as the ratio of the standard deviation of the differences between the two artsens measurements to the average of the means was calculated from the data . the calculated coefficients of variability shown in table 5 are comparable to the previously reported results on carotid stiffness measurements performed using artsens and other ultrasound imaging systems , , , , . this indicates the ability of the instrument to give precise measurements of stiffness irrespective of the field conditions.table 5coefficients of repeatability for artsens measurements.parameter measuredcoefficient of repeatability ( variability ) in % \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta_{d}$ \end{document } ( mm)8.45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{d}$ \end{document } ( mm)10.52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta$ \end{document}15.31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{{\mathrm { p}}}$ \end{document } ( kpa)13.99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac ( { \mathrm { mm}}^{2}/{\mathrm { kpa}})$ \end{document}16.59 to investigate the ability of artsens to detect subtle changes in arterial stiffness , the data was analyzed to examine if artsens could pick up trends in arterial stiffness associated with age . to eliminate the influence of other factors that may affect stiffness , a total of 102 subjects were included in this age - trend analysis . to study the trend in variation of each parameter with age , the mean and standard deviation of stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } , pressure strain elastic modulus ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } and arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } in sitting posture were calculated for different age bins . as the age of the subjects recruited for the study was slightly skewed towards the younger age group , a classification based on age quartiles will not properly reflect the age related changes in arterial stiffness . hence age bins selected for analysis were chosen to ensure nearly uniform distribution of the overall subject pool among the various age bins . a strong positive trend between arterial stiffness measures and age could be inferred from these plots . this illustrates the ability of artsens to detect subtle changes in arterial stiffness that occurs due to ageing , thereby indicating potential use in vascular screening and diagnosis . figure 26.age related increase in stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } , measured using artsens . figure 27.age related increase in pressure strain elastic modules ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured using artsens . figure 28.age related decrease in arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measured using artsens . age related increase in stiffness index ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ) $ \end{document } , measured using artsens . age related increase in pressure strain elastic modules ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{p})$ \end{document } measured using artsens . age related decrease in arterial compliance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ac)$ \end{document } measured using artsens . unlike the commercially available image - based systems , artsens has completely automated measurement and is the only image free system available for direct measurement of arterial dimensions to evaluate stiffness . because of its image free technology , an expert ultrasonographer is not required for performing the measurement . the time taken for measurement ( typically 5 minutes ) is much less than that required for image - based systems , and the measurement procedure is simple and less laborious . the portable nature of the instrument and the ability of the intelligent algorithms to quickly perform the measurement without any operator input make it very amenable for field deployment . artsens measurements could not be performed on few volunteers , where the artery was very superficial due to which the near wall could not be identified clearly . the hardware of the device is being improved to reduce the apparent blind spot close to the probe surface , by increasing the transducer damping . in all subjects whose data were eliminated as outliers in this study , the quality of the echo signal was found to be very low , as the artery walls could not be distinguished clearly . the enforcement of a stricter protocol of probe positioning and angulation to ensure high quality echo signals during measurement will eliminate such cases . an automated signal quality evaluation algorithm is being developed to avoid this in screening scenarios . a novel image free instrument for non - invasive evaluation of arterial stiffness , called artsens was presented . artsens uses a single element ultrasound probe , and is an affordable , compact , user friendly device for measurement of arterial stiffness . the hardware and software architecture of the system was explained . the ability of artsens to easily perform in - vivo measurement of carotid artery stiffness was verified by the study conducted on 125 subjects . the accuracy of the measurements provided by artsens was verified by comparing with a reference , state of art ultrasound imaging system . the stiffness estimates provided by artsens strongly correlated with those obtained using the imaging system . bland altman analysis demonstrated significant degree of agreement between artsens measurements and imaging system measurements . the results show that artsens is at par with state of art technology for measurement of arterial stiffness . measurements of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } stiffness index , considered to be better estimate , as it characterizes stiffness independent on distending pressure , was found to be repeatable and accurate in artsens . artsens device demonstrated enough sensitivity to detect age related trends in arterial stiffness , thereby indicating potential for use in large scale screening studies . artsens allows quick and easy measurement of arterial stiffness even in sitting posture , and its automated measurement algorithms allow it to be used by any operator with minimal training . artsens can thus be deployed in primary or secondary care facilities , and even in field scenarios , thereby making vascular stiffness measurements feasible in large scale epidemiological studies as well as for early detection and screening . the relative significance of various measures of stiffness , and normative values in indian population would be explored in future epidemiological studies using artsens .

vascular stiffness is an indicator of cardiovascular health , with carotid artery stiffness having established correlation to coronary heart disease and utility in cardiovascular diagnosis and screening . state of art equipment for stiffness evaluation are expensive , require expertise to operate and not amenable for field deployment . in this context , we developed arterial stiffness evaluation for noninvasive screening ( artsens ) , a device for image free , noninvasive , automated evaluation of vascular stiffness amenable for field use . artsens has a frugal hardware design , utilizing a single ultrasound transducer to interrogate the carotid artery , integrated with robust algorithms that extract arterial dimensions and compute clinically accepted measures of arterial stiffness . the ability of artsens to measure vascular stiffness in vivo was validated by performing measurements on 125 subjects . the accuracy of results was verified with the state - of - the - art ultrasound imaging - based echo - tracking system . the relation between arterial stiffness measurements performed in sitting posture for artsens measurement and sitting / supine postures for imaging system was also investigated to examine feasibility of performing artsens measurements in the sitting posture for field deployment . this paper verified the feasibility of the novel artsens device in performing accurate in vivo measurements of arterial stiffness . as a portable device that performs automated measurement of carotid artery stiffness with minimal operator input , artsens has strong potential for use in large - scale screening .

Introduction Artsens: System Architecture Validation of Artsens Results and Discussion Conclusion

PMC4640439

in the development and patterning of embryonic and adult tissues , secreted signaling molecules of the wnt , fgf , hedgehog , and transforming growth factor ( tgf- ) families can act as morphogens to activate different transcriptional programs along a signaling gradient ( perrimon et al . , 2012 ) . ideas of how morphogens impart spatial information have been dominated by the assumption that these molecules form concentration gradients by diffusion , inducing dose - dependent responses in the receiving field of cells . however , it is becoming increasingly clear that for some ligands , for example , hedgehog , wnt , and fgf , other mechanisms , such as short - range signaling activation , transcriptional feedback , and cellular rearrangements , underlie morphogen function ( alexandre et al . regulation of nodal signaling in the zebrafish embryo has long served as a paradigm for understanding how morphogens pattern tissues ( schier , 2009 ) . nodals are secreted ligands that belong to the tgf- superfamily of growth and differentiation factors . during vertebrate development , nodal is required for stem cell maintenance , specification of mesoderm and endoderm ( mesendoderm ) , and establishment of left - right asymmetry ( shen , 2007 ) . recent work has also suggested that nodal signaling is reactivated in advanced cancers , where it may be important for self - renewal of cancer stem cells ( wakefield and hill , 2013 ) . nodal ligands signal through serine / threonine kinase receptor complexes comprising two type i receptors ( acvr1ba [ taram - a ] ) , two type ii receptors ( acvr2a / b ) , and the co - receptor tdgf1 ( cripto / oep ) ( schier , 2009 , shen , 2007 ) . ligand binding activates the receptors , after which the type i receptor phosphorylates the intracellular signal transducers smad2 and smad3 , which then bind smad4 ( massagu , 2012 ) . these smad2/3-smad4 complexes accumulate in the nucleus , where , together with transcription factors such as foxh1 , mixer , and oct4 , they regulate gene transcription ( gaarenstroom and hill , 2014 ) . at zebrafish blastula stages , two nodal - related ligands , ndr1 ( squint ) and ndr2 ( cyclops ) , specify mesendoderm in marginal cells around the circumference of the embryo by inducing a smad2-smad4-foxh1-dependent transcriptional program ( feldman et al . , 1998 , gritsman et al . , 1999 ) . ndr1/2 are thought to form a signaling gradient by diffusion , extending up to about ten cell tiers from the margin ( dubrulle et al . , 2015 , indeed , expression of presumed long - range nodal target genes such as ta ( ntla ) and fscn1a suggests low - level signaling up to ten cell tiers from the margin ( bennett et al . , 2007 ) . this appears supported by bimolecular fluorescent complementation experiments ( harvey and smith , 2009 ) . however , other nodal target genes are expressed in up to five to six cell tiers from the margin , which coincides with nuclear accumulation of smad2-gfp fusion protein ( dubrulle et al . , 2015 ) . importantly , other signaling pathways , such as bmp , wnt , and fgf , are also active at the margin , which can potentially co - regulate nodal target genes and thus contribute to their expression domains . formation of the nodal signaling domain at the correct time and of appropriate dimensions is thought to be controlled by a reaction - diffusion system ( meinhardt , 2009 , schier , 2009 ) . this model requires positive and negative feedback , which is provided by nodal - induced expression of both the ligands ndr1/2 and the antagonists lefty1 ( lft1 ) and lefty2 ( lft2 ) ( chen and shen , 2004 , cheng et al . , 2004 ) . besides these feedback mechanisms , the model requires lft1/2 to be more diffusible than ndr1/2 ( mller et al . , 2012 , schier and talbot , 2005 ) . these conditions are thought to allow ndr1/2 to activate signaling at the margin , whereas lft1/2 proteins would inhibit signaling in more distal cells . overexpression studies have shown that ndr1/2 and lft1/2 can differentially diffuse and that ndr1 , but not ndr2 , can diffuse over a distance to activate signaling ( chen and schier , 2001 , chen and schier , 2002 , mller et al . , 2012 ) . however , the importance of diffusion of endogenous ndr1/2 remains unclear , as mesendoderm can develop normally in zygotic ndr1 mutants ( dougan et al . , 2003 , feldman et al . , 1998 , lim et al . , in addition to the negative feedback provided by lft1/2 , nodal signaling is regulated by the mir-430/427/302 family of micrornas ( mirnas ) ( bassett et al . , 2014 , choi et al at blastula stages , the mir-430 family is the most abundant family of mirnas in the zebrafish . importantly , mir-430 regulates ndr1 , lft1 , and lft2 , but not ndr2 , and this is thought to dampen nodal signaling ( choi et al . , 2007 ) . however , to what extent mir-430s contribute to the formation of the nodal signaling domain is unknown . to develop a specific readout for endogenous nodal signaling , avoiding overexpression of any pathway components , we generated a transgenic zebrafish nodal reporter line . using this line combined with immunofluorescence for phosphorylated smad2 ( p - smad2 ) , we show that nodal signals exclusively in cells that express ndr1/2 , up to five to six cell tiers from the margin . this prompted us to revisit the mechanism underlying the formation of the nodal signaling domain . our data do not support the reaction - diffusion model , but instead , we propose that nodal activates signaling in a temporal window that is defined by a mir-430-mediated delay of lft1/2 translation . in this way , temporal information is converted into spatial information in the developing embryo . the range of activity of the nodal signaling pathway in the blastula margin has mainly been inferred from the expression of endogenous target genes , such as fscn1a and in particular ta ( ntla ) ( bennett et al . , 2007 , gritsman et al . , 1999 ) . however , in addition to nodal , fgf signaling is also known to regulate ta expression ( griffin et al . , 1995 , rodaway et al . , 1999 , schier and talbot , 2005 ) . genes encoding fgf ligands , such as fgf3 and fgf8a , are expressed in the margin ( figure s1a ) and are known nodal targets ( mathieu et al . , 2004 ) , suggesting that fgf signaling at the margin of blastula - stage embryos is downstream of nodal signaling ( rodaway et al . , 1999 ) . this is clearly demonstrated by treating embryos with the nodal inhibitor sb-505124 ( hagos and dougan , 2007 ) , which results in a near complete loss of phosphorylated erk ( p - erk ) , a readout for fgf pathway activity ( dorey and amaya , 2010 ) ( figures 1a and 1b ) . given that both nodal and fgf signaling are active at the margin , we examined to what extent fgf signaling regulates endogenous nodal target genes , focusing on the expression of ta and fscn1a as examples of long - range genes and lft1 and lft2 as examples of short - range target genes ( bennett et al . to inhibit fgf signaling , wild - type ( wt ) embryos were treated with the fgf receptor ( fgfr ) inhibitor su-5402 ( mohammadi et al . , 1997 ) or were injected with mrna encoding a dominant - negative fgfr ( dnfgfr ) ( amaya et al . , 1991 ) both treatments resulted in a reduction in the size of the expression domains of ta and fscn1a in the margin of 40%50% epiboly embryos , but not of lft1 or lft2 ( figure 1d ) . similarly , morpholinos ( mos ) against fgf3 and fgf8a resulted in a reduction of ta expression , but not of lft1 ( figure s1b ) . qpcr on su-5402-treated 50% epiboly embryos confirmed the whole - mount in situ hybridization ( wish ) results ( figure 1e ) , and as expected , inhibition of nodal signaling by sb-505124 led to reduction in expression of all four genes ( figure 1e ) . importantly , fgfr inhibition had no effect on c - terminal phosphorylation of smad2 ( p - smad2 ) or overall smad2 levels , demonstrating that nodal signaling is not affected by fgf signaling inhibition ( figure 1f ) . to quantitate the effect of inhibiting fgf signaling on the ta expression domain , we performed serial sectioning on ta - stained embryos at 40% epiboly . in control embryos , ta is expressed in an average of about 10 cell tiers from the margin , whereas expression was reduced to six cell tiers in su-5402-treated embryos ( figures 1 g and 1h ) . this indicated that ta expression beyond six cell tiers was due to fgf signaling and not directly dependent on nodal . in support of this idea , injection of increasing doses of fgf8a mrna into a maternal zygotic ( mz ) tdgf1 background confirmed that nodal signaling is not required for induction of ta by fgf , excluding a requirement for synergism between nodal and fgf signaling for ta expression beyond six cell tiers ( figure s1c ) . this was further confirmed by the observation that inhibition of nodal signaling from the 16-cell stage resulted in the loss of both ta and lft1 expression , but when nodal signaling was inhibited from dome stage , only lft1 expression was severely reduced , whereas the expression of ta was unaffected ( figure s1d ) . thus ta expression is not dependent on nodal activity after fgf signaling has been initiated . together these data demonstrate that fgf signaling regulates presumed long - range endogenous nodal target genes beyond six cell tiers . to exclusively monitor nodal signaling without inputs from other pathways an egfp reporter gene under the control of three foxh1 and smad binding sites , termed activin response elements ( ares ) ( germain et al . , 2000 ) , was inserted into the zebrafish genome using tol2-mediated transgenesis ( tg[are : egfp ] ) ( figure 2a ) . we chose this reporter , which we have extensively characterized in a number of cell culture and developmental contexts ( germain et al . , 2000 , inman and hill , 2002 , randall et al . , 2004 ) , because foxh1 is the primary transcription factor required for immediate early nodal target gene expression ( pogoda et al . , in addition , smad2 is the predominant receptor - regulated smad during blastula stages ( figure s2a ) , and indeed mz deletion of smad2 results in a phenotype identical to mztdgf1 embryos ( dubrulle et al . , 2015 ) . in four independent tg(are : egfp ) lines , wish for egfp mrna revealed that the reporter was activated in identical domains , excluding any effects caused by different integration sites of the transgene ( figures 2a and s2b ) . the egfp expression domains correspond to the expression domains of nodal ligands ( schier , 2009 ) and include the embryonic margin at blastula stages , axial mesoderm during gastrulation , and the left lateral plate mesoderm during somitogenesis ( figures 2a and s3 ) . incubation of developing tg(are : egfp ) embryos with sb-505124 resulted in a strong reduction of egfp staining ( figure 2b ) . this was also the case when foxh1 mos were injected or when the tg(are : egfp ) embryos were bred into a tdgf1 background ( figure 2b ) . the tg(are : egfp ) reporter is therefore specific for smad2-smad4-foxh1-mediated nodal signaling during blastula stages . to confirm the inducibility of the reporter gene , we exposed dissociated cells from tg(are : egfp ) blastula - stage embryos to increasing concentrations of recombinant nodal and measured gene expression by qpcr . egfp expression was induced at identical nodal concentrations compared with ta , suggesting equal sensitivity of the reporter compared with this target gene ( figure 2c ) . historically the sensitivity of nodal target genes , such as ta and noto ( flh ) , has been assessed by ectopically expressing nodal ligands in the animal pole of blastula - stage embryos and using wish to assay gene expression ( chen and schier , 2001 ) . using nodal - coated beads in such an assay , we found that the reporter was induced in a domain of similar size to that of ta and noto ( figure 2d ) . finally , we found no differences in the expression of downstream nodal genes in 40%50% epiboly embryos by qpcr and wish when we compared wt and tg(are : egfp ) embryos , demonstrating that the introduction of the transgene had no impact on nodal signaling ( figures s2c s2e ) . in conclusion , the tg(are : egfp ) zebrafish line is a specific and sensitive biosensor for early nodal signaling . we next used the tg(are : egfp ) reporter line to determine how nodal signaling is initiated in the embryo . at the 8-cell and 1,000-cell stages , no egfp mrna could be detected ( figure 2e ) , indicating that egfp mrna is not maternally contributed . at sphere stage , however , signaling cells are detected as a localized cluster of egfp - positive cells ( figure 2f ) . from 30% epiboly until the onset of gastrulation , nodal signaling is detected in the entire margin , with a shallow staining gradient running from dorsal to ventral ( figure 2f ) . as expected , the overall expression of egfp correlated well with increasing levels of p - smad2 ( figure 2 g ) . we compared the spatial and temporal activation of nodal signaling in tg(are : egfp ) embryos with the expression of core components of the pathway ( figure s3 ) . the genes encoding the receptor acvr1ba , the co - receptor tdgf1 , and transcription factors smad2 and foxh1 are ubiquitously expressed during blastula stages . the activation of signaling therefore depends exclusively on ligand expression ( figure s3 ) . the discrete signaling domain at sphere stage likely corresponds to the future dorsal side of the embryo , given the known dorsal localization of maternal ndr1 mrna ( figures s3 and s4a ) ( gore et al . we could show that ndr1 transcripts are polyadenylated before the maternal - to - zygotic transition ( figure s4b ) , suggesting that maternal ndr1 is translated and may signal during blastula stages . this was demonstrated by knocking down the mix - like transcription factor mxtx2 , which is required for zygotic ndr1 and ndr2 expression in the yolk syncytial layer ( ysl ) ( fan et al . , 2007 , hong et al . , 2011 , xu et al . , 2012 ) . tg(are : egfp ) mxtx2 morphants retained only a small , dorsal domain that expressed ndr1 , ndr2 , and egfp ( figure s4c ) . loss of the ventrolateral expression of ndr1/2 in the blastoderm in mxtx2 morphants suggested that this ndr1/2 expression was initiated by nodal ligands secreted by the ysl and resulted from the ability of ndr1/2 to induce their own expression . indeed , embryos incubated with the nodal inhibitor sb-505124 from the 32-cell stage exhibited complete loss of expression of ndr1/2 in the blastoderm of 40% epiboly embryos , whereas expression of ndr1/2 in the ysl was not affected ( figure s4d ) ( fan et al . , 2007 ) . thus , maternally provided ndr1 activates nodal signaling in dorsal - most embryonic cells before the initiation of signaling in the entire margin by ndr1/2 synthesized in the ysl ( figure s4e ) . the tg(are : egfp ) zebrafish line provides an ideal tool to investigate the dimensions of the nodal signaling domain at the margin . to initially investigate whether there was any nodal signaling beyond the ligand - expressing domain , we performed double fluorescent wish for ndr2 and egfp in 30% epiboly embryos , when the nodal signaling domain is expanding ( dubrulle et al . , 2015 , harvey and smith , 2009 ) . although individual signaling cells were occasionally observed directly adjacent to ndr2-expressing cells , no egfp - positive cells were detected further beyond the ligand expression domain ( figure s5a ) . to determine the extent of nodal signaling more rigorously , 40%50% epiboly tg(are : egfp ) embryos were stained for egfp , ndr1 , ndr2 , lft1 , and lft2 expression , sectioned , and quantitated ( figures 3a and 3b ) . the expression of egfp , ndr1 , ndr2 , and lft1 was limited to an average of about five cell tiers , whereas lft2 expression was detected in only two to three cell tiers from the margin . to confirm that these findings were not due to a lack of sensitivity of the wish , we analyzed the expression of sox3 , which is repressed by nodal signaling in the margin ( bennett et al . significant repression was seen in up to six cell tiers from the margin ( figure 3a ) . importantly , staining for all induced nodal target genes , including egfp , was consistently stronger in a cluster of cells directly proximal to the ysl , where signaling originates ( figure 3a ) . to corroborate the data obtained from the sections , we used a direct readout of the nodal pathway , fluorescent immunostaining of whole - mount 50% epiboly embryos with an antibody against p - smad2 . for comparison , we also stained for p - erk . nuclear p - smad2 staining was observed exclusively in the margin and , as expected , in a smaller domain than the p - erk staining ( figure 3c ) . to quantitate the size of the p - smad2-positive domain , we imaged the embryos at the margin , used metamorph software to generate normalized nuclear staining intensities , and measured the distance of each nucleus from the margin in control- and sb-505124-treated embryos . p - smad2 staining above background was found up to five to six cell tiers ( 8090 m ) from the margin in a steep gradient , which was abolished in sb-505124-treated embryos ( figures 3d , 3e , and s5b s5d ) . the staining was strongest in the nuclei of cells nearest the ysl . taken together with our observations from the tg(are : egfp ) reporter line , these data demonstrate that nodal signaling occurs in five to six cell tiers from the margin , closely mirroring the expression of the ligands . our data demonstrate that the nodal ligands and antagonists are co - expressed at the margin of late blastula - stage embryos , and moreover , nodal activity is restricted to these cells . first , if the ligands and antagonists are co - expressed , how does signaling occur at all ? second , given that all cells are competent to signal at blastula stages and nodal signaling induces the expression of the ligands , why does signaling not spread throughout the embryo ? to address these questions , we first determined to what extent lft1/2 regulate nodal signaling in early and late blastula - stage embryos . the lft1/2 proteins are known to inhibit signaling by sequestering tdgf1 and possibly also nodal itself ( chen and shen , 2004 , cheng et al . , 2004 ) . as expected , injection of lft1/2 mos led to an expansion of the nodal signaling domain at 50% epiboly , which was confirmed by qpcr ( figures 4a and 4b ) . at dome stage , however , there was no increase in the size of the nodal signaling domain , suggesting that lft1/2 do not regulate nodal signaling at early stages . although there is an offset in the appearance of ndr1/2 and lft1/2 mrna due to the presence of maternal ndr1 transcripts , the lack of an early role for lft1/2 is not explained by an absence of lft1/2 mrna at sphere and dome stages ( figures 4c and s3 ) . we hypothesized therefore that a delay in translation of the lft1/2 proteins could account for the inability of lft1/2 to regulate nodal signaling at dome stage . both lft1 and lft2 transcripts were readily polyadenylated by sphere stage ( figure 4d ) , suggesting that lack of polyadenylation could not account for any delay in lft1/2 translation . to measure endogenous protein levels directly , we raised polyclonal antibodies for lft1 and lft2 and thoroughly characterized them ( figure s6 ) . although both antibodies recognized their corresponding target protein when overexpressed ( figure s6b ) , only the lft1 antibody was able to detect endogenous protein , so we focused on this family member . the major band detected by western blot corresponding to endogenous lft1 migrated by sds - page with a molecular weight of 40 kd ( figure s6c ) . mutation analysis indicated that this product arose from cleavage at the first furin cleavage site ( marked as c1 in figure s6a ) , and we could demonstrate that this product was active ( figures s6d s6f ) . the same 40 kd band was also detected in embryos injected with ndr1 mrna ( figure s6 g ) . levels of endogenous lft1 were barely detectable by western blotting at dome stage and 30% epiboly but increased at 50% epiboly ( figures 4e and 4f ) . thus , endogenous lft1 protein levels remain low until 50% epiboly , despite readily detectable mrna levels at all these time points . this suggested that the lack of an early role for lft1/2 could be due to low protein abundance and led us to hypothesize that repressed lft1/2 translation creates a window of opportunity for nodal signaling to become established . during blastula stages , the mir-430 family of mirnas have been reported to block translation , without affecting polyadenylation ( bazzini et al . we reasoned therefore that the activity of mir-430 could be responsible for the repressed translation of lft1/2 proteins to create a temporal window for nodal to activate signaling . mir-430 pri - mirna is expressed in the nuclei of all cells in the blastoderm , immediately after activation of zygotic transcription ( figure 5a ) . we also confirmed a ubiquitous expression pattern for mature mir-430a and mir-430b at 50% epiboly ( figure 5b ) and demonstrated that mature mir-430a and mir-430b are directly processed upon expression ( figure 5c ) . mir-430c was not detected by either wish or northern blotting , but rna sequencing data demonstrated that mir-430c is much less abundant than mir-430a and mir-430b ( unpublished data ) . thus , the mir-430 family is abundant , ubiquitously expressed , and readily processed during mid to late blastula stages . to determine the role of mir-430 in the regulation of lft1/2 protein translation , we designed three mos that prevented processing of mature mir-430a , mir-430b , and mir-430c . injection of these mos into one - cell - stage embryos resulted in a phenotype resembling mz dicer mutants at 22 hpf ( figure s7a ) ( giraldez et al . , 2005 ) . furthermore , they abolished mir-430a staining at 50% epiboly and reduced mir-430a , mir-430b , and mir-430c expression , as determined by qpcr , by 89% ( figures s7b and s7c ) . co - injection of mir-430 mos with a gfp reporter containing either three mir-430 binding sites or a gfp reporter with the lft2 3utr , which contains a single mir-430 binding site , resulted in increased translation compared to control mos ( figure s7d ) . we next injected equal amounts of control or mir-430 mos and performed western blotting for p - smad2 and lft1 at several blastula stages . in control mo - injected embryos , lft1 protein was not detectable until 30% epiboly and increased at 50% epiboly , as observed for lft1 protein expression in wt embryos ( compare figure 4e with figure 5d ) . importantly , this was accompanied by a gradual increase of p - smad2 over time . in contrast , injection of mir-430 mos led to premature translation of lft1 from dome stage , and this coincided with lower overall accumulation of p - smad2 ( figure 5d ) . interestingly , the level of lft1 protein in dome - stage mir-430 morphants was similar to the maximal level of lft1 protein measured at 50% epiboly in control mo - injected embryos , suggesting that there may be a threshold level of lft1 that is inhibitory . to determine whether reduced signaling in the mir-430 morphants was due to premature translation of lft1/2 and to investigate the spatial consequences for nodal signaling of the loss of mir-430 , we injected mir-430 mos and/or lft1/2 mos into tg(are : egfp ) embryos and assayed nodal activity ( egfp ) and ndr1 levels at 30% epiboly . injection of lft1/2 mos alone resulted in a modest increase in egfp and ndr1 staining ( figure 5e ) , whereas injection of mir-430 mos led to a reduction in egfp staining in the blastoderm , consistent with the inhibition of the nodal signaling pathway we observed using p - smad2 levels as a readout ( figure 5d ) . when lft1/2 mos and mir-430 mos were co - injected , signaling was activated in the entire blastoderm , and this was accompanied by a similar expansion of ndr1 expression ( figure 5e ) . importantly , this spreading of egfp staining in lft1/2 and mir-430 mo - co - injected embryos was due to nodal signaling , because it was completely blocked by incubating double morphants with sb-505124 ( figure 5f ) . the further spreading of signaling following combined knockdown of lft1/2 and mir-430 is readily explained by the regulation of ndr1 translation by mir-430 in the absence of lft1/2 ( see figure 7a ) ( choi et al . , 2007 ) . together , these experiments demonstrate that mir-430 delays lft1/2 translation to create a temporal window for nodal to activate signaling . our data demonstrate that a temporal window for nodal signaling activation determines the size of the nodal signaling domain and predict that once lft1/2 levels reach a certain threshold , nodal signaling is unable to spread to adjacent cells . we therefore tested if blocking signaling activation by recombinant mouse lefty1 ( mlefty1 ) is dose dependent in dissociated embryonic cells . a 5-fold excess ( calculated by mass ) of mlefty1 over human recombinant nodal led to a near complete inhibition of signaling activation as read out by western blotting for p - smad2 and endogenous zebrafish lft1 , which monitors the transcriptional output of the pathway ( figure 6a ) . thus , lft1/2 proteins can reach an inhibitory concentration at which signaling can no longer be activated . next we determined if duration of exposure to nodal directly corresponds to increasing levels of signaling . when blastula - stage cells were exposed to 50 ng / ml nodal and then inhibited with a blocking concentration of 500 ng / ml mlefty1 at different time points , p - smad2 and endogenous lft1 levels were indeed proportional to the duration of ligand exposure ( figure 6b ) . finally , we investigated how rapidly signaling is blocked when lft1/2 levels reach inhibitory concentrations . this is a crucial issue , as we observe that nodal signaling in vivo ( as read out by egfp , ndr1 , ndr2 , lft1 , and lft2 ) is sustained in the margin for several hours after lft1 levels reach an inhibitory concentration at around 50% epiboly ( figure s3 ) . dissociated embryonic cells were therefore exposed to nodal for 1 hr , and then signaling was inhibited by addition of mlefty1 for 2 hr . we observed that p - smad2 levels decreased slowly , compared with the rapid termination of signaling with sb-505124 ( figure 6c ) . this demonstrated that although signal activation is blocked by lefty , nodal signaling is sustained for some time , presumably because of continued signaling from internalized receptor complexes in early endosomes ( jullien and gurdon , 2005 , vizn et al . , 2013 ) . in conclusion , our data show that activation of nodal signaling in blastula - stage cells can occur until lft1/2 levels reach inhibitory concentrations . moreover , the levels of p - smad2 , and as a result transcription , are proportional to the duration of signal activation . this can be maintained for some time after inhibitory lft1/2 concentrations are reached , while no new signaling is activated . here we describe a specific and sensitive nodal reporter line that has enabled us to visualize endogenous nodal signaling in developing zebrafish embryos , without overexpression of any pathway components . we show that signaling is initiated on the dorsal side because of maternally provided ndr1 . ventral and lateral signaling arises as a result of ndr1/2 expression in the ysl , which then spreads toward the animal pole as a result of autoregulation . nodal signaling in the margin reaches a maximum of six cell tiers , which we demonstrate by p - smad2 immunostaining and reporter activity . we find no evidence of signaling beyond the cells that express the ligand , and moreover , these same cells additionally express the nodal antagonists lft1/2 . spreading of presumed long - range nodal target genes , such as ta , that are activated beyond the ligand expression domain , our data support a model whereby a temporal window for nodal signaling activation dictates the dimensions of the nodal signaling domain ( figure 7 ) . thus , temporal information is translated into spatial information in the developing embryo . the crucial determinant of the temporal window is the delayed translation of the lft1/2 proteins , which is mediated by mir-430 . in addition , maternally provided ndr1 transcripts and the production of ndr1/2 by the ysl allow nodal signaling to be initiated in the blastoderm , before transcription of lft1/2 ( figure 7c ) . because of positive feedback , the blastoderm cells produce more ndr1/2 , while lft1/2 levels remain relatively low because of the ubiquitous synthesis of mir-430 . in these conditions , nodal signaling can be activated in neighboring cells until extracellular lft1/2 levels reach inhibitory concentrations . therefore , the duration for which lft1/2 levels are repressed dictates the size of the nodal signaling domain . how the repressive action of mir-430 is lifted at 50% epiboly to allow lft1/2 translation is not yet known and requires further investigation . although inhibitory lft1/2 levels prevent further activation of signaling , and hence additional spreading of nodal signaling , cells already responding to nodal will continue to signal for several hours , because this occurs from internalized receptors that are refractory to lft1/2 inhibition . a consequence of our proposed mechanism is that cells directly adjacent to the ysl activate nodal signaling for the longest duration . this likely explains the more intense p - smad2 staining in these cells relative to those further from the margin . we therefore propose that nodal signaling at the margin at blastula stages is dictated by an interplay among ligand , ligand antagonist , and a mirna , with a differential in timing between ligand and antagonist production being the key determining factor . the size and shape of the nodal signaling gradient had previously been thought to be regulated by a reaction - diffusion system ( meinhardt , 2009 , mller et al . , 2012 , schier , 2009 ) . here we propose an alternative mechanism whereby the size of the domain is dictated by the delay in lefty translation . although the nodal / lefty ligand / antagonist pair has many features of a reaction - diffusion system , we have uncovered one aspect that is incompatible . in reaction - diffusion models , which were originally conceived as pattern - forming chemical reactions , a homogeneous distribution of activator and inhibitor are unstable , and a local elevation of activator initiates formation of a gradient ( meinhardt , 2009 ) . integral to this model is the ability of the diffusing antagonist to immediately inhibit activator function at a distance . for nodal and lefty , this can not happen , because once nodal signaling is activated it occurs from internalized receptors and is therefore insensitive to lefty inhibition , except over prolonged time frames . our data suggest that spatially graded activity of the nodal signaling pathway is mainly the result of different durations of exposure to ndr1/2 over time , as opposed to exposure to different concentrations . we observe two graded signaling domains in tg(are : egfp ) embryos that are both explained by timing of ligand exposure . during blastula stages , a shallow signaling gradient runs from dorsal to ventral . this is readily explained by the fact that dorsal cells are exposed to nodal for a longer period than ventrolateral cells , because maternally provided ndr1 signals dorsally before ndr1/2 produced in the ysl induces signaling in the blastoderm margin . in addition , we found a clear vegetal - to - animal gradient within the ligand - expression domain using p - smad2 immunostaining in late blastula - stage embryos , and we also observed that cells directly adjacent to the ysl expressed higher levels of all nodal target genes , including the egfp reporter gene , reflecting higher levels of signaling . again , cells directly adjacent to the ysl are exposed to ndr1/2 for the longest period of time . the importance of duration of exposure is further supported by our ex vivo experiments with dissociated embryonic cells . the long - term functional consequences of this were demonstrated in previously published work , which linked cell fates to the duration of exposure to nodal signals ( hagos and dougan , 2007 ) . thus , our model explains how concentration and duration of signaling can be translated into positional information . finally , the importance of timing of signaling activation also rationalizes the normal development of ndr1 mutants ( feldman et al . , 1998 , heisenberg and nsslein - volhard , 1997 , lim et al . , 2013 ) . this is explained by the fact that ndr2 compensates for the lack of ndr1 , but its expression is delayed , because there is no maternal ndr2 . from the sectioning of tg(are : egfp ) embryos and immunostaining for p - smad2 , it is clear that smad2-smad4-foxh1-dependent nodal signaling is confined to 56 cell tiers from the margin , which could imply that all mesendodermal cells arise from this domain . this finding seems to contradict lineage - tracing studies that show that some mesodermal precursors are located up to 12 cell tiers away from the margin ( e.g. , see dougan et al . although these lineage - tracing studies have provided valuable insight into the overall spatial distribution of mesendoderm precursors , they can not themselves determine the extent of nodal signaling . this is because although nodal signaling is required for mesendoderm formation , not all cells that become mesendoderm have necessarily experienced nodal signaling directly . our understanding of how morphogens activate graded signaling in tissues has been dominated by the pre - molecular era assumption that secreted ligands diffuse from a source to form concentration gradients , and this assumption has naturally progressed into the formulation of models that include diffusion as a major determinant in patterning by morphogens . although in some contexts , such as the establishment of left - right asymmetry , nodal can act at long range ( shiratori and hamada , 2014 ) , the work presented here shows that the formation of the nodal signaling domain at the blastula margin is explained by short - range signaling activation , signaling dynamics , and transcriptional / translational regulation . human nodal ( 3218-nd / cf ; r&d ) was dissolved in 4 mm hcl at 100 g / ml , aliquoted in non - stick tubes , and used at 40 ng / ml ( unless stated otherwise ) without freeze - thawing . recombinant mlefty1 was dissolved according to the manufacturer s instructions ( 994-lf / cf ; r&d ) . the inhibitors sb-505124 ( 3263 ; tocris bioscience ) and su-5402 ( 572631 ; calbiochem ) were dissolved in dmso and used in embryos at 50 and 10 m respectively . in dissociated embryonic cells , up to 1,000 embryos were dechorionated using 2 mg / ml pronase ( 11459643001 ; roche ) in 10 ml e3 medium . the dechorionated embryos were washed extensively in e3 medium and once with calcium - free ringers buffer to remove the pronase ( link et al . , 2006 ) . the embryos were manually disrupted in calcium - free ringers buffer using a p200 pipette in 6 cm bacterial dishes , collected by centrifugation at 1,000 g for 5 min , and then resuspended at 50 embryos / ml in leibovitz s l15 medium ( 11415 - 064 ; gibco ) supplemented with 3% fetal bovine serum ( fbs ) . cells were plated in 24-well tissue culture plates coated with poly - l lysine ( p4707 ; sigma ) and allowed to attach for 30 min . embryos were fixed in 4% paraformaldehyde in pbs overnight , dehydrated to 100% methanol , and stored at 20c until processing . for whole - mount immunofluorescence , embryos were rehydrated to pbs and incubated in cold acetone at 20c for 20 min . blocking and antibody incubations were performed in 10% fbs and 1% triton x-100 in pbs , and washes were performed in pbs/1% triton . the following primary antibodies were used : -p - smad2/3 ( 8828 ; cell signaling technology ) and -p - erk ( m8159 ; sigma ) . note that because of the lack of smad3 at blastula stages , we solely detect p - smad2 with the -p - smad2/3 antibody . dapi was used to stain nuclei , and images were acquired on a zeiss lsm 780 confocal microscope . for quantification of p - smad2 intensity in deep cells , metamorph software ( molecular devices ) was used to generate p - smad2 to dapi ratios from at least three single optical slices per embryo , in three individual dmso or sb-505124 treated , 50% epiboly embryos . care was taken not to select mitotic or overlapping nuclei or nuclei from the enveloping layer . to measure the distance of a nucleus to the margin , a line was drawn laterally at the vegetal edge of the margin , and the distance to the nucleus perpendicular to the margin was measured using the metamorph software . to normalize the staining intensity for each embryo , the average ratio of an area further than 11 cell tiers from the margin ( > 165 m ) was subtracted from each measurement . the data were divided in 15 m bins , representing the average size of a deep cell at 50% epiboly ( unpublished data ; dubrulle et al . , the averages of the binned data for each cell tier , comparing dmso and sb-505124 treatment , were used for testing for statistical significant differences using paired t tests with a 95% confidence interval . all the zebrafish work was carried out under a uk home office license under the animals ( scientific procedures ) act 1986 . the license underwent full ethical review and approval by the cancer research uk london research institute animal ethics committee . , c.h . , and m .- c.r . performed the experiments and analyzed the data with the help of r.k.s .

summarymorphogen signaling is critical for the growth and patterning of tissues in embryos and adults , but how morphogen signaling gradients are generated in tissues remains controversial . the morphogen nodal was proposed to form a long - range signaling gradient via a reaction - diffusion system , on the basis of differential diffusion rates of nodal and its antagonist lefty . here we use a specific zebrafish nodal biosensor combined with immunofluorescence for phosphorylated smad2 to demonstrate that endogenous nodal is unlikely to diffuse over a long range . instead , short - range nodal signaling activation in a temporal window is sufficient to determine the dimensions of the nodal signaling domain . the size of this temporal window is set by the differentially timed production of nodal and lefty , which arises mainly from repression of lefty translation by the microrna mir-430 . thus , temporal information is transformed into spatial information to define the dimensions of the nodal signaling domain and , consequently , to specify mesendoderm .

Introduction Results Discussion Experimental Procedures Author Contributions

PMC4358047

the ratio of copper ( cu ) to other dietary components ( e.g. , zinc ( zn ) , iron , sulfate , and molybdenum ) may be as important as the actual cu levels in the diet . cu / zn ratios may be important to the adequate metabolism of cholesterol , with low ratios resulting in hypercholesterolemia . previous studies have focused on acute severe cu deficiency , which is relatively rare in humans and animals on typical varied diets . the determination of cu needs and marginal deficiency is complicated by the fact that while cu deficiency does not necessarily lower the level of cu - dependent enzymes , it does significantly lowers their activity . superoxide dismutase ( sod ) functions as an antioxidant by catalyzing the conversion of superoxide radicals ( free radicals or reactive oxygen species ( ros ) ) to hydrogenperoxide , which can subsequently be reduced to water by other antioxidant enzymes . superoxide radicals may react with other ros such as nitricoxide to form highly toxic species like peroxynitrite , in addition to its direct toxic effects . peroxy nitrite reacts with the tyrosine residues in proteins resulting with the nitrotyrosine production in plasma proteins , which is considered as an in direct evidence of peroxynitrite production and increased oxidative stress . although nitrotyrosine was not detectable in the plasma of the healthy controls , nitrotyrosine was found in the plasma of all type 2 diabetic patients ( type 2 diabetes mellitus ( t2 dm ) ) examined . previous studies correlated plasma nitrotyrosine values with plasma glucose concentrations and found a significant positive correlation . furthermore , exposure of endothelial cells to high glucose level leads to an augmented production of superoxide anion , which may quench nitric oxide level resulting in impaired endothelial functions , vasodilation , and delayed cell replication . alternatively , superoxide can be dismutated to a much more reactive hydrogen peroxide , which through the fenton reaction can then lead to a highly toxic hydroxyl radical formation . two forms of sod contain cu : i ) cu / zn - sod is found within most cells of the body , including red blood cells , and ii ) extracellular ( ec)-sod is a cu - containing enzyme found in high levels in the lungs and low levels in blood plasma . almost all of the cu in our bodies is bound either to transport proteins ( ceruloplasmin and cu - albumin ) , storage proteins ( metallothioneins ) , or cu containing enzymes . intracellular metallothionine normally stores little cu providing protection from the harmful effects of free cu . ceruloplasmin may function as an antioxidant in two different ways : by binding to cu , ceruloplasmin prevents free cu ions from catalyzing oxidative damage . the other way is through the oxidation of ferrous iron by ceruplasmin , facilitating iron load into its transport protein , transferrin , and preventing free ferrous ions from participating in harmful free radical generating reactions . major reason for the decreased sod activity is the glycosylation of cu / zn - sod which has been shown to lead to enzyme inactivation both in vivo and in vitro . also cu / zn - sod cleavage and release of cu in vitro resulted intransition metalcatalyzed ros formation . erythrocyte cu / zn - sod activity correlated inversely with indices of glycemic control in dm patients . however , red cell cu / zn - sod activity has also been found to be decreased in dm patients . glycation may decrease cell - associated ec - sod , which could predispose to oxidative damage . earlier reports found decreased red cell cu / zn - sod activity in dm patients with retinopathy compared to dm patients without microvascular complications and nondiabetic control subjects . this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university . ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) . type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) . patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital . the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs . all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity . the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92 8.9 years ) . the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy . a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting . two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay . the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection . all assays were obtained by running duplicates for the test , control , and the standard . random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio . no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements . a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution . urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al . b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al . a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve . urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution . cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame . serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) . serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) . glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) . statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression . this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university . ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) . type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) . patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital . the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs . all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity . the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92 8.9 years ) . the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy . a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting . two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay . the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection . all assays were obtained by running duplicates for the test , control , and the standard . random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio . no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements . a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution . urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al . b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al . a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve . urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution . cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame . serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) . serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) . glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) . statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression . all groups were closely age - matched , and the two diabetes groups were well - matched for duration of disease [ table 1 ] . microabuminuric diabetic group shows a significant increase in the mean urinary cu / creatinine ratio when compared with controls ( p < 0.05 ) , while normoal buminuric diabetic patients shows an insignificant increase , p > 0.05 in mean urine cu / creatinine ratio when compared with controls [ table 2 ] . urinary copper excretion and serum superoxide dismutase enzyme levels in microal buminurics , normoal buminurics , and control subjects serum sod was significantly decreased in the diabetes microal buminuric group compared to the control group ( p < 0.05 ) , such a significant correlation was not seen with the diabetes normoal buminuric group [ figure 1 and table 2 ] . studies showed a reduction in erythrocyte sod and catalase ( cat ) activities in subjects with impaired glucose tolerance ( igt ) , early hyperglycemia , and type 2 dm patients . however , other studies showed that the activities of these enzymes were within normal range in t2 dm patients in poor glycemic control . ec - sod activity was found to be similar in t1 dm patients despite some what higher plasma ec - sod levels . red cell cu / zn - sod activity was similar in t1 dm and t2 dm patients compared to normal subjects , irrespective of microvascular complications . leukocyte sod activity was similar between type 2 dm patients and healthy control subjects , despite increased lipidperoxidation and decreased ascorbate levels . while the role of adequate cu levels in maintaining cardiovascular health is well - established , there are still inconsistent data about the correlation of cu with persisting hyperglycemia . some studies showed an elevation of serum cu , while other studies showed a significant reduction of cu in diabetes . many researchers have considered this elevation of serum cu to play a role in the pathogenesis of cardiovascular disease , although other researchers have strongly disagreed with this hypothesis . an animal study , however , seems to have explained this relationship between cu levels and cardiovascular disease . this study examined the effects of diet - induced atherosclerosis on the cu levels and status of numerous tissues . it was found that serum cu levels increase significantly , while aorta and liver cu levels decrease significantly , in rats with experimental atherosclerosis . so instead of assuming that these elevated cu levels contribute to the formation of atherosclerosis , these researchers examined the effects of increasing the dietary cu levels in these animals . administration of additional cu resulted in a further increase in serum cu , a significant decrease in serum cholesterol , and an increase and normalization in aorta and liver cu levels . however , instead of increasing the incidence of atherosclerosis , additional cu significantly decreased the incidence of atherosclerosis in the aorta and coronary arteries . further , it has been shown that excess dietary cholesterol causes cardiovascular disease by lowering the absorption of cu , an effect that is preventable by increasing the cu level in the diet . however , increased urinary albumin loss has been postulated to be a marker of a generalized increase in vascular permeability , which might predispose to greater penetration into the arterial wall of atherogenic lipoprotein particles . urinary cu concentrations significantly increased only in microal buminuric patients , similar results were found by , but with macroal buminuric diabetic patients . in diabetic patients with advanced nephropathy , urinary cu excretion may be due to dissociations from both cu - albumin and ceruloplasmin - cu complexes filtered through the damaged glomerulus . overloading of urinary cu to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy . therefore , regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy . however , there is still a lot to know about the mechanism of cu homeostasis at the cellular level . if reduction of serum cu can be shown to have a protective effect against oxidative stress in dm , this may have a direct impact on the use of cu chelators as a safe therapeutic modality in diabetes .

background : copper ( cu ) is essential both for its role in antioxidant enzymes , like cu / zinc ( zn ) superoxide dismutase ( sod ) and ceruloplasmin , as well as its role in lysyl oxidase , essential for the strength and integrity of the heart and blood vessels . with such a central role in cardiovascular health , cu has been generally overlooked in the debate over improving our cardiovascular health . cu deficiency has produced many of the same abnormalities present in cardiovascular disease . it seems almost certain that cu plays a large role in the development of this killer disease , not because of its excess in the diet , but rather its deficiency.aim:this study was undertaken to investigate the cardiovascular effects of cu deficiency on the activity of sod in patients with type 2 diabetes mellitus ( t2 dm ) with and without diabetic nephropathy.materials and methods : fifty - five patients with t2 dm were recruited in this study which were divided into two subgroups based on the presence of microalbuminuria , the first group ( microal buminuric group , n = 31 ) had a microalbuminuria between 30 and 299 g / mg . the second group ( normoal buminuric group , n = 29 ) had an albumin level less than 30 g / mg . the two diabetic groups were compared to the control group ( n = 37).results : the results of our study showed a significant reduction in the levels of sod enzyme associated with an increased urinary cu excretion in microalbuminuric group compared to the control group at p < 0.05.conclusions:the current study illustrates that the regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy .

Introduction Materials and Methods Study protocol and participants Blood samples Urine samples Parameters of the study Statistical analysis Results Discussion Conclusion

PMC3334224

corneal transplantation for corneal endothelial diseases is undergoing a paradigm shift from penetrating keratoplasty to endothelial keratoplasty.1 descemet s stripping automated endothelial keratoplasty ( dsaek ) , which is essentially endothelial keratoplasty that involves donor tissue preparation using an automated microkeratome , is rapidly becoming the preferred alternative to conventional penetrating keratoplasty for endothelial dysfunction , such as fuchs endothelial keratoplasty and pseudophakic bullous keratopathy.2 in the united states , the number of donor corneas for endothelial keratoplasty has exponentially increased from 3% ( 2005 ) to 33% ( 2007 ) and 42.8% in 2009 ( eye banking statistical report 2009 , eye bank association of america data ( http://www.restoresight.org/donation/statistics.htm ) . the advantages of dsaek over penetrating keratoplasty include better tectonic stability , essentially sutureless surgery , and faster postoperative visual rehabilitation with more predictable refractive changes.37 however , early studies mainly in caucasian eyes suggest equivalent or higher cell loss in dsaek compared to penetrating keratoplasty at 6 months , albeit nonsignificant by 2 years.3,812 the main aim of this study was to compare our dsaek results in terms of endothelial cell loss with those of penetrating keratoplasty with at least 1-year follow - up in asian eyes using our previously described dsaek technique.13 we conducted a retrospective study of patients who underwent dsaek or penetrating keratoplasty for which the surgical indication was either fuchs endothelial dystrophy or pseudophakic and aphakic bullous keratopathy in 20062008 . we excluded any patients who did not have a minimum pos - surgical follow - up of 1 year . our subjects and clinical data were obtained from the ongoing cohort of the singapore corneal transplant study , an audited longitudinal prospective study which contains preoperative , intraoperative , and yearly postoperative follow - up clinical data.14 this study followed the principles of the declaration of helsinki , with ethics approval obtained from our institutional review board . a total of 241 patients met our inclusion criteria , of whom 68 underwent dsaek and 173 underwent penetrating keratoplasty by the five corneal surgeons at our center , as well as inclusion of cases which were partially performed by corneal fellows in training under direct supervision . our main outcome measure was endothelial cell count and the derived percent endothelial cell loss at 1 year follow - up . our secondary outcome measures included graft success and visual acuity at 1 year follow - up . visual acuity was measured using the snellen visual acuity chart and we analyzed the results using logarithm of the minimum angle of resolution ( logmar ) equivalent units , including manifest refraction , spherical equivalent , and cylindrical error.15 the singapore eye bank provides all donor corneas , stored in optisol in cold storage , with standard internal guidelines for penetrating keratoplasty and dsaek grafts and we obtained all donor information from their database , including donor endothelial cell counts.14 preoperative specular microscopy of the donor tissue was performed either by certified technicians in an eye bank association of america- certified eye bank or by a certified eye bank technician at the singapore eye bank . postoperative specular microscopy measurements of endothelial cell density were performed using a noncontact specular microscope ( konan medical corporation , hyogo , japan ) at 12 months postoperatively , by ophthalmic technicians trained in specular microscopy . calibrations and magnifications were standardized automated measurements with a mean value derived , as previously described.16 the incidence of postoperative complications was obtained from the singapore corneal transplant study database , which tracks all graft complications . graft failure was defined as irreversible loss of optical clarity , with the date of onset of corneal clouding selected as the time point of graft failure . penetrating keratoplasty surgeries were performed using a standard technique based on a hanna vacuum trephine system ( moria inc , antony , france ) . in summary , the recipient cornea was excised using the hanna trephine . a 0.250.50 mm oversized donor cornea was then punched out endothelial side up and sutured on to the recipient with 10 - 0 nylon , using either an 8-bite , 10 - 0 nylon double continuous running suture or a combination of a single 8-bite 10 - 0 nylon continuous and eight interrupted sutures . a bandage contact lens was placed at the end of the surgery , and subconjunctival dexamethasone 0.1% ( decadron ; merck and co , inc , rahway , nj ) , gentamicin 14 mg / ml ( garamycin ; schering ag , berlin - wedding , germany ) , and cefazolin 50 mg / ml ( ancef ; glaxosmithkline , research triangle park , nc ) was injected . dsaek was performed using our previously described technique.13,16 essentially , after descemet s stripping under air,17 a paracentesis was first made in the peripheral cornea opposite the scleral tunnel wound for insertion of kawai intraocular capsulorhexis forceps ( asico , westmont , il ) or tan dsaek forceps ( asico).13 a standard anterior chamber intraocular lens sheets glide ( bd visitec ) was trimmed to 4.5 mm and inserted into the eye through a 5 mm temporal scleral tunnel incision while the anterior chamber was maintained via an anterior chamber maintainer with a balanced salt solution infusion . the donor was prepared by the surgeon using an automated lamellar therapeutic keratoplasty system ( altk ; moria sa , antony , france ) aiming for a donor cornea of thickness of approximately 150 microns . four preplaced corneal venting incisions were made on the recipient cornea . a dispersive ophthalmic viscosurgical device ( viscoat ; alcon laboratories inc , hnenberg , switzerland ) was liberally applied over the endothelial surface of the donor cornea and on the anterior surface of the glide , taking care not to have the ophthalmic viscosurgical device on the stromal donor surface . the donor cornea was gently inverted , corneal endothelial surface - down , onto the ophthalmic viscosurgical device covered portion of the glide . kawai or tan dsaek forceps were passed through the nasal paracentesis , over the sheets glide , and out through the scleral incision , grasping the donor cornea stromal edge and pulling the donor cornea through the scleral incision , whilst the anterior chamber maintainer was infusing balanced salt solution at a medium to slow rate . with this technique , a small air bubble was injected under the donor cornea with a 30-gauge canula to prevent descent of the donor cornea , the sheets glide retracted , and the donor cornea was released from the forceps . the scleral tunnel was then sutured with three 10/0 nylon interrupted sutures , the anterior chamber maintainer was removed and the port sutured , the donor was adjusted centrally by gentle massage through the cornea surface , and full air tamponade was achieved with a large bubble in the anterior chamber for 8 minutes . following this , some air was replaced with balanced salt solution , leaving a smaller air bubble approximating the size of the endothelial keratoplasty graft in the anterior chamber . all patients were examined approximately one hour after surgery to ensure air was still present in the anterior chamber , and no donor dislocation or pupillary block was present . we used similar postoperative medication regimens in both groups , ie , predforte ( prednisolone acetate ophthalmic suspension , usp ) 1% every 3 hours for 1 week , three times a day for 6 months , twice daily for 3 months then once a day for up to 1 year . statistical analysis included descriptive statistics , whereby the mean and standard deviation were calculated for the continuous variables , while the frequency distribution and percentages were used for categorical variables . comparisons between categorical variables were conducted using fisher s exact tests , whereas the one - way analysis of variance test was used for means . meier survival analysis was conducted to determine survival probabilities of penetrating keratoplasty and dsaek groups . the survival period of failed grafts was defined as the time between the date of surgery and recorded date of survival or failure . penetrating keratoplasty surgeries were performed using a standard technique based on a hanna vacuum trephine system ( moria inc , antony , france ) . in summary , the recipient cornea was excised using the hanna trephine . a 0.250.50 mm oversized donor cornea was then punched out endothelial side up and sutured on to the recipient with 10 - 0 nylon , using either an 8-bite , 10 - 0 nylon double continuous running suture or a combination of a single 8-bite 10 - 0 nylon continuous and eight interrupted sutures . a bandage contact lens was placed at the end of the surgery , and subconjunctival dexamethasone 0.1% ( decadron ; merck and co , inc , rahway , nj ) , gentamicin 14 mg / ml ( garamycin ; schering ag , berlin - wedding , germany ) , and cefazolin 50 mg / ml ( ancef ; glaxosmithkline , research triangle park , nc ) was injected . dsaek was performed using our previously described technique.13,16 essentially , after descemet s stripping under air,17 a paracentesis was first made in the peripheral cornea opposite the scleral tunnel wound for insertion of kawai intraocular capsulorhexis forceps ( asico , westmont , il ) or tan dsaek forceps ( asico).13 a standard anterior chamber intraocular lens sheets glide ( bd visitec ) was trimmed to 4.5 mm and inserted into the eye through a 5 mm temporal scleral tunnel incision while the anterior chamber was maintained via an anterior chamber maintainer with a balanced salt solution infusion . the donor was prepared by the surgeon using an automated lamellar therapeutic keratoplasty system ( altk ; moria sa , antony , france ) aiming for a donor cornea of thickness of approximately 150 microns . four preplaced corneal venting incisions were made on the recipient cornea . a dispersive ophthalmic viscosurgical device ( viscoat ; alcon laboratories inc , hnenberg , switzerland ) was liberally applied over the endothelial surface of the donor cornea and on the anterior surface of the glide , taking care not to have the ophthalmic viscosurgical device on the stromal donor surface . the donor cornea was gently inverted , corneal endothelial surface - down , onto the ophthalmic viscosurgical device covered portion of the glide . kawai or tan dsaek forceps were passed through the nasal paracentesis , over the sheets glide , and out through the scleral incision , grasping the donor cornea stromal edge and pulling the donor cornea through the scleral incision , whilst the anterior chamber maintainer was infusing balanced salt solution at a medium to slow rate . with this technique , a small air bubble was injected under the donor cornea with a 30-gauge canula to prevent descent of the donor cornea , the sheets glide retracted , and the donor cornea was released from the forceps . the scleral tunnel was then sutured with three 10/0 nylon interrupted sutures , the anterior chamber maintainer was removed and the port sutured , the donor was adjusted centrally by gentle massage through the cornea surface , and full air tamponade was achieved with a large bubble in the anterior chamber for 8 minutes . following this , some air was replaced with balanced salt solution , leaving a smaller air bubble approximating the size of the endothelial keratoplasty graft in the anterior chamber . all patients were examined approximately one hour after surgery to ensure air was still present in the anterior chamber , and no donor dislocation or pupillary block was present . we used similar postoperative medication regimens in both groups , ie , predforte ( prednisolone acetate ophthalmic suspension , usp ) 1% every 3 hours for 1 week , three times a day for 6 months , twice daily for 3 months then once a day for up to 1 year . statistical analysis included descriptive statistics , whereby the mean and standard deviation were calculated for the continuous variables , while the frequency distribution and percentages were used for categorical variables . comparisons between categorical variables were conducted using fisher s exact tests , whereas the one - way analysis of variance test was used for means . meier survival analysis was conducted to determine survival probabilities of penetrating keratoplasty and dsaek groups . the survival period of failed grafts was defined as the time between the date of surgery and recorded date of survival or failure . overall , there were more patients whose main surgical indication was bullous keratopathy as compared with fuchs dystrophy ( 153/241 , 63.5% ) and more of the patients with bullous keratopathy had undergone a penetrating keratoplasty as compared with dsaek ( 120/173 , 69.4% versus 33/68 , 48.5% ; p = 0.003 ) . however , there were no significant differences in demographics or characteristics of patients with bullous keratopathy or fuchs dystrophy between the treatment groups . the mean preoperative donor endothelial cell density was 101 cells / mm greater in the dsaek than in the penetrating keratoplasty group ( 2792 327 versus 2691 360 cells / mm ; p = 0.0412 ) . donor size was significantly larger in patients undergoing dsaek as compared with penetrating keratoplasty ( mean donor size 8.75 0.49 versus 7.87 0.34 mm ; p < 0.001 ) . postoperative endothelial cell density was obtainable in 120 eyes ( 60 dsaek - treated eyes and 60 penetrating keratoplasty - treated eyes ) performed at 1-year follow - up . in this subanalysis , there were no significant differences in baseline characteristics , such as mean recipient age ( 64 10 versus 67 11 years ; p = 0.08 ) sex ( p = 0.06 ) , race ( p = 0.16 ) , or diagnosis ( 51% versus 49% fuchs dystrophy ; p = 0.86 ) between the dsaek and penetrating keratoplasty groups . we found that mean endothelial cell density was greater at 1 year in the dsaek group as compared with the penetrating keratoplasty group ( 2174 66 versus 1555 76 ; p = 0.001 ) and overall percentage of endothelial cell loss after 1 year was 40.9% 2.9% in penetrating keratoplasty - treated and 22.4% 2.3% in dsaek - treated eyes ( p < 0.001 ) . in patients with fuchs dystrophy , dsaek - treated eyes had significantly less percentage of endothelial cell loss compared with penetrating keratoplasty - treated eyes ( 30 eyes each group , mean percentage endothelial cell loss ; 20.4% 1.4% versus 37.7% 2.3% ; p = 0.001 ) . this was similar to patients with bullous keratopathy ( 30 eyes each group , mean percentage of endothelial cell loss ; 24.2% 2.0% versus 41.8% 2.2% ; p = 0.002 ) . we also compared percentage of endothelial cell loss at 1 year between patients with fuchs dystrophy and bullous keratopathy . at 1 year , the percentage of endothelial cell loss did not differ significantly between patients with fuchs dystrophy and those with bullous keratopathy for either procedure ( 39% versus 44% in penetrating keratoplasty - treated eyes ; p = 0.40 ; and 20% versus 24% in dsaek - treated eyes ; p = 0.45 ) . in our study cohort , 26 patients had concomitant eye disease , which significantly impacted on final visual outcome , while 17 patients had late graft failure at 1 year . all these patients were excluded from our final visual acuity analysis ( table 2 ) . we compared the remaining patients ( 61 dsaek with 137 penetrating keratoplasty - treated eyes ) with respect to visual acuity . amongst these patients there were fewer patients who had fuchs dystrophy in the penetrating keratoplasty group ( 35/61 [ 57.3% ] dsaek ; 51/137 [ 37.2% ] penetrating keratoplasty ; p = 0.011 ) . patients who underwent dsaek had better visual outcomes at 1 year when compared with penetrating keratoplasty - treated patients ( table 3 ) . overall , dsaek - treated eyes had significantly superior uncorrected visual acuity ( ucva ) and best spectacle - corrected visual acuity ( bscva ) as compared with penetrating keratoplasty - treated eyes at 1 year ( mean difference ucva , 0.42 0.0059 ; p < 0.001 ; bscva , 0.14 0.032 ; p < 0.001 ) . in addition , there were more eyes with bscva better than 20/40 in dsaek - treated group as compared with the penetrating keratoplasty - treated group ( 67.2% versus 38.7% ; p < 0.001 ) . when comparing visual outcomes between surgical indications , we found that visual outcome was significantly better in eyes with fuchs dystrophy for both dsaek ( mean difference bscva , 0.36 0.011 ; p = 0.002 ) and penetrating keratoplasty ( mean difference bscva , 0.40 0.0079 ; p < 0.001 ) . overall , patients with penetrating keratoplasty - treated eyes had higher astigmatism at 1 year as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 , respectively ; p < 0.001 , table 1 ) . we also observed this difference in patients with fuchs dystrophy ( 2.6 1.8 versus 1.7 0.8 ; p = 0.023 ) and bullous keratopathy ( 3.2 2.2 versus 1.7 0.8 ; p = 0.001 , table 3 ) . overall , dsaek - treated eyes were more hyperopic as compared with penetrating keratoplasty - treated eyes at 1 year ( spherical equivalent + 1.5 3.3 versus 0.25 1.8 , respectively ; p < 0.001 ) . we had one case of primary graft failure in each of the dsaek and penetrating keratoplasty treatment groups ( 1/68 , 1.5% versus 1/173 , 0.5% respectively ; p = 0.31 ) . at 1 year , 66/68 ( 97.0% ) eyes that underwent dsaek had clear grafts while 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479 ) . there were no significant differences in late graft failure between the groups ( penetrating keratoplasty 8% versus dsaek 3% ; p = 0.118 ) . reasons for graft failure in the penetrating keratoplasty group were infection - related ( n = 10 ) and immune - related ( n = 5 ) ; and for the dsaek group were immune - related ( n = 2 ) . the kaplan meier probability of survival at 1 year was 95.3% for the penetrating keratoplasty - treated group , which decreased to 89.6% at 18 months , while it was 98.4% at 1 year and 93.2% for dsaek cases up to 18 months of follow - up ( p < 0.001 ) . complications during the 1-year follow - up period were recorded according to our singapore corneal transplant study guidelines.14 we scored complications as events and made comparisons between the penetrating keratoplasty and dsaek groups ( table 4).18 epitheliopathy was significantly higher in penetrating keratoplasty - treated eyes ( p = 0.014 ) , while transient episodes of intraocular pressure elevation > 21 mmhg ( defined in terms of short - term , ie , 3 months use of antiglaucoma medications ) were seen in 20/68 ( 29.4% ) and 52/173 ( 30.0% ) for dsaek and penetrating keratoplasty , respectively ( p = 0.93 ) . of note , most of these patients ( 16/20 [ 80.0% ] for dsaek and 49/52 [ 94.2% ] for penetrating keratoplasty ) had an underlying history of glaucoma ( p = 0.07 ) . only four eyes ( three penetrating keratoplasty , one dsaek ) had had trabeculectomy performed previously , and there were no significant differences in graft outcomes . all eyes with transiently raised intraocular pressure were treated successfully with intraocular pressure - lowering topical and/or systemic medications . one patient who underwent dsaek had an acute graft rejection episode successfully treated with topical steroids , as compared with 11 ( 6.3% ) in the penetrating keratoplasty - treated group . of note , we had no graft dislocations in our dsaek - treated eyes in this series of patients . overall , there were more patients whose main surgical indication was bullous keratopathy as compared with fuchs dystrophy ( 153/241 , 63.5% ) and more of the patients with bullous keratopathy had undergone a penetrating keratoplasty as compared with dsaek ( 120/173 , 69.4% versus 33/68 , 48.5% ; p = 0.003 ) . however , there were no significant differences in demographics or characteristics of patients with bullous keratopathy or fuchs dystrophy between the treatment groups . the mean preoperative donor endothelial cell density was 101 cells / mm greater in the dsaek than in the penetrating keratoplasty group ( 2792 327 versus 2691 360 cells / mm ; p = 0.0412 ) . donor size was significantly larger in patients undergoing dsaek as compared with penetrating keratoplasty ( mean donor size 8.75 0.49 versus 7.87 0.34 mm ; p < 0.001 ) . postoperative endothelial cell density was obtainable in 120 eyes ( 60 dsaek - treated eyes and 60 penetrating keratoplasty - treated eyes ) performed at 1-year follow - up . in this subanalysis , there were no significant differences in baseline characteristics , such as mean recipient age ( 64 10 versus 67 11 years ; p = 0.08 ) sex ( p = 0.06 ) , race ( p = 0.16 ) , or diagnosis ( 51% versus 49% fuchs dystrophy ; p = 0.86 ) between the dsaek and penetrating keratoplasty groups . we found that mean endothelial cell density was greater at 1 year in the dsaek group as compared with the penetrating keratoplasty group ( 2174 66 versus 1555 76 ; p = 0.001 ) and overall percentage of endothelial cell loss after 1 year was 40.9% 2.9% in penetrating keratoplasty - treated and 22.4% 2.3% in dsaek - treated eyes ( p < 0.001 ) . in patients with fuchs dystrophy , dsaek - treated eyes had significantly less percentage of endothelial cell loss compared with penetrating keratoplasty - treated eyes ( 30 eyes each group , mean percentage endothelial cell loss ; 20.4% 1.4% versus 37.7% 2.3% ; p = 0.001 ) . this was similar to patients with bullous keratopathy ( 30 eyes each group , mean percentage of endothelial cell loss ; 24.2% 2.0% versus 41.8% 2.2% ; p = 0.002 ) . we also compared percentage of endothelial cell loss at 1 year between patients with fuchs dystrophy and bullous keratopathy . at 1 year , the percentage of endothelial cell loss did not differ significantly between patients with fuchs dystrophy and those with bullous keratopathy for either procedure ( 39% versus 44% in penetrating keratoplasty - treated eyes ; p = 0.40 ; and 20% versus 24% in dsaek - treated eyes ; p = 0.45 ) . in our study cohort , 26 patients had concomitant eye disease , which significantly impacted on final visual outcome , while 17 patients had late graft failure at 1 year . all these patients were excluded from our final visual acuity analysis ( table 2 ) . we compared the remaining patients ( 61 dsaek with 137 penetrating keratoplasty - treated eyes ) with respect to visual acuity . amongst these patients there were fewer patients who had fuchs dystrophy in the penetrating keratoplasty group ( 35/61 [ 57.3% ] dsaek ; 51/137 [ 37.2% ] penetrating keratoplasty ; p = 0.011 ) . patients who underwent dsaek had better visual outcomes at 1 year when compared with penetrating keratoplasty - treated patients ( table 3 ) . overall , dsaek - treated eyes had significantly superior uncorrected visual acuity ( ucva ) and best spectacle - corrected visual acuity ( bscva ) as compared with penetrating keratoplasty - treated eyes at 1 year ( mean difference ucva , 0.42 0.0059 ; p < 0.001 ; bscva , 0.14 0.032 ; p < 0.001 ) . in addition , there were more eyes with bscva better than 20/40 in dsaek - treated group as compared with the penetrating keratoplasty - treated group ( 67.2% versus 38.7% ; p < 0.001 ) . when comparing visual outcomes between surgical indications , we found that visual outcome was significantly better in eyes with fuchs dystrophy for both dsaek ( mean difference bscva , 0.36 0.011 ; p = 0.002 ) and penetrating keratoplasty ( mean difference bscva , 0.40 0.0079 ; p < 0.001 ) . overall , patients with penetrating keratoplasty - treated eyes had higher astigmatism at 1 year as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 , respectively ; p < 0.001 , table 1 ) . we also observed this difference in patients with fuchs dystrophy ( 2.6 1.8 versus 1.7 0.8 ; p = 0.023 ) and bullous keratopathy ( 3.2 2.2 versus 1.7 0.8 ; p = 0.001 , table 3 ) . overall , dsaek - treated eyes were more hyperopic as compared with penetrating keratoplasty - treated eyes at 1 year ( spherical equivalent + 1.5 3.3 versus 0.25 1.8 , respectively ; p < 0.001 ) . we had one case of primary graft failure in each of the dsaek and penetrating keratoplasty treatment groups ( 1/68 , 1.5% versus 1/173 , 0.5% respectively ; p = 0.31 ) . at 1 year , 66/68 ( 97.0% ) eyes that underwent dsaek had clear grafts while 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479 ) . there were no significant differences in late graft failure between the groups ( penetrating keratoplasty 8% versus dsaek 3% ; p = 0.118 ) . reasons for graft failure in the penetrating keratoplasty group were infection - related ( n = 10 ) and immune - related ( n = 5 ) ; and for the dsaek group were immune - related ( n = 2 ) . the kaplan meier probability of survival at 1 year was 95.3% for the penetrating keratoplasty - treated group , which decreased to 89.6% at 18 months , while it was 98.4% at 1 year and 93.2% for dsaek cases up to 18 months of follow - up ( p < 0.001 ) . complications during the 1-year follow - up period were recorded according to our singapore corneal transplant study guidelines.14 we scored complications as events and made comparisons between the penetrating keratoplasty and dsaek groups ( table 4).18 epitheliopathy was significantly higher in penetrating keratoplasty - treated eyes ( p = 0.014 ) , while transient episodes of intraocular pressure elevation > 21 mmhg ( defined in terms of short - term , ie , 3 months use of antiglaucoma medications ) were seen in 20/68 ( 29.4% ) and 52/173 ( 30.0% ) for dsaek and penetrating keratoplasty , respectively ( p = 0.93 ) . of note , most of these patients ( 16/20 [ 80.0% ] for dsaek and 49/52 [ 94.2% ] for penetrating keratoplasty ) had an underlying history of glaucoma ( p = 0.07 ) . only four eyes ( three penetrating keratoplasty , one dsaek ) had had trabeculectomy performed previously , and there were no significant differences in graft outcomes . all eyes with transiently raised intraocular pressure were treated successfully with intraocular pressure - lowering topical and/or systemic medications . one patient who underwent dsaek had an acute graft rejection episode successfully treated with topical steroids , as compared with 11 ( 6.3% ) in the penetrating keratoplasty - treated group . of note , we had no graft dislocations in our dsaek - treated eyes in this series of patients . in this study we found that 1-year percentage of endothelial cell loss and visual outcomes were superior in dsaek - treated eyes as compared with penetrating keratoplasty - treated eyes from the same study cohort , which confirms the results of our preliminary study on dsaek in asian eyes.16 this is in contrast with reports in the literature that dsaek has greater endothelial cell loss compared with penetrating keratoplasty , but few of these studies reported data on endothelial cell loss beyond 12 months of follow - up and none were directly compared with penetrating keratoplasty from the same study cohort.3,812 one study compared subjects from their prospective trial on dsaek with a separate study on penetrating keratoplasty from the specular microscopy ancillary study cohort.19 another study found that 1-year endothelial cell density was lower with dsaek compared with penetrating keratoplasty albeit but the difference was not statistically significant.20 this led to the conclusion in a recent review article that there is currently insufficient evidence to conclude whether endothelial cell loss is greater in dsaek.21 the results from this study are encouraging because our endothelial cell loss at 1 year ( 22.4% 2.3% ) using a sheets glide nonfolding technique was comparable with other non - folding techniques , and we were able to show less endothelial cell loss comparable with penetrating keratoplasty from a same cohort of patients . although our endothelial cell loss is less than that using a taco - folding technique,22 longer follow - up is required to confirm if we have a similar endothelial cell loss trend . another advantage of performing dsaek over penetrating keratoplasty is the ability to use a larger donor size , and thus transplant more endothelial cells.4 in our study , the mean donor size was 10% larger and endothelial cell density was greater in the dsaek group than in the penetrating keratoplasty group ( p < 0.001 ) . the percentage endothelial cell loss , visual outcomes , and astigmatism in eyes with fuchs dystrophy and bullous keratopathy treated with penetrating keratoplasty in our study of asian eyes were comparable with those of previous reports in caucasian eyes.3,2325 however , it may not be useful to compare visual outcomes at 1 year between dsaek and penetrating keratoplasty due to the longer visual rehabilitation and use of sutures in penetrating keratoplasty , thus it was only a secondary outcome measure . nonetheless , in our study , dsaek had better postoperative ucva and bscva as compared with penetrating keratoplasty , which was also found in caucasian eyes but with fewer eyes ( 20 in each group).3,20 while this is somewhat expected , our results confirm that visual rehabilitation takes much longer after penetrating keratoplasty , even when comparing bscva at 1 year follow - up . in our study cohort there was a higher proportion of pseudophakic eyes amongst patients who had undergone dsaek for fuchs dystrophy . this is because we adhere to the general policy that lens removal prior to dsaek obviates the risk of subsequent cataract formation and also creates more space in the anterior chamber during dsaek surgery . moreover , more of the penetrating keratoplasty - treated eyes had complicated surgeries that required anterior vitrectomy and anterior chamber intraocular lens insertion . these factors could contribute to the poorer visual outcome and lower endothelial cell counts in the penetrating keratoplasty - treated group . the main limitations of our study are due to its retrospective nature . although our patients differed in their characteristics , such as demographics and phakic status , we matched subjects in each group for donor and recipient characteristics . we acknowledge that case selection between dsaek and penetrating keratoplasty was dependent on surgeon choice , and that more severe or advanced cases of corneal decompensation were likely to have received penetrating keratoplasty surgery . however , we do routinely attempt dsaek in severe cases of bullous keratopathy , as long as there is only moderate anterior stromal scarring present . this may have affected our postoperative visual outcome comparisons , although this would have minimal effect on differences in endothelial cell loss postoperatively between dsaek and penetrating keratoplasty . our study also had a limited follow - up period of 1 year in order to compare graft survival and record reliable endothelial cell count data , which was only obtained in 120 eyes . however , we analyzed eyes from each group matched for surgical indication to minimize selection bias and there were no significant baseline differences in each group . nevertheless , due to the inherent success of dsaek , it would be challenging and unethical in our institution to conduct a randomized controlled study given the clinical advantages of dsaek over penetrating keratoplasty , hence a historical cohort of penetrating keratoplasty cases in the same population group using the same postoperative regime provided the best comparative cohort . we found that performing dsaek using the described technique in asian eyes resulted in a lower 1-year endothelial percent cell loss as compared with penetrating keratoplasty for patients with fuchs dystrophy and bullous keratopathy . graft survival was comparable in both groups at 1-year follow - up , although visual outcomes were superior in the dsaek group with fewer complications . longer follow - up will provide more data on endothelial cell loss with these two techniques . improvements in donor insertion devices may reduce the initial ecc loss further and improve long - term ecc outcomes.26

backgroundthe purpose of this study was to compare endothelial cell counts after descemet s stripping automated endothelial keratoplasty ( dsaek ) and penetrating keratoplasty in asian eyes.methodsthis was a retrospective study of patients from our prospective singapore corneal transplant study cohort who received corneal transplantation in 20062008 . we compared eyes that underwent dsaek or penetrating keratoplasty for fuchs endothelial dystrophy or pseudophakic and aphakic bullous keratopathy . clinical data , and donor and recipient characteristics were recorded . of 241 patients who met our inclusion criteria , 68 underwent dsaek and 173 underwent penetrating keratoplasty . the main outcome measure was endothelial cell loss at 1 year . secondary outcome measures were graft survival and visual outcomes at 1-year follow-up.resultsthere were no significant differences in baseline characteristics of patients between the treatment groups . percent endothelial cell loss at 1-year follow - up was greater in penetrating keratoplasty eyes ( 40.9% 2.9% ) compared with dsaek eyes ( 22.4% 2.3% ; p < 0.001 ) . dsaek - treated eyes had significantly superior uncorrected visual acuity ( mean difference = 0.42 0.0059 ; p < 0.001 ) and best spectacle - corrected visual acuity ( mean difference = 0.14 0.032 ; p < 0.001 ) as compared with penetrating keratoplasty - treated eyes . penetrating keratoplasty - treated eyes had worse astigmatism as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 ; p < 0.001 ) . graft survival at 1 year was comparable in both groups , ie , 66/68 ( 97.0% ) dsaek - treated eyes versus 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479).conclusionwe report lower percent endothelial cell loss comparing dsaek and penetrating keratoplasty at 1-year follow - up in asian eyes , with comparable graft survival rates in both groups .

Introduction Materials and methods Surgical technique Statistical analysis Results Recipient characteristics Endothelial cell counts Visual acuity outcomes at 1 year Refractive outcomes at 1 year Graft success Complications Discussion Conclusion

PMC4306294

although the statistical - mechanical foundations of free - energy calculations were laid a long time ago , their practical applications became possible only with the advent of modern computers . from the inception of computer - based free - energy calculations it has been clear to theorists that direct boltzmann sampling of rugged energy landscapes is inefficient . the subsequent development of the field is a history of efforts to remedy this problem . in free - energy calculations , the quantity of interest is almost always the free - energy difference between physical states of the system rather than the absolute free energy of a given state . from this standpoint , calculations can be categorized on the basis of variables used to transform the system between states of interest . then , two main classes can be distinguished , namely alchemical and geometrical transformations . they rely , respectively , on changes of a parameter in the hamiltonian or a function of atomic coordinates . the first class encompasses structural modifications of chemical species that rest upon the remarkable malleability of the potential energy function in molecular - mechanics - based simulations , reminiscent of the fabled ability of alchemists to transmute base metals into noble ones . alchemical transformations are often associated with the free - energy perturbation method on account of the progressive and perturbative nature of the change incurred by the system of interest , although , strictly speaking , alchemical free - energy calculations can be carried out by way of alternate approaches , such as thermodynamic integration . the very first application of alchemical transformations to a nontrivial chemical problem was published nearly 30 years ago by william jorgensen , to whom the present contribution is dedicated . in noteworthy agreement with experiment , this pioneering simulation reproduced the relative hydration free energy of methanol with respect to ethane . the second class of transformations embraces virtually any geometric modification in a molecule or a collection of molecules by means of selected collective variables tailored to address the problem at hand , which could vary from changes in the internal degrees of freedom in a molecule to intricate recognition and association phenomena . such collective variables form the transition coordinate , a low - dimensional representation of a multidimensional mathematical object . the distinction between the two types of transformations is theoretically important . for geometric transformations , the transition coordinate is , in effect , a generalized coordinate , the evolution of which is usually described by hamilton s equations of motion . in contrast , for a parameter in the hamiltonian , no equations of motion naturally exist , although it is possible to extend the formalism of dynamics to include such a parameter . as a consequence , a number of methods for calculating free energy by way of geometric transformations can not be applied to alchemical transformations without such extension . a considerable number of ingenious techniques have been developed to improve the efficiency of mapping free - energy landscapes associated with geometrical transformations along a transition coordinate . a common feature of these methods is their reliance on importance - sampling techniques . the central idea of these techniques is to depart from sampling from the boltzmann distribution defined by the original hamiltonian and , instead , sample from another distribution that favors regions of phase space that would be visited only infrequently but are important to achieving reliable free - energy estimates . because this procedure is clearly biased , it is essential to know how to correct , or unbias , it to recover the true underlying distribution . importance sampling is commonly used not only in statistical mechanics of condensed phases but also in other fields of science , usually as a variance reduction technique most frequently combined with the monte carlo method . probably the most popular , and also the oldest importance - sampling technique used in free - energy calculations is umbrella sampling . it relies on introducing a bias in simulations that favors states corresponding to large values of the free energy along the transition coordinate . local elevation , conformational flooding , metadynamics , and the wang landau algorithm are examples of more recent importance - sampling algorithms , united by the common denominator that a memory - dependent potential disfavors regions of conformational space that have already been frequently visited . in a sense , the adaptive biasing force method rewove the fabric of free - energy calculations of geometrical transformations , as it is characterized by both conceptual and practical simplicity and requires , at least in principle , little intervention from the end user . in spite of apparent similarities with the local - elevation and conformational - flooding strategies in its aim to sample efficiently all values of the transition coordinate , its theoretical underpinnings are quite different , as we will argue further in this paper . furthermore , in contrast with seemingly similar strategies , the adaptive biasing force algorithm requires no prior knowledge of the free - energy landscape at hand . at its core , the adaptive biasing force method is an adaptive importance - sampling strategy in which the quantity being adjusted is the average force acting along the transition coordinate . it helps the system under study escape from kinetic traps in which it would otherwise have remained for a very long time . the method constitutes a highly efficient route to estimating free energies , which , since its inception , has been used to tackle a number of challenging problems of chemical and biological interest , such as mechanical proteins , transport phenomena , or protein ligand and protein protein recognition and association . more generally , it is a versatile , adaptive , importance - sampling strategy that can be utilized in many fields , whenever sampling of a probability measure is thwarted by metastability of the sampling dynamics . in this self - contained contribution , the multiple facets of the adaptive biasing force algorithm are discussed in an exhaustive manner , tackling a number of issues that have not been addressed so far , or only rarely so . in the following section , we present the theoretical foundations of the method , discussed in the context of other free - energy approaches . next , we briefly address a number of practical issues related to a proper choice of the transition coordinate . then , an analysis of the convergence properties of the method and approaches to calculating and controlling statistical errors associated with the calculated free - energy values are presented . the discussion of convergence and errors continues with the focus on nonergodicity scenarios , and ways to identify and circumvent them . subsequently , we examine for the first time how the adaptive biasing force algorithm is used in conjunction with geometrical restraints , which have to be enforced in many problems of interest . finally , we discuss some new strategies for combining thermodynamics and kinetics in importance - sampling simulations , before closing with recommendations for good practices in applying the adaptive biasing force method and an outlook toward further promising statistical mechanical and algorithmic developments . in this section , the essential idea behind the adaptive biasing force algorithm is first explained in terms appealing to physical intuition , followed by the theoretical underpinnings of the method presented in a more formal language . then , the reader is guided through the common expressions for the mean force , and the adaptive algorithm , both of which are at the core of the method . the adaptive biasing force method is aimed at improving the efficiency of molecular dynamics simulations in which the potential energy surface is sampled ineffectively due to free - energy barriers . in practice , these barriers appear as bottlenecks in the dynamics of certain privileged coordinates that describe the transitions between physically important states ( transition coordinates ) . they also cause the system to become trapped in some states for durations exceeding the time scale of the simulation , resulting in incomplete sampling . the free energy along a transition coordinate can be seen as a potential resulting from the average force acting along the coordinate ( i.e. , the negative of the gradient of this potential ) , hence the name potential of mean force . in the formalism of thermodynamic integration , on which the adaptive biasing force is based , the instantaneous force acting along the coordinate may be decomposed into the sum of the average force ( which depends only on the value of the transition coordinate ) and a random force with zero average , reflecting fluctuations of all other degrees of freedom . hence , in a low - dimensional view of the process , the transition coordinate evolves dynamically in its time - independent potential of mean force , and this evolution is driven by the random force . in many instances , the random force can be satisfactorily approximated as diffusive , leading to a simple physical picture in which the system diffuses along the transition coordinate in the potential of mean force . the idea behind the adaptive biasing force algorithm is to preserve most characteristics of this dynamics , including the random fluctuating force , while flattening the potential of mean force to remove free - energy barriers , and thus accelerate transitions between states . this is done adaptively , without any prior information about the potential of mean force . to accomplish this , the instantaneous force acting along the coordinate is calculated , and its running time average is recorded , thus providing an on - the - fly estimate of the derivative of the free energy at each point along the pathway . at the same time , an external biasing force is applied , exactly canceling the current estimate of the average force . over time , as the estimate converges to the average force at equilibrium , the total , biased average force stabilizes at values very close to zero . then , the system experiences a nearly flat potential of mean force and displays accelerated dynamics along the transition coordinate . the fact that the biasing force is exactly equal to the mean force is actually not crucial . what is important is that the biasing force yields sufficiently uniform sampling of the transition coordinate that the remaining barriers can be easily traversed in response to thermal fluctuations . the adaptive biasing force algorithm is not inherently tied to any specific type of dynamics but does rely on sampling of the canonical ensemble . in explicit - solvent simulations , langevin dynamics with sufficiently soft damping and small stochastic forces becomes a mere perturbation of hamiltonian dynamics and may be used as one simple way to achieve canonical sampling . for convenience , but without loss of generality langevin dynamics can be written as1where ( xt , pt ) denotes the positions and momenta of the particles at time t , m is the mass tensor , v : is the potential energy function , is the friction coefficient , wt is the wiener process that underlies the random force ( white noise ) , and = kbt , where kb is the boltzmann constant and t is temperature . the dynamics of eq 1 is ergodic ( under mild conditions on v ) with respect to the canonical measure zp1 exp[pmp/2 ] dp (dx ) , where (dx ) = zx1 exp[v(x ) ] dx . ergodicity means that long - time averages converge to canonical averages:2and that the law at time t of the stochastic process converges to the canonical measure in the long - time limit:3where is the expected value . the first limit in eq 2 is of particular interest for practical applications because it allows for computing canonical averages from trajectory averages . for a typical potential v , the associated boltzmann measure is multimodal : high - probability regions are separated by low - probability regions . the former correspond , for instance , to the most likely conformations of a biological object , which are typically separated by transition regions of very low probability . for these reasons , estimating averages with respect to the probability measure is , in general , a difficult task . in particular , the ergodic properties of the dynamics of eq 1 are not sufficient to devise reliable numerical methods , because under these premises , the stochastic process remains trapped in large - probability regions , and , as a consequence , the long - time asymptotic regime t is very difficult to reach in the ergodic limits in eqs 2 and 3 . the fact that the system remains for a very long time in some region of phase space before hopping to another region is called metastability , and the corresponding states of the system are called metastable . the inability to reach the ergodic limit is often called quasi nonergodicity ; the system appears nonergodic on the time scales of the simulations . a typical example of a metastable state is a local free - energy minimum in the conformational space of a protein . the adaptive biasing force method relies on modifying the potential v in such a way that the energy landscape is flattened along a given transition coordinate : . here , we restrict ourselves to a one - dimensional transition coordinate , leaving generalization to high - dimensional transition coordinates for the section expressions for the mean force . at[(x ) ] and at is updated in such a way that it converges to the free energy a , defined ( up to an additive constant ) by4where the measure (x)z ( dx ) is supported by the subset { x , (x ) = z } and is such that (x)z ( dx ) dz = dx . in practice , the bias is only applied in a window [ zmin , zmax ] as explained in the section justification of a stratification strategy . notice that , by the definition of a , the canonical measure associated with the biased potential v(x ) = c , where c is a constant independent of z. therefore , if the biased potential v(x ) a[(x)]1(x)[zmin , zmax ] is used , the marginal along is a uniform law over [ zmin , zmax ] . here , 1(x)[zmin , zmax ] is an indicator function , which is one when (x ) [ zmin , zmax ] and zero otherwise . let us recall that the marginal law is defined as follows : if x is distributed according to a probability distribution , then the law of (x ) is called the marginal of along . if we knew the free energy a , it would be a good idea to use a as a biasing potential , because sampling along would be easier . this is illustrated in simple two - dimensional examples in figures 1 and 2 . notice in particular that the free energy seems to be a good biasing function for efficient sampling of both energetic barriers ( figure 1 ) and entropic barriers ( figure 2 ) . upper row : ( a ) original two - dimensional , double - well potential displayed as level sets ; ( c ) time trajectory of the first coordinate x in the stochastic process , showing oscillations between the two metastable wells . lower row : ( b ) level sets of the same potential biased by the free energy associated with the transition coordinate (x , y ) = x ; ( d ) time trajectory of the transition coordinate x in the adaptively biased dynamics , showing no metastability . upper row : ( a ) the original two - dimensional potential is zero inside the hourglass shape , and + outside ; ( c ) time trajectory of the first coordinate x in the stochastic process , showing oscillations between the two metastable wells . lower row : ( b ) free energy along the transition coordinate (x , y ) = x , featuring a purely entropic barrier ; ( d ) time trajectory of the transition coordinate x in the free - energy - biased dynamics , showing no metastability . the main ingredient that we now need is an update rule for at such that limtat = a. this is based on the following formula , which defines the mean force , namely , the negative of the derivative of the free energy . more general formulas can be derived , which give rise to many variants of the adaptive biasing force method ( see section expressions for the mean force),5where the instantaneous force is defined by6 equation 5 is a consequence of the definition of the free energy in eq 4 and of the co - area formula ( which is a generalization of the fubini theorem ) ; see for instance ref ( 8) . from eq 5 it follows that a is the conditional average of the instantaneous force , f , with respect to the canonical measure conditioned to a fixed value of the transition coordinate : a(z ) = [ f(x)(x ) = z ] . an important observation is that eq 5 remains true if the potential v is changed to v at : for any function at7 in other words , a biasing potential at depending solely on leaves averages conditioned by unchanged . this is the intuition behind the adaptive biasing force dynamics , which can be written as8 indeed , if ( xt , pt ) were at equilibrium with respect to the biased canonical measure zp1 exp(pmp/2 ) dpzt1 exp[(v at)(x ) ] dx , then at would be equal to a. in practice , of course , the sampling is not sufficiently fast for the process to be instantaneously at equilibrium with respect to the time - varying biased potential v at , and this is why this heuristic is not sufficient to fully understand the convergence of the adaptive biasing force dynamics ( see the section convergence and error analysis ) . however , this simple reasoning is sufficient to check that if at converges to some limit a , then necessarily , a = a. from a practical viewpoint , the conditional expectation in eq 8 can be computed using two procedures : either time averages over a single long trajectory or averages over many replicas run in parallel . these procedures will be discussed in ample detail in the section multiple - walker strategies below . intuitively , the adaptive biasing force dynamics thus consists of adding a force at[(x)](x ) that exactly compensates the average of the original force , v(x ) , along a given transition coordinate . if is well - chosen , the hope is to observe a fast convergence ( at least compared to the original dynamics embodied in eq 1 at equilibrium ) of at to the mean force a. given a transition coordinate (x ) , the mean force is a well - defined quantity , yet its expression as an ensemble average of an instantaneous force f is not unique , as we will see below . in adaptive biasing force simulations , the choice of a convenient expression for f is driven by practical considerations , notably ease of implementation and numerical behavior , such as variance . the classic expression for the instantaneous force involves an explicit coordinate transformation from cartesian to generalized coordinates , which include the transition coordinate . that is , : , with 1 = and components i for i > 1 are generalized coordinates of no particular physical significance , but necessary to the mathematical framework . a valid expression for the instantaneous force is then9which in the physics literature is more commonly written as10where |j| is the determinant of the jacobian matrix ( ij1)(i , j ) . from the arbitrary choice of i , i>1 is derived a ( somewhat arbitrary ) function f , whose -restricted ensemble average nevertheless yields the uniquely defined mean force ( eq 5 ) . equation 10 provides an intuitive view that different choices of and f correspond to different ways of projecting the cartesian forces , v , onto the transition coordinate . the direction along which the forces are projected in this expression is the vector 1 , which we call inverse gradient . as the gradient of can be seen as the changes in corresponding to infinitesimal changes in x , the inverse gradient is the vector along which a change in is propagated in cartesian coordinates , other generalized coordinates ( i , i>1 ) being constant , hence the dependence of the inverse gradient on the explicit coordinate transformation . the alternate notation for the inverse gradient , x/ , has the drawback of hiding this dependence on the choice of . numerically , eq 10 is impractical for two reasons . one is that defining i , i>1 explicitly can be exceedingly difficult , especially if is a collective coordinate ( e.g. , the radius of gyration of a set of particles ) . supposing that this step is done , the second difficulty comes with the numerical computation of the jacobian derivative , as it involves second derivatives of whose analytical derivation and numerical implementation may be cumbersome , again , depending on the nature of the transition coordinate . to circumvent this issue , the original adaptive biasing force method was introduced with an instantaneous force estimator based on a constraint force that is calculated iteratively , but never applied . in the initial implementation of the adaptive biasing force algorithm in the popular molecular dynamics program namd , eq 10 was used because the small set of implemented coordinates made it practical . as this set was greatly extended in the framework of the collective variables module , more versatile expressions of the instantaneous force were required . den otter put forward the idea that the inverse gradient can be replaced with an arbitrary vector field ( satisfying certain requirements ) . in other words , changes in may be propagated along an arbitrary direction in cartesian coordinates , without explicitly defining a complete set of generalized coordinates . consider a vector transition coordinate ( i ) , in the presence of a set of constraints of the form k(x ) = 0 . for each coordinate i , let vi be a vector field satisfying , for all j and k:1112 the ith partial derivative of the free energy can then be calculated as the conditional average of the following instantaneous force:13of which eq 6 is a special case , limited to a single coordinate and choosing v = /|| , which satisfies the condition ( eq 11 ) above . note that this estimator still requires the calculation of second derivatives , in the form of the divergence of the vector field v , although the relative freedom in choosing v can be taken advantage of to make the divergence calculation practical . the choice v = /|| is always valid , and as such , convenient for theoretical purposes , but certainly not always optimal when implementing specific generalized coordinates . expressions that were chosen in practice for those coordinates implemented in the collective variables module are listed in ref ( 39 ) . described an estimator that does not require second derivatives , but rather first derivatives with respect to time and space , and is valid for multidimensional adaptive biasing force calculations . this estimator resembles a statistical form of newton s equation of motion : instead of relating acceleration to the potential energy gradient , it relates the mean acceleration to the gradient of the free energy . in a considerable simplification , only the first derivative of the force with respect to needs to be derived . the time derivative is calculated numerically in the same fashion and at the same level of accuracy as time derivatives of other quantities in molecular dynamics . other ways of simplifying calculations of instantaneous forces will be discussed in the context of extended adaptive biasing force simulations , or eabf , in the section the extended adaptive biasing force method . the final , essential ingredient of the theory underlying the adaptive force method is an algorithm for deriving the current estimate of the average force as simulations progress . in its generic , one - dimensional implementation , the transition coordinate , , connecting two end points , is divided into m equally sized bins of width in which forces are accrued in the course of the simulation . in a nave approach , the approximation to the average force , f(nstep , k ) in bin k after nstep molecular dynamics steps is just the simple , unweighted average of all force samples in this bin14provided that the bin has already been visited at least once . nstepk is the number of samples accrued in bin k after nstep steps and fk abbreviates the th force sample in this bin . however , when only a few samples are available in a given bin k , the running average might be a poor estimate of the actual average force in this bin . large fluctuations in the running estimate of the average force are undesirable , as they may drive the system away from equilibrium , thus slowing the convergence of the algorithm and reducing the efficiency of the method . to control these effects , a procedure is needed to reduce variations in early estimates of f(nstep , k ) . a number of schemes can be applied for this purpose . in current implementations , the biasing force in bin k at time t is applied in full only if the number of samples ntk is above a threshold , nfull . it is ramped up smoothly as ntk varies from 0 to nfull . in one implementation , the ramp is linear and the force is proportional to ntk / nfull ; in another , the biasing force is zero for ntk < nfull/2 and is ramped linearly above that value , proportionally to 2ntk / nfull 1 . both implementations have proven to be efficient in a number of applications , but other , more advanced schemes are possible . so far , there have been no systematic studies on the efficiency of different adaptive algorithms , but it is anticipated that it may be strongly system - dependent . for a sufficiently large nstep , f(nstep , k ) approaches the correct average force in each bin . then , the free - energy difference , a , between the end point states can be estimated simply by way of summing the force estimates in individual bins.15if the average force is a rapidly changing function of , a more sophisticated integration algorithm may be required . it might be also possible to develop binless integration algorithms , similar to those proposed for some other free - energy calculation methods . a common trait of importance - sampling algorithms is the discretization of the transition coordinate , , in bins of width in which statistical information is accrued in the course of the simulation . in the umbrella - sampling scheme , for instance , a histogram is constructed , corresponding to the biased probability of occurrence of the molecular assembly of interest at the different values of the transition coordinate . in the adaptive biasing force algorithm , bins are utilized to store instantaneous values of the thermodynamic force that acts along the transition coordinate . as has been observed in practice previously for diffusive dynamics , the instantaneous force in any given bin obeys a normal distribution . at thermodynamic equilibrium , by definition , its average is exactly equal to a(z ) , that is , the gradient of the free energy along the transition coordinate . insisting upon being at thermodynamic equilibrium is pivotal here , as application of a poorly estimated time - dependent bias , i.e. , from a distribution out of equilibrium , will not yield a hamiltonian bereft of a mean force acting along the transition coordinate . the adaptive algorithms described above contain two adjustable parameters : bin width , , and the number of samples , ninit , below which r(nstepk ) is not equal to 1 or , equivalently , averaging does not follow eq 14 . the choice of these parameters should be done with some care . at constant , large values of ninit yield better estimates of the average force once the number of samples collected in a given bin reaches this threshold value and conventional averaging begins , at the price of delayed application of the full averaging and slow , initial progress along the transition coordinate . small values of ninit , in turn , tend to drive the system out of equilibrium . typically , setting ninit in the range between 200 and 500 appears to be a good compromise that allows for avoiding both types of problems . at constant ninit , large values of this may have adverse effects on the accuracy of integration in eq 15 . on the other hand , small values of require longer simulation times to collect sufficient force statistics in every bin . if the transition coordinate is a distance in an atomistic system , the choice of as 0.1 or 0.2 usually represents a satisfactory trade - off . in the last four decades a number of strategies have been developed for computing free - energy changes as a function of geometrical transformations , each endowed with advantages , as well as limitations . to a large extent , umbrella sampling , whether in its original formulation or variants thereof , remains one of the most widely utilized routes to address rare events in molecular simulations . in its original form , umbrella sampling referred to incorporating an external biasing potential in the simulation , i.e. , an umbrella , ideally the negative of the free energy , which would yield a broad , if not uniform exploration of the transition coordinate . in other words , in ideal circumstances , the system would evolve in the collective - variable space on a flat free - energy hyperplane , as is also the case for the adaptive biasing force method . under most circumstances , however , the form of the optimal umbrella is unknown . thus , for any qualitatively new problem , the end - user must resort to an educated guess regarding the shape of the biasing umbrella potential , usually on the basis of prior knowledge of this and related problems . this may constitute a daunting task . poorly predicted biasing potentials yield nonuniform probability distributions across the transition coordinate . this decreases the efficiency of umbrella sampling , which , in extreme cases , may reduce rather than improve the efficiency compared to results with unbiased calculations . this common shortcoming led to the development of an adaptive variant of the umbrella - sampling algorithm , wherein the initial guess of the biasing potential is progressively refined in light of a series of short simulations . adaptive umbrella sampling is one member of a broader family of techniques called adaptive biasing potential methods . local elevation , conformational flooding , or its more recent avatar , metadynamics , adaptive biasing molecular dynamics , and the wang the idea is to penalize the already visited states by changing the potential v to v at , at(z ) being related to the occupation time of the value z of the transition coordinate up to time t. the longer the time spent in a bin { x , (x ) [ z0 , z1 ] } , the larger the biasing potential at(z ) , z [ z0 , z1 ] . the biasing potential at is built as a sum of gaussian kernels that are periodically added to the hamiltonian along the variable . this pushes the system away from states that have already been visited and , by doing so , improves sampling . it ought to be noted that the potential at , rather than its derivative , is computed in these two cases , hence , the name adaptive biasing potential methods . one important downside of the adaptive biasing force algorithm , compared to the class of adaptive biasing potential methods , lies in its inability to handle discrete transition coordinates , for instance , coordination numbers . this drawback can be understood by considering that the free energy is now a map from integers to reals , and , thus , has no derivative and , hence , no mean force . from a mathematical viewpoint , the adaptive biasing force method , just like adaptive biasing potential methods , is an adaptive importance - sampling procedure . there is , however , a salient difference between these two techniques . in the latter , the potential of mean force or , equivalently , the corresponding probability distribution along the transition coordinate is being adapted . in contrast , the former relies on biasing the force , i.e. , the gradient of the potential . this difference is more important than it might appear at first sight , as potentials and probability distributions are global properties whereas gradients are defined locally . in terms of probability distributions , it means that the count of samples in the neighborhood of a given value of the transition coordinate is insufficient to estimate probability . knowledge of the underlying probability distribution over a much broader range of is required . this may considerably impede efficient adaptation . in contrast , all that is needed to estimate the gradient is the knowledge of local behavior of the potential of mean force . thus , in many instances , adaptation proceeds markedly faster . using a common metaphor , the difference between the adaptive biasing potential and adaptive biasing force methods can be compared to inundating the valleys of the free - energy landscape as opposed to plowing over its barriers to yield an approximately flat terrain , conducive to unhampered diffusion . for example , in metadynamics and its ancestors , the widths and weights of gaussian functions and the frequency with which the biasing potential is updated have to be carefully chosen , which often requires considerable experience . in the adaptive biasing force method , estimating the biasing gradient happens automatically by way of a simple algorithm , described in the previous subsection . another technical concern about adaptive methods is to ensure that adaptation vanishes once at approaches the converged free energy . there are a number of ways to fulfill this condition more - or - less automatically , but adaptive biasing force and adaptive biasing potential techniques remain intrinsically different from this point of view . in the adaptive biasing force algorithm , if the correct free energy is given as an initial guess ( namely if v is replaced by v a in eq 8) , then the biasing force will not be updated ( at in eq 8 will be constant over time ) . moreover , if the derivative of the biasing potential is needed ( for example to bias the langevin dynamics as in eq 8) , the advantage of the adaptive biasing force algorithm is that at is directly computed , whereas in adaptive biasing potential algorithms , one needs to differentiate the evaluated biasing potential at , which may lead to very noisy results because at is estimated along a stochastic trajectory . because the basic quantity calculated , and subsequently integrated , in the adaptive biasing force method is the force , this approach belongs to the thermodynamic integration class of methods . however , in contrast to conventional implementations of thermodynamic integration and its generalizations , such as the blue - moon ensemble approach , the adaptive biasing force algorithm does not rely on constrained molecular dynamics , but instead is based on unconstrained simulations ; i.e. , the free - energy difference is not determined at discrete values of the transition coordinate through solving constrained equations of motion . sampling of the transition pathway proceeds in a continuous , unhampered fashion , guided by the diffusion properties of the system of interest , obviating the need for re - equilibration at fixed , predefined values of the transition coordinate , even in stratified simulations . as will be discussed further in this paper it is also of interest to compare the adaptive biasing force method with reconstructions of free - energy landscapes from nonequilibrium trajectories that represent repeated pulling experiments . the latter are based on the groundbreaking jarzynski identity , or its extension to bidirectional transformations , combined with steered molecular dynamics . even though both approaches involve molecular dynamics trajectories that are initially away from equilibrium , there is a fundamental distinction between them . in the adaptive force method , only the average , or systematic force is removed to erase the ruggedness of the free - energy landscape , preserving the random force responsible for diffusion . once a good estimate of the average force becomes available , the equilibrium behavior of the system is restored . in pulling experiments , the transformation always proceeds away from equilibrium at constant velocity and , therefore , the instantaneous force acting along the transition coordinate is nil . the random force actually appears in the formalism after averaging over the ensemble of pulling experiments . moreover , achieving convergence in calculations based on jarzynski s identity usually requires large numbers of independent realizations , which comes at a significant computational cost . taken together , when a geometrical transformation can be undertaken at equilibrium , it is not clear whether there is any practical advantage of handling the problem at hand by means of nonequilibrium work experiments rather than the adaptive biasing force method . an important advantage of gradient - based methods is the possibility of formally decomposing the free - energy change into physically meaningful contributions , thereby helping to dissect qualitatively the nature of the intermolecular interactions at play . it is worth noting that different energy terms contribute to the mean force both explicitly through force terms and implicitly through the boltzmann weights in the canonical average ; contributions can only be separated numerically at the former level , not at the latter . decomposition of the free energy is generally handled a posteriori through computing the thermodynamic force between , for instance , groups of atoms of interest . this force is then projected onto the transition coordinate determined for each stored configuration , prior to the construction of a histogram from which the average force is inferred . integration of the latter yields the desired contribution to the total free - energy change across the entire transition pathway . among many options for reconstructing free - energy landscapes along a transition coordinate , which one is the best ? considering that the efficiency of different methods strongly depends on their implementation in software packages and , very likely , on a system of interest , attempts to answer this question appear somewhat misguided and unproductive . that said , one aspect of the adaptive biasing force algorithm pleading in its favor is its simplicity . how the algorithm operates is physically intuitive , requiring , in principle , very little prior knowledge of the free - energy landscape , or input from the end - user , even for qualitatively new problems . central to geometric transformations is the concept of a transition coordinate . in this section , this concept is illuminated in the context of free - energy calculations aimed at tackling rare events . specifically , we will discuss how the transition coordinate is explored with the adaptive biasing force algorithm and delve into the practical aspects of defining this coordinate . stratification , a common technique for improving the efficiency of free - energy calculations by partitioning the reaction pathway into ranges of the transition coordinate , will be discussed with the focus on the justification for and limitations of this strategy . for any transition from a macrostate , a , to another macrostate , b , of the same system there exists an exact one - dimensional transition coordinate : the committor probability . in most cases , this coordinate is difficult to calculate and usually offers very limited insight into the nature of the process of interest . for these reasons , it is often more useful to employ a transition coordinate that is only an approximation to the committor probability but is physically more meaningful and easier to handle . sometimes it might be helpful to extend the reduced representation of the transition to a transition coordinate that extends beyond one dimension . not only physical significance but also efficiency of sampling , given a transition coordinate , is of concern . as in any enhanced sampling method based on a reduced representation , adaptive biasing force sampling relies on stochastic exploration along the transition coordinate , , enhanced by the adaptive bias , combined with equilibration of other , orthogonal degrees of freedom . equilibration in the orthogonal space is critical in two respects : it affects the mobility along ( see figure 1b for a diffusive example ) , and it determines the rate of convergence of a(z ) = f(x) , which is an average over the orthogonal degrees of freedom . in the ideal situation of time scale separation , all slow degrees of freedom are captured by the transition coordinates , and relaxation in the orthogonal space is comparatively fast . in other words , the adaptive biasing force algorithm removes metastability along the transition coordinates , provided that other degrees of freedom are not metastable . complex systems such as biological macromolecules , however , possess many slow , coupled degrees of freedom , making time scale separation difficult or impossible to achieve . fortunately , empirical results suggest that time scale separation is not an absolute requirement of adaptive biasing force sampling . one reason behind it might be that enhanced diffusion in the transition coordinate space reduces metastability in the orthogonal space , by letting the dynamics sidestep orthogonal barriers rather than cross them , making some multichannel cases ( see the section hidden barriers and other challenges to obtaining accurate results ) tractable with standard adaptive biasing force simulations . more encouraging still , convergence in such multichannel cases can be markedly accelerated by multiple - walker formulations of the adaptive biasing force algorithm ( see the section multiple - walker strategies ) . yet as will be extensively discussed further in this paper , the end - to - end distance of the -helical deca - alanine peptide provides a good example of an inadequate transition coordinate . depending on the range of values , the coordinate exhibits completely different behavior . for values corresponding to the stable -helix ( 14 ) and larger , separation of time scales is obeyed and the adaptive biasing force converges well . in contrast , smaller values of the end - to - end distance correspond to a rich set of metastable , compact states that are not resolved by the transition coordinate . as a result , adaptive biasing force dynamics becomes trapped in these states , and the free - energy estimator does not converge in accessible simulation times . attempts to resolve these metastable states by two- and three - dimensional coordinates give improved results , allowing the exploration of all metastable basins , yet those basins are not all resolved even in three dimensions . these difficulties are not due to specific deficiencies of the adaptive biasing force method , but rather , to shortcomings of the reduced representation , which would constitute an obstacle to any sampling method . in practice , finding an effective reduced representation is still a process largely guided by physical intuition about the process of interest , as well as trial - and - error . more systematic and robust approaches for this dimension reduction step are an area of active research . a limiting factor is often the availability of usable numerical implementations of the generalized coordinates of choice . the collective variables module is an attempt to overcome this limitation , by providing a rich and flexible toolbox to define many types of coordinates , in particular those useful for the description of biological macromolecules . in this module , once an intuitive understanding of the relevant generalized coordinate has been obtained , some technical choices remain to be made to express this coordinate as a function of atomic cartesian coordinates . though these decisions may seem ancillary , they have a strong influence on the accuracy , convergence , and computational performance of the adaptive biasing force algorithm . when objects of interest are composed of many atoms , there are often several nearly equivalent ways to define the transition coordinate . in sufficiently long simulations , different definitions produce nearly identical potentials of mean force , but the efficiency might vary considerably . for example , the distance between two proteins could be defined by selecting one central atom in each protein , or by selecting the centers of mass of large groups of atoms . the largest contribution to the instantaneous force on each atom in a molecule is due to rapidly oscillating bonded interactions ; more generally , in all applications , forces on the particles will contain some background noise . if many atoms contribute to the projected force ( e.g. , the first right - hand - side term in eq 13 ) , contributions from those noisy terms average out , which lowers the variance of the instantaneous force estimator . in the initial stage of an adaptive biasing force simulation , nonequilibrium effects occur if the biasing force applied to some degrees of freedom varies faster than coupled degrees of freedom can relax . collective variables that involve many cartesian coordinates with smoothly varying contributions to the gradient ( hence , to the biasing force vector ) . at equilibrium , a smooth motion may be described by a nonsmooth variable and this may make the convergence of the adaptive bias more difficult . in the deca - alanine stretching toy example , the geometric process of interest involves all atoms in the peptide , yet our classic approach uses the end - to - end distance as a biasing coordinate , with the implicit assumption that biasing forces exerted on the terminal atoms propagate , and that the entire peptide relaxes rapidly , so that the biased trajectory remains close to equilibrium . a more robust approach is to replace that coordinate with the radius of gyration of the peptide . the gradient of the radius of gyration has components on each atom proportional to its distance from the center of the group , so that atoms close to the center also experience moderate biasing forces and do not lag behind the terminal atoms when the biasing force varies rapidly over time . a more elaborate discussion of this problem can be found in ref ( 42 ) . in some cases , however , coordinates involving large collection of atoms will have less resolution than more local ones . a biophysical example is permeation through an interface , such as a lipid bilayer . the common choice of transition coordinate is the distance of the permeant molecule to the center of the bilayer center , projected onto the bilayer normal . in such a case , the physically relevant phenomenon is interaction of the permeant with the membrane surface , on a local scale . if the bilayer patch is large enough , it will experience fluctuations away from planarity , thus the local position of the interface will fluctuate with respect to the bilayer center . in turn , this will cause spurious fluctuations in the transition coordinate . a comparable situation arose in a study of glycerol permeation through the water channel protein glpf . interaction of the permeant with the protein depended on distances to neighboring pore - lining residues , which fluctuated with respect to the bulk of the protein . therefore , the global coordinate measured between the protein and glycerol molecule had insufficient resolution on a local scale , and a more local coordinate had to be defined to resolve the structure of the free - energy profile in the constricted region of the selectivity filter . depending on system size and implementation details , choices of coordinates may impact performance noticeably . a common application case is a biomolecular simulation performed with the namd package , with the adaptive biasing force algorithm implemented in the collective variables module . namd is highly parallelized and can simulate large systems on supercomputers with nearly linear scaling . the current implementation of the adaptive biasing force algorithm is not parallelized and runs on one node , leading to two potential bottlenecks : ( 1 ) poor serial performance on the master node : for the most expensive variables ( e.g. , those involving sums on atom pairs ) , the bias calculation on the master node might take longer than the force calculation on other nodes . ( 2 ) scaling may suffer even for computationally simple coordinates , if too many atom coordinates and forces have to be communicated across nodes , increasing latency . this second case may affect any highly parallel application with coordinates defined on many atoms . in practice , one often has to find an acceptable trade - off between variance and performance by selecting a reasonable number of cartesian coordinates that are most representative of the quantity of interest . to describe conformational fluctuations of a protein , for example , the root - mean - square deviation of carbon coordinates is often a good compromise . to increase the efficiency of exploring the transition coordinate in adaptive biasing force or umbrella sampling , it is common to break down the transition path into a series of sequential strata or windows . this idea arises from the intuition that the time to convergence grows as the square of the range of the transition coordinate . convergence of the free energy over the entire range is achieved after t0 . let us now divide the transition path into n nonoverlapping windows of lengths , for which convergence is attained after t1 , ... , tn. as shown in the supporting information , t0 > iti. we illustrate this result in a simple example of a tagged water molecule diffusing in a bulk environment over a stretch of 20 . the transition coordinate is the projection of the distance separating the centers of mass of the tagged water molecule and the simulation cell along a given direction of cartesian space . translational invariance due to the isotropic nature of the liquid imposes the condition that the free - energy change along the transition coordinate be zero . for this system , the potential of mean force was determined in a single , 20 long stratum , two 10 strata , four 5 strata , and eight 2.5 strata . the simulations continued until the root - mean - square deviation between the computed potential of mean force and the reference zero free - energy profile was less than 0.1 kcal / mol . as can be observed in figure 3 , t0 , the time necessary to attain convergence within this preset tolerance without stratification is on the order of 100 ns . when the transition coordinate is divided into two strata , convergence in each 10 stratum is reached in approximately 40 ns , i.e. , in 80 ns over the full 20 range . the effect of stratification increases further , as the reaction coordinate is decomposed into four and eight windows . in these cases , convergence is achieved in approximately 6 and 2 ns per window , respectively , which corresponds to 24 and 16 ns for the complete 20 range . stratification strategies for the translationally invariant toy model of a tagged water molecule diffusing in a bulk aqueous medium . the transition path spans 20 and is handled in a single window ( a ) , in two 10 windows ( b ) , in four 5 windows ( c ) , and in eight 2.5 windows ( d ) . for each stratification strategy , a potential of mean force calculation is carried out until the root - mean - square deviation with respect to the accurate zero free - energy profile is less than 0.1 kcal / mol . both theoretical considerations and a simple example given above appear to suggest that extensive stratification should be always preferred . first , simulations in each window require initial equilibration , which may erase benefits gained from stratification into many windows . perhaps more importantly , extensive stratification may impede ergodic sampling of the phase space . this behavior , shared with the standard thermodynamic integration , will be discussed in section addressing nonergodicity scenarios . in contrast to umbrella sampling and its adaptive variants , the adaptive biasing force algorithm does not require that consecutive windows of a dissected transition coordinate overlap . provided that convergence has been achieved in each window , the gradient , a( ) , can be reconstructed merely by joining the gradients from individual windows at the boundaries . this improves efficiency , as the requirement for overlap between windows may frequently add as much as 50% to the total simulation time . if the gradient between consecutive windows is not continuous to within statistical error , this is usually a sign of difficulties with ergodic sampling . again , this problem will be considered in section addressing nonergodicity scenarios . in this section , we examine the convergence properties of the adaptive biasing force algorithm and the reliability of the computed free - energy estimates . first , the reader is invited to follow a demonstration that the numerical scheme formally converges . then , we discuss how statistical errors associated with the reconstructed free - energy landscapes can be measured and managed . the aim of this section is to explain convergence properties of the adaptive biasing force algorithm . for more details , we consider the overdamped langevin dynamics,16where xt is defined in the n - dimensional torus ( namely , in [ 0 , 1 ] with periodic boundary conditions ) and (x1, ... we direct the reader to refs ( 67 ) and ( 68 ) for extensions to more general situations of the results presented below . starting from eq 16 and using the above choice of the transition coordinate , the adaptive biasing force dynamics can be represented as17where xt1 denotes the first coordinate of the vector xt , e1 is the vector with coordinates ( 1 , 0 , ... , 0 ) , and 1v denotes the partial derivative of v(x1, ... ,xn ) with respect to x1 . as explained in the section theoretical backdrop , it is clear at least formally that the only possible stationary state for at is the free energy ( up to an additive constant ) . indeed , if at converges to some stationary state a , then the law of xt converges to z1 exp({v(x ) a[(x ) ] } ) dx , which implies that ( 1v(xt)xt1=x1 ) is a(x1 ) . yet , this does not provide a proof of the convergence of at to a , and it does not explain why the adaptive biasing force dynamics of eq 17 indeed converges faster to equilibrium than the original , unbiased dynamics of eq 16 . one way to understand for the original dynamics of eq 16 , the density satisfies the fokker planck equation:18for the adaptive biasing force dynamics , the density satisfies19 it ought to be noted that eq 19 is a nonlinear partial differential equation ( pde ) , which makes the study of its long - time behavior much more complicated than for the linear fokker using appropriate mathematical tools ( namely , entropy techniques , see the supporting information ) , one can show that if the transition coordinate is well chosen , the convergence to equilibrium for the adaptive biasing force dynamics of eq 19 is much faster than for the original unbiased dynamics of eq 18 . roughly speaking , the assumption on the transition coordinate is that the canonical measure z exp[v(x ) ] dx is more multimodal than the conditional measures at a fixed value x1 of the transition coordinate this is typically the case for the simple illustrative two - dimensional potentials in figures 1 and 2 if (x1,x2 ) = x1 . this can be fully quantified , and it actually gives a way to measure the quality of the transition coordinate . in the analysis outlined above , it is assumed that the conditional expectation appearing in the adaptive biasing force dynamics is computed exactly . this analysis is therefore well adapted to discretizations that involve many replicas in parallel , which indeed converge to the adaptive biasing force dynamics with the exact conditional expectation ; see ref ( 69 ) . analysis of the adaptive algorithms ( adaptive biasing force or adaptive biasing potential ) with estimates of the conditional expectations based on trajectory averages along a single path are much more complicated . see refs ( 70 ) and ( 71 ) for preliminary results for the wang just like any experimental measurement , free - energy calculations , of either alchemical or geometrical nature , ought to be reported with the associated error bars . in the absence of an error estimate , a free - energy difference is generally of limited utility , making direct comparison with experiment difficult and speculative . although much effort has been devoted in recent years to the characterization of errors that are associated with free - energy calculations , estimating the reliability of such calculations remains an intricate task . this explains why it is not unusual that calculated free energies are still being published without error estimates . ideally , any free - energy difference should be determined from a series of n independent simulations . if this were , indeed , the case , the best possible estimate of the target free - energy difference would be the expected value over the n simulations , i.e. , , where denotes the estimate of the exact quantity , a , inferred from one individual free - energy calculation . then , the associated mean - square error can be written as20the first term of eq 20 , = , is the variance , or the precision of the free - energy calculation . in other words , it is a measure of its statistical error . the second term , , is the square of the bias of the free - energy estimator , i.e. , the square of the difference between the expected value of the estimator and the actual free - energy change , a . the bias , also referred to as the accuracy of the free - energy calculation , is a measure of systematic error of the latter . how to estimate statistical error for the adaptive force method will be discussed in the next section . one important source of bias arises from incomplete sampling in finite - length simulations due to quasi nonergodic behavior of the system . although this behavior can not be quantified , in many instances it can be detected . then , a number of remedial tools are at our disposal . how to recognize and remedy problems with quasi nonergodicity will be discussed in section addressing nonergodicity scenarios . other common sources of bias are inaccurate treatment of intermolecular interactions and algorithmic artifacts arising primarily from imprecise numerical integration of the equations of motion . these contributions to bias , which are common to all simulation methods , will not be discussed here . the adaptive biasing force method is typically applied to nanoscale molecular processes under physiological conditions , a realm dominated by thermal noise . thus , estimating free - energy differences using adaptive biasing force requires careful consideration of statistical error . within the adaptive biasing force framework , free - energy differences are determined by integrating the estimated mean force of the system exerted along the transition coordinate . namely , a free - energy difference on the interval za zb can be expressed as21where fz is the average of the instantaneous force on the transition coordinate at the position (x ) = z. thus , to determine the statistical error of the free - energy differences , we must delve into the statistics of the mean system force . we assume that the transition coordinate , , is discretized and instantaneous forces calculated during the course of the simulation are collected in appropriate bins along . as derived in the supporting information , one way to estimate the error of the mean force in bin i is given by22where f(xt ) = f(xt ) fi is the random component of the instantaneous force , ni is the number of samples accrued in bin i , t is the time step of the simulation , and i and f2i are the autocorrelation time and variance of f(xt ) in bin i. given a reliable estimate of the error of the mean force in each bin , we are prepared to analyze how these estimates are propagated to yield free - energy differences . on a discrete grid along the transition coordinate , the integral in eq 21 becomes23where ia and ib are bin indices delimiting the -interval [ za , zb ] . assuming independent behavior in each bin , the error of a sum of mean forces is approximated from from the bienaym formula as equal to the square root of the sum of squares of the errors of these mean forces,24 a notable property of this formula is that the error increases with the size of the interval over which the free - energy difference is calculated . for concreteness , we now consider the error in free - energy differences for the reversible folding of deca - alanine in vacuum . we define the transition coordinate of interest as the end - to - end distance of the peptide , specifically the distance between the carbonyl carbon atoms of the first and the tenth residue . below we discuss the behavior of the three quantities that enter eq 22 , namely . the number of samples in each bin ni , the standard deviation of the random force ( f2i ) , and the autocorrelation time of this force i . the black curve in figure 4a is the number of samples in each bin of width = 0.1 for the adaptive biasing force calculated in the range from 12 to 32 ( the red curve will be discussed later in the paper ) . the number of samples , ni , in this range varies from 17 000 to 36 000 . thus , as expected from the adaptive biasing force algorithm , the number of samples in each bin approaches uniformity . in this case , nonuniformity of sampling exceeds only slightly a factor of 2 , which corresponds to the variations of the biased free energy not exceeding 0.5 kcal / mol . for comparison , the unbiased free energy changes in the same range by approximately 30 kcal / mol . in figure 4b , we show the distribution of instantaneous forces in four different bins . the forces in each bin are approximately normally distributed , with similar standard deviations of about 20 kcal mol . this point is underscored in figure 4c , which illustrates that , for deca - alanine in vacuum with langevin dynamics emulating buffeting of the molecule by solvent , the standard deviation of the force acting along the transition coordinate changes very modestly , even though the peptide explores structures that are as disparate as can be imagined . the peptide courses through different compact forms for < 10 , remains mostly -helical for 10 < < 16 , and forms extended structures with diminishing helical fractions as increases beyond 16 . the approximate uniformity of f2i seen here is also characteristic of many other systems . for example , it has been previously found that the standard deviation of the instantaneous force on the center of mass of a water molecule is about 2 kcal mol , irrespective of whether the molecule lies in the bulk aqueous phase or in the hydrophobic core of a lipid bilayer . on the other hand , the transfer of a solute across a liquid vapor interface is an example of a transition for which f2i is expected to vary considerably with . coordinate dependence of sampling and the system force distribution in reversible folding of deca - alanine . ( a ) samples in each bin for a 10 ns adaptive biasing force calculation on the domain [ 4 , 32 ] ( black curve ) and a 100 ns calculation on the domain [ 4 , 32 ] ( red curve ) . note the logarithmic scale on the vertical axis . ( b ) distribution of the -component of the instantaneous system force for different ranges of . ( c ) standard deviation of the -component of the instantaneous system force as a function of . note that f2i is considerable . because this term enters prominently the expression for statistical error in eq 22 , there are significant merits in reducing the dispersion of instantaneous forces . first , because the expression for the ensemble average of instantaneous force is not unique we can , in principle , choose one that reduces f2i . second , variation of forces can be also reduced by a thoughtful choice of the transition coordinate . the third variable in eq 22 , the correlation time of the instantaneous system force i , is also the most difficult to calculate . the sampling in a single bin is rarely sufficient to obtain a converged autocorrelation function of the system force ; thus , in figure 5a we plot the autocorrelation function averaged over 40 bins along different regions of . the correlation time for each region , , is determined by fitting an unscaled exponential function e to the positive values of the autocorrelation function . coordinate dependence of correlations in the system force in reversible folding of deca - alanine . ( a ) autocorrelation functions of the random component of the instantaneous system force for different ranges of . the functions are normalized by the variance f2i ( b ) correlation time for -ranges in panel a. using the calculated values of ni , f2i and i , we now estimate the uncertainties of free - energy differences between deca - alanine structures with different end - to - end distances . the mean system force , with uncertainties calculated by eq 22 , is shown in figure 6a . because only free - energy differences between two points along , rather than free energies at single points , have a clear physical meaning , we focus on errors associated with these differences . to compute the error in the free - energy difference at points za and zb , we must accumulate the uncertainties of the force between these two points , in accord with eq 24 . if stratification is used and points za and zb are in different windows , then the bienaym formula needs to be used across all windows separating these points . propagation of error in the mean force to the error of free - energy differences . ( a ) mean system force on for a 10 ns adaptive biasing force calculation , with error bars determined according to eq 22 . we show the estimated error of a between the -helical minimum free - energy state ( za = 14 ) and other states within the interval zb [ 12 , 32 ] . if we want to calculate , for instance , the difference in free - energy between the minimum and = 16 , we obtain 3.6 1.8 kcal / mol . larger distances in yield larger error : the free - energy difference between the minimum and the plateau at 25.5 is 19 4 kcal / mol . it is expected that , for sufficiently long total simulation time , t , statistical errors of both the average forces and free - energy differences will decay proportionally as t. the same dependence on t should apply to deviations from nonuniform sampling . if force statistics is collected in m bins , then the root - mean - square deviation from the uniform distribution can be defined as the square root of the variance , var(t),25where nstepk and nstep are the number of samples in bin k and the total number of samples at time t , respectively . for large t , the variance is expected to be proportional to 1/t or , equivalently to 1/nstep . in other words , an example of how var(t ) behaves in a typical simulation is shown in figure 7 . errors that clearly deviate from such behavior are a sign of insufficient sampling . if the problem persists with increasing t , and especially if errors exhibit large fluctuations , then most likely problems with quasi nonergodic behavior have been encountered . var(t ) as a function of the number of molecular dynamics steps , nstep . this quantity was calculated for the permeation of k through a transmembrane hexametric channel of a peptaibol , trichotoxin in a window along the normal to the membrane spanning the range between z equal to 15 and 9 . note that for nstep between 0.6 10 and 2.3 10 , nstep var(t ) differs from its average value in this range by no more than 10% . the inset : the number of force samples along z in the window as a function of nstep . a common manifestation of pathological free - energy calculations , in particular those of geometrical nature , is quasi nonergodicity , wherein sampling along the selected transition coordinate appears to be hampered . here , we inspect closely the effects that impede accurate results to be obtained from free - energy methods , including the adaptive biasing force scheme ( notably hidden barriers in the slow manifolds ) , and discuss how to identify these effects and outline possible remedies , by increasing the dimensionality of the transition coordinate , improved stratification , or sampling aided by multiple - replica strategies . the primary objective of importance - sampling schemes is to facilitate exploration of the transition pathway with a uniform probability . among these schemes , as has been previously emphasized , the adaptive biasing force algorithm uses a local estimate of the gradient , a , acting along the transition coordinate , to erase progressively the original ruggedness of the free - energy landscape . as has already been discussed in section formal convergence of the adaptive biasing force algorithm , this feature is valid from a theoretical standpoint . how true is this in practice ? in most instances , satisfactorily uniform sampling is achieved quite efficiently . occasionally , however , the adaptive biasing force algorithm does not perform as expected . the reminder of this section is devoted to explaining and identifying these special , yet important cases . potential difficulties in applying the adaptive biasing force algorithm are intimately related to the choice of transition coordinate . a basic , yet seldom verified assumption that underlies this choice is the separation between time scales of motions along the transition coordinate and orthogonal degrees of freedom ( see section transition coordinates and rare events ) . for complex , rugged free - energy landscapes , notably those formed by parallel valleys separated by considerable barriers in the direction orthogonal to the transition coordinate ( figure 8) , assuming time scale separation may turn out to be unwarranted . because the adaptive biasing force algorithm exerts no direct action in the orthogonal space , it will not improve sampling at constant . returning to the foundational expression for the adaptive biasing force method , eq 8 , which relates the gradient of the free energy to an ensemble average at constant value of the transition coordinate , the inability to cross hidden barriers in the orthogonal space is tantamount to incomplete ensemble averages and , hence , poor estimates of free - energy changes . common free - energy landscape featuring parallel valleys , collinear to the transition coordinate , . these valleys are separated by substantial free - energy barriers in the direction , orthogonal to . can be interpreted as a slow degree of freedom coupled to and hampering progression along the latter direction . excessively stratified reaction pathways preclude spontaneous crossing of high barriers , typically a1 . wider windows should allow diffusion toward values of , where the barrier separating valleys in the direction is smaller , typically a2 . their presence is a guaranteed sign of flawed free - energy calculations , but their absence is not a sufficient condition to ensure that such calculations converged to the correct value . as has been discussed previously , sampling along the transition coordinate in well - behaved simulations should approach uniformity with time , and the statistical error associated with the biasing force or , equivalently , the free - energy differences should decrease in a predictable fashion . if this is not the case , difficulties in equilibrating the system along orthogonal degrees of freedom are , most likely , at play . to illustrate this point , we return to the toy model of deca - alanine reversibly folding in vacuum and to figure 4a . in the range of between 12 and 32 , in contrast , in the [ 0 , 12 ] range , sampling remains quite nonuniform , even after 100 ns ( red curve ) . as has already been pointed out in the section transition coordinates and rare events , there are a number of metastable states in this region , all corresponding to similar values of . difficulties in properly averaging over these states markedly impedes equilibration along . in the context of stratified simulations , the presence of hidden barriers along degrees of freedom orthogonal to often leads to discontinuous biasing forces between adjacent windows . another strategy for exposing apparent nonergodic behavior is to carry out bidirectional calculations , i.e. , initiate the adaptive biasing force simulation from both end points along the transition coordinate . just as in free - energy perturbation calculations , the resulting hysteresis is a good indicator ( although not necessarily a measure ) of error . if the hysteresis markedly exceeds statistical error , the calculated free - energy values are , most likely , poorly converged . in such a case , simply combining data from both directions is not likely to improve accuracy significantly , as their proper weighting remains unknown . when quasi nonergodicity scenarios are encountered , additional simulation strategies , such as multiple walkers or multidimensional transition coordinates , should be brought to bear . formally , the adaptive biasing force algorithm does not prescribe a particular window size nor does it require that consecutive windows along a stratified transition coordinate overlap . provided that convergence has been achieved in each window , the gradient , a( ) , can be reconstructed merely by joining the gradients from individual windows at the boundaries . as we have already argued , from the efficiency point of view it might appear that using small windows is always beneficial . one concern about extensive stratification is that efficient sampling of rugged landscape along orthogonal degrees of freedom may require temporary excursions beyond the window s boundaries , where the barrier separating adjacent valleys is smaller . the barrier between the minima along the orthogonal degree of freedom , , may be difficult to cross at in the range of [ 0 , 0.2 ] , but not in the [ 0.8 , 1 ] range . in this case , stratification will create kinetic traps , most likely reducing rather than improving efficiency . a similar problem was observed in simulations aimed at determining the potential of mean force for a small , proline - rich peptide , p41 , bound to the sh3 domain of abl kinase using the root - mean - square deviation ( rmsd ) with respect to the native conformation as the transition coordinate . there , a second minimum was only discovered through creation of a window that did not encompass the native state ( see figure 2a in ref ( 89 ) ) . this second minimum represents a shift in register of the peptide in its binding site , and although it would occur spontaneously given sufficiently long time , its probability can be enhanced by first driving the peptide to higher values of rmsd , which disrupts some of the bonds characteristic of the native state , and then letting it come back . because it is often not possible to predict orthogonal barriers a priori , determining an appropriate windowing scheme typically requires an adaptive procedure . the basic criterion here is the continuity of the biasing force across consecutive windows . if one is concerned about quasi nonergodicity , a good strategy is to start with large windows . if the continuity of forces appears to be satisfactory , one might attempt to improve efficiency through further stratification . the advantage of this strategy is that the approximation to the biasing force acquired from the large - window simulation can be used in smaller windows . if no windowing scheme yields continuous forces , then other strategies , described in the next section , should be employed . simulations involving multiple replicas are perhaps the most powerful strategies to accelerate ergodic sampling along degrees of freedom orthogonal to the transition coordinate . beyond the embarrassingly parallel strategy of running independent simulations to obtain more sampling than is possible with a single simulation in the same real time , a number of schemes have been devised that significantly enhance sampling , at the cost of transferring information between replicas . in a prototype example of multiple - replica algorithms , originating with monte carlo simulations , replicas are run at different temperatures and exchanges of system configurations are attempted periodically between replicas , so as to maintain a canonical ensemble at each temperature . the advantage of this method is that replicas at higher temperatures cross energetic barriers more quickly and can pass the resulting configurations to replicas at lower temperatures , preventing the latter from remaining in metastable states . exchange of replicas with different umbrella - sampling potentials , known as hamiltonian exchange , has also been widely successful . with the adaptive biasing force method , each replica multiple - walker strategies range from simply running similar independent adaptive biasing force calculations in parallel , to more complex ones , involving communication between replicas . here we consider two communication strategies that can be used in concert . in the first strategy , which we refer to as shared adaptive biasing force , the instantaneous forces sampled from each walker are collected in a single shared buffer as the simulations progress simultaneously . in the current implementation , the shared buffer is merely conceptual : each walker retains its own buffer , which is synchronized with all the others at fixed intervals . regardless , shared adaptive biasing force can result in significantly faster exploration of the transition coordinate and improved convergence of the free energy , as compared to the case for independent walkers . a second strategy , complementary to the first , is the application of so - called walker selection rules . these selection rules eliminate replicas with values of the transition coordinate that are already well sampled , while duplicating replicas in relatively unexplored regions , enforcing more uniform sampling . selection rules may be implemented using a so - called resampling procedure and weights that are associated with each replica . there are many ways to choose the weights and to implement this idea in practice . the basic requirement is that the selection mechanism automatically vanishes at equilibrium , namely when the biasing force is the mean force . here , we calculate the weight of each walker in a somewhat simpler way than in previous studies , while obtaining similar results . the weight assigned to each walker is the inverse of the number of samples accrued in a neighborhood of bins near the walker . specifically,26where j is the bin occupied by walker i at the time of application of the selection rules , nj(t ) nj(tlast ) is the number of samples that have been accrued in bin j since the last selection and resampling step , and h defines the number of bins surrounding j that are included in the sum . the walkers are resampled on the basis of these weights , and the selection mechanism is switched off when the smallest and largest ni(t ) differ by less than 20% , i.e. , when [ max(1/wti ) we consider reversible unfolding of deca - alanine as a model system for testing different multiple - walker strategies . in figure 9a we compare the results of adaptive biasing force calculations of 8 ns total simulated time to a converged free - energy profile . a single , 8-ns long simulation gave the poorest results , whereas 16 short , independent calculations perform somewhat better , even though the region 31.432 has not been sampled at all . in contrast , shared adaptive biasing force yields complete and more uniform sampling , and a smoother , more reliable free - energy profile . the best free - energy profile , almost indistinguishable from the converged one , was obtained from shared adaptive biasing force combined with walker selection . note that , as expected , the free energies converge to the same values for sufficiently long simulations , irrespective of the multiple - walker strategy , as demonstrated in figure 9b . an implementation of the shared adaptive biasing force algorithm with the selection rules used here is expected to be available in future releases of the molecular dynamics program namd . ( a ) potentials of mean force obtained for different multiple - walker strategies and total simulation times of 8 ns . for reference , the black curve shows the result for a total simulation time of 128 ns . single refers to a single long 8 ns simulation , and independent denotes the result of combining force samples for 16 walkers after they ran independently for 0.5 ns . for the curves marked shared and selection , force samples were synchronized among walkers every 20 ps . also included the application of walker selection , as described in the text , on the same interval . ( b ) potentials of mean force obtained for different multiple - walker strategies and total simulation times of 128 ns . there are several reasons to perform adaptive biasing force calculations with a transition coordinate of dimension greater than one . first , we may be simply interested in how the free energy varies as a function of more than one coordinate . for example , to study the interaction between two molecules , it may be of interest to obtain the free energy as a function of both their distance and relative orientation . as described in section hidden barriers and other challenges to obtaining accurate results , sampling along a single collective variable may be hampered by barriers in orthogonal dimensions . if one can identify these orthogonal dimensions , the application of adaptive biasing force along them will remove the barriers and improve sampling . by itself , obtaining a multidimensional free - energy landscape requires more sampling than is needed to calculate a one - dimensional profile . for example , if one uses n bins along collective variable and m bins along collective variable , each component of the gradient over both collective variables , a( , ) , requires mn bins . with one sample obtained per time step , the time required to generate a statistically significant number of samples in all bins will increase approximately proportionally . despite the additional , substantial computational effort , adding a second or third dimension may improve efficiency , as it accelerates convergence and increases uniformity in sampling by allowing the system to more rapidly cross between multiple parallel valleys subsequently , one can recover the potential of mean force along a single dimension through integrating out the additional dimensions . however , when one wishes to include a large number of dimensions , the generalized adaptive biasing force algorithm ( see section the generalized adaptive biasing force algorithm ) may be more appropriate . if the transition coordinate involves angular degrees of freedom that span their full range ( e.g. , [ 0 , 2 ] ) , an additional complication develops . the exact forces and free energies are periodic in these variables , but this is not necessarily true for these quantities burdened with statistical errors . one way to restore the required periodicity is to approximate the free energy as a function of angular variables with spline functions , coefficients of which are computed to distribute errors smoothly across the whole hyperspace spanned by these variables . how to do this has been described by darve et al . as an example application of multidimensional adaptive biasing force , we consider the free - energy landscape of deca - alanine as a function of the root - mean - square displacement ( rmsd ) from three reference structures . figure 10a shows these reference structures : an -helix , a 310-helix , and a particular compact conformation that we refer to as the conformation due to its visual similarity to this greek letter . in agreement with the one - dimensional calculations , extensively discussed above , the free - energy minimum occurs when the structure is close to the -helix , i.e. , when rmsd = 0.3 . in figure 10b the three - dimensional potential of mean force the violet region on the left corresponds to the -helix , whereas the region on the right is closer to the conformation than to the other two structures . the minimum in the latter region is only 1.2 kcal / mol higher than the minimum for -helix and lies at ( rmsd rmsd310 rmsd ) = ( 4.3 5.0 2.7 ) . in figure 10c , the multidimensional free - energy landscape thus yields more insight into the correspondence between the free energy and molecular conformations , revealing , in particular , a minimum for a compact structure that was not identified when the one - dimensional transition coordinate was used . the root - mean - square deviation of selected atoms from their positions in each of the three reference structures defines each of the three transition coordinates . ( b ) free - energy isosurfaces as a function of the three transition coordinates . the violet , pink , and gray surfaces contain all points for which the free energy is less than 5 , 10 , and 20 kcal / mol , respectively . in all cases the tick marks represent a root - mean - square deviation of 1 . the minimum value of the free energy , which occurs at ( rmsd rmsd310 rmsd ) = ( 0.3 2.6 2.7 ) , is defined to be zero . ( c ) another view of the surface shown in panel b , with representative structures shown for the two energetically favorable regions . previously , we considered one particular approach to multidimensional adaptive biasing force . here first , it is easy to check that if the dimension of the transition coordinate is larger than one , the biasing force can not be in general derived from a gradient ( it is not conservative ) . on the other hand , this is of course true of the expected long - time limit . a natural idea is , therefore , to project , using , for example , the classical helmholtz hodge projection : change the biasing force ft to t where t = arg min ft l . because this is consistent with the expected stationary state , this should not alter the convergence properties . moreover , the interest of this projection is that the variance of the force is reduced , because the nonconservative part is set to zero . second , it would be interesting to develop efficient adaptive biasing force techniques for higher dimensional transition coordinates . the standard implementation of the adaptive biasing force algorithm is currently limited to a dimension up to 3 , because it relies on a cartesian grid of the transition coordinate values , whose complexity is exponential with respect to the dimension of the transition coordinate . it would not be very useful , however , to develop a technique that yields a flat energy landscape along the transition coordinate if the dimension is high , as mapping a high - dimensional cube would be quite time - consuming . one alternative idea , explored in ref ( 101 ) , is to use a bias of the form i=1matii , where ( 1 , ... , m ) denotes an m - dimensional transition coordinate . under appropriate assumptions , one can show the convergence of the method , and preliminary numerical results are encouraging ; see ref ( 101 ) . another route would be to consider a bias of the form i=1matii , or even following greedy algorithms and tensor product approaches used in nonlinear approximation theory ( see , for instance , ref ( 102 ) ) , k1i=1mati , ki , where the functions ( a , ... , a)k1 would be iteratively computed . the objective of this section is to assess the influence of geometrical restraints on the free - energy landscape and how such restraints ought to be treated in the context of adaptive biasing force simulations . toward this end , the nature of the forces at play , either thermodynamic forces or forces arising from external harmonic potentials , will be clarified , and the current strategy for handling the latter , using an extended - lagrangian formalism , will be outlined . one subtlety of the adaptive biasing force algorithm that is often overlooked is its dependence on the measure of the thermodynamic force , which is not necessarily synonymous with the total force acting on the transition coordinate . for most biomolecular simulations , for example , hydrogen - bond lengths are often constrained in a simulation via the rattle algorithm . if only the heavy atom of the bonded pair is involved in the transition coordinate , then the adaptive biasing force algorithm will include the constraint force emanating from the hydrogen in addition to the thermodynamic force , thus contaminating its estimate . a straightforward solution is to include both the hydrogen and its parent atom in the collective variable(s ) defining the transition coordinate , causing the constraint forces to cancel each other . in other words , if the constraint / restraint force is zero in the collective variable s center of mass , it will not contribute to the measured potential of mean force . alternatively , if restraints nonuniformly affect atoms in the collective variable(s ) but are not accounted for in the thermodynamic force used by the adaptive biasing force algorithm , then convergence is impossible ; the adaptive biasing force scheme can not remove forces that it does not measure . though it may seem apparent that one will always want to calculate what the potential of mean force would be in the absence of artificial restraints , there are cases in which externally imposed restraints are meant to be included . ligand binding free energies , which utilizes a staged procedure involving a series of geometrical restraints . by design of the procedure , these restraints must involve the same atoms also being biased at each stage and their contributions are individually determined and tabulated at the end . in this case , to ensure that all necessary forces are included , a procedure such as extended adaptive biasing force is required . as mentioned above , it may be cumbersome to write an analytical expression for the instantaneous force in dimensions larger than one or for complicated transition coordinates . one possible way around this issue is to extend configurational space with a fictitious degree of freedom and define an extended potential,27where k > 0 is a ( large ) force constant that couples to the transition coordinate , and proceed utilizing the extended transition coordinate , (x, ) = , instead of . it can be checked that the free energy associated with this extended system is the convolution of the original free energy with a gaussian kernel , a( ) = dz k(z ) a(z ) , where k(z ) = z exp[(/2k)|z| ] is the gaussian kernel with variance k. the constant k should thus be chosen sufficiently large to ensure that a is a good approximation of a. this enables quick convergence to equilibrium . the adaptive biasing force algorithm can be applied to the extended system , the potential energy being v and the transition coordinate being . notice that the instantaneous force on the extended degree of freedom , corresponding to free energy a , is trivial to compute , because it is equal to the harmonic spring force . the original free energy a can then be recovered by using either deconvolution procedures or simple unbiasing techniques based on formulas such as eq 34 . provided that a good estimate of the biased marginal of the transition coordinate , (z ) , has been collected , the following unbiased estimator of the free - energy derivative can be used,28where z is the conditional average value of for (x ) = z. here , the biased marginal of is used as a correction to the inaccurate biasing force . it should be noted that this is valid in a more general context , including the absence of biases ( removing the second term of the right - hand side ) , in which case one recovers the trivial histogram - based estimator of the potential of mean force . although the adaptive biasing force algorithm is often considered merely a free - energy calculation technique , it is , at its core , an importance - sampling scheme that can have applications beyond obtaining free energies . one such application is to calculate kinetic parameters , such as the diffusivity along a transition coordinate , where an importance - sampling scheme is often essential to obtain reliable estimates in all regions of the coordinate , particularly near all - important free - energy barriers . together , the diffusivity and free energy as functions of the transition coordinate can be used to construct a kinetic model of a process of interest , which yields insight beyond the static picture of molecular phenomena given by free - energy calculations . in this section , we describe a recently developed scheme leveraging adaptive biasing force to compute simultaneously the free energy and diffusivity along a transition coordinate . this scheme is just an example , as a number of other approaches in which the adaptive biasing force is used to improve kinetic descriptions of different systems are being investigated . also note that , although this scheme was designed with the adaptive biasing force method in mind , it can be straightforwardly applied to other importance - sampling techniques . determining kinetic parameters can be more subtle than mapping the free - energy landscape , because kinetic descriptions are usually approximations and can not be exactly derived from statistical mechanics . a commonly invoked diffusive model is overdamped langevin dynamics , in which the following equation of motion is assumed for a set of m collective variables z = ( z , z , ... , z),29where ti is the time derivative of the ith collective variable at time t , dij(zt ) is the ( i , j ) component of the diffusivity tensor for the configuration of the collective variables at time t , fj(zt , t ) is the total force on collective variable j for the configuration at time t , j = /z , and i(t ) is a stochastic variable with i(t ) j(t) = 2dij(zt ) (tt ) . the total force on variable j , fj(zt , t ) , is the sum of the biasing force , fjbias(t ) , and the system force , which can be expressed as the negative of the gradient of the potential of mean force fj(z ) = ja(z ) . here , we will focus on diffusive models along a single collective variable , but note that multidimensional transitions have been analyzed by similar approaches . in one dimension , the overdamped langevin model has two free parameters , the free - energy profile a(z ) and the position - dependent diffusivity d(z ) . the adaptive biasing force method , as well as other techniques , provides a route to a(z ) , whereas a number of methods have been applied to determining position - dependent diffusivity . however , some of the most basic methods are not compatible with nonuniform free - energy landscapes , and others are based on the assumption that the free energy can be approximated as a harmonic well . in methods specifically designed for compatibility with the adaptive biasing force method , it is assumed that the dependence of diffusivity on position is weak . to parametrize a diffusive model , a(z ) and d(z ) should be determined consistently , because , for example , coarsening a(z ) results in a reduction of the associated d(z ) . furthermore , to save computational resources , it would be desirable to obtain both the free energy and the diffusivity in the same calculation . however , in many methods cited above , equilibrium or steady - state statistics is assumed , which is likely to yield erroneous results with time - dependent biasing forces . solutions to the problem of calculating diffusivity in simulations with time - dependent biases take advantage of statistical tools such as maximum likelihood or bayesian inference . conceptually , these methods rely on optimizing the parameters of the diffusive model such that the observed trajectory has the greatest likelihood of occurring . the bayesian inference scheme described below begins with assuming a particular dynamical model for the collective variable ( or collective variables ) of interest . in practice , we represent the functions dij(z ) and fi(z ) by piecewise cubic interpolation from a discrete grid in m - dimensional z - space . we seek the optimal parameters comprising the values of the functions dij(z ) and fi(z ) at each grid node that best correspond to the simulated trajectory , denoted by t. for simplicity , we represent these optimal parameters as h*. we consider the trajectory as a set of discrete hops of duration t . given trial parameters h , as well as the biasing forces f(t ) , we compute for each hop p ( { z+1 , t+1}{z , t } , f(t ) , h0 ) , the conditional probability of arriving at transition - coordinate configuration z+1 at time t+1 , given that the system occupied the configuration z at time t. the probability of the complete trajectory given the parameters is the product of the probabilities at each step30this equation yields the probability of the trajectory given an assumed set of parameters . using bayes theorem , we can infer the probability of the parameters given the trajectory:31 an advantage of the bayesian approach is that it permits inclusion of any prior knowledge about the form of the parameters in a consistent way by defining the prior probability pprior(h ) of the parameters . for example , one can assume scale invariance of the function values or smoothness of the functions . finding the optimal parameters then becomes a problem of finding the set of parameters h * that maximizes posterior probability p(h*|t ) , which can be carried out by generating a markov chain of states hk using the metropolis hastings algorithm . in the one - dimensional case ( or for independent motions along multiple dimensions ) , we can simplify and discretize eq 29 to yield32where gt is a random variable with a standard normal distribution . from the properties of gt , it is evident that eq 30 becomes33below , using this formula and eq 31 , we are able to reconstruct d(z ) and f(z ) for deca - alanine , yielding a complete diffusive model . indeed , we may even set f = 0 and construct the diffusive model from an equilibrium simulation . however , the accuracy of the results of a bayesian scheme are wholly dependent on the quality of sampling near each point along z , necessitating importance sampling for rugged free - energy landscapes . in the supporting information , we discuss assessment of the precision and reliability of the results of the bayesian scheme , which includes estimating the statistical error , as well as checking the consistency of the diffusive model with itself and the output of adaptive biasing force . the z - dependent diffusivity for reversible unfolding of deca - alanine is shown in figure 11b . the variation with z could not have been inferred in any simple way from knowledge of the system force or free - energy profiles . diffusion along z , which can be thought of as the rate at which the end - to - end distance randomly changes , is highest near z = 16 , corrsponding to slightly unfolded -helical structures . the ensemble of diverse compact structures in the range z [ 5 , 10 ] seems to be associated with the lowest diffusivity values , whereas a secondary minimum appears near z = 22 . given these data , one might hypothesize that the diffusivity is inversely related to the conformational degeneracy at z. note that figure 11b shows considerable dependence of the calculated diffusivity on t , the time over which the trajectory is discretized in eq 33 , which implies that the motion on the observed times is not well modeled by overdamped langevin dynamics , and that a more sophisticated model of motion along z may be needed . ( a ) comparison of the system force as determined by adaptive biasing force and that calculated from the bayesian scheme on the same trajectory . ( b ) diffusivity as a function of the end - to - end distance of deca - alanine for different observation time intervals t . free - energy calculations have become standard tools of statistical mechanics applied to a wide variety of problems in chemistry and biology . this has been possible due to significant theoretical advances in this area , their highly efficient implementations in software packages developed for modern , parallel computers , and remarkable improvements in computational power available for free - energy calculations . yet , these calculations are still not sufficiently mature to be carried out without careful supervision of the end user . this may be fortunate , as there is no substitute for physical insight of the researcher . this also implies that a number of good practices should be followed in free - energy calculations in general , and in applying the adaptive biasing force in particular . these good practices are usually quite simple and involve careful design of simulations , monitoring their progress and postprocessing analysis . in favorable circumstances , they will simply increase confidence that the calculated free energies are reliable . in less favorable cases , they are even more important , as they allow for identifying and correcting shortcomings of the calculations that might adversely affect the results and their interpretation . below , we recapitulate these good practices , which have been already discussed in a considerable detail in the preceding sections.(1)careful choice of the transition coordinate is a key step toward successful free - energy calculations . in making this choice , it is desirable not only to capture the physical nature of a problem of interest but also to ensure small variance and smoothness of the biasing force . ignoring the latter issues may adversely affect efficiency and even correctness of the calculation . reducing the variance should be balanced with the cost of calculating the instantaneous value of the transition coordinate , as usually the former will decrease and the latter will increase with the number of atoms involved . this balance will depend on implementation details and , therefore , some familiarity with the underlying code might be required.(2)a suitable stratification strategy should be planned in advance . if there are concerns about possible quasi nonergodic behavior of the system , and in particular the existence of parallel channels between the end point states , it is recommended to use large windows . if these concerns prove to be unjustified , it is always possible to switch to smaller windows without losing information about the biasing force that has already been accrued . in other instances it is usually more efficient to stratify the transition coordinate into smaller windows throughout a simulation.(3)free - energy calculations should always be accompanied by estimates of statistical errors . for the adaptive biasing force algorithm , a formula to do so exists and should be applied whenever possible . without error estimates the reliability of the calculated free - energy values is questionable and the ability to compare them with experimental measurements is seriously hampered.(4)in stratified simulations , lack of continuity in the biasing force across consecutive windows that clearly exceeds statistical errors is a sure sign of problematic free - energy calculations . this issue should not be ignored , and any attempt to circumvent it by way of some sort of averaging is unlikely to succeed . instead , remedial steps aimed at improving ergodic behavior of the system , such as applying the multiple - walker strategy , should be taken.(5)it is usually very useful to monitor the behavior of the total , biased force , as it should converge to zero in long simulations . moreover , the rate of convergence at sufficiently long times t should become proportional to 1/t . if the biased force along the transition coordinate or within a window exhibits large deviations from nonuniformity , which clearly does not decrease with time , or the convergence rate is erratic , we have , again , a likely indication of quasi nonergodicity in orthogonal degrees of freedom . then , there is no basis for assuming that a reliable free - energy dependence on the transition coordinate can be extracted from the calculated forces . instead , in such circumstances , techniques for removing quasi nonergodicities along orthogonal degrees of freedom should be brought to bear . careful choice of the transition coordinate is a key step toward successful free - energy calculations . in making this choice , it is desirable not only to capture the physical nature of a problem of interest but also to ensure small variance and smoothness of the biasing force . ignoring the latter issues may adversely affect efficiency and even correctness of the calculation . reducing the variance should be balanced with the cost of calculating the instantaneous value of the transition coordinate , as usually the former will decrease and the latter will increase with the number of atoms involved . this balance will depend on implementation details and , therefore , some familiarity with the underlying code might be required . if there are concerns about possible quasi nonergodic behavior of the system , and in particular the existence of parallel channels between the end point states , it is recommended to use large windows . if these concerns prove to be unjustified , it is always possible to switch to smaller windows without losing information about the biasing force that has already been accrued . in other instances it is usually more efficient to stratify the transition coordinate into smaller windows throughout a simulation . free - energy calculations should always be accompanied by estimates of statistical errors . for the adaptive biasing force algorithm , a formula to do so exists and should be applied whenever possible . without error estimates the reliability of the calculated free - energy values is questionable and the ability to compare them with experimental measurements is seriously hampered . in stratified simulations , lack of continuity in the biasing force across consecutive windows that clearly exceeds statistical errors is a sure sign of problematic free - energy calculations . this issue should not be ignored , and any attempt to circumvent it by way of some sort of averaging is unlikely to succeed . instead , remedial steps aimed at improving ergodic behavior of the system , such as applying the multiple - walker strategy , should be taken . it is usually very useful to monitor the behavior of the total , biased force , as it should converge to zero in long simulations . moreover , the rate of convergence at sufficiently long times t should become proportional to 1/t . if the biased force along the transition coordinate or within a window exhibits large deviations from nonuniformity , which clearly does not decrease with time , or the convergence rate is erratic , we have , again , a likely indication of quasi nonergodicity in orthogonal degrees of freedom . then , there is no basis for assuming that a reliable free - energy dependence on the transition coordinate can be extracted from the calculated forces . instead , in such circumstances , techniques for removing quasi nonergodicities along orthogonal degrees of freedom should be brought to bear . following these simple good practices guarantees the improved quality of free - energy calculations carried out by way of the adaptive biasing force method but does not ensure that all problems encountered in such calculations , especially those related to quasi nonergodicity , have been identified . to this end , the toughest challenges to practitioners of the adaptive biasing force algorithm and related methods are linked to multiple slow degrees of freedom leading to orthogonal barriers . one way to address these problems is through a multiple - walker strategy , which has proven effective in a test system that exhibits metastability in the orthogonal space , a significant challenge to the classic adaptive force method . along the same lines , the basic method can be integrated with other enhanced sampling techniques that involve multiple - copy schemes , such as parallel tempering and hamiltonian exchange . another promising research direction is to increase the dimensionality of the bias , as is done in an implicit way in the generalized adaptive biasing force scheme . real - life applications would , however , require an accessible and well - documented implementation , now available as part of the independent collective variables module . although better sampling of orthogonal degrees of freedom is the most promising direction for increasing the efficiency of the adapting biasing force method , there is also room for improvement in achieving convergence along the transition coordinate . for example , it would be of interest to develop adaptive algorithms for the early stages of simulations that would converge faster than the currently used step or ramp functions . one possibility along these lines is to employ a kernel function along . in other words , adaptation in a given bin would depend not only on samples accrued in this bin but also on samples in neighboring bins . another avenue to improve convergence along the transition coordinate , which has not been explored so far , is to exploit the freedom in defining the ensemble average of instantaneous force and identify choices that reduce the variance . for example , the error in integrating force to calculate free energy that is due to binning of force values could be reduced by using improved , smooth interpolation schemes . further , we observe that the best accuracy is obtained not when the free energy is flat but rather when the statistical error of the average force is the same everywhere along the transition coordinate . to achieve this , the number of samples , nk , in bin k should be such that k/(nk ) rather than nk is constant for all bins , where k is the standard deviation of estimated average force in bin k. this is realized if the biasing potential due to the average force used in the standard adaptive biasing force method is supplemented by the term 2kbt ln(k/0 ) , where 0 is the standard deviation at a reference point . in practice , k and 0 are estimated from their running values or their approximations during the course of a simulation . the additional term becomes important whenever the friction coefficient changes markedly with or parts of the system undergo large fluctuation , for example because they are near phase transition . a number of extensions to the adaptive biasing force method have not been explored yet . for example , the method could be straightforwardly extended to transformations along a parameter of the hamiltonian if an equation of motion was associated with this parameter , as is done in metadynamics . it would be of interest to check whether such application of the adaptive biasing force method were more efficient and reliable than other , related methods currently used for this purpose , such as conventional thermodynamic integration , metadynamics , or the wang another extension of the method that was outlined in one of the original studies on the adaptive biasing force but has not been pursued so far , is to add to the biasing force another contribution that would depend only on and would favor certain states of particular interest to the user or drive the dynamics in a specified direction . the latter would make the adaptive biasing force method a more efficient and better controlled alternative to steered dynamics . finally , we emphasize that the adaptive biasing force is not just a free - energy calculation technique but can also be seen as a general adaptive biasing scheme ( see , for instance , ref ( 124 ) for applications in bayesian statistics ) . indeed , once a correct sampling of the biased measure zp1 exp(pmp/2 ) dp zt01 exp{[v at0](x ) } dx has been obtained ( t0 being a fixed time , and the bias being fixed from time t0 : t t0 , at = at0 , it is easy to recover canonical averages using standard unbiasing procedures:34 although the adaptive biasing force dynamics has been applied so far almost exclusively to chemical and biological systems , it could also be very useful for sampling a multimodal measure ( namely , a probability measure for which high - probability regions , called modes in this context , are separated by low - probability regions ) in other fields , e.g. , among others , free - energy computations in material sciences or markov chain monte carlo techniques for bayesian inference . in summary , the combination of simplicity , versatility , and strong mathematical underpinnings makes the adaptive biasing force method an attractive target for a wide variety of extensions in statistical mechanics and beyond .

in the host of numerical schemes devised to calculate free energy differences by way of geometric transformations , the adaptive biasing force algorithm has emerged as a promising route to map complex free - energy landscapes . it relies upon the simple concept that as a simulation progresses , a continuously updated biasing force is added to the equations of motion , such that in the long - time limit it yields a hamiltonian devoid of an average force acting along the transition coordinate of interest . this means that sampling proceeds uniformly on a flat free - energy surface , thus providing reliable free - energy estimates . much of the appeal of the algorithm to the practitioner is in its physically intuitive underlying ideas and the absence of any requirements for prior knowledge about free - energy landscapes . since its inception in 2001 , the adaptive biasing force scheme has been the subject of considerable attention , from in - depth mathematical analysis of convergence properties to novel developments and extensions . the method has also been successfully applied to many challenging problems in chemistry and biology . in this contribution , the method is presented in a comprehensive , self - contained fashion , discussing with a critical eye its properties , applicability , and inherent limitations , as well as introducing novel extensions . through free - energy calculations of prototypical molecular systems , many methodological aspects are examined , from stratification strategies to overcoming the so - called hidden barriers in orthogonal space , relevant not only to the adaptive biasing force algorithm but also to other importance - sampling schemes . on the basis of the discussions in this paper , a number of good practices for improving the efficiency and reliability of the computed free - energy differences are proposed .

Introduction The adaptive biasing force algorithm TRANSITION COORDINATE ADDRESSING NONERGODICITY SCENARIOS Combining the adaptive biasing force algorithm with geometrical restraints Combining thermodynamics and kinetics Summary and outlook

PMC3394281

genome - wide association studies ( gwas ) have successfully discovered nearly 1500 genetic loci associated with over 200 common disease and quantitative traits ( http://www.genome.gov/gwastudies/ ) . gene interactions ( epistasis ) that could be a potential source of missing heritability evidenced in these studies ( 1,2 ) . a recent study demonstrated that epistasis could exist widely in biological pathways and create substantial phantom heritability undetectable via conventional gwas ( 3 ) . indeed , additional tests of interactions involving gwas loci with significant marginal effects have successfully discovered epistasis in several studies ( 47 ) . however , despite enormous effort , so far studying epistasis in gwas has been far more challenging and less fruitful than conventional gwas focussing on single locus effects ( 3,8 ) . one major challenge in studying epistasis in gwas ( typically with hundreds of thousands of snp markers ) is the need to scrutinize billions of pairwise snp combinations in order to consider all possible interactions . the challenge is increasing as more sequencing data ( and consequently more snps ) become available in future gwas . these methods are confined to either binary disease or quantitative traits and several are designed specifically for computers equipped with particular graphical processing units . substantial computing time is still required in some of these methods , especially when widely available computer systems are used ( 11,13 ) . there is therefore great demand for high - throughput tools that can run on widely used computer platforms to analyse both quantitative and disease traits , making the analysis of epistasis routine in gwas and ultimately improving our understanding of the role of epistasis in complex traits . another major challenge is that many current gwas populations may have only limited power to detect and replicate significant epistasis signals due to their relatively small sample sizes ( 8,14 ) . the approach of meta - analysis of multiple gwas ( 15 ) may enhance the power of detection of epistasis but this is not yet applicable due to the lack of powerful computational tools to support epistasis analysis in such data sets using imputed ( not categorical ) genotype data . pathway - based approaches may narrow the search space and also enhance power , for example , by seeking pathway pathway interactions ( 3 ) or by identifying common pathways enriched in epistatic genes with modest interaction signals ( i.e. strong but not necessarily genome - wide significant ) detected from multiple gwas populations ( 16 ) . in either case , fast screening of pairwise interactions in individual gwas populations appears to be critical in order to provide information for pathway - based analyses . we have developed the biforce toolbox to address the challenges of high - throughput detection of epistasis . biforce toolbox is programmed in java to support large scale analysis of pairwise epistasis in quantitative and disease traits on commonly used computer systems ( e.g. running either windows , osx or linux operating system ) via a graphical user interface ( gui ) or the command line . it integrates available algorithms and advanced computing technologies such as bitwise computing technologies first adopted in boost ( 12 ) and fastepistasis ( 11 ) and multi - threaded parallelization into one software package to offer rapid and comprehensive pairwise genome scans . biforce toolbox is built on concepts and algorithms we previously developed to maximize the power of detection of different forms of epistasis while controlling false positive rates ( 9,17,18 ) . it has been rigorously tested and successfully identified interesting interaction signals and pathways in multiple studies using large gwas data sets ( 7,16 ) . here , we describe the main features and functionality of the biforce toolbox using the body mass index ( bmi ) trait in the northern finland birth cohort 1966 ( nfbc1966 ) ( 16,19 ) as an example . the nfbc1966 gwas data were provided by the database of genotype and phenotype ( dbgap ; http://www.ncbi.nlm.nih.gov/gap ) via specific data use certification . biforce toolbox is named to reflect its main features : fast screening of pairwise interactions in gwas of complex disease and quantitative traits , using the brute force computational power of bitwise computing and multi - threaded parallelization . it is implemented in java to enable its use on most commonly used computer systems , allowing local secure analysis of gwas data sets and comprehensive fast genome - wide scans for epistasis . it has been designed to be user friendly and benefits from an intuitive gui as well as command line access for automated submission of jobs . biforce toolbox uses a combined search algorithm ( 17,18 ) that integrates a full pairwise genome scans with specific tests of interactions involving snps with marginal effects that are genome - wide significant ( marginal - snps ) to increase the power of detection . biforce toolbox is free and the binary files compiled for the three operating systems ( mac osx , windows and linux ) can be downloaded from http://bioinfo.utu.fi / biforcetoolbox/. biforce toolbox takes ordinary gwas data ( in genotype and phenotype files ) as input , where snp genotypes need to pass normal quality control procedures and phenotypes are recommended to be adjusted for covariates and relatedness for quantitative traits ( 7,16 ) or post biforce analysis for disease traits . after data input , it converts the snp genotype data into boolean bit values and the data are stored in memory - efficient java bitset arrays that allow missing snp genotypes to be handled easily and boolean bitwise operations ( e.g. logical and ) to be applied to the arrays of bit values , which makes the association tests ( see below ) extremely fast . further , biforce toolbox partitions the whole pairwise genome scan automatically into smaller tasks and feed them evenly to available processing threads to compute in parallel and store results appropriately . the combined search algorithm implemented in biforce toolbox includes two consecutive genome scans : single snp - based genome - wide association tests and pairwise epistatic interaction tests of all snp combinations . marginal - snps are identified in the first scan and used to test interactions involving them . by default the 5% genome - wide significance thresholds are derived based on the bonferroni correction for the total number of tests performed . given n to be the total number of snps in a gwas with k ( k > 0 ) marginal - snps being identified , the thresholds are : p = 0.05/n for marginal - snps , p = 0.05/[(n 1 ) * k ] for marginal - snp interactions and p = 0.05/[n * ( n 1)/2 ] for a pairwise genome scan . alternatively , user specified significance thresholds can be specified in the analysis . association tests are based on linear regression models , where the genotypes of each snp ( i.e. homozygote of the minor allele , homozygote of the major allele and heterozygote ) are fitted as fixed factors . pairwise snp interactions are assessed using contingency tables which makes biforce toolbox applicable to both quantitative and binary disease traits . briefly , a saturated model ( fitting two snps and interactions ) is tested against a reduced model ( fitting the two snps without interactions ) with four degrees of freedom and then the f ratio for quantitative traits or log - likelihood ratio for binary traits is calculated . p values are then derived according to specific test statistic distributions and appropriate degrees of freedom ( assuming fixed four degrees of freedom in interaction tests for disease traits ) . for disease traits , biforce toolbox adopts the approximation filtering algorithm developed in boost ( 12 ) as a default option to accelerate the exhaustive pairwise genome scan which can be dismissed when necessary ( i.e. using only log - likelihood ratio tests in the scan ) . on completion of the search process , biforce toolbox can be used to generate summary information and examine the identified snp pairs ( e.g. allele frequencies and contingency tables ) after reloading the input data files and the retained results ( figure 1 ) . furthermore , it can perform a generic analysis of pathways enriched within groups of genes showing interaction signals to provide an initial view of the potential biology underlying these signals . such an analysis involves two steps : annotating snps ( with reference snp i d numbers ) to the nearest genes and then performing overrepresentation analysis based on hypergeometric tests against a variety of gene - centred functional data such as gene ontology ( go ) and kegg pathway annotation ( 20 ) . further details of such an analysis can be found in the manual of biforce toolbox . gwas data are generated by microarray or next - generation sequencing platforms ( a ) . snapshot of the biforce toolbox gui after loading and conversion of the bmi data of the nfbc1966 cohort ( note the multi - tab options such as working with subsets of snps or snp - pairs and performing single snp - based genome - wide association ) ( d ) . snapshot of the biforce toolbox gui running the pairwise genome scan of the bmi data with progress reporting at the bottom ( note snp information , genotype counts , contingency table are showed for a retained pair of snps ; the retained results of the pairwise genome scan can be exported as tab - separated text file or excel compatible spreadsheet for further analyses by users ) ( e ) . a graphic view of positions of interaction signals in bmi of the nfbc1966 cohort generated by a third party tool circos , where chromosome ideograms are shown around the outer ring and are oriented pter snapshot of the biforce toolbox gui running pathway enrichment analysis after the pairwise genome scan using epistatic genes with interaction log10 p values greater than 7.3 ( i.e. p < 5.0e ; note the member epistatic genes and associated snps are displayed for a pathway selected ) ( g ) . gwas data are generated by microarray or next - generation sequencing platforms ( a ) . snapshot of the biforce toolbox gui after loading and conversion of the bmi data of the nfbc1966 cohort ( note the multi - tab options such as working with subsets of snps or snp - pairs and performing single snp - based genome - wide association ) ( d ) . snapshot of the biforce toolbox gui running the pairwise genome scan of the bmi data with progress reporting at the bottom ( note snp information , genotype counts , contingency table are showed for a retained pair of snps ; the retained results of the pairwise genome scan can be exported as tab - separated text file or excel compatible spreadsheet for further analyses by users ) ( e ) . a graphic view of positions of interaction signals in bmi of the nfbc1966 cohort generated by a third party tool circos , where chromosome ideograms are shown around the outer ring and are oriented pter snapshot of the biforce toolbox gui running pathway enrichment analysis after the pairwise genome scan using epistatic genes with interaction log10 p values greater than 7.3 ( i.e. p < 5.0e ; note the member epistatic genes and associated snps are displayed for a pathway selected ) ( g ) . we use bmi of the nfbc1966 cohort to illustrate a typical analysis procedure using biforce toolbox . the genotype and phenotype data were pre - processed following the instructions given in the original gwas ( 19 ) . bmi was further corrected for covariates and relatedness and normalized to prevent spurious associations as detailed elsewhere ( 16 ) . in total 323 697 autosomal snps and the remaining analysis is summarized in figure 1 , with an example list of the retained results of the pairwise genome scan ( none is genome - wide significant ) shown in table 1 . table 1.a short list of the results of the pairwise genome scan of bmi in nfbc1966 cohortsnp1snp2anova_pairanova_intpair_log10pint_log10pgenoclassnors9873966rs9506047.70414.8509.58711.3419rs9873966rs44063737.64614.7859.49611.2879rs2310173rs96914727.61514.3119.44810.8939rs7536830rs111690637.64413.9289.49210.5769rs1453405rs22890257.23713.7448.86210.4239rs2962896rs68906736.81013.4668.20310.1939rs983936rs10746517.38013.4659.08310.1929rs983936rs15416947.32413.4478.99710.1779rs10490096rs178160027.30516.7898.01810.1248rs3980965rs117639727.26013.0018.8979.8089snp1 ( snp2 ) : the first ( second ) snp ; anova_pair ( anova_int ) : f ratio of a whole pair of snps with interaction ( interaction between a pair of snps ) ; pair_log10p ( int_log10p ) : log10 p value of a whole pair of snps with interaction ( interaction between a pair of snps ) ; genoclassno : the number of joint genotype classes ( 9 in total ) with samples . a short list of the results of the pairwise genome scan of bmi in nfbc1966 cohort snp1 ( snp2 ) : the first ( second ) snp ; anova_pair ( anova_int ) : f ratio of a whole pair of snps with interaction ( interaction between a pair of snps ) ; pair_log10p ( int_log10p ) : log10 p value of a whole pair of snps with interaction ( interaction between a pair of snps ) ; genoclassno : the number of joint genotype classes ( 9 in total ) with samples . we measured the biforce toolbox performance in analysing disease ( 50% cases and 50% controls ) and quantitative traits of gwas datasets with 500 000 snps and 5000 samples , using one thread and eight threads on a single workstation ( 2.8 ghz intel core imac with 4 gb ram , 4 cpu cores each with 2 threads ) and 256 threads on a computer cluster ( 32 nodes each with 4 cpu cores each with 2 threads ) , respectively . biforce toolbox took 118.18 , 30.8 and 0.46 h , respectively , for the disease trait , and 293.24 ( using 8 threads on the workstation ) and 6.81 h ( using 256 threads on the cluster ) for the quantitative trait . in contrast , fastepistasis , a parallel extension of plink ( 21 ) took 29 , 4 or 0.5 days to analyse a quantitative trait in a gwas data set of the same size using 8 , 64 or 512 mpi - bound processors , respectively ( 11 ) ; gboost , a graphical processing unit version of boost ( 12 ) took 1.34 h to analyse a disease trait in a smaller gwas data set ( 351 542 snps and 5003 samples ) on a computer with a nvidia geforce gtx 285 display card ( i.e. 240 cpu cores ) ( 13 ) . biforce toolbox is a powerful and accessible tool to support high - throughput analysis of epistasis in gwas of disease and quantitative traits on general computer platforms . it is hoped that with biforce toolbox analysis the study of epistasis in gwas will become a routine exercise and hence improve our understanding of the role of epistasis in the architecture of complex traits . the biotechnology and biological sciences research council ( bbsrc ) [ bb / h024484/1 to w.h.w . ] ; the medical research council core fund ( to c.a.s . , c.s.h . and w.h.w . ) . funding for open access charge : bbsrc [ bb / h024484/1 ] .

genome - wide association studies ( gwas ) have discovered many loci associated with common disease and quantitative traits . however , most gwas have not studied the gene gene interactions ( epistasis ) that could be important in complex trait genetics . a major challenge in analysing epistasis in gwas is the enormous computational demands of analysing billions of snp combinations . several methods have been developed recently to address this , some using computers equipped with particular graphical processing units , most restricted to binary disease traits and all poorly suited to general usage on the most widely used operating systems . we have developed the biforce toolbox to address the demand for high - throughput analysis of pairwise epistasis in gwas of quantitative and disease traits across all commonly used computer systems . biforce toolbox is a stand - alone java program that integrates bitwise computing with multithreaded parallelization and thus allows rapid full pairwise genome scans via a graphical user interface or the command line . furthermore , biforce toolbox incorporates additional tests of interactions involving snps with significant marginal effects , potentially increasing the power of detection of epistasis . biforce toolbox is easy to use and has been applied in multiple studies of epistasis in large gwas data sets , identifying interesting interaction signals and pathways .

INTRODUCTION PROGRAM OVERVIEW A TYPICAL ANALYSIS PROCEDURE PERFORMANCE PROFILE CONCLUSIONS FUNDING

PMC2915663

ghrelin , a 28-amino acid peptide , is produced mainly in the stomach and to a lesser extent in the small intestine , pancreas , and hypothalamus and is the endogenous ligand for the growth hormone secretagogue receptor ( ghs - r ) [ 19 ] . serine-3 of ghrelin is acylated with an octanoic acid , the process catalyzed by a recently discovered ghrelin o - acyltransferase ( goat ) [ 10 , 11 ] . the acylation is thought to be required for its biological activity , although desacyl ghrelin has been reported to exert several effects . administration of pharmacological doses of acylated ghrelin (= ghrelin ) to intact animals increases food intake , induces body weight gain , and causes obesity [ 1318 ] . the orexigenic and body fat promoting properties of ghrelin and growth hormone ( gh ) secretagogue ( ghs ) are thought to be independent of gh and mediated primarily by the hypothalamic neuropeptide y ( npy ) and agouti - related protein ( agrp ) systems [ 1416 , 1922 ] . although pharmacologic effects of ghrelin are well documented , physiological role of endogenous ghrelin is poorly understood . it has recently been reported that knockout of either the ghrelin gene or ghrelin receptor gene exerts no or minor effects on body weight and body composition [ 2325 ] . however , the lack of phenotypic changes in knockout mice might reflect compensatory mechanisms that are known to operate occasionally . in humans , gastrectomy results in loss of body weight of about 10% within the first six months after surgery , primarily due to reduced body fat . in addition , gastrectomized patients often complain of loss of appetite , general fatigue , and in some cases impaired bone quality such as osteopenia and osteomalacia [ 9 , 27 ] . at present there are no satisfactory mechanistic explanation and treatment for any of these symptoms . the effects of gastrectomy on food intake and body composition have been poorly documented in rodents . loss of ghrelin could be implicated in these symptoms , since as much as 80% of circulating ghrelin is lost following surgical removal of the glandular stomach or the acid producing part of the stomach in rats and humans [ 4 , 28 , 29 ] . in this study , we performed total gastrectomy in rats and examined whether the recovery from gastrectomy - associated anorexia depends on the circulating ghrelin level or other factors and studied temporal changes in the biosynthesis and orexigenic ability of ghrelin following gastrectomy . male 4-week - old wistar rats ( slc , japan ) were maintained on a 12-hour light / dark cycle and given conventional food and water for 2 weeks and not deprived of food before gastrectomy . they were operated at 6 weeks of age with body weight around 130180 g. rats were anesthetized with an intraperitoneal injection of pentobarbital ( 40 mg / kg ) and a median abdominal incision was made . after the stomach was separated from the greater and lesser omentum , the duodenal bulb was ligated and transected . after the left gastric artery was ligated , the esophagus was clamped above the esophagogastric junction and the stomach was resected . an end - to - side anastomosis between esophagus and jejunum at the 4 - 5 cm anal side from treitz ligament was performed with 70 monofilament polyglyconate synthetic absorbable string ( maxon ; johnson & johnson inc . usa ) using interrupted suture ( billroth ii ( b - ii ) reconstruction ) . on the other hand , jejunum was transected at the 2 - 3 cm anal side from the treitz ligament . then , an end - to - side anastomosis between the esophagus and jejunum was performed with 70 maxon using an interrupted suture . jejunojejuno anastomosis was also done by end - to - side method ( roux - en - y ( r - y ) reconstruction ) . the abdominal wall and skin closure was made by 50 nylon ( johnson & johnson inc . after the operation , the rats were allowed only clear water without food for 3 days , dry milk from the 3rd day , and from the 7th day a conventional pellet diet with free access to water . after the operation , food intake for 24 hours and body weight were measured once a week for 3 months . the rats were sacrificed at three months after operation . to measure plasma ghrelin concentrations , duodenum samples of about 3 cm were taken from 1 cm anal side of duodenal stump . jejunum samples of about 3 cm were taken from 1 cm anal side of esophagojejunostomy . one antiserum was raised against the cooh - terminally cys - extended rat ghrelin ( position 1 - 11 ) in new zealand white rabbits ( # g606 ) that was shown to specifically recognize ghrelin with n - octanoylated ser 3 ( acylated ghrelin ; ghrelin ) . by the radioimmunoassay ( ria ) using this antiserum , designated nh2-terminal ria ( n - ria ) , the concentration of acylated ghrelin was obtained . the other antiserum was raised against the nh2-terminally cys - extended rat ghrelin ( position 13 - 28 ) ( # g107 ) that was shown to recognize both acylated ghrelin and desacyl ghrelin . by the ria using this antiserum , named as cooh - terminal ria ( c - ria ) these two ghrelin - specific rias were used to measure acylated ghrelin and desacyl ghrelin contents in several tissues . the bound and free ligands were separated using a second antibody . acylated ghrelin is expressed as ghrelin and acylated ghrelin plus desacyl ghrelin is expressed as plasma concentrations of acylated ghrelin and desacyl ghrelin were measured using elisa kits ( mitsubishi kagaku iatron , tokyo , japan ) . ghrelin ( peptide institute , osaka , japan ) or saline was administered subcutaneously to control normal rats ( eight - weeks - old ) and those received gastrectomy with b - ii reconstruction . for ghrelin treatment at 2 and 6 weeks after gastrectomy control and gastrectomized rats were divided into two groups ; one group was treated with ghrelin and the other with saline , and food intake and body weight were measured . the injection of ghrelin ( 10 nmol / kg body weight ) was carried out once a day for seven days , and food intake and body weight were measured . for ghrelin treatment at 12 weeks after gastrectomy , the following procedures were used . single injection of ghrelin or saline was followed by measurements of food intake for the subsequent 24 hours . in the next week , two groups were changed : one group that had first received ghrelin was injected with saline , and the other group that had first received saline was injected with ghrelin . food intake after the first injection and that after the second injection were pooled and averaged . statistical analysis was performed using student 's paired and unpaired t - tests . values of p < .05 were considered statistically significant . food intake for 24 hours and body weight were measured in total gastrectomized and control rats during 12 postoperative weeks . gastrectomy with b - ii ( n = 40 ) and r - y reconstruction methods ( n = 35 ) both markedly decreased food intake . in both b - ii and r - y groups , at the 1st week after operation food eaten for 24 hours decreased to a level around 50% of that in control rats ( figure 1(a ) ) . in b - in contrast , food intake in r - y group increased gradually during 4 postoperative weeks . the daily food intake averaged for postoperative 12 weeks was significantly ( p < .05 ) reduced in b - ii and r - y groups ( figure 1(a ) ) . food intake in r - y group was significantly ( p < .05 ) greater than in b - ii group throughout postoperative 12 weeks . body weight significantly decreased after gastrectomy in both b - ii and r - y groups ( figure 1(b ) ) . body weight in r - y group was significantly greater than that in b - ii group from the 2nd through 12th postoperative week ( p < .05 ) . the correlation between body weight , 24 hours food intake , and plasma ghrelin concentrations at the 12th postoperative week was studied . food intake and body weight were correlated with each other irrespective of reconstruction methods and were plotted on a single regression line ( figure 2(a ) ) . in contrast , there was no correlation between food intake and plasma concentrations of total ghrelin ( figure 2(b ) ) . we next examined whether body weight correlates with plasma concentrations of ghrelin or total ghrelin , by using n - ria and c - ria . body weight did not correlate with plasma ghrelin and total ghrelin levels ( figures 2(c ) and 2(d ) ) . furthermore , no significant difference between r - y and b - ii groups was observed in averaged ghrelin levels ( b - ii : 46.3 7.2 fmol / ml ( n = 40 ) , r - y : 35.9 4.6 fmol / ml ( n = 35 ) ) and total ghrelin levels ( b - ii : 393.0 40.0 fmol / ml ( n = 40 ) , r - y : 349.8 23.2 fmol / ml ( n = 35 ) ) , though some rats in b - ii groups showed higher levels of ghrelin and total ghrelin . these results indicate that plasma ghrelin levels are significantly reduced in r - y and b - ii groups and that greater food intake in r - y than in b - ii is related to the method of reconstruction but not plasma ghrelin levels . plasma ghrelin concentrations were markedly reduced in both r - y and b - ii groups to the levels of about 30% of those in control rats ( figure 3(a ) ) . therefore , the reduction of plasma ghrelin levels is due to lack of release of ghrelin from stomach . we examined whether the reduction of ghrelin due to gastrectomy could influence the production of ghrelin in the intestine and pancreas , the tissues known to produce ghrelin . ghrelin concentrations in the duodenum increased approximately by 100% in r - y and b - ii groups ( figure 3(b ) ) , while those in the jejunum did not change ( figure 3(c ) ) . ghrelin concentrations in the pancreas were much smaller than those in the intestine , however , they dramatically increased approximately by 500% in b - ii and by 400% in r - y groups ( figure 3(d ) ) . on the other hand , plasma concentrations of total ghrelin were approximately five times higher than those of ghrelin , and they were markedly reduced by gastrectomy in both groups ( figure 3(e ) ) . contents of total ghrelin in the duodenum and pancreas , but not jejunum , increased in both r - y and b - ii groups ( figures 3(f)3(h ) ) . the gastrectomy - associated relative changes of total ghrelin in the circulation , duodenum and pancreas were similar to those of ghrelin . these results suggest that gastrectomy - induced reductions in circulating ghrelin levels promote synthesis of ghrelin in the duodenum and pancreas , which may have compensatory roles including the restoration of circulating ghrelin levels . in consistence with this inference , plasma levels of ghrelin and total ghrelin that were markedly reduced at 2 weeks after gastrectomy with b - ii reconstruction ( ghrelin : 25.0 5.3 fmol / ml ( n = 4 ) , total ghrelin : 295.8 34.6 fmol / ml ( n = 4 ) ) were significantly ( p < .05 ) elevated at the 12th postgastrectomy week ( ghrelin : 46.3 7.1 fmol / ml ( n = 40 ) , total ghrelin : 393.0 40.0 fmol / ml ( n = 40 ) ) . we examined whether reduced levels of plasma ghrelin after gastrectomy could be altered by fasting , the physiological regulator of ghrelin release . at the 12th postoperative week , blood samples were taken from ad - lib fed and 48 hour fasting rats . effects of fasting on plasma levels of ghrelin and desacyl ghrelin were examined by using elisa , since ria did not allow us to determine exact levels of desacyl ghrelin . .05 ) elevated by fasting approximately by 3 times in both b - ii and r - y groups ( figures 4(a ) and 4(c ) ) . desacyl ghrelin concentrations were also significantly ( p < .05 ) elevated by 3 - 4 times by fasting in b - ii and r - y groups ( figures 4(b ) and 4(d ) ) . thus , fasting induced similar fold increases in both ghrelin and desacyl ghrelin levels in the circulation in gastrectomized rats . rats aged 8-weeks were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for seven continuous days . injection of ghrelin increased food intake , in which significant ( p < .05 ) difference was obtained at the 2nd , 4th and 5th days ( figure 5(a ) ) . the cumulative food intake during the treatment period of 7 days was significantly ( p < .05 ) increased by ghrelin ( figure 5(b ) ) . at 2 weeks after total gastrectomy , rats were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for 7 continuous days . the 24 hours food intake averaged for the treatment period of 7 days was not significantly different between ghrelin - and saline - treated groups ( figure 5(c ) ) . essentially the same results were obtained in rats at 6 weeks after total gastrectomy ; 24 hours food intake averaged for the treatment period of 7 days was not different between ghrelin - and saline - treated groups ( figure 5(d ) ) . at the 12th week after total gastrectomy one group received daily subcutaneous injection of saline for a week followed by that of ghrelin for the next week . vice versa , the other group received ghrelin for the first week followed by saline for the next week . the amount of 24 hours food intake in rats injected with ghrelin was significantly ( p < .05 ) greater than that with saline ( figure 5(e ) ) . these results indicate that ghrelin injection increases food intake in the later stage of 12 weeks after gastrectomy but not earlier . ghrelin is secreted primarily from the stomach [ 9 , 30 ] , and stimulates food intake and body weight gain . gastrectomy commonly decreases food intake , body weight , fat mass and bone mass , and is also accompanied by a marked reduction in circulating ghrelin levels . however , whether ghrelin levels are causally related to food intake and/or body weight after gastrectomy is not well understood . in the present study , we produced total gastrectomized rat models and examined postoperative changes in ghrelin levels , food intake and body weight . plasma ghrelin levels were markedly reduced by gastrectomy irrespective to whether the reconstruction was performed by b - ii or r - y method . however , the recovery from gastrectomy - induced reductions in food intake and body weight was much greater in r - y than in b - ii group . the results indicate that the recovery from reduced food intake and body weight after gastrectomy is not correlated with circulating ghrelin levels but strongly depends on the reconstruction method . the mechanism for the better recovery of food intake and body weight with r - y method after total gastrectomy remains unclear . ghrelin contents in the duodenum , jejunum and pancreas at 12th postoperative week were not different between b - ii and r - y methods . it is speculated that in b - ii method bile juice may directly reflux the esophagojejunostomy and consequently decrease food intake . alternatively , shorter distance between esophagojejunostomy and jejunojejunostomy in r - y method than in b - ii method may yield smaller reflux of bile juice . it is suggested that selecting the reconstruction method with less reflux of bile juice may contribute to better recovery from the gastrectomy - induced anorexia . at the 12th week after gastrectomy , ghrelin production was markedly enhanced in the duodenum , the organ known to produce the second largest amount of ghrelin . this result was essentially the same between b - ii and r - y groups , suggesting that the enhanced ghrelin production is not related to the reconstruction method but likely due to the removal of gastric ghrelin . it is therefore suggested that the enhanced ghrelin production in the duodenum could partly compensate for gastrectomy - associated reductions of the circulating ghrelin and its endocrine functions . in another line of experiments , at 12th postoperative week fasting markedly increased plasma ghrelin levels in gastrectomized rats , indicating that the tissue other than stomach , most likely the duodenum , secretes ghrelin in response to fasting , the physiological regulator of ghrelin secretion [ 9 , 32 ] . furthermore , the feeding response to peripheral ghrelin administration was once eliminated by gastrectomy but restored at 12th postoperative week . these data suggest that ghrelin can be released and stimulate food intake under fasted conditions at this later period after gastrectomy . collectively , the markedly upregulated production of ghrelin in the duodenum could contribute to the recovery of food intake and body weight in the later postoperative period . in this study , since the ghrelin content in the pancreas and its increment due to gastrectomy are much smaller than those in the duodenum , they may neither significantly contribute to the circulating ghrelin levels nor operate endocrine functions . however , previous studies using ghrelin receptor antagonists and ghrelin - deficient mice have demonstrated that ghrelin in the pancreatic islets inhibits insulin release in an autocrine / paracrine manner and consequently regulate blood glucose levels [ 33 , 34 ] . the late postprandial dumping syndrome accompanying gastrectomy is characterized by hypoglycemia principally due to excessive insulin release . the mechanism for excessive insulin release remains unclear , lack of the stomach - derived ghrelin could be implicated . if so , it is possible that upregulated pancreatic ghrelin compensates lack of stomach - derived ghrelin and attenuate insulin release , thereby counteracting the hypoglycemia in the late dumping syndrome . further study is necessary to elucidate the role and mechanism for the upregulation of ghrelin production in the pancreas after gastrectomy . it has been well documented that administration of pharmacological doses of ghrelin to intact animals increases food intake , induces weight gain , and causes obesity [ 9 , 1318 ] . ghrelin injection failed to stimulate food intake in the early stages of 2nd to 6th week after operation , but increased it in the late stage of 12th week . the results suggest that some machinery that links ghrelin reception to feeding dysfunctions after gastrectomy but can be restored . it has been reported that the peripheral injection of ghrelin does not increase food intake after cutting vagus nerve [ 15 , 30 , 35 ] . it is thought that peripheral ghrelin signal is transmitted to the feeding center at least partly via afferent vagal nerves [ 15 , 30 , 35 ] . in our study , it is speculated that regeneration of vagus nerves takes place not immediately but time - dependently , allowing reception of injected ghrelin and transmission of its signal to the feeding center . in addition , chronic hypoghrelinemia in gastrectomized status was reported to induce hypersensitivity to ghrelin as judged by secretion of growth hormone . if this is also the case for the orexigenic action of ghrelin , the hypersensitivity to ghrelin could make the ghrelin injection more potent and/or efficacious in correcting anorexia after gastrectomy . collectively , in the certain period when vagus nerves are regenerated and sensitivity to ghrelin is elevated , ghrelin treatment is expected to effectively stimulate feeding . in fact , the fractional increase of food intake in response to ghrelin injection at 12th postoperative week was similar to that in control rats ( figures 5(e ) versus 5(a ) ) . thus , our results raise a possibility that ghrelin replacement therapy given in later stages could be effective in promoting food intake and correcting some symptoms associated with gastrectomy in humans . an effective treatment of severe obesity is bariatric surgery , in which gastric bypass is a representative method . gastric bypass , as well as gastrectomy , markedly reduces appetite and body weight , suggesting that changes in plasma ghrelin levels are involved . however , studies on changes in plasma ghrelin levels after gastric bypass have yielded inconsistent results ; decreased , unchanged , or increased [ 3840 ] . in our study , better restoration in feeding and body weight after gastrectomy was obtained with r - y than b - ii method , while plasma ghrelin levels were similar . these observations by us and others collectively suggest that feeding and body weight in the earlier period after gastric surgery , are not associated with plasma ghrelin levels but determined by other factors , possibly reconstruction methods associated with specific histological conditions . in summary , recovery from gastrectomy - induced reductions in food intake and body weight is not related to plasma ghrelin levels but depends on the reconstruction method in rats . at postoperational 12th week , ghrelin production is increased in the duodenum and pancreas , and circulating ghrelin level is elevated by fasting , suggesting that ghrelin could be released from extra - stomach tissues and play a compensatory role . ghrelin administration stimulates feeding at postoperational 12th week but not earlier , suggesting that ghrelin treatment in later periods could be effective in treating patients with gastrectomy - induced anorexia and associated symptoms .

gastrectomy reduces food intake and body weight ( bw ) hampering recovery of physical conditions . it also reduces plasma levels of stomach - derived orexigenic ghrelin . this study explored changes in orexigenic ghrelin system in rats receiving total gastrectomy with billroth ii ( b - ii ) or roux - en - y ( r - y ) method . feeding and bw were reduced by gastrectomy and subsequently recovered to a greater extent with r - y than b - ii while plasma ghrelin decreased similarly . at postoperative 12th week , ghrelin contents increased in the duodenum and pancreas , plasma ghrelin levels increased upon fasting , and ghrelin injection promoted feeding but not in earlier periods . in summary , gastrectomized rats partially recover feeding and bw , in a reconstruction - dependent manner . at 12th week , ghrelin is upregulated in extra - stomach tissues , plasma ghrelin levels are physiologically regulated , and orexigenic effect of exogenous ghrelin is restored . this time - related recovery of ghrelin system may provide a strategy for promoting feeding , bw , and thereby physical conditions in gastrectomized patients .

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

PMC3769079

the technical convenience of milk as a source of dna can be expected to increase the field of application of the markerbased methods for genetic analysis of goat genome . it has a remarkable importance for several reasons related to governmental regulation and public health . mixture in dairy products and species substitution should be observed to be a cause of human adverse reactions ( bottero et al , 2003 ) . the source of dna in milk could be from somatic cells ( fahr et al , 1999 ) . goat milk is similar to cow milk with around 87 percent water , 67 percent energy , 3.3 percent protein , 4.0 percent fat , and 4.6 percent carbohydrates ( belanger and jerry , 2001 ) . goat milk differs from cow and human milk in several ways , among them higher digestibility and lower lactose ( larson , 1978 ) . one difference is that goat milk has smaller fat globules present due to the lack of the enzyme that aggregates the globules in the milk ( belanger and jerry , 2001 ) . the objective of the present study was to evaluate milk as a source of goat dna and as a substrate for pcr amplification using mitochondrial cytochrome - b gene as a target dna for pcr amplification . this was prospective study in which samples are collected throughout the study this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010 25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 , 12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml . the primers were used at a concentration of 20mole / l which appears to 2 l . next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing . this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units . the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes . thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) . the 10x tris borate edta ( tbe ) buffer was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml . 15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed . this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010 25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included . before collection , the teats were cleaned with alcohol to avoid samples contamination from skin . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . before collection , the teats were cleaned with alcohol to avoid samples contamination from skin . milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital . the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna . extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes . the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute . the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes . the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c . maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature . for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences . gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna . the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 , 12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml . the primers were used at a concentration of 20mole / l which appears to 2 l . next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing . this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units . the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes . thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) . all pcr amplification product samples were visualized using agarose gel electrophoresis . the 10x tris borate edta ( tbe ) buffer was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml . 15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed . the dna yield varies with the different stages of lactation as compared with the yield from peripheral blood leukocytes . the pcr - based assay described in this study afforded sensitive and specific identification of goat mtcyt - b dna . using the pair of primers ( gsl1 , gsr2 ) . the sensitivity studies indicate that , the 428bp pcr products were detected from not < 1.0 pg of goat mtcyt - b gene ( fig.1 ) . the specificity studies for goat primers indicate that the described pcr assay failed to amplify the specific pcr product from dna extracted from other animal species including cattle , sheep , swine , camel , deer , horse , donkey , and human ( figure 2 ) . using primers gsl1 , gsr2 , amplification of the goat - specific 428 bp pcr product was produced from different milk samples collected from goats included in this study ( figure 3 , 4 , 5 ) . in goat , however , obvious technical difficulties exist in the collection of blood samples from large numbers of individuals among widely separated herds , limiting the application of marker - based methods for genetic analysis and for genetic improvement of economic traits in dairy goat . the scientific data presented in this study indicate that dna extracted from goat milk could serve as substrates for pcr amplification of the full length of the caprine mitochondrial cytochrome - b gene or a fragment of the gene . since collection of milk is a non invasive procedure , it can often substitute for blood as a source of dna . the technical convenience of milk as a source of dna can be expected to increase the field of application of the marker - based methods for genetic analysis of goat genome . it has a remarkable importance for several reasons related to governmental regulation and public health . protection against species substitution or admixture in dairy products is of significant importance ( bottero et al , 2003 ) . it was found that most milk proteins , even at low concentration , are potential allergens ( sampson , 2003 ) . also , cow s milk was reported as the main dairy product responsible for human adverse reaction ( rance et al , 2005 ) . thus , the counterfeiting of goat s milk with cow s milk may be considered as a health risk making species identification an important issue in current food safety requirement . the common fraudulent practice found in the dairy production line is the use of a cheaper type of milk in substitution of more expensive ones . currently , different methods are used for species identification in milk and milk products including immunological ( addeo et al , 1995 ) , electrophoretic ( cartoni et al , 1998 ) , and chromatographic techniques ( pellegrino et al , 1991 ) . among these methods , capillary electrophoresis , two dimensional electrophoresis , isoelectric focusing of milk caseins which is the european community reference method for cow s milk detection ( ecr , 1996 ) , however these methods ca nt always distinguish milk from closely related species and not suitable for heat treated milk . the pcr amplification technology , described in this study , provides a simple , rapid , reliable and sensitive method for species identification and differentiation . the time required for the pcr amplification was approximately 3 hours ; this means that confirmatory diagnosis could be obtained within the same working day . ( 1993 ) reported that milk is less reliable source of dna than is blood because it requires large size of sample and high concentration of somatic cells ( e. lipkin et al , 1993 ) . but in our study we used only 200l of milk sample for extraction of dna , and the pcr products were the same in length and approximate quantity for milk , for dna extracted from milk , and for dna extracted from blood , this difference may reflect the detection procedures described above . the intensity of dna signal in gel electrophoresis , show that , there is a decline in the dna content in the late lactation periods . this may indicate the milk dna content is high in the first and second lactation periods and then declines . for more significant results dna quantification by spectrophotometer these non - nuclear targets possess several advantages over nuclear genes ( unseld et al,1995 ) . they are generally more abundant in any given sample than single - copy nuclear genes , and , because mitochondrial dna has a relatively high mutation rate compared with the nuclear dna , they contain a greater accumulation of point mutations that can be used to better define species differences . moreover , mitochondrial dna tends to be inherited through the maternal germ line , and the resulting lack of heterozygosity in the alleles under study simplifies analysis ( kocher et al , 1989 ) . the mtcyt - b dna was selected in this work as the target sequence for species identification . pcr - based assay described in this study would be advantageous in the variety of conditions including comparative genomics , species identification in milk and milk products , experimental physiology and can be recommended in the quality control departments in order to support policies and regulation of import / export of milk and milk products .

conflict of interest : none declared.introductionthis study was carried out to evaluate pcr - based method for detection of dna in goat milk . it utilized primers targeting the mitochondrial cytochrome b ( mtcyt - b ) gene , which was used as a target dna for pcr amplification.methodsfor the specific identification of goat mtcyt - b gene , pair of primers ( gsl1 , gsr2 ) , were used , which produced a 428 base pair ( bp ) pcr product from milk samples as well as from peripheral blood . amplification products were visualized on ethidium bromide - stained agarose gels.results and discussionamplification products were not detected when the pcr was applied to dna from animal species including cattle , sheep , swine , camel , deer , horse , donkey , and human , which indicates that the 2 pairs of primers are specific for goat.conclusiondna can be extracted from goat milk and would be advantageous in the variety of application such as species identification in milk and milk .

1. INTRODUCTION 2. MATERIAL AND METHODS 2.1. Study design 2.2. Study Area 2.3. Study population 2.4. Data collections Milk samples collection from goats Blood samples collection DNA extraction from milk samples Selection of goat primers for PCR amplification 2.5. Polymerase chain reaction (PCR) 2.6. Visualization of PCR products 3. RESULTS AND DISCUSSION 4. CONCLUSION

PMC4590902

functional popliteal artery entrapment syndrome ( paes ) is an important and possibly underrecognized cause of exertional leg pain . it shares many features with other causes of exertional leg pain , and more than one condition can be present at once , confusing the clinical picture . an understanding of the typical presenting features of the common causes of exertional leg pain is essential , allowing the clinician to determine those suggestive of paes and requiring further investigation . investigating functional paes is fraught with potential problems and , if performed incorrectly , can result in false negative and false positive findings . the authors believe that currently accepted vascular investigations such as ankle - brachial indices and doppler ultrasound performed at rest are not accurate in investigating functional paes . rather , a review of the literature would suggest that investigations such as provocative doppler ultrasound and mri angiography are performed as soon as possible after reproducing symptoms to capture the occlusion while it is occurring . once the diagnosis of functional paes is confirmed , there are a number of treatment strategies available . until recently , definitive intervention was only available in the form of vascular surgery with variable myotomies and releases . we provide information on a pilot study suggesting a new less invasive intervention of guided botulinum toxin injection to the level of entrapment , as an alternative to surgical intervention . paes shares many clinical features with other causes of exertional leg pain , most of which are thought to be more common . adding to the complexity , more than one cause of exertional leg pain may be present in an individual patient at any one time . chronic exertional compartment syndrome ( cecs ) in particular has many of the same features of paes and the two conditions can be confused [ 2 , 3 ] . also , it is not possible to distinguish between anatomical and functional paes on clinical symptoms alone . a detailed history and examination provide very useful information and help determine whether further investigation is warranted . an outline of the more common differential diagnoses of exertional leg pain and their features follows and a summary is provided in table 1 . this condition is thought to represent the most common cause of exertional leg pain with an incidence between 13 and 42% . patients describe typically bilateral leg pain along a strip of the distal third of the posteromedial tibial border that comes on early with exercise and may warm up but usually will ache for some time after stopping . examination at rest will usually reveal palpable tenderness along the distal third of the posteromedial tibial border and pain with hopping . some studies suggest a possible predisposition of mtss in females . while stress fractures are not common in the nonexercising population , they are thought to represent 0.720% of sports medicine clinic presentations . patients typically complain of pain that is focal and palpable ( unless the site is covered by a significant muscle layer where symptoms can be vague and nondescript ) . with lower limb stress fractures , pain often presents early with impact related exercise and is worse during the landing phase of running . stress fractures are typically unilateral and can affect any bone in the lower limbs , although the tibia is most common [ 1 , 9 ] . female athletes are thought to be at greater risk of developing stress fractures than males and bennell et al . suggest that menstrual disturbances , caloric restriction , lower bone density , muscle weakness , and leg length differences are important risk factors for stress fracture . patients by definition are pain - free at rest and develop a steadily worsening pain with exertion . the pain will gradually build up over 1020 minutes and is not rapid in onset ( unlike some cases of paes ) . the pain is typically isolated to one or more compartments , most commonly anterior and deep posterior , with the least most common being the superficial posterior compartment ( where the majority of paes pain is described ) [ 5 , 9 ] . the pain will typically dissipate slowly over minutes to hours but will return quickly if the patient attempts to return to exercise again . the leg pain is overwhelmingly bilateral ( although unilateral cases can present ) and there is no difference in predisposition according to the sex of the individual [ 7 , 11 ] . paes can be further divided into two groups , anatomical and functional . in the case of anatomical paes , there is a clearly defined anatomical lesion that directly leads to entrapment and subsequent occlusion of the popliteal artery . the second and larger subgroup of anatomically normal or functional paes is particularly poorly understood . in functional paes , there is evidence of popliteal artery occlusion and subsequent claudicant symptoms , but no defined lesion can be found that directly causes the occlusion . patients with anatomical paes are thought to be older and more sedentary while functional paes patients are thought to be younger , more commonly female , and more active . baltopoulos et al . suggest that functional paes may be bilateral in 2576% of cases . the true incidence of functional paes is unknown , and it is described as being rare [ 8 , 13 , 14 ] . however , it is possible that this condition is underreported and the incidence may be greater than previously recognized . it occurs most commonly in the back of the calf but it can be anterior and lateral especially if the anterior tibial artery is involved [ 2 , 16 ] . patients can have pain with minimal exertion or provocative leg positioning and occasionally at rest . symptoms such as coldness and paraesthesia have also been reported . running or walking on an incline the pain follows a claudicant pattern similar to cecs but unlike cecs will resolve more quickly on cessation of exercise . provocative manoeuvres may result in the development of a reduced peripheral pulses and/or a popliteal bruit on auscultation of the popliteal fossa although this may also be possible in the asymptomatic occluder group as outlined later . when examining patients with suspected functional paes , it is important to diagnose and/or exclude other causes of exertional leg pain . examination includes palpation and percussion over the lower limb to look for signs of bone stress , be it focal at the site of a stress fracture or along a strip of the distal posteromedial tibial border in the case of mtss . some authors suggest that paes must have a combination of pain with hopping , in conjunction with the development of ischaemic signs like pallor , coldness , and reactive hyperaemia . in our experience , pain with hopping more likely indicates some form of bone stress , and the development of physical signs of ischaemia in younger individuals with suspected functional paes is extremely uncommon . some authors describe physical examination as unreliable and rely on clinical suspicion as justification for progressing to vascular investigations . some do not describe their clinical assessment , while others attempt to provoke symptoms by encouraging the patient to hop or climb stairs , followed by a cursory physical examination . most will assess resting peripheral pulses [ 12 , 18 ] which should be examined at true rest . the popliteal fossa should be auscultated to determine the presence of a resting bruit . in general , bruits are not audible until an artery is approximately 50% occluded . the sound increases in pitch as the lumen becomes more narrowed to a critical size . thereafter , the sound may no longer be detectable as the volume of blood flow becomes greatly reduced . if pulses are reduced or a bruit is present at rest , than this would indicate an underlying vascular malformation or significant luminal narrowing ( such as in advanced atherosclerosis ) and further radiological imaging is indicated . we have developed a simple clinical test that can be performed in the consulting rooms . after assessing for a popliteal fossa bruit and examining peripheral pulses at rest , the patient is required to perform 1520 single leg eccentric heel drops off the edge of a step , while asking about any developing or worsening leg pain . immediately after the test is performed , the popliteal fossa is again auscultated for a bruit , and peripheral pulses are examined for any reduction . in our experience , it is worth auscultating for a number of minutes after exertion , as more significant cases ( with complete occlusion ) may initially have no bruit , or at least delayed onset of a bruit as flow reestablishes . we believe that if patients do not develop pain or discomfort with this test , or if a bruit and/or pulse reduction is not evident , then it is unlikely that the patient is suffering from paes . unfortunately , the development of pain / bruit / pulse reduction does not mean the patient has paes , as it is possible that they may fall into the asymptomatic occluder group . provided that the patient has clinical features suggestive of paes , a positive result warrants further vascular investigation . despite paes being a well - defined condition , no clear - cut consensus regarding the diagnostic work - up of these patients exists . the general population can be divided into four groups , including asymptomatic nonoccluders , who presumably will not present for assessment or investigation . therefore , the three symptomatic groups that investigation must distinguish areanatomical paes , asymptomatic occluder ( i.e. , exertional leg pain due to another cause , but in whom the artery can incidentally occlude),symptomatic occluder , that is , functional paes . asymptomatic occluder ( i.e. , exertional leg pain due to another cause , but in whom the artery can incidentally occlude ) , symptomatic occluder , that is , functional paes . the abi is calculated by dividing the systolic blood pressure at the ankle by that at the arm . some authors recommend the use of ankle brachial indices in standard work - up for functional paes . however , as occlusion in functional paes occurs during exertion , testing after exercise is likely to result in a false negative result . there are also difficulties in obtaining abi measurements during exercise and pillai suggests that abis during forceful plantarflexion are difficult to interpret and not as helpful when assessing graded compression of a patent artery . some authors will routinely perform compartment pressure studies in addition to vascular studies in the work - up of suspected paes . this seems to be excessive and our review of the literature could find no compelling reason as to why this should be mandatory . for this reason we do not recommend routine compartment pressure studies , unless symptoms and history are strongly suggestive of possible concomitant cecs . doppler ultrasound provides a relatively cheap , noninvasive , and accessible procedure to assess flow through the popliteal artery , and it is generally recommended that this be the first line investigation for paes [ 20 , 21 ] . despite this , a review of the literature shows no definite consensus on how to perform doppler ultrasound in investigating paes . this assesses loss of the posterior tibial artery distal pulse during sustained passive dorsiflexion and plantarflexion of the foot . also , there is a risk of overcalling entrapment with movement of the artery , muscles , or probe during exercise giving the illusion of occlusion , but technique variations can usually overcome this . many authors researching paes suggest that the most important feature in its diagnosis is the reproduction of symptoms with the help of provocative manoeuvres and verification by duplex ultrasonography [ 17 , 18 , 21 ] . typically , investigations at rest will show a patent popliteal artery with normal distal pulses . in functional paes , occlusion will often only occur during exertion and can resolve almost immediately . even the 3060 seconds taken from getting off a treadmill to an examination bed and applying the ultrasound probe can be enough for arterial occlusion to cease . because occlusion can resolve very quickly , immediate assessment after reproduction of symptoms is essential . the position of most occlusions occurs when the patient has their knee extended , and they hold active plantarflexion . it is not possible to hold this position for sustained periods during a mri due to discomfort and fatigue . ultrasound is a useful modality , as it allows real time assessment whilst some form of provocation to reproduce symptoms is performed . despite the agreement that provocation is necessary , a clear and detailed description of the protocols to do this is generally not provided . we perform a resting anatomical study first , with the patient in the supine position . we record waveforms and velocities of the peripheral arteries as well as assess for evidence of intimal thickening or fixed arterial disease . the anatomy of the popliteal fossa is assessed , looking particularly for any popliteal artery or soft tissue structure variations or whether the anterior tibial artery has a high bifurcation . hoffman et al . found that the force of plantarflexion required to occlude the popliteal artery during provocative positioning is important , with the majority of asymptomatic occluders occluding the popliteal artery with sustained holding of 70% of maximal plantarflexion force . for this reason , we use a graded ultrasound protocol progressing throughprone patient holding a position of provocation ( knee extension and plantarflexion ) against no load , patient is prone and pushing against approximately 25% of maximal plantarflexion force ( figure 1(a)),dynamic loading by pushing against 50100% of maximal plantarflexion force ( figure 1(b ) ) . prone patient holding a position of provocation ( knee extension and plantarflexion ) against no load , patient is prone and pushing against approximately 25% of maximal plantarflexion force ( figure 1(a ) ) , dynamic loading by pushing against 50100% of maximal plantarflexion force ( figure 1(b ) ) . symptomatic patients who occlude in the first 2 categories are presumed to represent more severe cases of functional paes . if the initial 2 assessments are negative , the patient is assessed in an erect weight - bearing position while cycling through range of motion . this is thought to represent somewhere between 50 and 100% maximal plantarflexion force and may more reliably represent what is occurring functionally during activity . if the erect assessment is negative , the patient may attempt similar exertion that elucidates symptoms in a normal training experience , until they are symptomatic . at this point we will use cine loops to demonstrate the popliteal artery from resting patency through compression to occlusion and mark the site of compression on skin for mri correlation . we feel that doppler ultrasound does not adequately demonstrate the anatomy to rule out anatomical paes and confirmation of occlusion in a symptomatic patient means the patient may be in either the anatomical or functional paes groups . mri is a valuable noninvasive modality that allows optimal visualization of the popliteal artery as well as the surrounding structures [ 23 , 24 ] . mri can demonstrate a variety of findings including abnormal lateralized insertion of the medial head of gastrocnemius , medial displacement , and occlusion of the popliteal artery in the popliteal fossa and fat tissue filling the normal location of the medial head of gastrocnemius [ 23 , 25 ] . in this way , anatomical paes can be distinguished from functional paes and the muscles and anatomical boundaries contributing can be accurately delineated . this in turn can direct injection therapy ( as discussed in section 5 ) or the site of surgical intervention . mri angiography can demonstrate the level of occlusion , but limitations include the development of movement artifact with forceful plantarflexion positions and inability to hold such a position for prolonged periods . this is a significant limitation as for reasons outlined earlier ; investigating for occlusion in functional paes should occur during provocation . most papers reviewed do not describe provocative manoeuvres with mri angiography [ 2325 ] while one suggests holding sustained forceful plantarflexion continuously for 2029 seconds whilst the angiogram is performed . after positive ultrasound studies , a fiducial marker is placed on the patient 's skin where the popliteal artery is being occluded to help correlate between the ultrasound and mri site of occlusion . t1 weighted axial and coronal images are acquired to demonstrate the medial head and lateral heads of gastrocnemius , popliteus , and plantaris muscles and their alignment and associations with the femoral condyles and popliteal arteries and nerves . the patient is instructed to dorsiflex and plantarflex their feet whilst acquiring t2 weighted 2d steady state images axially across the popliteal region . before the final contrast mri angiogram is performed , the patient is instructed to alternate a neutral ankle position with maximal plantarflexion force until they stimulate the pain that they usually experience ( rather than a single sustained forceful contraction ) . once they experience this pain , they keep their ankles in plantarflexion whilst we inject the contrast and perform the angiogram ( figure 2 ) . one of the disadvantages of this technique is that there is an approximate 30-second delay before the contrast arrives at the popliteal artery and the angiogram can be performed and the artery may re - establish flow during this time . patients quite often are in pain and or exhausted during this last series and may shake because of this . based on our review of the literature , figure 3 represents a decision - making flowchart in the assessment , work - up , and treatment of suspected paes . given the described rapid progression of arterial injury , it is recommended that anatomical paes patients undergo surgery to remove the site of entrapment . this typically involves exploration , limited fasciotomy , myotomy to varying degrees , and possible excision of occlusive fibrous bands [ 2 , 17 ] . results of popliteal fossa exploration , bypass , or muscle detachment , or a combination of these , and fossa decompression are generally good . most series report a small number of patients , but > 90% appear to return to activities in sports 3 months with resolution of all previous symptoms . the pathogenesis and progression of functionalpaes are uncertain , but it may be that these patients develop arterial injury more gradually with onset of more significant symptoms later in life . turnipseed recommends resection of plantaris muscle and the crural band of soleus fascia that forms the outlet of the popliteal fossa as he feels that this fascial band is the fulcrum against which the neurovascular bundle is compressed in functional paes . however the site and amount of muscle necessary to be removed to prevent further occlusion is not always obvious . the popliteal artery will need exploration and there are increased risks of postoperative complications such as seroma ( 4.6% ) and infection ( 2% ) . also surgery for functional paes does not seem to be as successful as that for anatomical paes , with reports suggesting only 77% of patients after surgery reporting complete resolution of symptoms . the use of botulinum injections for paralysis of muscles to manage medical conditions is well established . there are several descriptions of the use of botulinum in the treatment of muscle spasticity , particularly in cerebral palsy patients [ 2729 ] , and piriformis injection of btx - a has been used successfully to treat sacral plexus and proximal sciatic nerve compression . described the use of btx - a injection into a crus of the hemidiaphragm to treat renal artery stenosis . the proposed mechanisms of action for intramuscular periarterial botulinum therapy for paes areparalysis of the muscular slip of muscle responsible for the dynamic arterial occlusion , localised muscle atrophy caused by the botulinum which may increase space for the vessel and would explain the prolonged effect of botulinum on this condition beyond the expected therapeutic effect of the medication , possible arterial smooth muscle relaxation of the popliteal artery resulting in vasodilatation . paralysis of the muscular slip of muscle responsible for the dynamic arterial occlusion , localised muscle atrophy caused by the botulinum which may increase space for the vessel and would explain the prolonged effect of botulinum on this condition beyond the expected therapeutic effect of the medication , possible arterial smooth muscle relaxation of the popliteal artery resulting in vasodilatation . unfortunately , to date , there is no published data on the efficacy of botulinum injection in the management of functional paes . we have commenced a pilot study using intramuscular periarterial injection of btx - a to treat functional paes with promising initial results . we hope to publish the outcomes as our cohort size increases , but at present this remains an unproven intervention . functional paes is a condition that is possibly underrecognized and , if left untreated , can result in progressive arterial damage and the risk of developing lower limb ischaemia . it shares many features with other causes of exertional leg pain ( especially chronic exertional compartment syndrome ) and may coexist with one or more of these . a suggestive clinical history includes features of pain aggravated by exercise , but also possibly at rest with positions of knee extension and plantarflexion . the pain will typically resolve quickly once provocative manoeuvres are ceased , although an ache may persist for hours . anatomical and functional paes can not be distinguished on clinical features alone , and possibly over half of the normal population can demonstrate some arterial occlusion with provocative manoeuvres . for this reason , specialized vascular investigations are indicated , particularly a doppler ultrasound protocol performed at rest and during provocation and immediately after , which can demonstrate real time arterial occlusion and the level it is occurring at . once occlusion is demonstrated , mri can demonstrate the definitive anatomy of the popliteal fossa , whether anatomical paes exists , and the site and extent of functional entrapment . from here , the best treatment can be provided , with consideration of guided botox injection for functional paes as a potential new intervention , or progression to surgical intervention .

functional popliteal artery entrapment syndrome ( paes ) is an important and possibly underrecognized cause of exertional leg pain ( elp ) . as it is poorly understood , it is at risk of misdiagnosis and mismanagement . the features indicative of paes are outlined , as it can share features with other causes of elp . investigating functional paes is also fraught with potential problems and if it is performed incorrectly , it can result in false negative and false positive findings . a review of the current vascular investigations is provided , highlighting some of the limitations standard tests have in determining functional paes . once a clinical suspicion for paes is satisfied , it is necessary to further distinguish the subcategories of anatomical and functional entrapment and the group of asymptomatic occluders . when definitive entrapment is confirmed , it is important to identify the level of entrapment so that precise intervention can be performed . treatment strategies for functional paes are discussed , including the possibility of a new , less invasive intervention of guided botulinum toxin injection at the level of entrapment as an alternative to vascular surgery .

1. Introduction 2. Clinical Features and Differential Diagnosis of Exertional Leg Pain 3. Examination Findings in Functional PAES 4. Investigations in Functional PAES 5. Treatment 6. Summary

PMC4939207

its raw or processed roots are the only organ source for clinical uses of traditional chinese medicine , and the leaves are not utilized at all . however , r. glutinosa leaves have the pharmacological effects and can nourish yin , tonify qi and kidney , and promote blood circulation . in the folk , the fresh leaves are often applied externally to treat malignant sore and tinea manus and pedis . as a perennial herb , r. glutinosa has to be annually cultivated and harvested since its fleshy roots easily decay during the winter dormancy or are consumed when new plantlets come out from them in the next season . due to its severe continuous cropping obstacle , however , r. glutinosa plants could not grow well on the same land after the first cropping , decreasing the root yield greatly . thus , exploitation of r. glutinosa leaves not only provides another potential medicinal source for extraction of important bioactive compounds present in its roots but also complements the shortage of r. glutinosa roots due to the limited land and continuous cropping obstacle . a lot of pharmacologically bioactive secondary metabolites [ 4 , 5 ] including iridoid glycosides , phenylethanoid glycosides , and polysaccharides have been isolated in r. glutinosa , of which iridoid glycosides are thought to be main bioactive constituents . till now , more than thirty iridoid glycosides have been separated and identified [ 46 ] , including catalpol , aucubin , geniposidic acid ( gpa ) , rehmanniosides a , b , c , and d , and rehmaglutosides a k . for example , as the main active iridoid glycoside in r. glutinosa , catalpol was found to play the important roles in treatment of many diseases including kidney diseases , neurodegenerative diseases , and diabetes [ 9 , 10 ] . it is revealed that aucubin had pharmacological effects such as antifungals , anti - inflammation and antioxidation , and hepatoprotection . similarly , gpa had therapeutic effects in anti - inflammation , liver disorders , and antinociception [ 14 , 15 ] . among these three compounds , only catalpol was assayed in detail to reveal its spatiotemporal expression profiling among different developmental stages of r. glutinosa leaves and between leaf and root [ 16 , 17 ] . it is shown that catalpol content varied among different developmental stages of r. glutinosa leaves and within the whole growth stage . aucubin and gpa were found to accumulate in roots of r. glutinosa [ 18 , 19 ] , and only the former was quantified and much lower than catalpol in the root [ 18 , 20 ] . therefore , in this paper , we aimed to determine the content of these three iridoid glycosides of two r. glutinosa cultivars in different developmental stages of leaves using hplc - uv method and compared their changing trend . 1 , were grown in around mid - april in 2014 at the implad ( institute of medicinal plant development ) , beijing , china . normal field management was applied during the growth period . on 11th september , 2014 , the top eight positions of leaves from each plant were collected separately , washed to remove the surface soil , and then stored at 80c till use . hplc grade acetonitrile ( acn ) and formic acid were purchased from thermo fisher ( usa ) . the standard catalpol ( 98% ) was obtained from the national institute for food and drug control , china ( http://www.nifdc.org.cn/ ) . standards of aucubin ( gr-133 - 140104 ) and geniposidic acid ( gr-133 - 140423 ) were purchased from nanjing guangrun biotechnology co. ltd . , china ( http://www.grbiology.com/ ) . the concentrations of catalpol , aucubin , and geniposidic acid in the mixed working stock standard solution were , separately , 0.5025 mg , 0.0375 mg , and 0.0020 mg in 1 ml of purified water . to make calibration curves , six series concentrations , ranging from 0.01570~0.5025 mg / ml for ca , 0.001172~0.0375 mg / ml for au , and 6.281e 05~0.002010 mg / ml for gpa , calibration curves were constructed by plotting the logarithm of the hplc - uv peak areas versus the logarithm concentration of each standard . the frozen samples were homogenized with liquid nitrogen and one gram of each sample powder was extracted twice with 25 ml of 30% methanol in an ultrasonic water bath at 25c for 20 min . the supernatants from two cycles of extraction were combined and evaporated to dryness in a rotary evaporator at 50c under the reduced pressure , redissolved in 50 ml of pure water , and then filtered with a 0.22 m millipore membrane filter prior to hplc analysis . for hplc - uv quantification , ten microliters of filtered extracts and standards was run at 30c on a waters 600e system equipped with a phenomenex kinetex c18 column ( 4.6 mm 100 mm , 2.6 m ) , waters 2487 dual wavelength detector , and 2707 autosampler ( usa ) . the isocratic mobile phase contained acetonitrile ( 5% ) and 0.1% formic acid in water ( 95% ) and was delivered at 0.4 ml / min . catalpol and aucubin were monitored at 210 nm and geniposidic acid was monitored at 240 nm . the presence of the three iridoid glycosides in r. glutinosa leaves was determined by comparing both retention time and spectral data with those of their corresponding authentic standards . concentrations of three iridoid glycosides in the samples were determined from the below linear standard calibration curves . are not heat - stable , the batch of leaf samples was used to calculate the dry weight of each stage leaf . hplc - uv method has been successfully applied to quantify catalpol ( ca ) , aucubin ( au ) , and gpa in medicinal plants [ 16 , 17 , 2022 ] . the hplc conditions that researchers used varied in terms of the columns , mobile phase system , and detection wavelengths . therefore , we optimized hplc - uv conditions in terms of column type , uv wavelength , mobile phase composition , addition of formic acid in aqueous phase , and ratio of acn and water in our early work ( table 1 ) . several uv wavelengths ( e.g. , 203 nm , 205 nm , 206 nm , and 210 nm ) were used to detect the presence and content of catalpol and ( or ) aucubin . we found that there was no significant difference in detecting these two iridoid glycosides under 203~210 nm and thus chose 210 nm as the detection wavelength , which was used in china pharmacopoeia ( 2010 ) . there was no much difference of the wavelengths ( 230~240 nm ) in peak shape and separation of gpa except that less miscellaneous peaks and stable baselines were produced at 240 nm . as ji et al . revealed , adding or not adding formic acid ( fa ) in the mobile phase did not obviously influence the peak shape and separation of catalpol and aucubin , except for gpa . while investigating the difference of different concentrations of acn in the mobile phase system on the chromatogram , we found that the elution times of the analytes decreased with acn concentration increased ( table 2 ) . when acn was higher than 5% , catalpol peak was overlapped with the solvent peak . however , when less than 5% of acn was used in the mobile phase , more late au and gpa were eluted . all the three analytes can be eluted within 10 minutes under 5% of acn . in order to shorten the running time for compounds behind them the final hplc conditions that we adopted to determine ca , au , and gpa in our study were summarized in table 1 . using the optimized conditions , we further tested our hplc - uv conditions by checking its linearity , precision , stability , and recovery ( table 3 ) . figure 1 showed that standards of ca , au , and gpa were eluted separately at 3.412 min , 4.857 min , and 9.850 min . their corresponding compounds in the sample extract were detected at 3.436 min , 4.941 min , and 9.813 min , respectively . the linear regression equations obtained were as follows : ca , y = 0.9707x + 6.7723 , r = 0.9997 ; au , y = 0.9952x + 6.8458 , r = 0.9999 ; and gpa , y = 1.0136x + 7.5797 , r = 0.9996 . the precisions were evaluated as the relative standard deviation ( % , rsd ) and , calculated for ca , au , and pga , were 0.14% , 2.65% , and 2.05% , respectively . 25% , 3.08% , and 2.41% , respectively , which indicated that our extraction method was reproducible . to check the stability of extracts , the same sample extract was assayed separately at 0 , 2 , 4 , 8 , 12 , 16 , and 24 h after extraction . the rsd values for ca , au , and pga were 1.46% , 2.38% , and 0.43% , respectively , indicating that our sample extract remained stable at least till 24 hours . the extraction recovery was determined by comparing the content of the compound extracted from the samples with the content of compound from nonextracted standard solutions at equivalent concentrations . to test the sample extraction recovery , one ml of the standard solution containing 4.824 mg of ca , 0.225 mg of au , and 0.0134 mg of gpa , respectively , the recovery rates for standards ca , au , and pga were 102.59% , 98.29% , and 101.96% , respectively . the calculated lod ( limit of detection ) at s / n > 3 was , separately , 1.3333e 04 mg / ml for ca , 1.8170e 04 mg / ml for au , and 4.9926e 05 mg / ml for gpa . the leaf biomass of r. glutinosa cultivars increased greatly from the seedling stage till 120 dap ( days after plantation ) and then deceased after mid - september ( 140 dap ) , partially due to ongoing senescence of bottom leaves ( figure 2 ) . however , the top eight positions of leaves still stayed green and varied in leaf size , which can represent the different developmental stages of leaves ( figure 3 ) . the 8th leaves , counted downwards from the uppermost leaves , looked similarly in their size and appearance status as the lower positions of unsenescent leaves . however , their dry weight ( dry weight per gram fresh weight ) changed dynamically with leaf development ( figure 4 ) . overall , leaf dry weight decreased with the leaf development and increased for l5 and then decreased in the rest of older leaves . l1l8 had about 0.140.18 grams of dry biomass per gram fresh biomass . in this paper , we just focused on the contents of ca , au , and gpa in the top eight positions of leaves and investigated their relationship with the leaf developmental stages . although the contents of ca , au , and gpa were somehow different between wen 85 - 5 and beijing no . 1 , the changing trends of these three iridoid glycosides with the leaf developmental stage were very similar between the two cultivars , indicating no genetic difference in developmental regulation of these three iridoid glycosides ( figures 5 and 6 ) . generally , catalpol , a major bioactive iridoid glycoside of r. glutinosa , was higher in younger leaves and decreased with the leaf development , which was consistent with the result of ji et al . . likewise in r. glutinosa root , aucubin in the leaf was still much lower than catalpol , which was found by pitczak et al . . the changing trend of aucubin among different developmental stages of r. glutinosa leaves was distinct from catalpol ; that is , the older the leaves , the higher the aucubin in them . it was found that aucubin was one of the intermediates for catalpol biosynthesis in scutellaria albida and paulownia tomentosa . the opposite metabolic profiling of aucubin and catalpol in r. glutinosa leaf indicated that catalpol might not be synthesized via aucubin ; however , this needs to be further investigated . gpa was detected to be present in r. glutinosa leaves and the lowest analyte in all the developmental stages of r. glutinosa leaves . like aucubin , gpa was increased with the leaf development and higher in older leaves . in this paper , we determined contents of catalpol , aucubin , and geniposidic acid in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions and further compared their changing trend with the leaf development . our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and had the increasing trend with the leaf development , which was different from catalpol . this work provided an important basis for future exploitation of r. glutinosa leaves such as isolation of interesting metabolites , for example , the high concentration compound present at 4.16 , which was further identified by hplc - lc - ms method . it also provided a model to study the relationship of aucubin and catalpol metabolisms in r. glutinosa .

although r. glutinosa roots are currently the only organ source in clinics , its leaves are a potential supplement for the roots especially in extraction of some important bioactive compounds . our early work found that the contents of catalpol and total iridoid glycosides varied among different developmental stages of r. glutinosa leaves . aucubin and geniposidic acid , the abundant major bioactive compounds in eucommia ulmoides and gardenia jasminoides , respectively , were found present in r. glutinosa roots , however , and have not been analyzed in its leaves . in this paper , we aimed to determine contents of these three iridoid glycosides in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions . our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and showed the increasing trend with the leaf development , which was different from catalpol . this work provided the important information for future exploitation of r. glutinosa leaves as a potential supplement for its roots in extraction of some important bioactive compounds and studying the relationship of aucubin and catalpol metabolism .

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

PMC3927558

functional fitness not only accounts for the traditional physical fitness parameters such as muscle strength , cardiorespiratory endurance , and flexibility , but also includes balance . however , it is well known that , with age , functional fitness declines , resulting in a reduced ability to perform activities such as rising from a chair or climbing stairs , which can be viewed as functional components of activities of daily living ( adl ) performance , and may eventually compromise the ability to perform adl [ 35 ] . in addition , approximately a third of community - dwelling older adults fall at least once a year , and over 30% of fallers suffer injuries requiring medical attention . strategies to prevent or attenuate the decline in physical function and balance are important for promoting independence in the elderly and thereby enhancing quality of life . research over the past two decades clearly shows that regular physical exercise is effective for maintaining and promoting health , physical fitness , and functional independence in older adults , especially in terms of endurance , muscular strength , flexibility , and balance . physical training programs can be undertaken in the clinic , commercial fitness centers or gymnasiums , in the community or at home . in contrast to clinic and gym - based exercise , community- and home - based exercise does not require special facilities or expensive equipment . moreover , community- and home - based exercise is capable of reaching a broad audience and , importantly , the community setting provides an opportunity for social contact and support which may have an array of benefits , especially for those living in regional and rural settings . in order to maximize compliance to community- and home - based programs thus , community- and home - based exercises have become increasingly popular as an alternative to gym - based resistance exercise . providing feedback and prompting have been shown to be effective in increasing and maintaining physical activity as well as other positive health behaviors . moreover , feedback may also enhance the exercise response which may prove valuable in enhancing recovery of functional abilities after injury or illness [ 11 , 12 ] . however , a paucity of information is currently available regarding the effects of feedback on muscular performance and functional mobility improvement in older adults undertaking resistance training in the community- and home - based setting . hasegawa and tomiyama have previously reported , that in middle - aged and older persons participating in a community- and home - based setting , periodic feedback enhanced muscle performance compared to those not receiving feedback . consequently , the purpose of this study was to confirm and extend these findings by examining the effects of periodic task - specific test feedback on not only muscle performance but also functional mobility in older adults undertaking resistance training with elastic bands in the community- and home - based setting . in response to a public relations magazine advertisement , 80 older adults from tokai city , aichi prefecture , japan , volunteered to participate in the study . inclusion criteria were 65 years of age , community residing , functionally independent , and able to perform physical exercise . participants were excluded if they had been advised by their physician to refrain from exercise . prior to acceptance into the study , a brief health examination was performed by an occupational therapist and questionnaires regarding medical history were completed . current presence of disease was determined using self - reported physician - diagnosed disease information . three others were excluded from the study because they had scheduling conflicts , transportation issues , or no further interest in joining the study . the remaining 75 participants ( 65 to 83 years ) were included in this study . they were divided into a muscular performance feedback group ( mpg ) or a functional mobility feedback group ( fmg ) by their residential area ( the two groups were from two separate communities within the same city ) . both groups participated in an identical 9-week community- and home - based resistance exercise program . all participants were asked to not alter their diet or physical activity patterns for the duration of the study . twenty - three participants did not complete follow - up measures due to travel - related reasons ( n = 18 ) and medically - related problems ( low back pain , n = 2 ; dizziness , n = 1 ; upper respiratory tract infection , n = 2 ) . regarding baseline characteristics , there was no difference between those who dropped out and those who completed the study within each group for age , weight or any functional fitness measure , with the only difference being in the mpg with more women than men dropping out ( due to travel ) and hence height of those who dropped out was lower than those who completed . the ethics committee of seijoh university approved the study and all participants provided written informed consent . instruction and progression of the exercise routine occurred at a community center once per week and participants were asked to exercise an additional two times each week at home . the community - based exercise sessions consisted of 15 min of warmup , 60 min of resistance exercise , and 15 min of cooldown . pictorial guidebooks were provided to all participants in order to assist them to perform the exercises correctly . the home - based exercise program also consisted of the same resistance and stretching exercises . participants were asked to record exercises they performed and submit a diary every week while attending the exercise classes . in order to train all major muscle groups , resistance exercises were prescribed as a combination of 3 upper body exercises , 6 lower body exercises , and 2 trunk exercises performed using an elastic resistance band ( thera - band , hygenic , usa ) . exertion was rated using borg 's rate of perceived exertion ( rpe ) scale [ 15 , 16 ] . participants were instructed to start resistance exercises at an intensity level of 13 on the rpe scale and then to progressively increase resistance to a level of 15 to 17 . participants were instructed to progressively increase resistance every two to four weeks by advancing to the next color of elastic band ( lower to higher resistance of bands in order : red , green , and blue ) or shortening the initial length of the band for increased resistance ( figure 2 ) . stretching exercises consisted of eight upper body exercises and seven lower body exercises . the participants were asked to stretch to the point where they felt moderate tension without feeling pain in joints or muscles . fortnightly assessments were performed by testers for three performance tests : arm curls , chair stands in 30 seconds , and the timed up and go ( tug ) test . task specific feedback , which is extrinsic or augmented information provided to a performer in regard to a specific task with the goal to enhance future performance of that task , was provided by the testers ( not instructors ) who were blinded to the participant 's previous results . mpg received immediate test feedback ( verbal and written ) only on the arm curls and chair stand test while fmg received immediate feedback only for the tug . in addition to the participant 's results for the respective tests ( muscle performance or balance ) , verbal feedback from the tester included statements ( in front of other participants ) such as you have improved in your performance and it shows in your current score and keep up the exercise and you are going to improve your performance . anthropometrics , physical symptoms by questionnaire , muscle performance , and functional mobility were evaluated at baseline and followup . participant 's height and weight were assessed and body mass index ( bmi ) was calculated as body weight ( kg ) divided by the square of height ( m ) . upper body muscle performance was assessed using the 30-second arm curl test ( arm curl ) [ 18 , 19 ] . on a signal , participants were instructed to flex and extend the elbow of the dominant hand , lifting a weight dumbbell ( men : 8-lbs [ 3.6 kg ] , women : 5-lbs [ 2.3 kg ] ) through the complete range of motion , as many times as possible in 30 seconds . a practice trial of one or two repetitions was given , followed by two test trials . the score was the number of repetitions completed with the best performance used for analysis . lower body muscle performance was assessed using the 30-second chair stand test [ 18 , 19 ] . the participant 's arms were crossed at the wrists and held against the chest . on a signal , participants rose to a standing position from a chair and then returned to a seated position and continued to complete as many full stands as possible in 30 seconds . a practice trial of one or two repetitions was given , followed by two test trials . the score was the total number of stands executed correctly with the best performance used for analysis . functional mobility was assessed using the 8-foot timed up and go ( tug ) test [ 1820 ] . participants sat in a chair with their hands on their thighs and feet flat on the floor . on a signal , participants stood from the chair without pushing off with the arms , walked as quickly as possible around a cone placed 8-feet ( 2.44 m ) ahead of the chair , and returned to a fully seated position in the chair . participants walked through the test one time as a practice and then were given two test trials with the best performance time used for analysis . comparisons between mpg and fmg at baseline were performed using an independent student 's t - test or chi - square test as appropriate . the effect of the intervention was determined using a repeated measures analysis of covariance ( ancova ) adjusted for gender and within group changes by a paired t - test . all tests were two tailed and a p value of less than 0.05 was considered statistically significant . there were no differences between groups at baseline for age , height , weight , and prevalent disease ( table 1 ) or muscle performance and functional mobility ( table 2 ) . the average adherence rates in the exercise class at the community center were 92 13% for mpg and 87 12% for fmg . both groups performed home - based exercises in addition to community - based classes for a total of 2.7 1.3 days / week in mpg and 2.1 1.0 days / week in fmg . no differences were observed between the two groups in adherence or home - based training frequency ( table 1 ) . there were no accidents or injuries during the exercise classes at the community center or at home . there was a significant group time interaction for the arm curl ( f = 15.2 ) and chair stand test ( f = 5.2 ) with mpg improving more than fmg , while fmg improved more than mpg for the tug ( f = 4.1 ) ( table 2 ) . in the mpg , the muscle performance measures for the arm curl and chair stand test increased by 31.4% ( p = 0.001 ) and 33.7% ( p < 0.001 ) , respectively , while tug performance time was reduced by 3.5% ( p < 0.001 ) . in the fmg , arm curl and chair stand test significantly increased by 15.9% ( p < 0.001 ) and 24.9% ( p < 0.001 ) , respectively , while time to perform the tug was reduced by 9.7% ( p = 0.049 ) . maintaining or enhancing muscle performance and functional mobility in older persons is critical for undertaking daily activities and for sustaining an appropriate quality of life . in the current study , we found significant improvements in muscle performance and functional mobility following a 9-week community- and home - based exercise regimen undertaken with elastic bands . importantly , gains were partly dependent on fortnightly test feedback suggesting that this is an important component of the training session in order to maximize gains resulting from the program . the muscular performance results are in agreement with other studies that have used elastic bands in a community setting . reported significant increases of 16% in arm curl and 18% in chair stand performance in older women who participated in a 12-week exercise program ( 3 d / wk ) using elastic bands . similarly , a 23% and 19% increase in arm curl and chair stand performance , respectively , was reported by rogers et al . who enrolled older women in a 4-week ( 3 d / wk ) elastic band resistance exercise program . however , gains resulting from home - based programs may not be of the same magnitude as that of supervised gym- or community - based programs with yamauchi et al . reporting improvements of 18% in the arm curl test and only 6% in the chair stand test for older adults following 12 weeks of a home - based exercise program . in the current study , the fmg had an average improvement for the arm curl test of 16% , which is comparable to other studies with a similar training period . however , the mpg improvement on this task was double that of the fmg with gains of 31% . these results suggest that task - specific feedback of serial testing may be an effective strategy to enhance performance in older persons . for the chair stand test , there was also a significant difference between the two feedback groups with the task - specific group enhancing their performance by ~34% while the fmg increased by ~25% . moreover , the results in mpg were higher than previous studies with comparable training programs [ 9 , 21 , 22 ] . the improvements in the chair stand test are particularly important given the role that lower body muscle performance , as does balance , plays in maintaining physical function . similarly , for the timed up and go test , which was used to assess functional mobility in our cohort , improvement was greater for the fmg who decreased their performance time by ~10% whereas the mpg experienced a reduction of only 3.5% . this suggests that improvements in functional mobility are also associated with the use of feedback in older persons . these results indicate that the magnitude of improvement in muscular function and functional mobility are associated with the feedback provided and would be beneficial to older persons in order to maximize program - related gains . the importance of data feedback has been previously described by mihalko et al . who reported that individual fitness feedback influences exercise attendance . in addition , it is suggested that relatedness is intensified in group work and exercise programs in which the participant 's interaction is increased . moreover , bourbonnais et al . reported that treatment of the lower limb in stroke patients based on muscle force - feedback produced an improvement in gait velocity , while feedback has also been found to improve pelvic floor muscle training in those with urinary incontinence . in athletes , providing verbal feedback has been associated with a modest enhancement in muscle performance and tuck - jump performance , although not in time trial cycling . feedback can be classified as either intrinsic or extrinsic with task - specific feedback categorized as knowledge of results . indeed , the observations of leventthal have documented the importance of providing relevant information in order to encourage an action . it may well be that awareness of muscular performance or functional mobility improvement and the encouragement provided by the tester ( in delivering the feedback in a face - to - face situation ) enhanced the participant 's self - efficacy for these respective tasks and their intrinsic motivation [ 33 , 34 ] and this contributed to task - specific performance differences in the mpg and fmg . in addition , the feedback may have reinforced or enhanced the participant 's goal - setting which in turn enhanced their intrinsic motivation . the finding that feedback of performance may have increased motivation is consistent with previous literature and cognitive evaluation theory which proposes that , when feelings of competence are enhanced , motivation increases . motivating older adults to perform exercises on a regular basis is an important factor in maintaining the effects of exercise . in the current study , we held community - based exercise classes once a week in addition to the home - based exercise program 2 days per week . the adherence to the community - based classes was over 85% and the frequency of the community- and home - based exercise was over 2 days per week . most participants mentioned that they were glad to make new friends and enjoyed exercising as a group in the community setting . this supports previous findings that community - based exercise classes have positive psychological effects as well as physical effects on community - dwelling older adults . after completion of this 9-week program , participants continued to exercise and currently attend exercise classes twice a month . enhanced self - efficacy resulting from participation may be one factor that contributed to the participation rates during the study period as well as with the participation postintervention . given the homogeneity of participants in the study ( age , socioeconomic status , and geographical location ) , caution should be taken when extrapolating our results to all older community - dwelling persons . moreover , the participants were not randomized to the treatment conditions as it was not possible to blind participants to the exercise program , leaving them vulnerable to a variety of tester and subject effects that may influence test results and introduce bias in our findings . our program was also of a relatively short - term nature and it is unclear if differences based on feedback would exist with a longer program , such as 6 or 12 months . nevertheless , our results show , even within a relatively short training period , that task - specific test feedback appears to have a beneficial effect in enhancing physical performance , and , regardless of the underlying mechanism for the improvement , this is an important practical outcome for those involved in the exercise training of older persons . a future direction would be in determining if task - specific feedback is a valuable strategy to enhance performance in those disabled or recovering from illness / disease or injury . in addition , a more extensive test battery could be employed to measure additional aspects of functional and ambulatory ability . lastly , it would be of interest to determine if performance was enhanced by motivation , whether it was motivation to train harder or to perform the tests or to the combination . this study was designed to determine the efficacy of test performance feedback to improve muscle performance and functional mobility in older adults . following the 9-week community- and home - based intervention , a significant interaction was noted for the muscle performance and functional mobility tests suggesting that periodic test feedback during resistance training may enhance task - specific physical performance in older persons . providing regular feedback on test performance in the community and , if possible , the home exercise setting may facilitate gains in muscle and balance performance resulting in enhanced physical function and a greater safety margin for functional thresholds .

the purpose of this study was to determine the effects of periodic task - specific test feedback on performance improvement in older adults undertaking community- and home - based resistance exercises ( chbre ) . fifty - two older adults ( 6583 years ) were assigned to a muscular perfsormance feedback group ( mpg , n = 32 ) or a functional mobility feedback group ( fmg , n = 20 ) . both groups received exactly the same 9-week chbre program comprising one community - based and two home - based sessions per week . muscle performance included arm curls and chair stands in 30 seconds , while functional mobility was determined by the timed up and go ( tug ) test . mpg received fortnightly test feedback only on muscle performance and fmg received feedback only on the tug . following training , there was a significant ( p < 0.05 ) interaction for all performance tests with mpg improving more for the arm curls ( mpg 31.4% , fmg 15.9% ) and chair stands ( mpg 33.7% , fmg 24.9% ) while fmg improved more for the tug ( mpg-3.5% , fmg-9.7% ) . results from this nonrandomized study suggest that periodic test feedback during resistance training may enhance task - specific physical performance in older persons , thereby augmenting reserve capacity or potentially reducing the time required to recover functional abilities .

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

PMC3153312

we used data from the 2006 brazos valley community health assessment ( bvha ) , which was developed by a collaboration of local and regional academic and community - based organizations in the brazos valley of central texas . participants were recruited from adult community residents who resided in one of six rural and one urban county by a professional independent survey research firm that identified 9,940 valid telephone numbers through random digit dialing . of these telephone numbers , more than 2,500 adults ( 19.4% minority , 71% female , and 61% rural residents ) who resided in the seven counties returned the mailed survey ; the response rate was 73.8% ( 25 ) . this study used data from 1,878 adult participants in the bvha who had complete responses for demographic characteristics , eating behaviors , and household food - related hardship ( experience of running out of food , without money to obtain more ) ( 26 , 27 ) ; 649 participants ( 25.7% ) were excluded due to missing data . there were no statistically significant differences between included and excluded participants with regards to demographic characteristics or rural residence . the texas a&m university institutional review board approved the study protocol and all participants provided informed consent . demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) . the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) . eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) . ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 . the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) . based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses . frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week ) . the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week . two questions from a validated , self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) . a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings . the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) . respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) . this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) . release 11 of stata statistical software was used for all statistical analyses ; p<0.05 was considered statistically significant . descriptive statistics were estimated for demographic characteristics , eating behaviors , and food - related hardship . the difference between rural and urban adults was assessed with contingency tables by using the statistic . bivariate correlations between theoretically selected variables ( demographic characteristics , eating behaviors , and food - related hardship shown in table 1 ) and ssb intake were estimated . correlations at p<0.10 were retained for inclusion in the logistic regression model that included rural and urban respondents ; excluded variables included sex , overweight , ages 4564 years , and employment status . using backward elimination of all variables with p>0.05 , a combined multivariable logistic regression model ( n=1,878 ) was estimated for high level of ssb consumption ( 3 cans / glasses per day vs. <3 cans / glasses ) . using the final model for the combined sample , separate multivariable logistic regression models were estimated for the 734 urban respondents and the 1,144 rural respondents . difference in demographic characteristics , eating behaviors , and household food - related hardship between urban and rural adults ( n=1,878)a comparisons were performed using test . statistically significant after using bonferroni correction for multiple comparison ( bonferroni - corrected p=0.002 ) . 1 can or glass of regular soda or sweet tea on an average day , compared with < 1 on an average day . 3 cans or glasses of regular soda or sweet tea on an average day , compared with <3 on an average day . we used data from the 2006 brazos valley community health assessment ( bvha ) , which was developed by a collaboration of local and regional academic and community - based organizations in the brazos valley of central texas . participants were recruited from adult community residents who resided in one of six rural and one urban county by a professional independent survey research firm that identified 9,940 valid telephone numbers through random digit dialing . of these telephone numbers , more than 2,500 adults ( 19.4% minority , 71% female , and 61% rural residents ) who resided in the seven counties returned the mailed survey ; the response rate was 73.8% ( 25 ) . this study used data from 1,878 adult participants in the bvha who had complete responses for demographic characteristics , eating behaviors , and household food - related hardship ( experience of running out of food , without money to obtain more ) ( 26 , 27 ) ; 649 participants ( 25.7% ) were excluded due to missing data . there were no statistically significant differences between included and excluded participants with regards to demographic characteristics or rural residence . the texas a&m university institutional review board approved the study protocol and all participants provided informed consent . demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) . the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) . eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) . ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 . the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) . based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses . frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week ) . the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week . two questions from a validated , self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) . a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings . the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) . respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) . this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) . demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) . the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) . eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) . ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 . the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) . based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses . frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week ) . the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week . two questions from a validated , self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) . a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings . the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) . respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) . this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) . release 11 of stata statistical software was used for all statistical analyses ; p<0.05 was considered statistically significant . descriptive statistics were estimated for demographic characteristics , eating behaviors , and food - related hardship . the difference between rural and urban adults was assessed with contingency tables by using the statistic . bivariate correlations between theoretically selected variables ( demographic characteristics , eating behaviors , and food - related hardship shown in table 1 ) and ssb intake were estimated . correlations at p<0.10 were retained for inclusion in the logistic regression model that included rural and urban respondents ; excluded variables included sex , overweight , ages 4564 years , and employment status . using backward elimination of all variables with p>0.05 , a combined multivariable logistic regression model ( n=1,878 ) was estimated for high level of ssb consumption ( 3 cans / glasses per day vs. <3 cans / glasses ) . using the final model for the combined sample , separate multivariable logistic regression models were estimated for the 734 urban respondents and the 1,144 rural respondents . difference in demographic characteristics , eating behaviors , and household food - related hardship between urban and rural adults ( n=1,878)a comparisons were performed using test . statistically significant after using bonferroni correction for multiple comparison ( bonferroni - corrected p=0.002 ) . servings of fruit and vegetables usually eaten each day . 1 can or glass of regular soda or sweet tea on an average day , compared with < 1 on an average day . 3 cans or glasses of regular soda or sweet tea on an average day , compared with <3 on an average day . rural respondents were older than urban counterparts ; a larger proportion were women , reported a household income 101199% fpl , and were obese ; and a smaller proportion were employed full - time outside the home for wages or had at least one child under the age of 18 years living in the household . compared with urban respondents , the prevalence and high level of ssb consumption ( 3 cans or glasses of ssb / day ) was greater among rural adults . a greater proportion of rural adults ate a regular breakfast meal less than three times a week and consumed fewer servings of fruit and vegetables . on the other hand , a larger proportion of urban adults ate fast food meals at least three times a week . finally , a larger proportion of rural adults reported household food - related hardship than urban counterparts ( 23.7% vs. 17.2% ) . several differences between urban and rural adults remained significant after correcting for multiple comparisons with a bonferroni - corrected level of statistical significance . several demographic variables were not correlated with ssb consumption ; namely sex , overweight status ( bmi 2529.9 kg / m ) , age category of participants 4564 years , and employment status . although statistically significant , the strength of individual correlations was weak ( r 0.15 ) . age category of participants 1844 years ( r=0.11 , p<0.001 ) , minority status ( r=0.10 , p<0.001 ) , poverty - level household income ( r=0.15 , p<0.001 ) , presence of 1 child in the household ( r=0.14 , p<0.001 ) , and obesity ( r=0.07 , p=0.005 ) were positively correlated with ssb consumption ; older age category ( 65 years ) was negatively correlated . among the variables for eating behaviors , frequency of fast food meals ( r=0.09 , p<0.001 ) , low fruit and vegetable intake ( r=0.15 , p<0.001 ) , and consuming <3 breakfast meals / week ( r=0.17 , p<0.001 ) were positively correlated with ssb consumption ; high fruit and vegetable intake of 5 servings / day was negatively correlated with ssb consumption ( r=0.12 , p<0.001 ) . food - related hardship was positively associated with ssb consumption ( r=0.21 , p<0.001 ) . minority status ( p=0.64 ) , employment status ( p=0.57 ) , age ( p=0.19 ) , and obesity ( p=0.17 ) were sequentially removed from the final model for the combined rural and urban sample , which adjusted for demographic characteristics , eating behavior , and household food - related hardship . independent of demographic characteristics , eating behaviors , and food - related hardship , rural residence was associated with greater odds for reporting a high level consumption of ssbs ( or 1.8 ; 95% ci 1.3 , 2.4 ; p<0.001 ) than urban residence . among all adults having a poverty - level household income ( or 2.2 ; 95% ci 1.6 , 3.1 ) , children in the household ( 1.8 ; 95% ci 1.3 , 2.3 ) , frequent consumption of fast - food meals ( 1.6 ; 95% ci 1.2 , 2.2 ) , infrequent breakfast meals ( 1.7 ; 95% ci 1.3 , 2.3 ) , low fruit and vegetable intake ( or 2.1 ; 95% ci 1.4 , 3.3 ) , and food - related hardship ( or 1.9 ; 95% ci 1.4 , 2.6 ) table 2 shows the results from the multivariable regression model for rural adults . among rural adults , a higher level of ssb consumption was associated with greater odds for respondents with poverty - level household income , presence of child in the household , frequent consumption of fast - food meals , infrequent consumption of regular breakfast , low fruit and vegetable intake , and food - related hardship . among urban adults ( table 3 ) , one eating behavior ( infrequent consumption of a regular breakfast meal ) , household food - related hardship , and one demographic characteristic ( children in the home ) were associated with ssb consumption . interestingly , frequency of fast - food meals and low fruit and vegetable intake were not associated with a high level of ssb consumption among urban adults . odds ratios and 95% ci from multiple variable logistic regression models correlating demographic characteristics , eating behaviors , and household food - related hardship with consumption of sugar - sweetened beverages among 1,144 rural adultsa dependent variable is consumption of 3 cans / glasses of regular soda or sweet tea on an average day compared with <3 cans / glasses . 1 child under 18 years living in the household with the adult respondent compared with no children . eat a regular breakfast meal <3 days a week compared with 3 days a week . in the last month , food bought did n't last and there was not enough money to buy more compared with food did last . odds ratios and 95% ci from multiple variable logistic regression models correlating demographic characteristics , eating behaviors , and household food - related hardship with consumption of sugar - sweetened beverages among 734 urban adultsa dependent variable is consumption of 3 cans / glasses of regular soda or sweet tea on an average day compared with <3 cans / glasses . 1 child under 18 years living in the household with the adult respondent compared with no children . eat a regular breakfast meal <3 days a week compared with 3 days a week . in the last month , food bought did n't last and there was not enough money to buy more compared with food did last . although research findings suggest a link between consumption of ssbs and health outcomes ( 1 , 2 , 12 ) , there are few studies that have examined the influence of less - healthy eating behaviors and food - related hardship on the consumption of high levels of ssb , especially among rural adults . this is critical considering the dramatic increase in prevalence of overweight and obesity ( 36 , 37 ) , ssb consumption ( 3 , 9 , 3840 ) , frequency of fast - food meal consumption ( 41 ) , and nutrition and health disparities associated with rural residence ( 25 , 4246 ) . however , studies of ssb consumption rarely have considered eating behaviors and adequacy of household food supplies as contributing factors . findings from this study of 1,878 rural and urban adults extend our understanding of the influence of less - healthy eating behaviors and household food - related hardship on higher levels of ssb consumption . first , the prevalence and high level of consumption of ssb were significantly greater among rural adults compared with urban counterparts . second , a high level of ssb consumption was associated with less - healthy eating behaviors , especially among rural adults . to our knowledge , this is apparently the first study that simultaneously evaluated the association of multiple eating behaviors and household food - related hardship among a large sample of rural adults . unlike primarily urban studies that used a single definition of ssb consumption such as once or more a week ( 17 ) , one 12-ounce serving of sugar - sweetened soda per day ( 6 ) , 1 ssb / day ( 47 ) , and > 1 bottle / day ( 48 ) , this study considered prevalence ( 1 can or glass of ssb / day ) and a high level of ssb consumption ( 3 cans or glasses of ssb / day ) . more than 52% of rural adults , compared with 43.7% of urban adults , consumed at least one ssb per day . this appears to be higher than a similar size study of rural adults ( n=1,817 ) in wyoming , montana , and idaho that defined ssb consumption as less than once / week versus once or more per week ( 17 ) or the large , primarily urban nurses health study ii that found that 9.5% of the sample consumed 1 ssb / day ( 47 ) . compared with previous studies of ssb consumption , our finding that rural adults consumed higher levels of ssb than urban adults is apparently new . one possible explanation may be that previous studies did not attempt to examine high levels of ssb consumption ; but chose lower levels of consumption , such as at least one ssb per day or week ( 6 , 17 , 47 , 48 ) . another explanation may be that rural residents have greater access to convenience and non - traditional food stores and fast - food opportunities where ssb are more available and affordable ( 43 , 45 , 4951 ) . preference and greater household availability for ssb such as regular soft drinks or sugar - sweet tea , which has been identified through household food inventories , may provide another explanation for high levels of ssb consumption ( 52 , 53 ) . in addition to consumption of ssb , three additional less - healthy eating behaviors that are associated with poor diet quality were examined ; namely , infrequent breakfast meals ( 28 , 54 , 55 ) , frequent consumption of fast - food meals ( 56 ) , and fewer portions of fruit and vegetables ( 57 , 58 ) . rural adults compared less favorably with urban adults in two of these three eating behaviors . a greater proportion of rural adults infrequently consumed a regular breakfast meal and ate less than three daily servings of fruit and vegetables . lower fruit and vegetable intake among rural adults may be the result of limited access to food stores that market fruit and vegetables store availability and transportation infrastructure ( 46 , 59 , 60 ) . in the united states , there has been an overall decline in breakfast consumption ( 61 ) . one explanation for less frequent breakfast meal consumption among rural adults may be that rural adults travel a greater distance in the morning to work and do not have the time for a regular breakfast . in both urban and rural areas , there are increased opportunities for fast food through traditional fast - food restaurants and marketing of fast food through convenience and other retail stores , often referred to as an explanation for greater utilization of fast - food meals by urban adults may be greater accessibility and availability . inadequate household food supplies or household food - related hardship are known to influence food choice and dietary intake ( 46 , 51 , 63 ) . we identified great nutritional disparity between rural and urban adults , which has been absent from the literature . more than 23% of rural adults compared with 17.2% of urban adults reported that in the past 30 days purchased food did not last and there was no money to buy more , which is supported by secondary analysis of national surveys ( 64 ) . one explanation for the higher prevalence of both household food - related hardship and ssb consumption for rural adults may be related to the coping strategies food - insecure individuals employ to mitigate the consequences of food - related hardship ( 6568 ) such as consuming inexpensive and inflationary - resistant energy - dense foods ( 69 ) . findings from multiple variable regression models confirmed geographic differences and similarities in the association of demographic characteristics , eating behaviors , and food - related hardship with high levels of ssb consumption . although poverty - level household income increased the odds for ssb consumption among rural adults and not urban adults , in both geographic groups the presence of a child in the household was associated with a high level of ssb consumption . all three eating behaviors frequent fast - food meals , infrequent breakfast , and low intakes of fruit and vegetables were associated with ssb consumption among rural adults , but only infrequent consumption was significant among urban residents . food - related hardship was associated with ssb consumption among both rural and urban adults ; the effect size was greater among the urban sample . thus , multiple less - healthy eating behaviors have a greater association with ssb consumption among rural adults than among urban adults . interestingly , two less - healthy eating behaviors were not independently associated with ssb consumption in our urban subsample . a prior rural study found an increased likelihood of overweight or obesity associated with greater frequency of ssb and fast food ( 17 ) . thus , it is critically important to understand individual and household contextual influences on high levels of ssb consumption . our findings revealed linkages among multiple less - healthy eating behaviors , which enhance results from a similar study of rural adults ( 25 ) . adults , especially rural adults who frequently ate fast - food meals , infrequently consumed a breakfast meal , or had fewer daily servings of fruit and vegetables were also more likely to consume high levels of ssb . just as healthier food patterns are associated with healthier beverage patterns ( 70 ) , the present study shows that consumption of ssb appears to be closely linked to less - healthy eating patterns ( 71 , 72 ) . as such , ssb consumption may serve as a marker of other less - healthy eating behaviors and overall poor nutrition . first , the self - reported measure of ssb consumption may understate actual frequency and amount of ssb consumed on a usual day . future work will include specific prompts for calorically sweetened beverages to include carbonated and non - carbonated soft drinks , fruit punch , fruit drinks , lemonade , sweetened powder drinks , bottled coffees , and coffees or teas with added sugar ( 73 ) . third , data were not available on the type and amount of fast - food items consumed or the source of fast - food meals or ssb . finally , measures on sedentary behaviors ( e.g. television viewing , computer use , video gaming ) should be included ( 74 ) . despite these limitations , this study advances our knowledge about less - healthy eating behaviors and household food - related hardship . results from this study provide impetus for understanding interactions among multiple eating behaviors especially among economically and geographically disadvantaged adults . considering that americans are consuming more total calories per day , with much coming from ssb and fast food ( 75 ) , new strategies are needed for educating consumers not only about how to moderate their ssb intake , but also how to simultaneously disrupt the co - occurrence of undesirable eating behaviors ( e.g. fast - food consumption and skipping breakfast ) and promote healthful behaviors ( e.g. eating a regular breakfast and increasing fruit and vegetable intake ) . challenges include the perception and observation that ssb are priced and promoted preferentially with meal deals at fast - food outlets and other venues that market fast - food items ( 49 , 76 ) , and that energy - dense foods are not only least expensive but also most resistant to inflation ( 69 ) . given the economic disincentive for consumers to make healthier selections at fast - food restaurants and other venues ( 49 , 76 , 77 ) and the reality of low - cost accessible energy - dense foods , strategies must consider convenience and cost ( 69 ) especially for low - income and/or rural families ( 51 ) . supported in part by the national institutes of health ( nih)/national center on minority health and health disparities ( # 5p20md002295 ) and by cooperative agreement # 1u48dp001924 from the centers for disease control and prevention , prevention research centers program through core research project and special interest project nutrition and obesity policy research and evaluation network . the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih and cdc .

backgroundsugar - sweetened beverage ( ssb ) consumption is associated with the increasing prevalence of overweight and obesity in the united states ; however , little is known about how less - healthy eating behaviors influence high levels of ssb consumption among rural adults.objectivewe assessed the frequency of ssb consumption among rural and urban adults , examined the correlates of frequent ssb consumption , and determined difference in correlates between rural and urban adults in a large region of texas.designa cross - sectional study using data on 1,878 adult participants ( urban=734 and rural=1,144 ) , who were recruited by random digit dialing to participate in the seven - county 2006 brazos valley community health assessment . data included demographic characteristics , eating behaviors ( ssb consumption , frequency of fast - food meals , frequency of breakfast meals , and daily fruit and vegetable intake ) , and household food insecurity.resultsthe prevalence of any consumption of ssb and the prevalence of high consumption of ssb were significantly higher among rural adults compared with urban counterparts . the multivariable logistic regression models indicated that a high level of ssb consumption ( 3 cans or glasses ssb / day ) was associated with demographic characteristics ( poverty - level income and children in the home ) , frequent consumption of fast - food meals , infrequent breakfast meals , low fruit and vegetable intake , and household food insecurity especially among rural adults.conclusionsthis study provides impetus for understanding associations among multiple eating behaviors , especially among economically and geographically disadvantaged adults . new strategies are needed for educating consumers , not only about how to moderate their ssb intake , but also how to simultaneously disrupt the co - occurrence of undesirable eating and promote healthful eating .

Methods Sample and study design Measures Demographic characteristics Eating behaviors Household food-related hardship Statistical analyses Results Discussion None Statement of authors contributions to manuscript Conflict of interest and funding

PMC4545169

type 2 diabetes ( t2d ) is one of the leading causes of mortality worldwide , largely due to vascular complications including myocardial infarction and stroke [ 1 , 2 ] . endothelial function , a key determinant of vascular health , is impaired in the diabetic vasculature primarily due to reduced nitric oxide ( no ) bioavailability that is brought about by a combination of uncoupling and inhibition of endothelial nitric oxide synthase ( enos ) and an excessive production of reactive oxygen species ( ros ) . loss of no bioactivity , leading to endothelial dysfunction , is known to predispose patients to developing secondary complications such as stroke . therefore understanding the mechanisms associated with vascular dysfunction is critical to the development of therapeutic targets in the hope of preventing t2d - related morbidity . the nad - dependent deacetylase , sirtuin 1 ( sirt1 ) , is abundantly expressed in the vasculature and deacetylates a number of substrates that are key modulators of vascular function including enos and foxo1 . sirt1 has been reported to be downregulated in peripheral blood mononuclear cells of obese humans and in the liver and pancreas of rodents on a high - fat diet . high - fat feeding resulted in a significant decrease in mouse aorta sirt1 protein expression , while no changes were seen in the cerebral vasculature in obese zucker rats . sirt1 interacts with specific double - stranded repeat sequences of dna at the extremities of chromosomes [ 5-(ttaggg)n-3 ] , known as telomeres . during each round of cell division , telomeres shorten until they reach a critical length . at this point , the cell can no longer replicate and it becomes either senescent or apoptotic , thereby creating an accelerated aging phenotype and increasing risk of secondary disease . telomere shortening can be prematurely accelerated in vitro in the face of oxidative stress , a hallmark of t2d . therefore restoring telomere although epidemiological studies have suggested an association between t2d and shortened leukocyte telomeres [ 17 , 18 ] , a direct cause and effect relationship has never been established . all animal procedures were carried out in accordance with the australian code for the responsible conduct of research and were approved by the flinders university animal ethics committee . male hooded wistar rats ( n = 17 ) were bred and housed under controlled room temperature with a 12 : 12 hour light - dark cycle in the school of medicine animal care facility at flinders university . after weaning at three weeks of age the diabetic group ( n = 9 ; t2d ) was placed on a high - fat diet which consisted of 45 kcal% fat , 35 kcal% carbohydrate , and 20 kcal% protein ( d12451 ; open source diet ) . the chow was custom - made by gordon 's specialty stockfeed ( yanderra , australia ) . the remaining group ( n = 8 ; control ) was maintained on a control diet which contained 10 kcal% fat , 70 kcal% carbohydrate , and 20 kcal% protein ( d12450h ; open source diet ) . after 18 weeks on the diet regimen , the diabetic group was given an intraperitoneal injection of streptozotocin ( 30 mg / kg body weight ) dissolved in saline while the control group was injected with the same volume of vehicle . previous studies have shown that a high - fat feeding followed by a low dose of streptozotocin induces a mild impairment of insulin secretion , similar to that seen in late stage t2d . five days prior to sacrifice , rats were anesthetized with 3% isoflurane and their body temperature was maintained on a heating pad at 37c . blood pressure was measured using mlt125/r pulse transducer / pressure cuff and in125 nibp controller ( ad instruments , australia ) and recorded using powerlab software ( ad instruments , australia ) . systolic pressure was determined from the average of three consecutive readings obtained from each rat . on the day of sacrifice blood glucose was measured from the tail vein using an accu - chek performa glucometer and test strips ( roche diagnostics , indianapolis , in , usa ) . the animals were then decapitated using a rodent guillotine and trunk blood obtained for measurement of serum insulin and free fatty acids , plasma triglycerides , and c - reactive protein ( crp ) and for extraction of dna using a dneasy blood and tissue kit ( qiagen ) according to the manufacturer 's instructions . the dna concentration of each extraction was measured in triplicate using a nanodrop 2000 ( thermo scientific ) . the left and right femoral artery were excised from animals , cleaned of connective tissue , and washed to remove blood . the size of each artery was 0.9 cm in length , 2 mm in en face width , and approximately 1 mg in weight . dna was extracted using a qiaamp dna micro kit ( qiagen ) according to the manufacturer 's instructions and stored at 80c until analysis . the brain was removed and the middle cerebral arteries were carefully harvested and pooled into 180 l modified radioimmunoprecipitation ( ripa ) buffer containing 50 mm tris , ph 7.8 with 150 mm nacl , 1% triton x-100 , 0.5% nonidet p-40 , 0.25% sodium deoxycholate , and 1 mm pmsf and protease inhibitor cocktail ( roche , indianapolis , in , usa ) . total protein was extracted as previously described and stored at 80c . total protein was measured using ezq protein quantitation ( roche diagnostics , indianapolis , in , usa ) . cerebral artery lysate was then combined with laemmli sample buffer and heated to 95c for 5 mins . equal amounts of protein ( 18 micrograms ) were loaded onto each lane of a stain - free gel ( bio - rad laboratories , hercules , ca , usa ) alongside dual color precision plus protein standard ( bio - rad laboratories ) . total protein loaded on the gel and then transferred to the low fluorescence polyvinylidene difluoride ( pvdf ) membrane was imaged ( chemidoc mp imager , bio - rad laboratories ) and analysed using image lab 4.0.1 software ( bio - rad laboratories ) . previous studies have shown that this method of protein normalization is superior to that of either total protein stains ( e.g. , sypro ruby , amido black , and coomassie blue ) or antibody loading controls . membranes were probed with an antibody directed against either sirt1 ( abcam ) , enos ( bd transduction labs ) , nox2 ( bd transduction laboratories ) , manganese superoxide dismutase ( mnsod ; millipore ) , p66 ( millipore ) , or 3-nitrotyrosine ( abcam ) overnight at 4c . the next day , membranes were washed and incubated with the appropriate secondary antibody ( jackson immunoresearch ) . bands were visualized using enhanced chemiluminescence , captured with a digital acquisition system ( fujifilm , japan ) and quantified ( carestream molecular imaging software , rochester , nt , usa ) . mean telomere length was measured using a singleplex assay similar to that described by cawthon . the telomere and single - copy gene ( -actin ) were amplified in separate singleplex reactions because the efficiency of each amplicon was optimal in different mastermixes ( assessed in pilot studies ) . the telomere primers were used as previously described by cawthon and used at a concentration of 900 nm . the single - copy gene ( -actin ) primer sequences ( written 5 3 ) were -actin - f , agg tca tca cta tcg gca atg a , and -actin - r , gag act aca act tac cca gga agg aa , and used at a final concentration of 2 m . each qpcr reaction was conducted in a total volume of 20 l using 384-well plates and performed on a viia 7 real - time pcr instrument ( life technologies , foster city , ca ) . the telomere assay was amplified in a custom - made mastermix as described by cawthon and the -actin assay was amplified in power sybr green pcr mastermix ( life technologies ) . for the telomere assay , a standard curve was run alongside the experimental samples consisting of a calibrator dna sample which was diluted serially by 5-fold to produce five concentrations of dna ranging from 515 to 0.824 ng / ml . for the -actin assay , a standard curve was run alongside the experimental samples consisting of the same calibrator dna sample which was diluted serially by 5-fold to produce five concentrations of dna ranging from 5150 to 8.24 ng / ml . the calibrator was rat dna extracted from either whole blood or femoral artery . the amount of telomere was calculated as t = e , where e was the efficiency of the telomere primer calculated from the standard curve , cqcalibrator was the cq of the 1 : 5 dilution on the standard curve , and cqsample was the cq of the experimental sample . the amount of -actin ( reference gene ) was calculated as s = e , where e was the efficiency of the -actin primer calculated from the standard curve , cqcalibrator was the cq of the 1 : 5 dilution on the standard curve , and cqsample was the cq of the experimental sample . relative telomere length ( t / s ) was estimated as the ratio between the amount of telomere ( t ) and reference gene ( s ) . the -actin and telomere primers were synthesized by geneworks ( thebarton , sa , australia ) and dissolved in water for injection ( glaxosmithkline ) . statistical comparisons were performed using graphpad instat version 3 software ( graphpad software , la jolla , ca , usa ) using an unpaired t - test . the initial power analysis was performed based on previous telomere length studies in rodents [ 24 , 25 ] . the experiment was powered at 80% to detect a 20% change in telomere length , assuming a two - tailed alpha level of 5% . rats fed a high - fat diet for 18 weeks weighed significantly more than age - matched control rats fed a control diet ( p = 0.029 ; see table 1 ) . animals then received either a low dose of streptozotocin or vehicle and the diet regimen was continued for another eight weeks . one rat died just prior to streptozotocin injection and two rats died the following week after streptozotocin injection . systolic blood pressure was similar in both t2d rats and control rats ( p = 0.16 ; 113 8 mmhg versus 105 5 mmhg , resp . ) . on the day of sacrifice , t2d rats weighed significantly less than the control group ( p = 0.0001 ; 358 12 g versus 432 7 g , resp . ) . fasting plasma glucose levels were significantly higher in t2d rats compared to control rats ( p < 0.0001 ; 24.2 1.3 mm versus 8.6 0.3 mm , resp . ) . circulating free fatty acid concentration was significantly elevated in t2d rats compared to control rats ( p = 0.012 ; 0.51 0.06 mm versus 0.34 0.02 mm , resp . ) . plasma insulin concentration was significantly lower in t2d rats compared to the control rats ( p = 0.0028 ; 0.5 0.1 ng / ml versus 2.8 0.5 ng / ml , resp . ) , a reflection of streptozotocin - induced pancreatic -cell dysfunction . as a marker of systemic inflammation , plasma crp was significantly elevated in t2d compared to control rats ( p < 0.0001 ; see table 1 ) . as shown in figure 2(a ) , arterial telomere length was comparable between t2d rats and control rats ( p = 0.28 ; 0.92 0.04 versus 0.81 0.08 , resp . ) . however leukocyte telomere length was significantly shorter in t2d compared to the control group ( p = 0.034 ; 0.49 0.14 versus 1.09 0.19 , resp . ) . the pcr amplification products were visualized by agarose gel electrophoresis , ethidium bromide staining , and uv transillumination ( see figure 2(b ) ) . the -actin pcr product was observed at the expected size of 81 base pairs while the telomere pcr product showed a main product at 82 base pairs ( the sum of the length of the two telomere primers ) and a smear to 150 base pairs , reflecting longer pcr products due to staggered annealing of the primers . vascular sirt1 and enos protein expression were significantly lower in t2d rats compared to their control counterparts ( p = 0.0082 and p = 0.011 , resp . ; see figure 3 ) . mnsod and p66shc protein levels were significantly lower in t2d rats compared to control rats ( p = 0.039 and p = 0.028 , resp . ) ; however total 3-nitrotyrosine - containing proteins and nox2 were comparable between groups ( figure 4 ) . firstly , we have demonstrated that t2d causes selective accelerated telomere shortening in circulating leukocytes but preserved telomere length in arteries . to our knowledge , this is the first direct evidence of leukocyte telomere shortening in t2d . secondly , although hyperglycemia leads to blunted sirt1 and telomere shortening , we have shown in vivo that hyperglycemia and a deficit in vascular sirt1 per se are not sufficient to prematurely shorten vascular telomeres . here we have shown that rats gained significantly more weight after an 18-week high - fat diet regimen ( see table 1 ) . injection of a low dose of streptozotocin significantly increased blood glucose levels in t2d rats , which is consistent with other reports . circulating insulin levels were significantly decreased , a feature of clinical late stage t2d when the pancreatic beta cells become impaired and insulin secretion is reduced . the animals also showed evidence of dyslipidemia that is in line with elevated plasma free fatty acids observed in human t2d that has been linked to the onset of insulin resistance . the t2d rats in our study had significantly elevated levels of crp , confirming that this animal model is a good representation of t2d in humans which is associated with prolonged low - grade inflammation . we found that leukocyte telomere length is shorter in t2d rats compared to control rats . our findings are in line with accumulating clinical studies showing a correlation between t2d and shorter leukocyte telomeres [ 17 , 18 , 34 , 35 ] . one possible explanation for the shorter leukocyte telomeres in t2d is that it reflects increased leukocyte turnover due to the systemic low - grade inflammation . however clinical studies have failed to show a significant inverse correlation between leukocyte telomere length and markers of inflammation [ 3537 ] . tentolouris and colleagues have previously reported a correlation between telomere length and nitrosative stress . taken together , we speculate that the observed leukocyte telomere attrition may be due , at least in part , to nitrosative stress which is absent within the vasculature in this animal model of t2d ( see below ) . shortened vascular telomere length has been previously documented in human coronary heart disease , atherosclerosis , and abdominal aortic aneurysms . because vascular biopsies can not be taken from obese or t2d patients without overt vascular pathology , leukocyte telomere measurements have been used as a surrogate for predicting vascular disease risk . we have shown for the first time that arterial telomere length is preserved in t2d rats compared to control rats . although sirt1 depletion and hyperglycemia have previously been shown to independently accelerate telomere shortening , our results have shown that hyperglycemia and blunted vascular sirt1 in vivo were not sufficient to prematurely shorten arterial telomeres . our experiments were sufficiently powered at 80% to detect a 20% change in telomere length . therefore a smaller change in telomere length would not have been detected and as a result we can not rule out the possibility that there are more subtle changes in telomere length within the t2d vasculature . nevertheless our data suggests that leukocyte telomere length may not be an optimal surrogate for vascular telomere length in t2d . sirt1 protein expression was significantly decreased in resistance - sized vessels harvested from t2d rats compared to control rats on a normal diet ( standard chow ) . our findings are in line with studies showing a decline in sirt1 levels at the level of the macrovasculature ( aorta ) after six months of high - fat feeding . however it is important to assess protein expression within the resistance - sized vessels , since these vessels are fundamental to the control of blood flow . we have now confirmed for the first time that sirt1 protein expression is blunted within the microvasculature in t2d . sirt1 activity has been shown to be controlled by both nutritional status and the cellular redox status ( nad / nadh ratio ) . in a nutrient rich environment , nad / nadh ratio is blunted due to an increased rate of reduction of nad to nadh , leading to a decrease in sirt1 activity . endothelial progenitor cells , human umbilical vein endothelial cells , and mouse brain endothelial cells ( sokoya et al . , unpublished findings ) cultured in high glucose recent studies have also shown that oxidative stress directly inhibits sirt1 activity via oxidative posttranslational modifications . therefore , both a reduction in cellular redox status and an increase in oxidant stress may be working together to reduce vascular sirt1 expression . the production of endothelial - derived no is key in mediating relaxation of the cerebral vasculature . our study has found that t2d leads to significantly lower enos protein levels within cerebral arteries . this may be mediated in part by crp , which has been shown to directly decrease enos protein expression and activity and/or hyperglycemia that promotes enos glycosylation thereby reducing the active form of no . a reduction in total enos production suggests that no bioavailability may be reduced , potentially leading to blunted endothelial - mediated relaxations , which have previously been shown in this model . our observation that vascular mnsod protein expression is blunted in t2d suggests that the antioxidant pathway is being overwhelmed , presumably due to an increase in superoxide production within the vasculature . this is consistent with other data showing downregulation of mnsod in the vasculature after high - fat feeding . vascular p66shc protein expression was also significantly lower in t2d rats compared to controls . as a redox mitochondrial enzyme however more recent studies have localized p66shc to the cytoplasm where it is regulated by the redox sensitive transcription factor , nfe2-related factor 2 ( nrf2 ) . nrf2 regulates the antioxidant response element ( are / epre ) mediated expression of antioxidant enzymes . in this way , our finding of blunted vascular p66shc protein levels in t2d is in accordance with a recent report of reduced nrf2 signaling in mouse brain after high - fat feeding . interestingly , sirt1 itself has been shown to play an important role in regulating antioxidant gene expression in endothelial cells by enhancing the stability of the foxo3a / pgc-1 complex , which promotes antioxidant gene expression . in this way , blunted vascular sirt1 in t2d could mediate , at least in part , the observed decrease in antioxidant protein expression . vascular nadph oxidases are one of the major sources of ros in the cardiovascular system . in our study , we examined a subunit of nadph oxidase , nox2 , which has previously shown to be significantly increased in mouse aorta after high - fat feeding . however , in our animal model of t2d , nox2 levels were uncompromised in cerebral artery homogenates . nevertheless there are additional sources of superoxide including cyclooxygenase , xanthine oxidase , and other nadph oxidases such as nox1 and nox5 . superoxide can react with no to form peroxynitrite which in turn promotes nitration of tyrosine residues in proteins leading to nitrotyrosine - containing proteins . in the present study , we found that the abundance of nitrotyrosine - modified proteins was comparable in cerebral artery lysate from t2d rats compared to control rats . this is in contrast to studies in mouse aorta , rat aorta , pig coronary artery , and rat epineurial arterioles that have shown increased nitrotyrosine expression following high - fat feeding . taken together , our data suggest that , although antioxidant defence capacity is reduced , there appears to be no vascular nitrosative stress in our experimental model of t2d . a number of caveats need to be recognised here in terms of interpretation of our data . firstly , markers of oxidative and nitrosative stress were measured within the cerebral vasculature while telomere length was measured in dna extracted from the peripheral vasculature . ideally , telomere length would have been measured within cerebral arteries ; however this was impossible given the small size of rodent cerebral arteries . secondly , the femoral artery comprises a single layer of endothelial cells and a few layers of smooth muscle cells . if there are changes in endothelial cell telomere length in the absence of smooth muscle cell changes , these would not be detected . thirdly , the qpcr assay as used in the present study measures mean telomere length . there is evidence that a cell may be triggered to enter senescence by a single critically short telomere . therefore it remains a possibility that there is telomere dysfunction within the t2d vasculature , driven by a small number of critically short telomeres . future studies could address this possibility using high - throughput quantitative fluorescence in situ hybridization . in summary , we have shown for the first time that leukocyte telomere length does not predict arterial telomere length in t2d rats . our findings highlight the importance of measuring telomere dynamics in tissues other than circulating immune cells .

vascular dysfunction is an early feature of diabetic vascular disease , due to increased oxidative stress and reduced nitric oxide ( no ) bioavailability . this can lead to endothelial cell senescence and clinical complications such as stroke . cells can become senescent by shortened telomeres and oxidative stress is known to accelerate telomere attrition . sirtuin 1 ( sirt1 ) has been linked to vascular health by upregulating endothelial nitric oxide synthase ( enos ) , suppressing oxidative stress , and attenuating telomere shortening . accelerated leukocyte telomere attrition appears to be a feature of clinical type 2 diabetes ( t2d ) and therefore the telomere system may be a potential therapeutic target in preventing vascular complications of t2d . however the effect of t2d on vascular telomere length is currently unknown . we hypothesized that t2d gives rise to shortened leukocyte and vascular telomeres alongside reduced vascular sirt1 expression and increased oxidative stress . accelerated telomere attrition was observed in circulating leukocytes , but not arteries , in t2d compared to control rats . t2d rats had blunted arterial sirt1 and enos protein expression levels which were associated with reduced antioxidant defense capacity . our findings suggest that hyperglycemia and a deficit in vascular sirt1 per se are not sufficient to prematurely shorten vascular telomeres .

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

PMC3146100

root canal preparation is performed with the use of endodontic instruments and auxiliary chemical substances , seeking to achieve cleaning , shaping , and disinfection , in order to fill the root canal later . the use of rotary nickel titanium ( niti ) instruments has increased considerably due to their safe use , when used in accordance with the recommendations of their manufacturers , enabling the canal to be prepared more quickly , when compared with manual instruments , and even with better quality , in case of canals with severe curvatures . cutting dentin is an important step during root canal preparation , as it is necessary to remove contaminated dentin and shape the canal to create conditions for it to be filled . due to elasticity , one could suppose that the cutting efficiency of niti instruments would be lower than that of steel instruments , because they undergo deflection or bend during contact with the dentinal surface . however , studies have shown that niti instruments have a more efficient cutting capacity when compared with steel instruments . although there have been significant advancements in rotary instrumentation , the influence of the rotary cutting instrument blade design is still controversial , with respect to the efficiency of their cleaning capacity . their cutting capacity results from a complex inter - relationship between different parameters , such as , the sectional design of the instrument , radial lands or active cutting blades , metallurgical properties,[911 ] and treatment of instrument surfaces with the incorporation of ions . the aim of this study is to evaluate the cutting efficiency of different rotary niti instruments ; that is , k3 , niti tee , profile , and quantec . forty instruments with taper size .04/25 were tested and divided into groups according to their brands ( n = 10 ) : k3 ( sybron - endo , orange county , ca , usa ) , niti tee ( sjding sendoline , kista , sweden ) , profile ( dentsply maillefer , ballaigues , switzerland ) , and quantec ( analytic endodontics , mexico ) . the characteristic of each instrument is shown in table 1 . different instrument designs forty acrylic resin blocks ( dentsply maillefer , petrpolis , rj , brazil ) , with simulated canals were used in this study , and for each block , a rotary instrument was used . the canals initially had a diameter of a caliber 10 instrument , having been manually prepared in this manner , up to a caliber 20 instrument , to their full extent , using the crown - down technique , under irrigation with 5 ml distilled and deionized water , at every change of instrument . before any intervention , the blocks were washed in an ultrasonic vat , with distilled water and detergent solution , in the proportion of 10 : 1 for 20 minutes , dried with air jets , and placed in an oven at 60c for 48 hours . instrumentation of each block with the analyzed instruments was performed by the same operator using a tc 3.000 engine ( nouvag , tcm endo , goldach , switzerland ) at a constant speed of 300 rpm . the instruments were introduced into the canal , inundated with 5 ml distilled and deionized water , until the working length was reached , which was 0.5 mm short of the total working length of the simulated canal . each instrument remained at the working length for 5 seconds , and was then removed , while it was still running . the procedure of washing and drying the acrylic resin blocks was performed before the instruments were used , was repeated after instrumentation , to determine the final weight ( fw ) , and also performed before the use of 04/25 instruments . the cutting efficiency of each instrument was measured by means of measuring the acrylic resin block mass before and after using the .04/25 instruments ( fw - iw ) , with the aid of a precision digital analytic balance ( bel engineering , italy ) , which was due to the quantity of material removed by instrumentation . after obtaining the results , having verified the normality of the sample , the analysis of variance ( p < 0.05 ) was performed . forty instruments with taper size .04/25 were tested and divided into groups according to their brands ( n = 10 ) : k3 ( sybron - endo , orange county , ca , usa ) , niti tee ( sjding sendoline , kista , sweden ) , profile ( dentsply maillefer , ballaigues , switzerland ) , and quantec ( analytic endodontics , mexico ) . the characteristic of each instrument is shown in table 1 . forty acrylic resin blocks ( dentsply maillefer , petrpolis , rj , brazil ) , with simulated canals were used in this study , and for each block , a rotary instrument was used . the canals initially had a diameter of a caliber 10 instrument , having been manually prepared in this manner , up to a caliber 20 instrument , to their full extent , using the crown - down technique , under irrigation with 5 ml distilled and deionized water , at every change of instrument . before any intervention , the blocks were washed in an ultrasonic vat , with distilled water and detergent solution , in the proportion of 10 : 1 for 20 minutes , dried with air jets , and placed in an oven at 60c for 48 hours . instrumentation of each block with the analyzed instruments was performed by the same operator using a tc 3.000 engine ( nouvag , tcm endo , goldach , switzerland ) at a constant speed of 300 rpm . the instruments were introduced into the canal , inundated with 5 ml distilled and deionized water , until the working length was reached , which was 0.5 mm short of the total working length of the simulated canal . each instrument remained at the working length for 5 seconds , and was then removed , while it was still running . the procedure of washing and drying the acrylic resin blocks was performed before the instruments were used , was repeated after instrumentation , to determine the final weight ( fw ) , and also performed before the use of 04/25 instruments . the cutting efficiency of each instrument was measured by means of measuring the acrylic resin block mass before and after using the .04/25 instruments ( fw - iw ) , with the aid of a precision digital analytic balance ( bel engineering , italy ) , which was due to the quantity of material removed by instrumentation . after obtaining the results , having verified the normality of the sample , the analysis of variance ( p < 0.05 ) was performed . the means and standard deviations of mass loss from the acrylic resin blocks during instrumentation are shown in table 2 . the results test showed that the acrylic resin blocks prepared with k3 , niti tee , and profile instruments presented the greatest mass loss , showing no statistically significant difference among them ( p < 0.05 ) . the lowest mass loss was found in the blocks prepared with quantec instruments ( p < 0.05 ) . biomechanical techniques for preparing root canals have evolved from manual methods to rotary techniques employing nickel - titanium instruments . using niti rotary instruments for root canal instrumentation has enabled clinicians to create consistently tapered preparations more predictably and efficiently , while minimizing procedural mishaps , especially in curved canals . the greater cutting capacity of k3 instruments , as compared to quantec , is possibly due to the unique cross - section design of k3 , which has a positive angle of inclination for greater cutting efficiencies and ample radial lands and relief at the posterior extremity of the blade , to reduce friction . unlike quantec , which has two cutting blades thus , the k3 instruments present excellent cutting capacity , as the debris resulting from its cutting action is easily displaced from the working area , and removed by the unique helical angle of this file . schafer and florek conducted a study in which they compared the efficiency of the k3 system and k - flexofile files for smear layer removal . the authors obtained excellent results with the k3 system , although complete root canal cleaning had not been obtained . the niti tee instruments showed a cutting efficiency similar to that of the k3 and profile instruments , and superior to that of the quantec instruments . these instruments had a rounded , non - cutting tip and a positive cutting angle , without the presence of radial land . in addition , they had two 90 cutting blades that caused grooves ( scratches ) on the canal wall , which allowed the removal ( drainage ) of dentin scrapings and other debris resulting from instrumentation . jodway and hlsmann , comparing k3 and niti tee instruments , observed that they were safe as regards their use , and had similar root canal cleaning and shaping capacities . the good cutting capacity of profile instruments could be attributed to the u - shaped , cross - sectional design , with radial lands and central parallel core . with a neutral and negative angle of inclination thus , the debris is transported up to the coronal portion and effectively removed from the root canals . some studies have indicated that the positive cutting angle improves the cutting efficiency of the instrument . nevertheless , this is not the only determining factor for the cutting capacity of instruments , as the profile instruments , which have a negative cutting angle , presented the same efficiency as the k3 and niti tee instruments and greater efficiency than the quantec instruments , fitted with a positive cutting angle . quantec instruments have two cutting blades and a helical angle combined with spaces created in the instrument , which progressively increase as they withdraw from the cutting edge , creating a larger area of escape and allowing rapid debris removal from the canal . thus , when activated , the instrument does not make a thread ; therefore it does not stick to the canal walls , diminishing the risk of instrument fracture , because it has a less aggressive cut , which possibly reduces its cutting power , as it presents the lowest cutting efficiency among the studied instruments . the cutting capacity of endodontic instruments is an important factor to evaluate , as the preparation of dentinal walls , by eliminating contamination and shaping endodontic space , is an important step in successful endodontic therapy . however , after debridement , in some oval and flattened root canals , the walls may not be prepared , irrespective of the debridement technique , leaving a smear layer , debris , and unprepared root canal walls . this complex anatomy may be considered as one of the main challenges in controlling the root canal infection , because the pulp tissue as well as the root dentin may conceal toxins and microorganisms , compromising the final result of endodontic treatment . the use of endodontic irrigant solutions and ultrasound agitation , or even the association of endodontic instruments are ways that may improve the cleaning of roots that have a complex anatomy . from the results of the present study it can be concluded that k3 , niti tee , and profile instruments present a better cutting efficiency than the quantec instruments .

aim : the aim of this study was to evaluate the cutting efficiency of rotary nickel - titanium ( niti ) instruments k3 , niti tee , profile , and quantec with taper size 04/25.materials and methods : the number of samples was 10 for each group ( n = 10 ) . the cutting efficiency was measured by the mass loss from each acrylic resin block after instrumentation of a simulated canal using the crown - down technique.results:the analysis of variance ( anova ) showed that there was a statistically significant difference among the studied groups . the tukey 's test showed that the acrylic resin blocks prepared with instruments k3 ( 0.00369 0.00022 ) , niti tee ( 0.00368 0.00023 ) , and profile ( 0.00351 0.00026 ) presented the greatest mass loss , showing no statistically significant difference among them ( p < 0.05 ) . the lowest mass loss was found in the blocks prepared with quantec instruments ( 0.00311 0.0003 ) ( p < 0.05).conclusions : it could be concluded that the k3 , niti tee , and profile instruments presented a greater cutting efficiency than the quantec instruments .

INTRODUCTION MATERIALS AND METHODS Instruments Cutting efficiency RESULTS DISCUSSION CONCLUSIONS

PMC4340444

uterine artery anatomic variants have been the subject of detailed study , since the beginning of last century , given fibromyoma role in the pathogenesis of arterial vasculature . according to various studies , there are certain classifications of uterine artery anatomical variants and paper proposes such a classification , the observations selected aspects of examinations performed angiographic fibromyoma embolization . 110 examinations were conducted in women aged between 20 and 47 years , with symptoms and clinical diagnosis of uterine fibromyoma . embolization procedure was performed by unilateral brachial vascular access , using seldinger technique with a sheath pressure mounting and placing a 4f or 5f cobra catheter tip , running through the axillary artery , subclavian artery , ( brachiocephalic arterial trunk ) descending aorta , iliac artery common internal iliac artery , uterine artery tracing is the purpose of selective catheterization of it . retrospective evaluation of imaging uterine artery arteriography allowed classification into four groups , depending on the origin of the uterine artery . 220 were observed in the uterine artery catheterization and were classified number 200 , which represents 90.1% of the total , the remaining 20 were not conclusive arteriogram . analysis arteriogram interpretation allowed a classification into 4 types , given the classifications listed in the literature as follows : - type i - the origin of the uterine artery inferior gluteal artery ram ( fig . 1 ) - type ii - the origin of the uterine artery bifurcation ram inferior gluteal artery ( fig . 2 ) - type iii - the origin of the uterine artery to the internal iliac artery trifurcation ram , with lower and upper gluteal artery ( fig . 3 ) - type iv - uterine artery proximal to the origin of the origin of the gluteal arteries top and bottom ( fig . 4 ) the origin of the uterine artery inferior gluteal artery ram the origin of the uterine artery bifurcation ram inferior gluteal artery the origin of the uterine artery to the internal iliac artery trifurcation ram , with lower and upper gluteal artery uterine artery proximal to the origin of the origin of the gluteal arteries top and bottom anatomy descriptive notes uterine artery origin from the internal iliac artery anterior trunk , with arteries bladder , rectal average obturatory artery , internal pudendal artery , sciatica , however note the variant of origin of uterine artery from a common trunk artery umbilical [ 5 - 8 ] . in fact , the origin of the uterine artery is variable , according to recent studies , which are based on angiographic examinations . currently there is a classification of the uterine artery origin : type i ( the origin of the uterine artery inferior gluteal artery ram ) in 45% , type ii ( the origin of the uterine artery bifurcation ram inferior gluteal artery ) 6% , type iii ( origin uterine artery to the internal iliac artery of the ram trifurcation , with the upper and lower gluteal artery ) 43% and 6% type iv ( proximal to the origin of the uterine artery to the origin of the upper and lower gluteal arteries ) . recent studies , which are based on angiographic examination , note the presence of two trunks of bifurcation of the internal iliac artery , anterior and posterior in 70% of cases , and uterine artery is inferior gluteal artery origin or branch of the internal iliac artery trifurcation . the present study shows different values of such a percentage , such type i is 24% of the total arteriograms classifiable and type ii is in a proportion of 10% . for type iii percentage different values are observed to specialized studies , with a much increased type iv also shows that the type iii and type i are halfway to the classification established values . this again demonstrates the variability of uterine artery origin in different percentages , but major practical importance . in addition to the above aspects of the present and the emergence of uterine artery dissection of the cadavers in department of anatomy , university of medicine craiova . this notice these issues . in fig.5 the origin of the uterine artery uterine artery dissection origin proximal to the superior and inferior gluteal arteries emerging ( arrow ) in fig.6 uterine artery arises from the internal iliac artery , making common core with superior gluteal artery and inferior gluteal . knowledge of emerging variants uterine artery has an important role in the success of fibromyoma embolization , thus avoiding damage to other vascular structures , also correct assessment of uterine artery origin is reflected in the reduction of radiation dose and shortening the time of intervention .

uterine artery embolization as a therapeutic method in fibromyoma requires a good knowledge of the origin of the uterine artery to the success of this procedure involving selective catheterization . this study presents a classification of anatomical variants of uterine artery as a retrospective review of consecutive arteriogram , complete with various aspects of the origin of the uterine artery in cadaver dissection , in the department of anatomy .

Introduction Matherial and Method Results Discussion Conclusions

PMC4779254

hepatitis a virus ( hav ) infection is considered as an important public health concern . it is estimated that about 1.4 million cases of hav infection occur every year worldwide ( 1 , 2 ) . healthcare workers ( hcws ) can always be exposed to hav infected patients and if they were non - immune , they will also be infected . hence hospital personnel can be proposed as a high risk group for hav infection and evaluation of a suitable preventive method for them seems to be reasonable . on the other hand , investigation of hav seropositivity among this group some countries have evaluated this rate and some of them proposed hav vaccination as preventive strategy for their hcws ( 2 - 5 ) . in this review article , we highlighted some points about virology , epidemiology , preventive strategies and importance of hav infection and then focused on available data regarding hav seroprevalence among iranian healthcare personnel to evaluate the need for using vaccination as a preventive method . for this purpose , scopus , pubmed and google scholar were comprehensively searched using all appropriate combinations of following keywords ; healthcare provider , healthcare worker , healthcare personnel , nurse , medical students , furthermore , to find related persian evidences we searched google scholar and scientific information database ( sid ) . it is a 27 nanometer rna virus , containing 7474 nucleotides , positive stranded , non - enveloped and icosahedral virus . four genotypes have been identified for hav in humans that are a single serotype without biological differences . the spread route for hav is fecal - oral and risk factors are travel to endemic areas , near contact with an infected person , homosexual activity , waterborne , being in a daycare center and injection drug use . the infection , as an important public health problem , occurs worldwide with more prevalence in low socioeconomic areas ( 1 , 2 , 9 ) . in a population study in tehran province of iran , the overall rate of anti - hav seroprevalence has been reported as 90% ( 10 ) . also a gradual shift for age of hav infection from childhood to adulthood has been reported . control of some of the aforementioned risk factors can be the reason of this gradual shift ( 9 ) . hav causes an acute illness that is self - limited , but it can cause fulminant hepatitis rarely in some patients . manifestation of hepatitis a infection could be silent or with non - specific symptoms in children , but it can cause a mild flu - like illness to hepatic failure in adults ( 11 ) . considering different causes of acute hepatitis , diagnosis of hav infection based on clinical manifestations detecting serum hav - rna using pcr amplification is an expensive method and usually used for research purposes . however , assessment of immunoglobulin ( ig ) antibody to hav has been suggested for diagnosis of hav infection . igm anti - hav anti - body emerges one to two weeks after exposure to virus and disappears after 3 to 6 months , so diagnose of acute phase of hav infection can be possible . positive results for testing igg hav anti - body also shows previous history of hav infection ( 8 , 13 ) . fulminant hepatic failure is an ominous complication of hepatitis a and in this case , liver transplantation is a suggested option . approximately 30% of symptomatic patients admitted to hospital for management of complications including dehydration , severe prostration , coagulopathy , encephalopathy and other evidences of hepatic decompensation ( 14 ) . as we explained before intramuscular administration of hav immune globulin provides passive immunization , which is short - term and temporary and usually takes from three to five months . passive immunization has its special indications like persons travelling to or working in countries and communities with intermediate and high rate of hav infection and etc . however , it is not related to the topic of this review and more information can be obtained from related review articles ( 8 , 15 , 16 ) . also there is another vaccine , a combined vaccine namely twinrix , which contains both hav and hepatitis b antigens ( 17 ) . the two mentioned types of vaccines has been approved and used in the united states . both of them are administered intramuscularly and as two separated injections with a six - month interval . it is said that after one month of receiving two doses of these vaccines , about 100% of immunocompetent vaccinated subjects can earn a probable lifelong immunity against hav infection ( 8 , 17 ) . nowadays , we know that vaccination is an important and effective preventive method for decreasing fulminant hepatitis . in a study in the united states , vaccination rate for hav among healthcare personnel was higher than general population , however overall protection has remained suboptimal ( 18 ) . though , selecting an appropriate preventive strategy in each area depends on evaluation of some issues related to that area and they are annual incidence rate of fulminant hepatitis due to hav infection , hav seropositivity in different age and occupation groups , costs for hospitalization and treatment of patients with hav infection . these are on one side of the equation and the costs for providing hav vaccines and running vaccination program are on the other side . furthermore , gross national income of each country is the major factor that influences this equation ( 19 - 22 ) . therefore , each country or territory needs to run some cost - effectiveness studies for making a decision about running hav vaccination program ( 23 ) . here is the other epidemiologic factor that should be considered in vaccination program of each country , especially developing countries like iran . controlling risk factors for hav infection with improvement in hygiene of food and water has led to a gradual shift of occurrence of hav infection from childhood to adulthood . in the first view it is good , but with this condition children remain without immunity to hav and we know that hav infection in adulthood is more severe than childhood ( 20 , 23 , 24 ) . combination of hav vaccine with hepatitis b virus vaccine , as a mandatory vaccine in some healthcare systems ( 25 ) and applying hav vaccination in non - immune healthcare personnel ( based on the previous screening ) are some of the cost - effective methods for hav prevention ( 26 ) . it is a 27 nanometer rna virus , containing 7474 nucleotides , positive stranded , non - enveloped and icosahedral virus . four genotypes have been identified for hav in humans that are a single serotype without biological differences . the spread route for hav is fecal - oral and risk factors are travel to endemic areas , near contact with an infected person , homosexual activity , waterborne , being in a daycare center and injection drug use . the infection , as an important public health problem , occurs worldwide with more prevalence in low socioeconomic areas ( 1 , 2 , 9 ) . in a population study in tehran province of iran , the overall rate of anti - hav seroprevalence has been reported as 90% ( 10 ) . also a gradual shift for age of hav infection from childhood to adulthood has been reported . control of some of the aforementioned risk factors can be the reason of this gradual shift ( 9 ) . hav causes an acute illness that is self - limited , but it can cause fulminant hepatitis rarely in some patients . manifestation of hepatitis a infection could be silent or with non - specific symptoms in children , but it can cause a mild flu - like illness to hepatic failure in adults ( 11 ) . considering different causes of acute hepatitis , diagnosis of hav infection based on clinical manifestations is not possible ( 12 ) . detecting serum hav - rna using pcr amplification is an expensive method and usually used for research purposes . however , assessment of immunoglobulin ( ig ) antibody to hav has been suggested for diagnosis of hav infection . igm anti - hav anti - body emerges one to two weeks after exposure to virus and disappears after 3 to 6 months , so diagnose of acute phase of hav infection can be possible . positive results for testing igg hav anti - body also shows previous history of hav infection ( 8 , 13 ) . fulminant hepatic failure is an ominous complication of hepatitis a and in this case , liver transplantation is a suggested option . approximately 30% of symptomatic patients admitted to hospital for management of complications including dehydration , severe prostration , coagulopathy , encephalopathy and other evidences of hepatic decompensation ( 14 ) . intramuscular administration of hav immune globulin provides passive immunization , which is short - term and temporary and usually takes from three to five months . passive immunization has its special indications like persons travelling to or working in countries and communities with intermediate and high rate of hav infection and etc . however , it is not related to the topic of this review and more information can be obtained from related review articles ( 8 , 15 , 16 ) . also there is another vaccine , a combined vaccine namely twinrix , which contains both hav and hepatitis b antigens ( 17 ) . the two mentioned types of vaccines has been approved and used in the united states . both of them are administered intramuscularly and as two separated injections with a six - month interval . it is said that after one month of receiving two doses of these vaccines , about 100% of immunocompetent vaccinated subjects can earn a probable lifelong immunity against hav infection ( 8 , 17 ) . nowadays , we know that vaccination is an important and effective preventive method for decreasing fulminant hepatitis . in a study in the united states , vaccination rate for hav among healthcare personnel was higher than general population , however overall protection has remained suboptimal ( 18 ) . though , selecting an appropriate preventive strategy in each area depends on evaluation of some issues related to that area and they are annual incidence rate of fulminant hepatitis due to hav infection , hav seropositivity in different age and occupation groups , costs for hospitalization and treatment of patients with hav infection . these are on one side of the equation and the costs for providing hav vaccines and running vaccination program are on the other side . furthermore , gross national income of each country is the major factor that influences this equation ( 19 - 22 ) . therefore , each country or territory needs to run some cost - effectiveness studies for making a decision about running hav vaccination program ( 23 ) . here is the other epidemiologic factor that should be considered in vaccination program of each country , especially developing countries like iran . controlling risk factors for hav infection with improvement in hygiene of food and water has led to a gradual shift of occurrence of hav infection from childhood to adulthood . in the first view it is good , but with this condition children remain without immunity to hav and we know that hav infection in adulthood is more severe than childhood ( 20 , 23 , 24 ) . combination of hav vaccine with hepatitis b virus vaccine , as a mandatory vaccine in some healthcare systems ( 25 ) and applying hav vaccination in non - immune healthcare personnel ( based on the previous screening ) are some of the cost - effective methods for hav prevention ( 26 ) . data about hav prevalence among iranian hcws are very limited . in a cross - sectional study in a hospital from babol ( a city of mazanadaran province of iran ) between 2011 and 2012 , 466 hcws ( paramedical technicians , nurses and nurses aid ) were tested for anti - hav antibody ( igg ) . it was reported that 330 ( 71% ) of participants had positive results for the mentioned test . on the other hand however , about 30% of healthcare personnel are not immuned against hav and in exposure with hav infected patients and need confident preventive strategy . as conclusion of this project , role of vaccination program in protecting susceptible hcws , 150 personnel of a military hospital in tehran were investigated regarding previous immunity to hav infection . this is a very considerable result in four years ago and suggests a wisdom preventive method for healthcare personnel against hav infection as authors of this project recommended hav vaccination as their solution ( 28 ) . comparison of hav seropositivity among hcws in different times can help making a better decision about the best hav preventive method . however , the literature has not enough data about this issue , especially in the past . in a cross - sectional project performed about 12 years ago in a hospital of sari ( another city of mazanadaran province ) , hav seropositivity rate was reported as 90.36% for hcws ( 29 ) . this is the maximum hav seropositivity rate for hcws among included studies in this project . perhaps this high rate of hav immunity in comparison with recent studies emphasizes on improvement in health status , controlling hav risk factors and finally gradual shift of hav infection from childhood toward adulthood . such important points can only be proved by cohort studies or only comparison of available cross - sectional studies with similar ones in the past . a study in one of the medical universities of tehran province in 2015 revealed that just 34% of evaluated patients ( n = 270 ) had positive results for anti - hav anti - body test ( 30 ) . another study in medical university of babol in 2014 showed that more than 30% of medical students were not immune against hav infection ( 31 ) . medical students in some other provinces of iran like isfahan , kermanshah and hamedan had been evaluated for hav prevalence . it has been reported that more than one third of these students in each university are hav seronegative ( 32 ) . as a result of occupational exposure , medical students , as future hcws , are at risk of hav infection and therefore all of these studies suggested vaccination as solution . regardless of this factor , there are some high risk groups like travelers to endemic countries , patients with chronic liver diseases , food handler , sewage workers and etc . that are suggested for hav vaccination ( 33 , 34 ) ; data about hav prevalence among iranian hcws are very limited . in a cross - sectional study in a hospital from babol ( a city of mazanadaran province of iran ) between 2011 and 2012 , 466 hcws ( paramedical technicians , nurses and nurses aid ) were tested for anti - hav antibody ( igg ) . it was reported that 330 ( 71% ) of participants had positive results for the mentioned test . on the other hand however , about 30% of healthcare personnel are not immuned against hav and in exposure with hav infected patients and need confident preventive strategy . as conclusion of this project , role of vaccination program in protecting susceptible hcws , 150 personnel of a military hospital in tehran were investigated regarding previous immunity to hav infection . this is a very considerable result in four years ago and suggests a wisdom preventive method for healthcare personnel against hav infection as authors of this project recommended hav vaccination as their solution ( 28 ) . comparison of hav seropositivity among hcws in different times can help making a better decision about the best hav preventive method . however , the literature has not enough data about this issue , especially in the past . in a cross - sectional project performed about 12 years ago in a hospital of sari ( another city of mazanadaran province ) , hav seropositivity rate was reported as 90.36% for hcws ( 29 ) . this is the maximum hav seropositivity rate for hcws among included studies in this project . perhaps this high rate of hav immunity in comparison with recent studies emphasizes on improvement in health status , controlling hav risk factors and finally gradual shift of hav infection from childhood toward adulthood . such important points can only be proved by cohort studies or only comparison of available cross - sectional studies with similar ones in the past . a study in one of the medical universities of tehran province in 2015 revealed that just 34% of evaluated patients ( n = 270 ) had positive results for anti - hav anti - body test ( 30 ) . another study in medical university of babol in 2014 showed that more than 30% of medical students were not immune against hav infection ( 31 ) . medical students in some other provinces of iran like isfahan , kermanshah and hamedan had been evaluated for hav prevalence . it has been reported that more than one third of these students in each university are hav seronegative ( 32 ) . as a result of occupational exposure , medical students , as future hcws , are at risk of hav infection and therefore all of these studies suggested vaccination as solution . regardless of this factor , there are some high risk groups like travelers to endemic countries , patients with chronic liver diseases , food handler , sewage workers and etc . that are suggested for hav vaccination ( 33 , 34 ) ; a gradual shift for occurrence of hav infection from childhood toward adulthood has been reported in iran . on the other hand , hav infection can be more severe when it occurs in adulthood . healthcare personnel are at a more risk to be infected with hav due to occupational exposure ( 23 , 35 ) . hav vaccination is an effective method for preventing morbidity and mortality due to fulminant hepatitis . it also helps to decrease the spread of hav in the community and subsequently prevent from hav outbreaks ( 35 ) . more original studies should be performed to investigate hav seroprevalence among hcws ( 15 ) . limited available studies show that a considerable number of hcws are hav seronegative and therefore vaccination in this high risk special group should be considered as a wisdom method ( 24 , 35 ) .

context : hepatitis a virus ( hav ) infection is an important public health problem . it is estimated that about 1.4 million cases of hav infection occur every year worldwide . non - immune healthcare workers ( hcws ) can be at higher risk of hav infection in comparison to general population and an appropriate preventive method should be considered for them.evidence acquisition : for finding related articles , a comprehensive search was performed in scopus , pubmed and google scholar and all appropriate combinations of following keywords were considered ; healthcare provider , healthcare personnel , healthcare worker , nurse medical students , iran , hepatitis a and vaccination . also we did a search in persian language in google scholar and scientific information database ( sid ) to find related persian literature.results:a gradual shift in age of hav infection has been seen from childhood toward adulthood . data about hav seropositivity among iranian hcws are very limited . however based on the recent studies , it seems that hav seropositivity has been reduced among hcws in comparison with the past . all recent studies have suggested hav vaccination for hcws.conclusions:available limited studies show that iranian healthcare personnel need hav vaccination . however , for selecting an appropriate preventive method for this high risk group , more original studies are still needed .

1. Context 2. Evidence Acquisition 2.1. Hepatitis A, Virology and Epidemiology 2.2. Hepatitis A, Diagnosis and Treatment 2.3. Hepatitis A, Immunization Strategies 2.4. Determining the Best Preventive Strategy 3. Results 3.1. Hepatitis A Seroprevalence in Iranian Healthcare Workers 4. Conclusions

PMC4009298

autologous bone grafts remain the gold standard for the treatment of congenital craniofacial bone disorders , such as alveolar cleft [ 18 ] . however , autologous bone grafts have potential problems , which include donor site morbidity and limitations to the amount of bone that can be harvested [ 913 ] . porous beta - tricalcium phosphate ( -tcp ) , which is now commercially available , is known for its osteoconductive and biodegradable properties . however , its use as a replacement for autologous bone grafts remains controversial [ 1418 ] . according to recent studies , various growth factors exhibit osteogenic properties [ 8 , 1923 ] , such as bone morphogenetic protein 2 ( bmp-2 ) [ 2436 ] , basic fibroblast growth factor ( b - fgf ) [ 3742 ] , platelet derived growth factor ( pdgf ) [ 4346 ] , transforming growth factor - beta 1 ( tgf-1 ) [ 4750 ] , and vascular endothelial growth factor ( vegf ) [ 5154 ] . in general , growth factors administered in solution form are readily diffused or degraded in vivo [ 26 , 50 , 55 , 56 ] . thus , their enhanced and prolonged bioactivity at the target site is necessary to reduce bolus dosage , especially in pediatric patients . recent studies suggest that granulocyte colony - stimulating factor ( g - csf ) promotes fracture healing or osteogenesis [ 5760 ] . because g - csf is an essential drug most frequently used to treat neutropenia secondary to chemotherapy , it is widely administered not only to adults but also to pediatric patients [ 6169 ] . accordingly , its biosafety is well established through extensive use in clinical contexts compared to other growth factors . commercially available -tcp ( superpore , pentax , tokyo , japan ) was used in the present study as an osteoconductive scaffold and space - maintaining material . to investigate the bone regenerative properties of g - csf , topical supplementation either in solution or in sustained release form with a gelatin hydrogel system the purpose of this study was to investigate whether g - csf with or without a controlled release system stimulates bone regeneration in combination with -tcp using a rat calvarial defect model [ 7073 ] . the present study was approved by the institutional committee of animal experiments at hokkaido university ( institutional animal care and use committee protocol number 12 - 0017 ) . fourteen wistar rats ( male , 13 weeks old ; weight , 250350 g ) were purchased from sankyo labo service corporation ( tokyo , japan ) . a total of 27 calvarial defects were randomly divided into nine treatment groups , with a total of three defects per treatment group . in solution - based treatment groups , defects were filled with a -tcp disc containing normal saline alone ( group a , control ) or 1 ( group b ) , 5 ( group c ) , or 20 g of g - csf ( group d ) . in controlled release groups , defects were filled with a -tcp disk with an overlaid gelatin hydrogel sheet incorporating normal saline alone ( group e ) or , 1 ( group f ) , 5 ( group g ) , or 20 g g - csf ( group h ) . the remaining defects were left empty to measure spontaneous healing ( group i ) ( table 1 ) . commercially available porous -tcp blocks ( superpore ) blocks were cut into discs 5 mm in diameter and 1 mm thick using a fine surgical saw and round bur . briefly , a mixed acidic gelatin - glutaraldehyde aqueous solution was cast into a polypropylene dish ( 80 80 mm ) and maintained at 4c for 12 hours . the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95 wt% . gelatin hydrogels were designed so that degradation would be complete in approximately two weeks under in vivo conditions [ 29 , 39 , 49 , 74 ] . hydrogel sheets were cut into discs 5 mm in diameter and 1 mm thick . human recombinant g - csf was kindly supplied by kyowa kirin co. ( tokyo , japan ) . to prepare gelatin hydrogels incorporating g - csf , 20 l of normal saline solution containing 1 , 5 , or 20 g g - csf similarly , 20 l of g - csf - free normal saline was dropped onto a freeze - dried hydrogel to obtain g - csf empty hydrogels . animals were anesthetized by intraperitoneal administration of pentobarbital sodium ( 50 mg / kg ) . subsequently , a skin incision was made and subperiosteal dissection was performed under a surgical microscope to raise the periosteal flaps . a bone defect 5 mm in diameter was then prepared on each side lateral to the sagittal suture using a fine surgical bur under copious sterile saline irrigation . defects were filled with bone substitutes according to the groups described above ( table 1 and figure 1 ) . periosteal flaps were repositioned using a 4 - 0 nylon suture , and the skin was closed with a running 4 - 0 nylon suture . specimens were prepared for decalcified sectioning by immersing them in 10% ethylenediaminetetraacetic acid ( edta ) for four weeks . embedded samples were then sectioned into 3 m slices parallel to the sagittal suture across the center of each calvarial defect using a microtome ( leica , sm2000r ) . hematoxylin and eosin ( he ) staining was used for histological analysis and aniline blue staining was used for histomorphometric analysis . each specimen was examined under a light microscope and digital photographs were obtained for histological evaluation of a region corresponding to the center of the calvarial defect ( figure 1(b ) ) . high magnification images with aniline blue staining ( 1.001 mm or 1360 1024 pixels ) of the most - central area of the defect were quantified to measure the percentage of newly formed bone and remaining bioceramics using imaging software ( adobe photoshop cs5 ) . statistical analysis was performed using kruskal - wallis one - way analysis of variance ( anova ) . figure 2 shows low magnification images of decalcified specimens stained with he along the midline of each calvarial defect . no remaining gelatin hydrogel or surgical site infections were observed . in group a ( control group ) , newly formed trabecular bone was observed focally but failed to occupy the entire defect . in groups b d ( solution - based treatment groups ) and in group e ( g - csf - free gelatin hydrogel group ) , newly formed trabecular bone was observable but failed to fill the defect . in group f ( 1 g g - csf gelatin hydrogel group ) , newly formed bone tissue nearly bridged the calvarial gap , whereas residual -tcp was also present . in group g ( 5 g g - csf gelatin hydrogel group ) , most of the defect was occupied with newly formed bone tissue ; moreover , sparse residual -tcp was observed . in contrast , group h ( 20 g g - csf gelatin hydrogel group ) showed focal formation of new bone surrounded by fibrous connective tissue at the superficial area of the defect with the presence of remaining biomaterials . in group i ( untreated defect group ) , the defect was filled with fibrous connective tissue with hardly any newly formed bone . figure 3 shows higher magnification images of groups e ( e ) and g ( g ) . in group g , newly formed bone was observed immediately below the periosteal flap and multinuclear giant cells were detected around the newly formed bone . in contrast , in group e , the formation of fibrous tissue and blood vessels was significant compared with newly formed bone in the subperiosteal region . figure 4 shows high magnification images of aniline blue staining in which matured bone tissue exhibits homogeneous dark blue and entrapped osteocytes . figure 5 shows the percentage of newly formed bone and remaining -tcp per high - powered field . in groups a , b , c , d , e , and h , defects had a tendency to be occupied by more remaining -tcp compared to newly formed bone tissue . in group a ( control ) , the percentages of newly formed bone and remaining -tcp were 20.77% 25.44% and 35.01% 2.01% , respectively . in contrast , in groups f and g ( 1 g and 5 g g - csf gelatin hydrogel groups ) , the percentage of newly formed bone ( 54.84% 9.46% and 69.53% 5.35% for groups f and g , resp . ) conspicuously exceeded values of remaining -tcp ( 20.47% 2.89% and 14.76% 7.36% for groups f and g , resp . ) . the values were significantly higher in groups f and g compared to the control group ( p < 0.01 ) . there was no significant difference between groups a ( control ) and i ( empty defect ) . values corresponding to groups b , c , d , e , and h showed no significant difference compared to that of group a. figure 7 shows the percentage of remaining -tcp , which was used to evaluate biodegradability in vivo . there was no significant difference between groups a , b , c , d , e , f , and h. in contrast , only in group g ( 5 g g - csf gelatin hydrogel group ) the percentage was significantly lower compared to group a ( 14.76% 7.36% versus 33.53% 0.80% , p < 0.05 ) . this result indicated a prominent enhancement of the biodegradable properties of -tcp , which was further accelerated by 5 g g - csf in sustained release form . in the present study , we demonstrated that the controlled release of low - dose ( 1 g and 5 g ) g - csf significantly enhanced bone regeneration when combined with a -tcp disc . moreover , administration of 5 g g - csf using a controlled release system significantly promoted the biodegradable properties of -tcp . according to our results , this tissue - engineering approach combining -tcp and the sustained release of g - csf is potentially feasible and promising for clinical use . to our knowledge , this is the first report which demonstrates the bone regeneration properties of g - csf at membranous ossification sites . because systemic administration of 510 g g - csf / kg / day is commonly used for pediatric malignancies [ 64 , 6668 ] , the results shown here indicate that notably low doses of g - csf ( 15 g / defect/2 weeks ) with controlled release can promote osteogenesis . in this study , we used -tcp as an osteoconductive scaffold and space - maintaining material . in the present study , although the control group ( -tcp alone ) showed a greater tendency for bone formation compared to the untreated defect group , there were no significant differences between the groups . furthermore , the defect in the control group had more residual -tcp than newly formed bone tissue . these results suggest that -tcp alone implantation is not sufficient to fill the defect with regenerated bone in the craniofacial region . some experimental studies have confirmed the osteoconductive properties of -tcp , which were comparable to autologous bone grafts [ 15 , 17 , 70 ] . however , other groups have emphasized versatility by combining -tcp with autologous bone fragments [ 16 , 7779 ] , growth factors [ 45 , 46 , 8084 ] , simvastatin , or stem cells [ 18 , 85 , 86 ] in both experimental and clinical studies . interestingly , ishida et al . reported that topical application of g - csf had bone regenerative properties via neovascularization and osteogenesis . that study revealed a significant increase in cd34 cells an endothelial and hematopoietic progenitor - enriched cell population in capillaries corresponding to the bone defect site . the study also showed that g - csf was responsible for mobilizing osteoblasts to the bone defect site . in addition , recent studies demonstrated the promotion of fracture healing by cd34 cells [ 58 , 8789 ] . kuroda et al . reported the first successful clinical case of a tibial nonunion treated with topically applied g - csf - mobilized cd34 cells . some reports have shown that cd34 cells play an important role in releasing angiogenic factors , including vascular endothelial growth factor ( vegf ) , hepatocyte growth factor ( hgf ) , and fibroblast growth factor 2 ( fgf2 ) [ 58 , 90 , 91 ] . moreover , the differentiation capacity of cd34 cells into osteoblasts has been shown in previous reports [ 59 , 92 ] . in the present study , the controlled release g - csf groups showed more newly formed bone immediately below the periosteum compared to the other groups . on the other hand , rojbani et al . reported that osteoprogenitor cells differentiate from the dura mater . presumably , the sustained release of g - csf may stimulate periosteal cells along an osteogenic lineage , resulting in enhanced bone formation . in the present study , we used gelatin hydrogel as a sustained release carrier of g - csf . various growth factors have been shown to have bone regenerative properties , such as bone morphogenetic proteins ( bmps ) [ 2436 ] , b - fgf [ 3742 , 93 ] , pdgf [ 4346 ] , tgf-1 [ 4750 ] , and vegf [ 5154 ] . bmp-2 has the strongest osteoinductive activity in promoting ectopic bone regeneration [ 26 , 29 ] and has been approved by the food and drug administration for use in orthopedics and oral surgery [ 30 , 34 , 35 ] . in general , growth factors administered in solution form are easily diffused or degraded prior to achieving full bioactivity [ 26 , 50 , 55 , 56 ] . therefore , commercially available bmp-2 in combination with a collagen sponge kit must contain milligram amounts of the growth factor ( 1.5 mg / ml ) [ 30 , 34 , 35 ] . potential risk for local inflammatory responses should be taken into consideration after topical application . in order to reduce bolus dosage , enhanced and one of the practical ways to control the in vivo release of growth factors is to use gelatin hydrogel , in which the growth factor is physicochemically immobilized and subsequently released in proportion to hydrogel degradation [ 74 , 94 ] . in the present study , the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95 wt% . the hydrogels were designed so that degradation would be complete in approximately 2 weeks under in vivo conditions [ 29 , 39 , 49 , 50 , 74 ] . gelatin is commercially available and its biosafety is well established through its long clinical use as a plasma expander and drug ingredient . in the present study , the controlled release of 5 g g - csf ( group g ) significantly promoted the osteoconductive properties and biodegradability of -tcp . improved biodegradability compared to hydroxyapatite is a major characteristic of porous -tcp [ 71 , 9597 ] . biodegradability is generally thought to occur in harmony with bone remodeling , in which -tcp allows tissue fluid dissolution and absorption by osteoclasts in vivo [ 72 , 95 ] . reported that g - csf increased both osteoclast activity and bone resorption in the bone marrow , triggering an increase in the number of mesenchymal precursor cells in the bone marrow using a mouse model . in another study , pdgf modified -tcp resorption , although the underlying mechanism was not provided . some studies have shown that bmp-2 does not facilitate -tcp resorption [ 70 , 99 ] . in group g of the present study , we can speculate from the results that the controlled release of 5 g g - csf may stimulate the mobilization and differentiation of mesenchymal precursor cells in the periosteum as well as osteoclast activation . in contrast , group h ( 20 g g - csf gelatin hydrogel group ) showed less new bone formation and -tcp resorption . this might be explained by the multidifferentiation potential of g - csf - mobilized progenitor cells , which is consistent with previously published reports [ 92 , 100102 ] . interestingly , ishida et al . stated that topical application of 50 g g - csf did not induce bone regeneration according to preliminary data . moreover , some reports have shown that sustained release of g - csf enhances tendon - bone integration with significantly more formation of sharpey 's fibers and microvessels . these results led us to speculate that a prolonged high concentration of topical g - csf drives progenitor cells toward fibrous tissue formation rather than osteogenesis . therefore , sustaining relatively low concentrations of topical g - csf can play an important role in inducing balanced bone regeneration and -tcp resorption . our findings suggest an optimal dose of 5 g per defect for controlled release of g - csf , which is consistent with previously published reports [ 59 , 103 ] . first , the study was designed using small animals and a limited number per experimental group . second , although some reports accept the calvarial defect rat model [ 7073 ] , the decortication procedure may not fully reflect clinical situations of congenital craniofacial anomalies , since some evidence suggests that fractures mobilize cd34 cells from the bone marrow into the peripheral blood [ 88 , 89 ] . third , we used histomorphometric analysis to characterize newly formed bone and biodegradation of -tcp ; however , we did not identify cd34 cells or evaluate the activity of osteogenic cells at the bone defect site . future studies should incorporate experimental models without decortication , larger animals , and immunohistochemical analysis . in conclusion , controlled release of 5 g g - csf using a gelatin hydrogel system the present results indicate that the combination of g - csf slow - release and -tcp is feasible and promising for the treatment of congenital craniofacial bone defects .

autologous bone grafts remain the gold standard for the treatment of congenital craniofacial disorders ; however , there are potential problems including donor site morbidity and limitations to the amount of bone that can be harvested . recent studies suggest that granulocyte colony - stimulating factor ( g - csf ) promotes fracture healing or osteogenesis . the purpose of the present study was to investigate whether topically applied g - csf can stimulate the osteoconductive properties of beta - tricalcium phosphate ( -tcp ) in a rat calvarial defect model . a total of 27 calvarial defects 5 mm in diameter were randomly divided into nine groups , which were treated with various combinations of a -tcp disc and g - csf in solution form or controlled release system using gelatin hydrogel . histologic and histomorphometric analyses were performed at eight weeks postoperatively . the controlled release of low - dose ( 1 g and 5 g ) g - csf significantly enhanced new bone formation when combined with a -tcp disc . moreover , administration of 5 g g - csf using a controlled release system significantly promoted the biodegradable properties of -tcp . in conclusion , the controlled release of 5 g g - csf significantly enhanced the osteoconductive and biodegradable properties of -tcp . the combination of g - csf slow - release and -tcp is a novel and promising approach for treating pediatric craniofacial bone defects .

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

PMC4049053

sacrococcygeal pilonidal sinus disease is a common and usually a minor disease . although wide excision has been a common practice , there are more simple alternatives , and its treatment remains controversial . numerous surgical procedures have been described , but treatment failure and disease recurrence are frequent , leading to considerable morbidity in these , otherwise healthy patients . the optimal surgical treatment option should be simple , inexpensive , and associated with low hospitalization periods and recurrence rates . total excision of the involved sinus tract to the post sacral fascia is the most frequently applied surgical option but the ideal type of reconstruction is disputed . the defect formed on the excised area might be primarily closed , partially closed as is the case in marsupialization , left open for secondary healing , or reconstructed by differing flap techniques . published literatures comparing the use of flap as off - midline procedure versus primary midline suture techniques found that primary closure would be as effective as the flap reconstruction . midline excision techniques are associated with high morbidity and recurrence due to wound 's placement in the natal cleft . conventional off - midline flap closures are associated with fewer serious complications , but they have less acceptable esthetic outcomes . in the present study , the author reported a novel simplified off - midline technique for closure of the defect after complete excision of the sinus tracts without flap reconstruction . all patients completed written informed consent and received an explanation of the operative procedure from the study staff . the study procedures were approved by the institutional ethics committee of port - fouad general hospital , represented by the hospital 's general director . a total of two hundreds patients of both sexes with primary , non - recurrent , sacrococcygeal pilonidal sinus disease were enrolled for this prospective study from january 2002 to december 2008 regarding our modified simple closure of sacrococcygeal pilonidal sinus disease . all procedures were performed at port - fouad general hospital , port - said , egypt by a single surgical team consisting of a consultant colorectal surgeon assisted by a general surgeon . the surgical team treated 1 - 2 patients with pilonidal sinus disease per week in both private and public health sectors i.e. , over 500 patients , in total , during the study period . patients were excluded from the study when they had a history of drained pilonidal abscess or recurrent disease , or declined the protocol . patients under general anesthesia were placed in a prone , jack - knife position with buttocks widely separated using adhesive tapes , and methylene blue was injected into the sinuses . modified d - shaped excisions were used [ figure 1a ] to include all sinuses and their ramifications to the presacral fascia and surrounding unhealthy tissues to achieve healthy , soft and supple wound margins [ figure 1b ] . a suction drain was placed under the flap and was delivered through a separate stab incision . the wound was closed using four or five deeply running sutures obliterating the dead space [ figure 2 ] . the sutures were tied in a way to obliterate the dead space and permit the incision just lateral to the midline , while two sutures were left long enough to be tied over a cigar - like dressing to promote further obliteration of the dead space [ figure 3a and b ] . the drain was removed after 48 hours and the two dressing sutures tying the dressing were cut short at the fourth day to exchange the dressing . outpatient clinic follow - up occurred two weeks , one month , and three months after surgery , and after six months when necessary thereafter . ( a ) an operative photograph showing planning of offmidline incision ( b ) an operative photograph showing complete excision of the sinus tracks with good hemostasis an operative photograph showing planning of wound closure with the deeply running sutures in situ ( a ) an operative photograph showing complete wound closure with two sutures long enough to be tied over the dressing ( b ) an operative photograph showing final step with dressing being tightly in place the primary endpoints of the study were early failure , defined as wound disruption within 10 days of surgery and recurrence of the disease , defined as a clinically detectable sinus . secondary endpoints were wound infection , time - off from work and painless walking and sitting time . patients under general anesthesia were placed in a prone , jack - knife position with buttocks widely separated using adhesive tapes , and methylene blue was injected into the sinuses . modified d - shaped excisions were used [ figure 1a ] to include all sinuses and their ramifications to the presacral fascia and surrounding unhealthy tissues to achieve healthy , soft and supple wound margins [ figure 1b ] . a suction drain was placed under the flap and was delivered through a separate stab incision . the wound was closed using four or five deeply running sutures obliterating the dead space [ figure 2 ] . the sutures were tied in a way to obliterate the dead space and permit the incision just lateral to the midline , while two sutures were left long enough to be tied over a cigar - like dressing to promote further obliteration of the dead space [ figure 3a and b ] . the drain was removed after 48 hours and the two dressing sutures tying the dressing were cut short at the fourth day to exchange the dressing . outpatient clinic follow - up occurred two weeks , one month , and three months after surgery , and after six months when necessary thereafter . ( a ) an operative photograph showing planning of offmidline incision ( b ) an operative photograph showing complete excision of the sinus tracks with good hemostasis an operative photograph showing planning of wound closure with the deeply running sutures in situ ( a ) an operative photograph showing complete wound closure with two sutures long enough to be tied over the dressing ( b ) an operative photograph showing final step with dressing being tightly in place the primary endpoints of the study were early failure , defined as wound disruption within 10 days of surgery and recurrence of the disease , defined as a clinically detectable sinus . secondary endpoints were wound infection , time - off from work and painless walking and sitting time . in the present study , there were 164 ( 82% ) male and 36 ( 18% ) female patients with age range from 15 - 33 years and male : female ratio was 4:1 . a large majority of patients was between the ages of 15 and 25 , and the incidence or decreased thereafter [ table 1 ] . patients with sex and subgroup distributions regarding the body mass index ( bmi ) , patients were classified as obese ( bmi > 30 kg / m ) , overweight ( bmi > 25 but < 30 kg / m ) and healthy weight ( bmi < 25 kg / m ) . by these definitions , 64 patients were obese ( 52 males and 12 females ) , 100 were overweight ( 80 males and 20 females ) and 36 patients were of healthy weight ( 32 males and 4 females ) . the average bmi for both sexes was 29.3 ( sd 2.8 , range 22.0 - 31.0 ) kg / m . wound infection was observed in 24 patients ( 20 males , 4 females ) necessitating daily dressing with injection antibiotics . the overall wound infection rate was 12% , ( 12.2% for males and 11.1% for females ) . wound disruption was observed in 12 patients ( 10 males , 2 females ) necessitating open - wound management . the overall wound disruption rate was 6% , ( 6.1% males , 5.5% females ) . the overall recurrence rate was 7% , ( 6.1% males , 11.1% females ) [ tables 2 and 3 ] . postoperative complications postoperative complications regarding the bmi the time required for pain - free walking after surgery was 20.5 3.6 days and the time required to achieve pain - free sitting on the toilet was 24 3.89 days . the time - off from work , defined as the number of days between the day of surgery and the first day a patient returned to work . all patients were admitted as day - surgery cases ; 174 cases ( 87% ) ultimately conformed to the day - case surgery protocol with mean hospital stay 1.125 0.34 days . after more than half a century , the best surgery for sacrococcygeal pilonidal sinus disease is still a subject of debate , and methods ranging from extensive excisions with complicated reconstructive procedures to limited debridement are being recommended with equal enthusiasm . the main problems with the primary closure technique appear to be high - recurrence rate and high - infection rate . on the other hand , patients generally complain about open - packing or marsupialization methods because of painful wound management and dressing changes . in spite of the recently accepted superiority of the flap reconstructions to the non - flap techniques , morbidity related to infection and recurrence has not been eliminated . despite the controversy about the best surgical technique for the treatment of pilonidal sinus , an ideal operation should minimize financial cost , allow patients to return earlier to work , be simple to perform , not require a prolonged hospital stay , inflict minimal pain , and have a low disease recurrence rate . however , there is no surgical procedure satisfies these requirements as various operative methods utilized in the treatment of pilonidal disease are associated with a number of advantages and disadvantages . postoperative complication rates of unroofing and marsupialization are low , but require long wound care . in addition , there were high complication rates in the primary closure and limberg flap groups . so , the best option is to explain the advantages and disadvantages of the available surgical methods and respect the patient 's decision . methods of simple primary closure can be broadly categorized as midline closure and off- midline techniques . karydakis was the first to advocate off - midline asymmetric closure of pilonidal wounds to decrease recurrence by avoiding placing a wound in the midline at the depth of the natal cleft . his off - midline procedure also flattens the cleft , reducing hair accumulation and mechanical irritation . a clear benefit was shown in favor of off - midline rather than midline wound closure so that , the closure of pilonidal sinuses is the desired surgical option , off - midline closure is now the standard strategy . regarding the postoperative complications in the primary closure techniques , the wound infection was reported as ( 17.2% ) , and wound dehiscence as ( 13.8% ) with no hematoma formation , whereas those in the flap repair , the wound infection was ( 4.5% ) , hematoma was ( 6.8% ) , and wound dehiscence was ( 4.5% ) . according to other series with simple closure technique , it was stated that wound dehiscence was 7.7% and recurrence rate was 5% . another study reported the postoperative complication rate in flap construction as wound infection 20% , wound dehiscence 10% and recurrence 10% . our data came in concordance with those studies regarding the wound infection , wound dehiscence and recurrence with the simplest and less invasive technique . it is now generally agreed that minimally invasive surgery should be used to treat pilonidal disease , whenever possible . ghnnam and his colleagues agreed the same opinion of less invasive surgery that pilonidal sinus could be treated with limited excision and primary closure when they performed excision and primary midline closure . the incidence of the wound infection , wound dehiscence and recurrence is directly proportional with value of bmi . two recent studies showed that among patients with recurrent pilonidal sinus , majority had bmi 30 and 25 to 30 kg / m . our data supported these results , where most of our patients with wound dehiscence or recurrence were of bmi 30 and bmi of 25 to 30 kg / m . the results favoring flap closure in the treatment of sacrococcygeal pilonidal sinus are low recurrence rates , shorter hospital stay , and time off from work but the disadvantages related to unfavorable cosmetic appearance and body image are troubling . the limitations of flap techniques are noteworthy : the unfavorable cosmetic appearance ; the surgical length and hospitalization . the flap surgery takes longer than primary closure , requiring an additional attempt of reconstruction , which is the most complex step of the surgical treatment . important goals in the surgical treatment of sacrococcygeal pilonidal sinus disease include minimized hospital stays , minimized time to return to work and daily activities , and esthetic satisfaction ; no single technique currently optimizes all of these criteria . for example , patients generally complain about open packing and marsupialization methods because of prolonged and painful wound management and dressing changes . primary closure techniques are more acceptable in those regards , but have the disadvantage of generally higher recurrence and infection rates . flap closure has important advantages and disadvantages relative to primary closure : considerations that favor flap closure include lower recurrence rates , shortened hospital stays , and less time - off from work . for example , a recent study compared excision with primary closure to limberg flap in terms of time required for pain - free walking ; pain - free sitting on the toilet after surgery ; and return to work . the limberg flap technique proved superior .. importantly , however , flap surgery takes longer than primary closure because it requires additional reconstruction ; the most complex aspect of the surgery . the simplified off - midline closure technique described in the present study may offer a favorable alternative to the flap closure : like flap closure , the simplified procedure was associated with less time required for pain - free walking , pain - free sitting on the toilet and time off from work than has been reported with other techniques . postoperative esthetic satisfaction has been reported to be higher in patients with simplified closures , an advantage that appears to be due to the simplified technique itself , rather than due to differences in rates of wound infection or recurrence . avoiding flap reconstruction , the cosmetic result was better in patients with simple closure and the flap construction turned out worst while neither wound infection nor recurrence had any influence on the cosmetic result . this means that the technique itself is responsible for the patient satisfaction of his body image after surgery . for simple non - recurrent pilonidal sinus , less invasive surgery with limited excision and primary closure could be enough . simple off - midline technique advocates asymmetric closure of the wound leading to decrease both wound infection and recurrence by avoiding placing a wound in the midline at the depth of the natal cleft . the present study represented data comparable with the more aggressive used excision methods regarding the wound infection , wound dehiscence and recurrence . for simple non - recurrent pilonidal sinus disease , our simplification of off - midline limited excision with primary closure may provide a favorable alternative to conventional off - midline excisions , which involve flap closure . our simplified procedure resembled conventional off - midline techniques in terms of favorable wound infection , wound dehiscence and recurrence rates , whereas advantages of our simplified procedure include potentially reduced surgery complexity , reduced surgery time , and improved cosmetic outcome .

background : numerous surgical procedures have been described for pilonidal sinus disease , but treatment failure and disease recurrence are frequent . conventional off - midline flap closures have relatively favorable surgical outcomes , but relatively unfavorable cosmetic outcomes.aim:the author reported outcomes of a new simplified off - midline technique for closure of the defect after complete excision of the sinus tracts.patients and methods : two hundred patients of both sexes were enrolled for modified d - shaped excisions were used to include all sinuses and their ramifications , with a simplified procedure to close the defect.results:the overall wound infection rate was 12% , ( 12.2% for males and 11.1% for females ) . wound disruption was necessitating laying the whole wound open and management as open technique . the overall wound disruption rate was 6% , ( 6.1% for males and 5.5% for females ) and the overall recurrence rate was 7%.conclusion : our simplified off - midline closure without flap appeared to be comparable to conventional off - midline closure with flap , in terms of wound infection , wound dehiscence , and recurrence . advantages of the simplified procedure include potentially reduced surgery complexity , reduced surgery time , and improved cosmetic outcome .

Introduction Patients and Methods Operative technique End points Results Discussion Conclusion

PMC3034921

a dietary supplement is a product , which contains a dietary ingredient intended to supplement the diet . in general , the natural dietary supplements for male sexual potency consist of different herbal extracts such as ginseng root , lychee seed , barbary wolfberry fruit , longan aril , and aweto . these dietary supplements could improve male sexual potency without causing danger , even when overdose occurred . recently , chemically synthetic phosphodiesterase-5 ( pde-5 ) inhibitors have been used to improve erectile dysfunction ( ed ) , one of the most popularly used is vardenafil hydrochloride ( levitra ) , 2-[2-ethoxy-5-(4-ethyl - piperazine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3h - imidazo[5,1-f]-triazin-4-one monohydrochloride , trihydrate ( figure 1 ) . vardenafil , approved by the united states food and drug administration ( fda ) in 2003 , is a new oral , potent , highly selective phosphodiesterase-5 ( pde5 ) inhibitor marketed for the improvement of ed in man . studies have shown that doses of vardenafil ( 1040 mg ) are rapidly absorbed following oral administration , reaching maximum plasma concentration in some men within 15 minutes . nevertheless , it has been proved that vardenafil poses a serious health risk , such as headache , low blood pressure , flushing , dyspepsia , and nasal congestion or rhinitis . thus , vardenafil could be legally obtained only with a doctor 's prescription . however , some illegal dealers add vardenafil into dietary supplement products for the sake of profit . for purposes of quality control and health safety , establishing sensitive and selective methods to detect illegal vardenafil addition in dietary supplements are necessary . as shown in the literatures , several methods have been reported for vardenafil determination in dietary supplements such as lc - ms and gc - ms method , lc - esi - ms method [ 1 , 4 ] , lc - esi - ms / ms method , mrm - esi - ms - ms method , and fticrms method . ms provides sensitive and reliable results , but it also implies highly sophisticated apparatus , careful control of conditions and relatively high operation cost . thus , simpler , automated and less expensive methods are still necessary . taking the advantages of perfect versatility and easy fluidic manipulation , flow injection ( fi ) the integration of fi and high - performance liquid chromatography ( hplc ) has also showed the power for the complex samples analysis . cl - based technique as a means of detection for hplc aroused the analysts interest in recent years [ 12 , 13 ] , although cl detection is not as universal as fluorescence detection or ultraviolet visible detection , it is more selective for fewer interfering species observed in the chromatogram and sensitive for a large cl signal - to - noise ratio ( s / n ) and wide linear working range . the combination of hplc high resolution , cl sensitive detection and fi manipulation would be one of the plausible techniques for a sensitive , precise , and facile determination of trace levels of compounds in the complex mixture of substances . recent studies witness rapid progress in hplc - cl method , which is widely used to perform diverse qualitative and quantitative analytical problems [ 1518 ] . our experiments indicated that in alkaline medium the cl of luminal - k3fe(cn)6 could be greatly enhanced by the presence of trace vardenafil . based on above phenomenon , an automated hplc - cl method for vardenafil determination in dietary supplements was developed . the proposed method exhibited the advantages of automated operation , high analytical throughput , simple instrumentation , wide linear range , and reagent - saving and was applied to oral liquid , wine , and capsule samples analysis . all the other reagents were of analytical reagent grade unless specified otherwise , and deionized and doubly distilled water was used throughout . vardenafil hydrochloride trihydrate ( purity , 99.6% ) was purchased from qiyu biology co. , ltd . other reagents including k3fe(cn)6 , h3po4 , ethylenediaminetetraacetic acid disodium salt ( na2edta ) , and naoh were supplied by xi'an chemical reagent factory ( xi'an , china ) . mol / l ) was prepared by ethanol and stored at 20c in a refrigerator . standard series from 8.0 10 ~ 1.0 10 mol / l were prepared daily by sequentially diluting stock solution with mobile phase solution . a 1.0 10 mol / l k3fe(cn)6 stock solution was prepared by dissolving 164.6 mg of k3fe(cn)6 in 50 ml water , and it was protected against light and stored at 4c in a refrigerator . a 1.0 10 mol / l stock solution of luminol was prepared in 0.1 mol / l naoh solution . the mobile phase consisting of ethanol and an aqueous solution containing 1.0 10 mol / l h3po4 and 2.0 10 mol / l na2edta ( 25 : 75 , v / v% ) was prepared . mobile phase was filtered through 0.45 m millipore membrane of polytetrafluoroethylene ( ptfe ) and further degassed in an ultrasonic bath before use . health food samples including one herbal oral liquid , one herbal medicated wine , and one herbal capsule claimed being capable of enhancing sexual performance for men that were under suspicion of adulteration , were purchased from local drug store and market . a portion of oral liquid and wine sample were , respectively , 10-fold diluted with mobile phase solution , filtered through a 0.45 m ptfe membrane filter ( shanghai xinya purifier devices factory , shanghai , china ) and finally degassed prior to use . for capsule sample preparation , one capsule weight powder was transferred to a 50 ml beaker , and vortically extracted with 10 ml mobile phase solution for 1 h. a portion of the extraction solution was filtered through a 0.45 m ptfe membrane filter and diluted with mobile phase solution for further analysis . as illustrated in figure 2 , the hplc - cl experimental setup consisted of a cl analyzer ( xi'an remex analyse instrument co. , ltd , xi'an , china ) equipped with a hamamatsu r456 pmt ( tokyo , japan ) and a negative high voltage ( nhv ) generator , a planar coiled transparent ptfe flow cell , a 1.8 ml / min constant flow rate cl pump ( shanghai instrument electric motor factory , shanghai , china ) , a lc-10at liquid chromatography ( shimadzu , tokyo , japan ) equipped with a rheodyne 7725i syringe - loading sample valve ( 20 l - loop , cotati , ca , usa ) , a waters c18 column ( i.d . : 4.6 mm 150 mm , particle size : 5 m , usa ) and a dt-230a column oven ( tianjin do - chrom technology co. , ltd , tianjin , china ) . stream a containing 1.0 10 mol / l luminol , 2.0 10 mol / l na2edta , and 1.0 mol / l naoh and stream b containing 1.0 10 mol / l k3fe(cn)6 delivered by cl pump merged in the t1 . then , the reagent mixture merged with mobile phase in the t2 and generated a stable cl baseline . subsequently , when the sample valve was switched to the injection position , 20 l standard or sample was injected into the mobile phase . outflow vardenafil reacted with the reagent mixture and generated the enhanced cl signal in the flow cell . the cl signal was recorded as a function of time and cl intensity by the remex analyzer with the pmt operated at 400 v. a microcomputer equipped with remex software running under windows xp was employed for data treatment . vardenafil peak identification was carried out by the standard addition method and the retention time ( tr ) of vardenafil . the peak height , i ( i = is i0 , where i0 was the cl of baseline , is was the peak cl of vardenafil ) was used to quantify vardenafil content by means of the related calibration equation . for vardenafil cl determination , we considered several known cl reactions including alkaline luminol - h2o2 , alkaline luminol - k3fe(cn)6 , alkaline luminol - kio4 , acidic kmno4 , acidic kmno4-hcho , acidic kmno4-na2so4 , and naclo - naoh . to prevent the possible interference from metal ions , 2.0 10 mol / l na2edta was used in all luminol concerned reactions . experimental results indicated that alkaline luminol - k3fe(cn)6 reaction contributed the strongest cl response to 1.0 10 mol / l vardenafil presence . the kinetic curve of the alkaline luminol - k3fe(cn)6-vardenafil cl reaction was studied with a static injection method . alkaline luminal and k3fe(cn)6 solution were mixed in the beaker placed in front of the pmt window . after a stable baseline was obtained , 1.0 ml 1.0 10 mol / l vardenafil standard was injected into the beaker by a syringe . the kinetic curve recorded as a function of time and cl intensity , and it could be concluded that vardenafil greatly enhanced luminol - k3fe(cn)6 cl , which reached a maximum within 1.5 s. thus , the studied cl reaction was a fast reaction . as a result , the t2 was placed as near as possible to the flow cell inside the dark box as shown in figure 2 . to obtain the sensitive and fast vardenafil detection with the described hplc - cl system , a series of experiments were conducted to establish optimum analytical performance . 1.0 10 mol / l vardenafil standard was used in all optimization experiments . both mobile phase and luminol and k3fe(cn)6 flow rates were controlled at 1.8 ml / min , respectively . experimental optimum analytical performances were selected based on the biggest cl s / n values and on the average of three injections for each test when the relative standard deviation ( rsd ) of each test point was less than 5.0% , unless specified otherwise . for hplc - cl analysis , the mobile phase should be , not only suitable for vardenafil separation in complex matrix but also compatible with cl detection . acetonitrile - acidic aqueous buffer [ 1 , 4 , 11 , 19 ] and methanol - acidic aqueous buffer [ 8 , 20 ] were the most commonly used mobile phases for vardenafil separations on c18 column . when investigating the compatibility of acetonitrile with cl detection , it was found that acetonitrile brought about very high cl background . similar experiments showed that when methanol and ethanol were used , relatively low cl backgrounds and smooth baselines could be obtained . thus , methanol and ethanol were selected to constitute the mobile phases for the posterior study . the effect of organic phase volume percentage ( v / v% , organic phase volume / organic + aqueous phase volume ) on tr , s / n value and column pressure ( p ) , were investigated in the range of 40%60% methanol and 20%30% ethanol , respectively . observing the experimental results listed in table 1 , it could be concluded that the higher the v / v% , the shorter the tr , and 30% ethanol contributed the biggest s / n value . however , 30% ethanol also caused relatively high p. thereupon , as a compromise among the bigger s / n value , shorter tr and suitable p , 25% ethanol was chosen in subsequent experiments . a number of organic or inorganic acids were employed to constitute mobile phase to improve vardenafil separation [ 1 , 4 , 8 , 11 , 19 , 20 ] . considering the possible reaction between organic acid and cl reagents , inorganic acid was considered to be more suitable for cl detection . experiment indicated that h3po4 could effectively suppress the peak - tailing , and a relatively narrow and symmetric chromatographic peak could be achieved ( figure 3 ) . what is more , as expected , the tr was shortened . with respect to obtaining the biggest cl s / n value , the optimum h3po4 concentration was investigated by varying its concentration in the range of 1.0 10 ~ 1.0 10 mol / l . experiments showed that s / n value increased with h3po4 concentration increasing . when h3po4 concentration was 1.0 10 mol / l , the ph of mobile phase was about 2.0 , the biggest s / n value was obtained . according to the column supplier 's suggestion , h3po4 concentration higher than 1.0 10 mol / 1.0 10 mol / l h3po4 concentration was employed as the optimum . as could be seen the experimental results summarized in table 2 , by varying the flow rate from 0.3 to 0.9 ml / min , the effect of mobile phase flow rate upon cl detection was investigated . it was found that the higher the mobile phase flow rate , the shorter the tr and the higher the p. lastly , a flow rate of 0.8 ml / min was chosen as a compromise between the biggest s / n value and relatively shorter tr . as was well known , the alkaline medium was necessary for luminal - k3fe(cn)6 reaction . in our experiments , the effect of naoh concentration on the cl detection was examined in the range of 0.2~1.3 mol / l . experiments showed that the s / n value increased with the naoh concentration increasing , and a leveling off could be found when the naoh concentration was higher than 1.0 the effect of luminol concentration on cl detection was evaluated in the range of 1.0 10 ~ 1.3 10 mol / l . it was found that the s / n value increased with luminol concentration increasing and reached maximum at 1.0 10 mol / l , whereas the s / n values decreased with further luminol concentration increasing . consequently , 1.0 10 mol / l luminol concentration was selected . as an oxidant of cl reaction , k3fe(cn)6 affected the cl signal directly . the effect of k3fe(cn)6 concentration on cl detection was assessed in the range of 1.0 10 ~ 3.0 10 mol / l . it was found that the s / n value increased with k3fe(cn)6 concentration increasing in the range of 1.0 10 ~ 1.0 10 mol / l . based on above results , 1.0 10 mol / l k3fe(cn)6 was selected . a series of standard solutions ( 1.0 10 ~ 3.0 10 mol / l ) was used to determine the analytical figures of merit under the optimum conditions . experiments showed that i to vardenafil concentration ( c ) was linear in the range of 8.0 10 ~ 1.0 10 mol / l with the regression equation of i = 3.17c + 9.91 ( c : 10 mol / l , n = 5 , r = 0.9980 ) and the detection limit was 5 10 mol / l ( 3 ) . the reproducibility was investigated by injecting 2.0 10 mol / l vardenafil standard for 11 times , which was demonstrated by the rsd . intraday and inter - day precisions of the described method were examined using the oral liquid sample spiked with 2.0 10 mol / l standard . the intraday precision , defined as the rsd of 5 times determinations to the same sample within one day , five injections one time , was less than 3.5% . the inter - day precision , defined as the rsd of five days determinations to the same sample , 5 injections one day , was less than 2.3% . it was concluded that the described hplc - cl method presented relatively good precision . following the procedures detailed in the experimental section , the proposed hplc - cl method was applied to determine vardenafil in oral liquid , wine , and capsule samples . the original and vardenafil standard spiked chromatograms of oral liquid , it could be seen from figure 4 that vardenafil was not detected in all three samples and there was no interfering from the endogenous substances at the tr of vardenafil . it was noticed that the tr of vardenafil was 6.4 min , which was much shorter than the tr reported in relative literatures [ 1 , 4 , 8 , 9 , 11 , 19 ] . this shorter tr also implied the time and mobile phase saving . to evaluate the applicability of the proposed hplc - cl method for the determination of vardenafil in oral liquid , wine , and capsule , recovery experiments were performed . these experiments were conducted by spiking 3 levels of vardenafil content into oral liquid and wine and adding identical levels of vardenafil content into capsule as shown in table 3 . the recovery percentages were estimated by comparing the concentrations detected from spiked samples with the nominal concentrations added . from experimental results summarized in table 3 , it was concluded that the proposed method afforded good precision and accuracy when applied to oral liquid , wine , and capsule samples . we had carried out the automated hplc - cl method on vardenafil determination in oral liquid , wine , and capsule samples . based on the obtained experimental results , the following conclusions could be drawn . a new mobile phase that is composed of 25% ethanol and 75% aqueous was found to be nontoxic , environment friendly , and compatible with the alkaline luminol - k3fe(cn)6 cl detection . furthermore , the cl detector was cheap , simple and no sophisticated technique required . in a word , our proposed method might be adopted as an alternative method for vardenafil determination of in dietary supplement .

a flow method of high - performance liquid chromatography ( hplc ) seperation and chemiluminescence ( cl ) detection for sensitive vardenafil analysis in dietary supplements was developed . the vardenafil separation was achieved on a c18 column at 30c using ethanol - h3po4 and ethylenediaminetetraacetic acid disodium salt ( na2edta ) aqueous solution ( 25 : 75 , v / v% ) as mobile phase . the followed continuous cl detection was conducted based on the strong cl enhancement by the presence of vardenafil to luminol - k3fe(cn)6 reaction in alkaline medium . at the flow rate of 0.8 ml / min , the vardenafil retention time ( tr ) was 6.4 min . factors that affected the hplc resolution and cl detection were studied and optimized . the calibration curve obtained for vardenafil standard was linear in concentration range of 8.0 107 ~ 1.0 104 mol / l . the relative standard deviations ( rsd ) of intraday and interday precision were less than 3.5% . the proposed method was applied to the vardenafil determination in oral liquid , wine , and capsule samples .

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

PMC3547245

mass spectrometry ( ms ) based glycomics techniques are broadly used to analyze free oliogsaccharides , glycosaminoglycans as well as the glycan portions of glycoproteins , proteoglycans and glycolipids . both matrix - assisted laser desorption - ionization ( maldi ) and electrospray ionization ( esi ) are commonly applied . ms may be used as a stand - alone technique , or coupled online to separation methods such as hplc [ 14 ] and capillary electrophoresis ( ce ) [ 57 ] . carbohydrate and glycoconjugate analysis by maldi - ms has been comprehensively reviewed by harvey [ 8 , 9 ] . other useful review articles , which cover a range of analytical techniques including tandem ms ( ms / ms ) of glycoconjugates have appeared in recent years [ 815 ] . this review aims at giving a concise overview of ms based glycomics technology , together with selected applications in clinical research . protein - linked n - glycans and o - glycans are typically released by enzymatic and chemical methods , respectively . also glycosaminoglycans are generally degraded by chemical or enzymatic means for subsequent analysis [ 5 , 17 ] . analysis of released ( or free ) glycans may be achieved by a variety of techniques such as mass spectrometry , hplc of reductively aminated glycans employing fluorescence or uv detection and capillary gel electrophoresis with laser - induced fluorescence detection ( cge - lif ) of labeled glycans [ 16 , 1821 ] . ms is particularly advantageous for analyzing very complex glycan mixtures containing unusual oligosaccharide structures for which the standardized migration positions in hplc or cge - lif have not yet been determined . importantly , the mass of the analyzed glycan when determined with sufficient accuracy or accompanied by a tandem ms experiment will directly provide information on the glycan composition in terms of hexoses , n - acetylhexosamines , deoxyhexoses , etc . by contrast , this direct link between the observed glycan species and its molecular composition is not inherently present for hplc and cge - lif experiments and additional efforts are required such as the use of glycan standards or exoglycosidase treatments for the determination of terminal monosaccharides [ 22 , 23 ] . on the other hand , separation - based methods for glycan analysis will often resolve structural isomers such as the 6-arm and 3-arm isomers of monogalactosylated biantennary glycans , while their distinction is not easily achieved by ms and requires additional efforts such as tandem ms analysis . therefore , while very complex pools of oligosaccharides can be analyzed by ms(/ms ) without separation , many researchers choose to perform glycan analysis by lc - ms [ 13 ] or - less frequently - ce - ms coupling [ 57 ] . ( normalized ) retention and migration times , precursor masses and fragmentation spectra may then be used for structural elucidation as in the case of o - glycan alditol analysis by porous graphitized carbon ( pgc ) hplc coupled online to ms [ 2729 ] . pgc - hplc appears to have a particularly high power in separating oligosaccharide structural isomers , which makes this method very useful for in - depth structural analysis of complex oligosaccharide mixtures . another popular separation technique hyphenated with ms for oligosaccharide analysis is hilic , which likewise features isomer separation [ 24 , 30 , 31 ] . high - performance anion - exchange chromatography ( hpaec ) coupled with online - desalting and online - esi - ms is another approach which is particularly useful for the analysis of underivatized oligosaccharides . for example , oligosaccharides may be reduced to alditols resulting in a 2 da mass tag on the innermost monosaccharide which facilitates fragment assignment in tandem ms . analysis of o - glycan alditols obtained by reductive beta - elimination may be achieved by porous graphitized carbon ( pgc ) hplc coupled via online , negative - mode electrospray ionization to ion trap - tandem mass spectrometry ( ms / ms ) [ 27 , 28 ] . an online database has been made available by the unicarb - db partners allowing structural assignment of o - glycan alditols on the basis of ms and ms / ms spectra in addition to retention times ( http://www.unicarb-db.com/ ) . similarly , n - glycans may be structurally assigned on the basis of mass and retention time in pgc - esi - ms . this approach has been introduced by altmann and coworkers . within the range of mass spectrometric techniques , negative - mode ms of glycans has recently obtained increased attention , both for maldi and esi ionization [ 33 , 34 ] . negative - mode ionization is particularly effective for acidic glycan structures . in this respect , labeling of glycans at the reducing end with an acidic tag such as 2-aminobenzoic acid ( anthranilic acid ; aa ) is advantageous , as it confers acidic properties to all glycans including neutral species , thereby allowing the efficient detection of both sialylated and non - sialylated aa - labeled oligosaccharides in negative - mode maldi - time of flight ( tof)-ms . in addition , negative - mode ms / ms of oligosaccharides has attractive features , for example that the glycosidic linkages of fucose are rather stable , in contrast to their labile behavior in positive - ion mode . harvey has described several diagnostic ions , which are observed in negative - ion mode ms / ms of n - glycans and allow the elucidation of antenna compositions as well as the differentiation between the 6-branch and 3-branch of the glycan [ 36 , 37 ] . moreover , the carboxylic acid groups of sialylated glycans are protected by methyl esterification , which stabilizes the sialic acids and enables maldi - tof - ms profiling of permethylated neutral and acidic glycans simultaneously . by contrast , sialic acids are labile when analyzing native glycans , leading to the observation of degradation products in maldi - tof - ms spectra [ 16 , 33 ] . analysis of the sodium adducts of permethylated glycans by tandem ms is a very useful approach for detailed structural characterization as - next to cleavages of glycosidic bonds - diagnostic cross - ring cleavages are observed , which reveal linkage positions . these analyses may be performed by high - energy collision - induced dissociation ( cid ) maldi - tof / tof - ms resulting in very complex yet informative fragmentation spectra . maldi - ion trap - ms of permethylated n - glycans released from total plasma glycoproteins has recently been established by guillard et al . . this approach allows in - depth analysis of glycans by multistage - tandem mass spectrometry as exemplified in fig . 1 : ms2 ( fig . 1b ) provided evidence for the occurrence of a sialyl - lewis x structure on a plasma n-glycan.fig . 1permethylated serum n - glycans measured by maldi - linear ion trap - ms ( a ) . fragments at m / z 1022 , 1143 , and 2690 are indicative of antenna fucosylation , whereas fragments at m / z 1113 , 1317 , and 2516 mark core fucosylation ( b ) . the inset in ( b ) shows an ms3 experiment of m / z 1022 confirming the proposed structurewith terminal sialic acid and antenna fucosylation . filled square , glcnac , empty circle , galactose ; filled circle , mannose ; filled triangle , fucose ; filled diamond , n - acetylneuraminic acid . taken from with permission permethylated serum n - glycans measured by maldi - linear ion trap - ms ( a ) . fragments at m / z 1022 , 1143 , and 2690 are indicative of antenna fucosylation , whereas fragments at m / z 1113 , 1317 , and 2516 mark core fucosylation ( b ) . the inset in ( b ) shows an ms3 experiment of m / z 1022 confirming the proposed structurewith terminal sialic acid and antenna fucosylation . filled square , glcnac , empty circle , galactose ; filled circle , mannose ; filled triangle , fucose ; filled diamond , n - acetylneuraminic acid . taken from with permission analysis of permethylated glycans in combination with esi - ion trap - ms when this approach is combined with multistage fragmentation of permethylated glycans , the combination of various characteristic fragmentation spectra of sub - structures of the precursor oligosaccharides allows the unambiguous structural assignment of large oligosaccharide structures as impressively demonstrated by reinhold and coworkers [ 25 , 26 ] . for example reductive amination or permethylation using deuterated or c13-labeled versions of the tag / chemicals have been shown to be advantageous for oligosaccharide quantification and the detailed comparison of glycan profiles . it has to be noted , however , that most of these isotope labeling strategies have not yet been applied to clinical glycomics research questions . in addition to the analysis of released glycans studying protein glycosylation at the level of glycopeptides is rapidly gaining importance [ 4044 ] . the peptide portion may be seen as a tag , which potentially allows the assignment of the glycan to a specific n- or o - glycosylation site on a specific protein . however , this approach is complicated by several obstacles . first , proteolytic cleavage is often hindered in highly glycosylated proteins , resulting in very large , highly and heterogeneously glycosylated peptide moieties , which are hardly accessible for ms analysis . second , a variety of glycans are generally found attached to one specific glycosylation site ( microheterogeneity of glycosylation ) , and different n - glycosylation sites on one protein often have different glycan patterns . therefore , glycopeptides generally occur substoichiometrically , making them difficult to analyze by ms in the presence of a majority of non - glycosylated peptides . various enrichment techniques including lectin affinity chromatography are available to purify glycopeptides for ms analysis [ 3 , 46 ] . a very promising technique for enriching n - glycopeptides is hydrophilic interaction liquid chromatography - solid phase extraction ( hilic - spe ) , which may be performed using silica - based or carbohydrate - based stationary phases [ 30 , 31 , 47 , 48 ] . third , depending on the size of the glycan moiety and the chosen ms / ms approach , it is often hard to obtain peptide sequence information , which is in most cases needed for unambiguous assignment of the glycan to a specific protein . popular approaches are electron capture dissociation ( ecd ) and electron transfer dissociation ( etd ) of glycopeptides as well as various types of ( multistage ) cid [ 4 , 43 , 44 ] . in ecd and etd the glycan portion is generally stable , and peptide backbone cleavages tend to provide ( some ) peptide sequence information . single stage low - energy cid ( as occurring on an ion trap ) is generally characterized by fragmentation of glycosidic bonds , and peptide backbone cleavages are usually minor , if detectable at all . fragmentation of the peptide portion may be achieved by performing ion trap - multistage ms / ms , and has been successfully applied in various cases for the identification of glycosylated proteins and glycosylation sites [ 41 , 43 ] . alternatively , fragmentation of glycopeptides at elevated energies in maldi - tof / tof - ms and maldi- or esi - quadrupole - tof - ms has been reported to provide peptide sequence information next to information on glycan composition and structure . it has been observed that under these conditions glycan moieties may undergo rearrangements in ms / ms , of which prominent examples are the migration of fucoses between n - glycan antennae , or from the core to outer portions of the n - glycan structure [ 49 , 50 ] . these rearrangements may not only be observed for n - glycopeptides , but also for o - glycopeptides . obviously , awareness of these processes is required for avoiding misinterpretation of glycopeptide fragmentation spectra . the major bottle - neck in glycopeptidomics - based proteomics of complex samples is data analysis . software supporting data analysis is desperately needed , and several promising approaches have recently been reported ( and references cited therein ) . yet additional , concerted efforts in developing data analysis tools are needed to boost the impact of this analytical approach . analyzing protein glycosylation at the glycopeptide level the analysis of the intact mass of glycoproteins , together with a bottom - up analysis , often allows the detailed structural assignment of protein species such as monoclonal antibodies [ 51 , 52 ] . in addition , top - down glycoproteomics , i.e. , the ms analysis of intact glycoproteins followed by their tandem ms analysis for the characterization of posttranslational modifications including glycosylation , has high potential but needs to be further developed [ 53 , 54 ] . igg glycopeptide profiling by maldi - tof - ms has been performed to determine the changes in igg1 and igg2 fc glycosylation features with pregnancy and rheumatoid arthritis as well as with longevity and healthy aging . maldi - fticr - ms was likewise evaluated for igg fc glycopeptides profiling and was found to be particularly useful for analyzing changes in sialylation . maldi - fticr - ms analysis of igg fc glycopeptides is characterized by reduced losses of sialic acid , which is most probably due to the higher pressure in the source and the resulting collisional cooling , in combination with the lower extraction voltages as compared to maldi - tof - ms . recently , using a sheath - flow esi sprayer , a robust nanolc - ms method for igg fc glycosylation profiling was established ( fig . 2 ) . notably , the sheath - flow esi sprayer setup was found to significantly increase the long - term stability of the system while keeping the sensitivity of the system in the same range as with conventional nano - esi - ms . high - sensitivity igg fc glycosylation analysis is particularly valuable when analyzing affinity - purified , antigen - specific iggs , which may be present at low concentrations . for the most common applications , however , such as glycosylation analysis of total plasma igg and biotechnologically produced igg the available sample amounts are generally plenty and sensitivity is not an issue . the sheath flow setup was used in combination with trifluoracetic acid containing running solvents resulting in the coelution of sialylated and non - sialylated igg fc glycopeptides . in contrast , conventional nano - lc - ms with formic acid - containing running solvents features early - eluting glycopeptides with neutral glycans and late - eluting ones with sialylated glycans . it was found that galactosylation , sialylation were increased whilst fucosylation and the incidence of bisecting glcnac were decreased during pregnancy . the observed glycosylation changes may contribute to the immune suppression occurring during pregnancy in order to protect the fetus from alloimmune reactions of the mother .fig . long - term stability of ms detection in nanolc - ms is achieved using a sheath - flow esi spray with a sheath flow of 2 l / min 50 % isopropanol , 20 % propionic acid ( a ) . the extracted ion chromatograms of igg1 , igg4 and igg2/3 tryptic fc glycopeptide species ( b ) , and the mass spectrum of the igg1 fc glycopeptides elution range is shown in ( c ) . glycopeptide signals observed below m / z 1200 are triple protonated , and signals above m / z 1200 are double protonated . the excellent repeatability of the overall sample preparation and analysis method is demonstrated in ( d ) for igg1 . blue square , n - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose ; purple diamond , n - acetylneuraminic acid igg fc - glycosylation profiling by lc - quadrupole - tof - ms . long - term stability of ms detection in nanolc - ms is achieved using a sheath - flow esi spray with a sheath flow of 2 l / min 50 % isopropanol , 20 % propionic acid ( a ) . the extracted ion chromatograms of igg1 , igg4 and igg2/3 tryptic fc glycopeptide species ( b ) , and the mass spectrum of the igg1 fc glycopeptides elution range is shown in ( c ) . glycopeptide signals observed below m / z 1200 are triple protonated , and signals above m / z 1200 are double protonated . the excellent repeatability of the overall sample preparation and analysis method is demonstrated in ( d ) for igg1 . blue square , n - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose ; purple diamond , n - acetylneuraminic acid next to glycoproteins , glycolipids play an important role in cellular interaction and cellular differentiation . the majority of glycolipids observed in humans have a ceramide portion ( sphingoid base carrying an amide - linked fatty acid ) and are , therefore , categorized as glycosphingolipids . they occur mainly in the outer leaflet of the plasma membrane and also in the inner membranes . glycosphingolipids show a marked tissue- and cell type - specific expression pattern [ 59 , 60 ] . this is for example reflected by the fact that various human cluster of differentiation ( cd ) markers , which are differentially expressed between leukocytes , are glycolipids such as cd60a ( gd3 ; neu5ac(2 - 8)neu5ac(2 - 3)gal(1 - 4)glc(1 - 1)ceramide ) , cd60b ( 9-o - acetyl gd3 ) , cd60c ( 7-o - acetyl gd3 ) , cd77 ( gb3 ; globotriaosylceramide ; gal(1 - 4)gal(1 - 4)glc(1 - 1)ceramide ) ) ( http://www.hcdm.org/ ) . recently , chip - based approaches for glycosphingolipid analysis have been reviewed [ 59 , 62 ] . importantly , there is also technology available to combine the most commonly applied separation technique in lipid , as well as glycolipid analysis , high - performance thin layer chromatography ( hptlc ) , with ms . for example , glycosphingolipids separated by hptlc can be probed by overlay detection using carbohydrate - binding proteins such as lectins , bacterial toxins , and antibodies , followed by the ms analysis of positive bands , either directly from the hptlc plate or after lipid extraction , as reviewed recently by meisen et al . . alternatively , glycolipids may be analyzed by hilic - nano - lc - ms using slightly adjusted solvent conditions when compared to methods used for glycan and glycopeptide separation . using this approach , it has been demonstrated that 2 - 3-sialylated and 2 - 6-sialylated isomers of lactoneotetraosylceramides can be baseline separated from complex mixtures and characterized individually by tandem ms . the importance of the above described techniques is illustrated by their application in clinical studies . glycosylation changes play important roles in the cellular mechanisms of health and disease , and glycans have a great potential as biomarkers for different types of cancer [ 66 , 67 ] . there is a vast range of studies of human glycobiology in healthy and diseased people employing ms , and some selected examples will be presented demonstrating the potential of mass spectrometric approaches for clinical glycomics . established an approach that relies on n - glycan release from total plasma , permethylation , and maldi - ion trap - ms measurement , allowing in - depth analysis of glycans by tandem ms ( fig . 1 ) . this approach was applied to determine plasma n - glycan profiles of congenital disorder of glycosylation ( cdg ) type ii patients , as well as controls . a total of 38 peaks were assigned in terms of molecular composition , and changes in the n - glycan profiles were found to be useful to distinguish between the patient groups . the authors also successfully addressed the challenge of differentiating cdg type ii diseases from other diseases with secondary causes of underglycosylation . this method is now being successfully applied in clinical research , including research on patients with defects in 1 - 4-galactosyltransferase i ( b4gat1 ) , which leads to the expression of largely truncated glycans on plasma proteins . lysosomal defects of glycoconjugate degradation may lead to the secretion of glycopeptides , glycolipids or oligosaccharides in patient urine . molecular analysis of these degradation products by ms often directly pinpoints to the genetic defect . in schindler s disease , which is a hereditary n - acetylhexosaminidase deficiency , characteristic o - glycosylated amino acids and o - linked glycopeptides were detected from patients urines . in fabry s disease , the causative enzymatic defect leads to elevated levels of globotriaosylceramide and lyso - globotriaosylceramide species in urine and plasma , which can be detected by lc - ms with good diagnostic sensitivity and specificity [ 71 , 72 ] . a very powerful approach for the analysis of urinary oligosaccharides is hpaec , which was applied in capillary - scale with online - desalting and esi - ion trap - ms / ms analysis to study urinary oligosaccharides of patients with gm1-gangliosidosis and galactosialidosis [ 32 , 73 ] . on the basis of literature knowledge of n - glycan biosynthesis , this approach allowed the structural assignment of chromatographically separated isomeric n - glycan degradation products in gm1-gangliosidosis ( fig . the observation of n - glycans with terminal galactose residues points to a deficiency of -galactosidase activity . when the same analytical setup was applied to study urinary glycans in galactosialidosis , novel degradation products were observed such as glycolipid - derived oligosaccharides , both in reducing form and with c1-oxidation of the innermost glucose . these results indicate the presence of an alternative glycolipid degradation pathway in galactosialidosis patients involving a hitherto not described endoglycoceramidase activity.fig . 3high performance - anion exchange chromatography with online ms detection of urinary oligosaccharides of a gm1-gangliosidosis patient ( a ) . next to the total ion chromatogram ( tic ) specific extracted ion chromatograms are given labeled with the composition of the oligosaccharide in terms of hexoses ( h ) and n - acetylhexosamines ( n ) . the ion trap tandem mass spectra obtained for the two detected h3n2 isomers are shown in ( b ) and ( c ) . green circle , mannose ; yellow circle , galactose ; blue square , n - acetylglucosamine . taken from with permission high performance - anion exchange chromatography with online ms detection of urinary oligosaccharides of a gm1-gangliosidosis patient ( a ) . next to the total ion chromatogram ( tic ) specific extracted ion chromatograms are given labeled with the composition of the oligosaccharide in terms of hexoses ( h ) and n - acetylhexosamines ( n ) . the ion trap tandem mass spectra obtained for the two detected h3n2 isomers are shown in ( b ) and ( c ) . green circle , mannose ; yellow circle , galactose ; blue square , n - acetylglucosamine . taken from with permission the analysis of protein degradation products from bio fluids has repeatedly led to the identification of glycopeptides , thereby shedding new light on protein glycosylation . apolipoprotein ciii - derived o - glycopeptides were found in the urine of schistosoma mansoni infected individuals . remarkably , these glycopeptides did not exhibit the sialylated t - antigen glycan structures found on apolipoprotein ciii from human serum , but instead carried larger o - glycan structures with a high degree of sialylation . in another study , an o - glycosylated peptide stemming from the c - terminus of the fibrinogen -chain was found to be increased in the urine during urinary tract infection with escherichia coli . recently , o - glycosylated amyloid -peptides representing a potential disease biomarker were characterized from cerebrospinal fluid of alzheimer patients using both cid and ecd fragmentation . cancer glycomics biomarker discovery has recently been reviewed [ 66 , 67 ] , and ms is becoming an important research tool in this field . novotny and mechref with coworkers chose to analyze serum n - glycan profiles after permethylation using maldi - tof - ms . using this approach , they demonstrated vastly different n - glycan profiles in metastatic prostate cancer as compared to healthy tissue . a variety of mainly fucosylated , complex - type n - glycans were found to be increased in cancer vs. control . in another study the relative abundances of a set of 8 complex - type serum n - glycans were found to be indicative of the progression of breast cancer . for example , maldi - ms of 2-aminobenzoic acid - labeled n - glycans showed that the n - glycan fucosylation of -1-acid glycoprotein is significantly increased in ovarian cancer . notably , most of the reported cancer glycomics studies focus on the analysis of the total plasma or serum n - glycome or certain acute - phase proteins [ 66 , 67 ] . while these approaches are promising , an increase in sensitivity and specificity may be expected when tumor - derived antigens isolated from body fluids are characterized together with their specific glycosylation profiles . still another glycomics application area for ms is the study of the genetic and environmental regulation and dysregulation of protein glycosylation in health and diseases . for example , various novel aspects of the regulation of immunoglobulin g fc glycosylation have only recently been revealed by high - sensitivity glycosylation profiling at the glycopeptide level . employing this analysis technique , in vitro studies have shown that soluble factors such as cytokines and toll - like receptor ligands modulate the degree of igg fc galactosylation , sialylation and the incidence of bisecting glcnac . likewise , fucosylation of igg fc glycans appears to be regulated in humans : igg fc glycan fucosylation in humans is known to be generally above 90 % , yet recently pathogenic alloantibodies with a low degree of fucosylation ( 50 % and below ) have been described for patients with fetal and neonatal alloimmune thrombocytopenia ( fnait ) . figure 4 shows the total serum igg1 fc glycosylation profile of a patient and the corresponding profile of the pathogenic anti - human plate antigen ( hpa ) 3a alloantibodies . while the total serum igg1 shows 9 % afucosylated structures ( a ) , the afucosylation is 38 % for the alloantibodies of this patient ( b ) . low fucosylation has been associated with enhanced cellular cytocoxicity , whilst high degrees of sialylation confer anti - inflammatory properties to iggs .fig . fc glycosylation of total serum igg1 ( a ) and anti - hpa3a alloantibodies from a patient with pregnancy complications ( fetal and neonatal alloimmune thrombocytopenia ; fnait ) ( b ) . glycopeptides were detected in triple protonated form by nanolc - esi - ion trap - ms carrying neutral n - glycan chains ( left panels ) and acidic n - glycan chains ( right panels ) . in blue square , n - acetylglucosamine ; yellow circle , galactose , green circle , mannose ; red triangle , fucose ; purple diamond , n - acetylneuraminic acid ; pep , tryptic peptide moiety ; asterisk , non - glycopeptide signal . taken from with permission low core - fucosylation of anti - hpa-3a alloantibodies . fc glycosylation of total serum igg1 ( a ) and anti - hpa3a alloantibodies from a patient with pregnancy complications ( fetal and neonatal alloimmune thrombocytopenia ; fnait ) ( b ) . glycopeptides were detected in triple protonated form by nanolc - esi - ion trap - ms carrying neutral n - glycan chains ( left panels ) and acidic n - glycan chains ( right panels ) . in blue square , n - acetylglucosamine ; yellow circle , galactose , green circle , mannose ; red triangle , fucose ; purple diamond , n - acetylneuraminic acid ; pep , tryptic peptide moiety ; asterisk , non - glycopeptide signal . taken from with permission in the coming years the field of mass spectrometric analysis of protein glycosylation is expected to show an increase in measurement sensitivity and precision as well as sample throughput , allowing the in - depth analysis of biological systems , with data analysis being a major challenge . despite the limitations mentioned in this review , glycoproteomics approaches focusing on the glycopeptide level will gain in popularity . mass spectrometric analyses of ( tryptic ) glycopeptides are rewarding as they have intrinsically the potential of assigning specific glycan structures to a specific site on a specific protein . this information is often of utmost importance , as the primary role of glycans is modulating the properties ( such as function , activity , stability , targeting ) of their carrier proteins . notably , approaches based on the analysis of released n - glycans often fail to provide this information on protein- and site - specificity and are , therefore , of limited value . another important aspect is the analysis of intact glycoproteins by ms , which is expected to gain importance in the next years . on the one hand , ms analysis of intact glycoproteins allows the integration of the information obtained at the glycopeptide and released glycan level to obtain an overall view of protein glycosylation . on the other hand intact protein analysis may be accompanied by top - down tandem mass spectrometric analysis for characterization of posttranslational modifications , including glycosylation . it is anticipated that the concept of a specific protein having specific functions will undergo refinement , and specific proteins will be perceived as an assembly of isoforms ( including glycoforms ) that are caused by a variety of posttranslational modifications including glycosylation . defining such protein species is of utmost importance for functional proteomics supporting systems biology and will require bioinformatics tools and databases to facilitate posttranslational modification analysis at the glycopeptide level .

mass spectrometry plays an increasingly important role in structural glycomics . this review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans , the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids . the given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation , lysosomal storage diseases , autoimmune diseases and cancer .

Introduction Analysis of free glycans Analysis of glycopeptides Analysis of glycolipids Clinical glycomics applications Perspectives

PMC1568514

computerized techniques for the evaluation of o'farrell two - dimensional electrophoretic gels have been applied to proteins derived from asbestos bearing macrophages . preliminary results indicate definite changes in the protein content of cells depending on fiber phagocytosis.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.figure 13.figure 14.figure 15.figure 22 . afigure 22 . bfigure 22 . cfigure 22 . d

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PMC3161204

chronic pelvic pain ( cpp ) in women is commonly described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or to lead to medical care.1 - 2 cpp may originate from one or more organ systems or pathologies and may have multiple contributing factors.3 the prevalence may vary from country to country . in primary care , the prevalence of cpp is found to be comparable to that of asthma and back pain , with values of 3.7% , 3.8% , and 4.1% , respectively.4 in a us study , the prevalence among women of reproductive age reached 24%,4 - 8 whereas in new zealand , grace , and zondervan identified a prevalence of 25.4% when considering a three - month duration of symptoms.9 this elevated prevalence was confirmed by latthe et al . in a recent review of this condition.10 the direct and indirect costs of this condition amount to over two billion dollars per year , and the condition is responsible for 10% of all gynecologic visits , 40 to 50% of all gynecologic laparoscopies and 12% of all hysterectomies.5 cpp has a direct impact on marital status as well as on the social and professional lives of women , and it thus constitutes an important public health problem . in brazil , international studies have demonstrated a high prevalence of persistent pain in women from brazil , including approximately 36% in rio de janeiro.11 according to the health ministry of brazil , in 1997 there were 1.8 million gynecologic visits and approximately 300,000 hospital admissions of women aged 15 to 69 with complaints associated with cpp.12 in a survey of diseases and/or health problems among working women in so paulo , gomes & tanaka13 reported that 13% of them mentioned abdominal and pelvic pain among their major complaints . recently , some studies have concluded that drug or alcohol abuse , abortions , inflammatory pelvic disease , cesarean sections , and psychological diseases are associated with cpp.14 the etiology of cpp in women is not clear , and cpp usually involves a complex interaction between the gastrointestinal , urinary , gynecologic , musculoskeletal , and neurologic and endocrine systems . it is also influenced by psychological and sociocultural factors.6 several other factors may be associated with the condition , including ( 1 ) neuroplastic changes occurring in the posterior horn of the spinal cord as a consequence of electrophysiological , biochemical , and metabolic changes promoted by the initial noxious stimulus , ( 2 ) cross - sensitivity between viscera that share the same innervation , and ( 3 ) development of a visceromuscular reflex that may culminate not only in dysfunctional repercussions but also in the development of myofascial syndrome and the generation of new pain points.15 - 16 although there is a consensus that cpp has a high prevalence among women , there are a limited number of studies in our country , which can be attributed to the lack of a precise diagnosis , the heterogeneity of the group of diseases that result in cpp , and the variation of primary diseases in different populations.17 considering that knowledge about the magnitude and the demographic and socioeconomic characteristics of cpp contributes to its prevention , control , and treatment , the aim of the present study was to estimate the community prevalence of cpp in women living in ribeiro preto ( a southeastern city in the state of so paulo , brazil with a population of about 700,000 people ) , as well as to identify independent factors associated with it . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate . the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . the data collected by the interviewers were entered during the interview and sent to an electronic database . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . five percent of the subjects were selected at random and re - interviewed to check data consistency . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale . women who had been pregnant in the previous 12 months were excluded from the analyses . the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis , we considered only moderate and intense dysmenorrhea to be positive . dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' . the dagostino and pearson test was used to determine whether data followed a gaussian distribution . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate . the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . the data collected by the interviewers were entered during the interview and sent to an electronic database . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . five percent of the subjects were selected at random and re - interviewed to check data consistency . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale . women who had been pregnant in the previous 12 months were excluded from the analyses . the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' . the dagostino and pearson test was used to determine whether data followed a gaussian distribution . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the prevalence of cpp was 11.5% ( 147/1,278 ) . considering only women of reproductive age , the prevalence was 15.1% ( 127/841 ) . 82.4% of healthy women ( 932/1,131 ) and 90.5% of women with cpp ( 133/147 ) stated that they make regular clinic visits at basic health centers ( at least two appointments per year ) . only 4.1% of women with the disease , however , knew their diagnosis before the interview ( n = 6/147 ) . the average duration of the pain was 51.861.2 [ 6 - 360 ] months : 6 - 12 months in 34.0% of the women ( 50/147 ) , 13 - 36 months in 28.6% ( 42/147 ) , and over 36 months in 37.4% ( 55/147 ) . the mean intensity of pain obtained with a visual analogue scale ( vas ) was 58.523.4 mm . the intensity was 30 - 50 mm in 52.4% of the women ( 77/147 ) , 51 - 70 mm in 17.7% ( 26/147 ) , and 71 - 100 mm in 29.9% ( 44/147 ) . in total , 44.9% of the women reported a spontaneous onset of pain ( 66/147 ) ; in 3.4% ( 5/147 ) , the pain was related to food intake ; in 8.5% ( 9/106 excluding those who were not sexually active ) , the pain was related to intercourse ; in 21.8% ( 32/147 ) , the pain was related to physical activity ; in 30.6% ( 30/98 excluding those who were menopausal ) , the pain was related to menstruation ; in 2.0% ( 2/98 ) , the pain was related to ovulation ; in 0.7% ( 1/147 ) , the pain was related to stress ; and in 1.4% , it was related to other factors ( 2/147 ) . the frequency of self - medication was 1.4% ( 16/1131 ) and 24.5% ( 111/147 ) among healthy and cpp women , respectively . the common occupations were maidservant , hairdresser , and seamstress , with no possibility of grouping the women according to their occupations . a univariate analysis of the variables investigated is presented in table 2 . in the logistic regression , the factors independently associated with cpp were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . in the present study , we detected a one - year prevalence of 11.5% for cpp among women from ribeiro preto and a prevalence of 15.1% in women of reproductive age . to our knowledge , this is the first time that a high prevalence of this disease has been reported in brazil . the presence of chronic pain was found to be 48.4% in women from salvador , brazil.20 although that study described pain at various sites , it made no reference to abdominal or pelvic pain . a recent review published by latthe et al.10 has shown that the worldwide prevalence of this condition ranges from 2% to 24% , which places brazil among the countries with a higher prevalence of cpp . zondervan et al . , for example , observed a community prevalence of 24.0% in women between 18 and 49 years of age.7 in that study , the questionnaire response rate was 74% . a higher response rate is natural among women with the disease , which may explain the difference in prevalence compared with our study , which had a response rate of 97.9% ( 1,278/1,306 ) . we believe that our data are representative of the entire ribeiro preto community because the social - demographic indicators of the covered area are similar to those of the general population of the city ( socioeconomic level , age distribution , sex , and ethnicity ) . a notable result was that only 4% of the women with cpp stated that they had previously received a specific diagnosis , even though 90% of them attended a basic health center . three different factors may have contributed to this finding : whether the physician is able to provide the correct diagnosis ; whether the relationship between the patient and the physician enables effective communication and explanation of the diagnosis ( which other studies have commented on21 ) ; and whether the women complain about their clinical conditions to physicians . studies have shown that gender is an important determining factor of pain complaints in women and might be the reason for diagnosis underestimation at primary care centers.22 the frequency of self - medication was also a notable finding . approximately one quarter of the women used some kind of medication ( especially analgesics and anti - inflammatory drugs ) for the relief of their symptoms in an indiscriminate way . this self - medication predisposes them to side effects and requires financial expenditures that do not guarantee clinical improvement , especially over the long term.23 we believe that this percentage may be underestimated because people responding to an interviewer may not want to report the use of their pain medication . additionally , more than one third of the women had symptoms consistent with a diagnosis of cpp for over three years , and the same proportion had symptoms that are considered severe ( vas>70 mm ) . although we have no data to support this speculation , it is plausible that the high prevalence of cpp is associated with high rates of absenteeism ( about half of these women have paid employment outside the home ) , which has a direct impact on social and economic life . we identified the following as factors independently associated with chronic pelvic pain : dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . dyspareunia is an important element of sexual dysfunction that ranges in prevalence from 7% to 75% depending on the diagnostic criteria and the associated clinical conditions , such as cpp.24 - 29 in a recent study , we observed that dyspareunia was associated with pelvic muscle tenderness.30 although pelvic muscle tenderness may be a primary cause of cpp,31 we hypothesize that it is more often secondary to cross - talk communication between the viscera and muscles32 through neurogenic inflammation caused by the release of inflammatory mediators at the periphery in response to the antidromic stimulus over time.33 similarly , current sexual activity may be linked in some way to dyspareunia , although this relationship is still unclear . another hypothesis is that women with dysmenorrhea have a lower pain threshold , which may favor the onset of illness.34 moreover , mechanisms of viscero - visceral hyperalgesia between organs probably involve the sensitization of viscero - viscero - somatic convergent neurons.35 the most notable factor observed in the present study is the association of cpp with abdominal surgery , particularly because we observed that two - thirds of the women studied had undergone a previous abdominal surgery , and more than 40% of the women had previously undergone a cesarean section surgery . considering the high rates of cesarean section surgery in our country36 - 37 ( including this sample of our community ) , and the previous detection of an association with cpp by our group38 and by latthe et al . in a recent systematic review,14 we stress the need for detailed studies regarding this possible causal relationship between cpp and cesarean section . some studies suggest the possibility of an association between cpp and adhesions.39 however , the correlation between pelvic pain and adhesions is uncertain because adhesiolysis has not been shown to be effective in achieving pain control.40 thus , we emphasize the importance of recognizing abdominal myofascial syndrome as a differential diagnosis.41 anxiety and depression disorders are frequently concomitant with chronic pain , particularly in women , in both developed and developing countries.42 - 43 we have also observed a direct relationship between depression , anxiety , chronic pelvic pain , and quality of life.44 in addition to being a risk factor for cpp , mood disorders may make it more difficult for a women to engage in cognitively or emotionally demanding rehabilitation.45 because the individual experience of pain is personal and subjective , it is probably affected by emotional states and , therefore , by psychosocial factors . there is some evidence that it is the stress of living with chronic pain , and not personal or family predisposition , that causes depression in these patients.46 the idea that pain , particularly chronic pain , can lead to feelings of frustration , worry , anxiety , and depression seems obvious . there is also evidence , however , for reverse causality , in which negative moods and emotions can lead to or exacerbate pain.47 it therefore remains uncertain whether depression / anxiety precedes or is a consequence of chronic pain.48 low back pain is usually comorbid with cpp . this association probably reflects the same pathophysiological mechanisms,49 although each can reduce the thresholds and thus contribute to the development of the other . painful bladder syndrome and constipation are nongynecologic conditions that may cause or exacerbate cpp ( level of evidence a).2 the prevalence of constipation identified in our sample of healthy women ( 22.7% ) was similar to that observed by oliveira et al.50 because the women reported that constipation was present before cpp , we may infer that this condition may at least contribute to the development of cpp . though this was beyond the scope of our study , some studies indicate that constipation may be secondary to the dysfunction of the levator ani muscle,51 - 52 which is a common condition in cpp . several studies in the literature have shown that a lower educational level is linked to a higher prevalence of chronic pain.20,53 because cpp increases with age and most education takes place in early life , it is likely that a lower educational level increase the risk for the development of cpp , or that both are the consequences of some other undescribed factors . we have not , however , reached final conclusions about the causal relationship between these factors and cpp . a thorough investigation and other different types of studies are necessary to corroborate our results . we conclude that the prevalence of cpp in women from ribeiro preto is very high , even though only 4% sufferers are aware of their diagnosis . this confirmation is crucial for the prevention , early diagnosis , control , and resolution of cpp . the authors are grateful to cnpq ( conselho nacional de desenvolvimento cientifico e tecnologico ) for financial support .

introduction : chronic pelvic pain is a disease that directly affects the social and professional lives of women.objective:to estimate the prevalence of this clinical condition and to identify independent factors associated with it in women living in ribeiro preto , brazil.methods:a one - year cross - sectional study was conducted in a population sample of 1,278 women over the age of 14 years . the target population was predominantly composed of women who are treated by the public health system . the questionnaire was administered by interviewers who were not linked to the city health care programs . the prevalence of the morbidity was estimated . first , we identified the significant variables associated with pelvic pain ( p<0.10 ) and then we attributed values of 0 or 1 to the absence or presence of these variables . logistic regression analysis was used to identify and estimate the simultaneous impact of the independent variables . the results were expressed by odds ratio and their 95% confidence interval with p<0.05.results : the disease was found in 11.5% ( 147/1,278 ) of the sample . the independent predictors were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level.conclusion:the prevalence of chronic pelvic pain in ribeiro preto is high and is associated with conditions that can usually be prevented , controlled , or resolved by improvement of public health policies and public education .

INTRODUCTION MATERIALS AND METHODS Study design and participants Questionnaire Definitions and grouping Statistical analysis RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENT

PMC3482083

an autogenous arteriovenous fistula ( avf ) is the optimal form of vascular access for hemodialysis ( hd ) due to the longer duration of patency , fewer infections , and lower mortality [ 1 , 2 ] . the presence of occlusive neointimal hyperplasia at the anastomosis and/or the outflow veins , which may be accelerated by chronic kidney disease , has been considered to be the leading cause of avf failure , while the major problem associated with the creation of an avf is the fact that fistulas do not necessarily mature into a usable vascular access , requiring subsequent revision and construction of another access . the caliber of the vessels used for fistula creation is a pivotal factor predicting the presumable maturation of the constructed fistula , and veins less than 2.5 mm in diameter have poor outcomes . this report describes the balloon - assisted creation and maturation of an autogenous radial - cephalic arteriovenous fistula ( rcf ) in a patient with small - caliber veins and a radial artery of approximately 2 and 1.5 mm in diameter , respectively . a 78-year - old female with end - stage renal failure caused by diabetic nephropathy had been undergoing hd treatment for 9 years . her medical history included arteriosclerosis obliterans , an old myocardial infarction , and complete atrioventricular block , which was managed by the implantation of a definitive pacemaker via the right subclavian vein . she first began undergoing hd through a left upper arm brachial - cephalic avf , since a forearm rcf created before initiating hd did not mature despite repeated surgical revisions . the fistula failed 2 years later because of a thrombotic occlusion presumably induced by frequent postdialytic hypotension resulting from excessive interdialytic weight gain . the patient underwent numerous vascular access interventions , including percutaneous transluminal angioplasty and stenting for thrombotic or stenotic lesions , not only in accessory veins , but also in the central venous system . the peripheral venous access in the left upper extremity seemed to be exhausted 5 years after the initial intervention , and the right brachial artery was superficially repositioned . the peripheral venous system in the right upper extremity also did not seem to be feasible to use as the return route , so the patient was dialyzed with a silicone tunneled cuffed catheter inserted through the right femoral vein . however , thrombotic catheter occlusion developed 5 months after the insertion , and the catheter could only be used for the return route . finally , a balloon - assisted procedure was used to try to create and expedite the maturation of the autogenous right forearm rcf . small - caliber veins of approximately 2 mm and a radial artery of 1.5 mm in diameter were confirmed by preoperative digital subtraction angiography ( dsa ) . the cephalic vein and radial artery were exposed through a transverse incision at the wrist using magnification glasses ( 3.0 ) . the vein was then freed by wide dissection , and the collateral branch was ligated , followed by a longitudinal venotomy of the posterior wall . the anterior wall of the radial artery was exposed without any dissection of its trunk , and a 5-mm longitudinal arteriotomy was made . a side - to - end , artery - to - vein anastomosis palpation just after the creation of the anastomosis failed to confirm the patency of the fistula , and subsequent dsa revealed the stenosis of the juxta - anastomotic vein . next , the stenotic lesion of the juxta - anastomotic radiocephalic vein was crossed with a 0.018-inch hydrophilic guide wire , and a 3-mm ( 40 mm long ) balloon catheter ( sterling balloon dilatation catheter , boston scientific , natick , mass . , usa ) was inflated to 6 atm for at least 1 min , with one overlapping angioplasty under direct visualization ( fig . the patient underwent subsequent balloon angioplasty on days 20 and 60 to aid in the maturation of the radial artery and the usable portion of the rcf . balloon angioplasty was performed under radiological guidance on day 20 , with a retrograde radial arterial approach using a 0.018-inch platform combined with a 4-mm balloon catheter for the radial artery ( inflated to 4 atm for 10 s ) , anastomotic region ( inflated to 6 atm for 20 s ) , and the juxta - anastomotic region ( inflated to 8 atm for 20 s ; fig . angioplasty was performed on day 60 , using a 5-mm balloon catheter from the anastomotic to the juxta - anastomotic lesion . follow - up fistulography was performed 3 months later , when the rcf became functional and the tunneled cuffed catheter was removed , revealing the matured radial artery and cephalic vein ( fig . the right rcf has been functional for more than 6 months since the final fistulography . a wrist avf including an rcf has been the first choice of vascular access for chronic hd , since it is simple to create and preserves more proximal vessels for future access placement with fewer complications than other fistula types . however , a high level of surgical experience appears to affect the primary success and patency rate of an rcf , thus suggesting that the construction of such access is technically demanding . on the other hand , the availability of a distal cephalic vein and radial artery will become even more limited for arteriovenous fistula construction with the increasing average age of the end - stage renal failure population . the survival rate of a successfully created forearm autogenous rcf is excellent , and the 2-year complication - free survival of such a fistula is approximately 75% , while another study by miller et al . demonstrated that the overall success rate of forearm rcf is rather poor . therefore , it is not unusual that the left forearm rcf created in the current patient before initiating hd did not mature despite repeated surgical revisions . several studies have evaluated factors that might predict fistula maturation , and preoperative vascular mapping has been shown to improve the rate of fistula placement and overall surgical success rate . in addition , the criteria for the placement of an autogenous fistula have included a minimum artery diameter of 2 mm , a minimum vein diameter of 2.5 mm , and a lack of stenosis or thrombosis in the draining vein or central veins [ 3 , 8 ] . the experience in the current patient with a suboptimal artery measuring approximately 1.5 mm in size and veins measuring 2.0 mm in diameter suggests the clinical benefit of balloon - assisted creation and maturation of an autogenous right forearm rcf . although one may argue that perioperative radiological intervention could be dangerous due to the risk of disruption of the anastomosis , intraoperative interventions are not exceptional and are occasionally indicated in clinical settings . the most common reason is limited percutaneous vascular access , such as with lesions proximal or distal to a stenosed or occluded portion that is simultaneously treated by vasculotomy . the directly visualized intraoperative procedure may avoid the development of several adverse events , such as vascular perforation or rupture variability in the techniques used for creating avfs has made it difficult to objectively analyze the results and develop best practice recommendations , despite a large amount of literature on the subject and continued technological advances . in addition , there are no clear protocols to address accessible vessel maturation with regard to an avf . the application of intraoperative balloon angioplasty and the subsequent interventional approach has allowed the maturation of an autogenous avf with suboptimal vessels that otherwise could not have been used [ 13 , 14 ] . the mechanisms of successful balloon angioplasty with vascular access stenoses involves vessel stretching and dissection , which are similar to the mechanisms that have been validated with morphological evaluation of arterial balloon angioplasty [ 15 , 16 ] . on the other hand , the vascular access portions dilated by repeated balloon angioplasty are thought to heal mostly by fibrosis , and eventually to become large - diameter fibrous segments [ 15 , 16 ] . further studies and more extensive clinical experience are required to better determine the optimal interval and appropriate duration of balloon angioplasty not only for post - dilation healing , but also for expediting the maturation of an autogenous avf with small - caliber vasculature .

the major problem associated with the creation of an arteriovenous fistula ( avf ) , which is the optimal form of vascular access for hemodialysis , is the fact that fistulas do not necessarily mature into a usable vascular access , requiring subsequent revision and construction of another access . the caliber of the vessels used for fistula creation is a pivotal factor predicting the presumable maturation of the constructed fistula , and veins less than 2.5 mm in diameter have poor outcomes . this report describes the balloon - assisted creation and maturation of an autogenous radial - cephalic avf in a patient with a small - caliber vein and a radial artery measuring approximately 2 and 1.5 mm in diameter , respectively . the clinical impact of percutaneous radiological intervention for expediting the maturation of an autogenous avf with small - caliber vessels is also discussed .

Introduction Case Report Discussion Disclosure Statement

PMC5018950

intraductal papillary neoplasms of the bile duct ( ipnb ) have been associated with papillary tumors of malignant potential in the extrahepatic and intrahepatic bile ducts.123 ipnb is classified as a distinct clinical and pathological entity in the 2010 world health organization classification.4 its malignant potential reportedly ranges from 19.5% to 83%.5 these tumors show papillary proliferation in the bile duct with or without mucin secretion and are considered as ipnb , the biliary counterpart of intraductal papillary mucinous neoplasm ( ipmn ) of the pancreas.467 some mucin - producing ipnbs can cause obstructive jaundice due to production of abundant high - viscosity mucin.4 herein , we presented two cases of mucin - producing ipnbs with obstructive jaundice who underwent resection of the intrahepatic primary lesions and bypass hepaticojejunostomy . a 69 year - old male was admitted to the emergency room due to obstructive jaundice . five years ago , he underwent lung resection for adenosquamous carcinoma of the lung , without recurrence to date . at the time of lung surgery , he was diagnosed with gallbladder stones and hepatolithiasis of the segment iii , vi and viii ducts ( b3 , b5 and b8 ) with corresponding ductal dilatation , suggestive of associated inflammatory stricture . yearly imaging study follow - up for gallstone lesions until the recent last visit to emergency room showed no interval changes of gallstone disease or evidence of malignant changes . imaging study findings of dynamic liver computed tomography ( ct ) and magnetic resonance ( mr ) imaging were as follows : mucin - secreting ipnb mainly in b3 with slight interval aggravation of biliary ductal dilatation ; and multiple intrahepatic duct stones , interval increased extent and gallbladder ( fig . cancer antigen 19 - 9 ( ca 19 - 9 ) was mildly elevated ( 38 u / ml ) . endoscopic retrograde cholangiography ( erc ) showed multiple filling defects in the biliary system , which was compatible with mucin - producing ipnb ( fig . two endoscopic nasobiliary tubes were inserted and retained for 10 days in order to mediate biliary decompression . 2a and 2b ) . after removing the very sticky mucin , the b3 cystic lesion identified as the source of the mucin was excised and a 2 cm - sized b3 opening was exposed ( fig . 2c and 2d ) . frozen - section biopsy revealed that the b3 cystic wall was involved with cancer , thus the whole b3 duct wall except its connecting part to the left intrahepatic duct was resected . intrahepatic duct stones at b5 and b8 were removed with curved stone forceps . due to some risk of residual tumor that could re - induce mucin production , we performed two separate biliary bypass drainages to the common bile duct and b3 orifice ( fig . the operation included segment iii hepatic resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy . pathologic findings were as follows : intrahepatic cholangiocarcinoma with intraductal growth type ; confinement to bile duct wall ; no lymphovascular invasion ; no perineural invasion : no involvement of b3 margin and hepatic parenchymal resection margins ; biliary intraepithelial neoplasia 2 at the common bile duct wall ; and chronic cholangitis with bile duct dilatation , extensive erosion , multifocal rupture and abscess and xanthogranulomatous inflammation . 3 ) . since this resection was regarded as r0 resection , no additional adjuvant treatment was scheduled . a 74 year - old female was referred for surgery due to suspected malignant mucin - producing ipnb and obstructive jaundice . she had undergone repeated episodes of erc and percutaneous transhepatic cholangiography ( ptc ) for recurrent intrahepatic and common bile duct stones for the last 11 years . findings of dynamic liver ct and mr imaging were as follows : mucin - producing ipnb with asymmetric disproportional dilatation in the segment iv duct ( b4 ) and mural nodule in b4 ; increased fluid collection due to bile duct rupture at the right subphrenic space adjacent to the segment iv ( fig . 4a and 4b ) . chest ct and positron emission tomography showed multiple mild hypermetabolic lymph nodes in right internal mammary chain , suggesting metastatic lymph nodes . ptc was performed for biliary decompression and tissue confirmation ; intra - operatively , the intraductal mass was diagnosed as ipnb of intermediate grade dysplasia ( fig . 4c and 4d ) . 5a ) . after removing the very sticky mucin , the b4 cystic lesion identified as the source of mucin was excised and two 3 cm - sized right and left hepatic duct openings were exposed ( fig . we decided to perform non - curative resection since residual scattered bile duct tumors at the hepatic confluence portion did not permit simple resection and left hepatectomy was contra - indicated due to patient safety . two large openings to the right and left hepatic ducts were unified with running sutures and then reconstructed with single large hepaticojejunostomy ( fig . 5c and 5d ) . the operation included segment iv hepatic resection , partial resection of the diaphragm and central hepaticojejunostomy . pathologic findings were as follows : well differentiated mucinous adenocarcinoma of nodular type arising from ipnb ; extension beyond bile duct ; serosal penetration and involvement of the diaphragm ; no lymphovascular invasion ; no perineural invasion ; involvement of the radial resection margin ; and no involvement of common and hepatic bile duct resection margins . this resection was regarded as r1/r2 resection with possibility of distant lymph node metastasis , but no additional adjuvant treatment was planned due to poor general condition of the patient . she has been doing well for 4 months post - surgery without any evidence of tumor recurrence or distant metastasis . a 69 year - old male was admitted to the emergency room due to obstructive jaundice . five years ago , he underwent lung resection for adenosquamous carcinoma of the lung , without recurrence to date . at the time of lung surgery , he was diagnosed with gallbladder stones and hepatolithiasis of the segment iii , vi and viii ducts ( b3 , b5 and b8 ) with corresponding ductal dilatation , suggestive of associated inflammatory stricture . yearly imaging study follow - up for gallstone lesions until the recent last visit to emergency room showed no interval changes of gallstone disease or evidence of malignant changes . imaging study findings of dynamic liver computed tomography ( ct ) and magnetic resonance ( mr ) imaging were as follows : mucin - secreting ipnb mainly in b3 with slight interval aggravation of biliary ductal dilatation ; and multiple intrahepatic duct stones , interval increased extent and gallbladder ( fig . cancer antigen 19 - 9 ( ca 19 - 9 ) was mildly elevated ( 38 u / ml ) . endoscopic retrograde cholangiography ( erc ) showed multiple filling defects in the biliary system , which was compatible with mucin - producing ipnb ( fig . two endoscopic nasobiliary tubes were inserted and retained for 10 days in order to mediate biliary decompression . 2a and 2b ) . after removing the very sticky mucin , the b3 cystic lesion identified as the source of the mucin was excised and a 2 cm - sized b3 opening was exposed ( fig . 2c and 2d ) . frozen - section biopsy revealed that the b3 cystic wall was involved with cancer , thus the whole b3 duct wall except its connecting part to the left intrahepatic duct was resected . intrahepatic duct stones at b5 and b8 were removed with curved stone forceps . due to some risk of residual tumor that could re - induce mucin production , we performed two separate biliary bypass drainages to the common bile duct and b3 orifice ( fig . the operation included segment iii hepatic resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy . pathologic findings were as follows : intrahepatic cholangiocarcinoma with intraductal growth type ; confinement to bile duct wall ; no lymphovascular invasion ; no perineural invasion : no involvement of b3 margin and hepatic parenchymal resection margins ; biliary intraepithelial neoplasia 2 at the common bile duct wall ; and chronic cholangitis with bile duct dilatation , extensive erosion , multifocal rupture and abscess and xanthogranulomatous inflammation . 3 ) . since this resection was regarded as r0 resection , no additional adjuvant treatment was scheduled . a 74 year - old female was referred for surgery due to suspected malignant mucin - producing ipnb and obstructive jaundice . she had undergone repeated episodes of erc and percutaneous transhepatic cholangiography ( ptc ) for recurrent intrahepatic and common bile duct stones for the last 11 years . findings of dynamic liver ct and mr imaging were as follows : mucin - producing ipnb with asymmetric disproportional dilatation in the segment iv duct ( b4 ) and mural nodule in b4 ; increased fluid collection due to bile duct rupture at the right subphrenic space adjacent to the segment iv ( fig . 4a and 4b ) . chest ct and positron emission tomography showed multiple mild hypermetabolic lymph nodes in right internal mammary chain , suggesting metastatic lymph nodes . ptc was performed for biliary decompression and tissue confirmation ; intra - operatively , the intraductal mass was diagnosed as ipnb of intermediate grade dysplasia ( fig . 4c and 4d ) . after removing the very sticky mucin , the b4 cystic lesion identified as the source of mucin was excised and two 3 cm - sized right and left hepatic duct openings were exposed ( fig . we decided to perform non - curative resection since residual scattered bile duct tumors at the hepatic confluence portion did not permit simple resection and left hepatectomy was contra - indicated due to patient safety . due to high risk of residual tumors that could re - induce mucin production , these lesions were destroyed with deep electrocautery and argon beam coagulator . two large openings to the right and left hepatic ducts were unified with running sutures and then reconstructed with single large hepaticojejunostomy ( fig . 5c and 5d ) . the operation included segment iv hepatic resection , partial resection of the diaphragm and central hepaticojejunostomy . pathologic findings were as follows : well differentiated mucinous adenocarcinoma of nodular type arising from ipnb ; extension beyond bile duct ; serosal penetration and involvement of the diaphragm ; no lymphovascular invasion ; no perineural invasion ; involvement of the radial resection margin ; and no involvement of common and hepatic bile duct resection margins . this resection was regarded as r1/r2 resection with possibility of distant lymph node metastasis , but no additional adjuvant treatment was planned due to poor general condition of the patient . she has been doing well for 4 months post - surgery without any evidence of tumor recurrence or distant metastasis . ipnb is considered a precursor lesion of cholangiocarcinoma.12 ipnb was proposed as a new disease entity because of striking similarities to ipmn of the pancreas , wherein the disease entity and clinicopathological features are well established.8 however , preoperative diagnosis of intrahepatic ipbn is usually difficult in practice . the common clinical manifestations of patients with intrahepatic ipnb are recurrent abdominal pain , repeated episodes of acute cholangitis and obstructive jaundice , as presented in our previous study.1 intrahepatic duct dilatation and intraductal mass are common abnormal findings in patients with intrahepatic ipnb . cholangiography , including indirect cholangiography ( mr cholangiography ) and direct cholangiography ( erc and ptc ) , is useful to show the entire bile duct to define the extent of the ipnb.910 mucin can not be detected on usual imaging studies . direct cholangiography is useful for the detection of mucobilia.8 however , erc and ptc are invasive procedures , which not only risk introducing bacteria into the bile duct , but more importantly may result in dissemination of tumor cells . mucinous obstruction of bile duct prevents visualization of the whole biliary tree as well as tumor detection by cholangiography . ptc and erc are usually performed in patients with obstructive jaundice , but it is often not possible to relieve the jaundice by draining the inspissated mucobilia.10 cholangioscopy including percutaneous transhepatic cholangioscopy ( ptcs ) and peroral cholangioscopy can approach the bile duct directly , confirm the histology , and assess the extent of the tumor including superficial spreading along the biliary epithelium , which facilitates appropriate treatment choice.58 we prefer ptcs to peroral cholangioscopy for reliable evaluation of the intrahepatic duct in patients with mucin - producing lesions ; in addition , percutaneous transhepatic biliary drainage enables biliary decompression in the environment of sticky mucin formation . ptcs examination is therefore an indispensable preoperative procedure for determining treatment modality and the appropriate extent of resection in intrahepatic ipnb.111 intrahepatic ipnb should not be regarded as a benign disease with low malignant potential but as a premalignant lesion with high malignant potential . in our previous study , low - grade intrahepatic ipnb was rather rare and the majority of intrahepatic ipnb cases were high - grade ipnb , and invasive ipnb with minimal and considerable invasion.1 ipnb with different malignant potentials can be ultimately diagnosed as adenoma , borderline tumor , non - invasive carcinoma or invasive carcinoma,12 a continuum of intraductal neoplastic progression . these neoplasms arise within the ductal system and show a predominantly intraductai growth pattern macroscopically and papillary proliferation with delicate fibrovascular cores.1314 their pathological similarities are summarized as follows : macroscopic growth pattern of intraductal papillary proliferation ; occasional association with mucin hypersecretion ; microscopic feature of papillary proliferation with fibrovascular cores ; occurrence of three types of tumor cells ; occasional association with multiple lesions ; possible progression to tubular adenocarcinoma and mucinous carcinoma ; and influence of histological types of invasive lesion on survival rate . their pathological differences are summarized as follows : high frequency of ck20 expression in ipnb than in pancreatic ipmn ; lower percentage of gastric type tumors in ipnb than in pancreatic ipmn ; and lower frequency of mucin hypersecretion in ipnb than in pancreatic ipmn.14 mucin is macroscopically identifiable in most cases of pancreatic ipmn but in only less than one - third of ipnb cases.61516 we previously reported that mucin pool formation was observed in only 7 of 43 cases ( 16.3%).1 mucin - producing ipnb is capable of secreting abundant quantities of mucus . the liquid is composed of mucin and albumin and is rich in electrolytes , but contains neither bile salts nor pigments . its external drainage is often contraindicated because it is followed by discharge of extraordinary quantities of mucobilia and leads to marked loss of proteins and electrolytes.17 because patients with intrahepatic ipnb often show favorable prognosis , curative surgical resection is regarded as the first - choice treatment for patients with intrahepatic ipnb without distant metastasis . we previously showed that surgical curability was the only reliable prognostic factor for tumor recurrence and patient survival.1 palliative treatments are recommended if curative resection is not feasible.17 especially in patients with mucin - producing tumors , hepaticojejunostomy can palliate the liquid loss and is also efficient , at least temporarily , in previously reported cases.18 in this study , the 2 patients were diagnosed with intrahepatic stone disease 5 and 11 years prior to surgery , respectively . it is likely that the pre - existing dysplastic lesions transformed to malignancy at a certain time , resulting in acceleration of mucin production . they were referred for surgical treatment only after obstructive jaundice was developed due to massive mucin production . in retrospect , closer follow - up may have led to earlier surgical treatment and better outcome , but poor general condition of these patients did not lead to surgery before manifestation of overt symptoms . the long - term clinical courses of our patients highlight that abundant production of highly viscous mucin might be associated with malignant transformation . in conclusion , we presented two rare cases of mucin - producing ipnb of malignant transformation that were treated with primary resection and biliary drainage . the case studies indicated that abundant production of highly viscous mucin inducing obstructive jaundice may be associated with malignant transformation in patients with ipnb .

intraductal papillary neoplasms of the bile duct ( ipnb ) leads to malignant transformation and mucin production . herein , we presented two cases of mucin - producing ipnb with obstructive jaundice who underwent resection of the intrahepatic lesions and bypass hepaticojejunostomy . the first case was a 69 year - old male patient with 5-year follow up for gallstone disease . imaging studies showed mucin - secreting ipnb mainly in the hepatic segment iii bile duct ( b3 ) and multiple intrahepatic duct stones for which , segment iii resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy were conducted . the second case was a 74 year - old female patient with 11-year follow up for gallstone disease . imaging studies showed mucin - producing ipnb with dilatation of the segment iv duct ( b4 ) and mural nodules for which , segment iv resection , partial resection of the diaphragm and central hepaticojejunostomy were conducted . both patients recovered uneventfully from surgery . these cases highlight that in patients with ipnb , abundant production of highly viscous mucin inducing obstructive jaundice may be associated with malignant transformation .

INTRODUCTION CASE Case 1 Case 2 DISCUSSION