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background : limited data exist on the association of serum gamma - glutamyl transferase ( ggt ) level within the reference range with the increased risk of coronary heart disease ( chd ) prediction in men . the study examined the association between serum ggt concentration within the reference range and the chd risk prediction in korean men.methods:the study employed data from korean national health and nutrition examination survey ( v-1 , 2010 and v-2 , 2011 ) where a total of 1301 individuals were analyzed . a 10-year chd risk prediction was computed using the framingham risk score ( frs ) modified by the national cholesterol education program ( ncep ) adult treatment panel iii ( atp iii).results : positive correlations were established between log - transformed ggt concentration and frs ( r = 0.237 , p < 0.001 ) . after adjustment of body mass index , the amount of alcohol intake and low - density lipoprotein - cholesterol , the odds ratio ( 95% confidence interval ) for intermediate risk and beyond of 10-year chd prediction ( 10-year risk 10% ) with lowest quartile of participants was 1.21 ( 0.781.87 ) for second quartiles , 1.39 ( 0.882.21 ) for third quartiles and 2.03 ( 1.233.34 ) for highest quartiles.conclusions:higher serum ggt within its reference range was significantly correlated with a 10-year chd risk prediction estimation using ncep atp iii in korean men .

there are various pathophysiologic reasons for coronary heart disease ( chd ) including atheroma , also known as atherosclerosis , which is critical for chd occurrence . of these , oxidative stress is one of the most significant aspects in the pathogenesis of chd , which is thought to play an important role in the progression of atherosclerosis . several studies suggest that gamma - glutamyl transferase ( ggt ) is at the pathophysiological background in the precipitation and progression of atherosclerosis . the ggt is regarded as a biomarker of hepatobiliary disease and alcohol consumption or abuse . however , it has been recently demonstrated that the activity of ggt in serum is a sensitive marker of oxidative stress associated with concomitant risk factors , such as obesity , fatty liver , hypertension , dyslipidemia , diabetes , and metabolic syndrome . serum ggt levels are also related positively to novel cardiovascular risk factors like c - reactive protein ( crp ) , and fibrinogen . many studies were executed in order to prove that the higher level of serum ggt within normal range is related to higher incidence of hypertension , diabetes mellitus , fatty liver , and metabolic syndrome , especially in men . however , limited data exist on the significance of serum ggt level within the reference range with the increased risk of chd prediction in men . this study aimed at determining whether serum ggt within its reference range in korean men is associated with the risk of chd prediction . the framingham risk score ( frs ) modified by the national cholesterol education program ( ncep ) adult treatment panel iii ( atp iii ) guidelines from the korea national health and nutrition examination surveys ( knhanes ) from 2010 to 2011 was employed for computation . the knhanes has been conducted periodically by the korea centers for disease control and prevention since 1998 and provides comprehensive information on health status , health behavior , nutritional status , and sociodemographics in 600 national districts . the cross - sectional analysis used samples containing serum ggt taken from knhanes data ( knhanes v-1 , 2010 and v-2 , 2011 ) . about 5932 out of 8983 people were excluded due to missing data on frs , smoking , or alcohol history . moreover , approximately 1750 subjects with diabetes mellitus , chd , hepatobiliary disease , positive tests for antibody to hepatitis b surface antigen , antibody to hepatitis b virus core antigen or anti - hepatitis c virus , and patients taking drugs influencing liver function and lipid - lowering drugs were excluded . subjects that had been smoking cigarettes regularly 1-year before the time of survey were considered as current smokers . the weekly alcohol intake was calculated and converted based on grams of ethanol consumed . a reference sample constituted by healthy subjects was obtained according to the following inclusion criteria : no smoking , no heavy alcohol intake ( < 70 g of ethanol per week ) , and absence of cardiovascular disease , hypertension , diabetes , obesity , renal diseases , metabolic syndrome , and dyslipidemia . the final reference serum ggt was drawn from the population , and reference range between 6 and 65 iu / l , which was a statistical interval representing 95% or 2 standard deviations . before collection of blood samples , each subject fasted for more than 10 h. after overnight fasting , a venous blood sample was obtained between 08:00 and 10:00 a.m. to measure ggt and fasting blood glucose ( fbg ) , liver enzymes , total cholesterol , triglycerides , high - density lipoprotein ( hdl ) cholesterol , and low - density lipoprotein ( ldl ) cholesterol . serum ggt was assayed by the standard method recommended by the international federation for clinical chemistry using l - f - glutamyl-3-carboxy-4-nitroanilide as substrate with a toshiba 200fr autoanalyzer . in addition , the fbg , liver enzymes , and lipid levels were assayed using a toshiba-200fr automatic analyzer ( toshiba medical systems , tokyo , japan ) . blood pressure ( bp ) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement , where the average measurement was recorded . hypertension was defined as a systolic bp ( sbp ) 140 mmhg or a diastolic bp 90 mmhg or by the use of antihypertensive medication . body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) . the distribution of ggt values and weekly alcohol consumption were right - skewed ; therefore , a natural log - transformation was applied . after assessment , the general characteristics were presented , and the study subjects were grouped into quartiles according to levels of serum ggt . analysis of variance ( anova ) trend analysis using polynomial contrasts was adapted to perform tests for trends . in order to evaluate the relationship between serum ggt activity within reference range and the individual components of the frs , spearman 's correlation analysis was employed . the frs was calculated from the ncep atp iii algorithm based on six coronary risk factors : age gender , total cholesterol , hdl - cholesterol , sbp , and smoking habit . among these factors , framingham risk equations were used to predict the risk of developing coronary disease events ( myocardial infarction or chd death ) over the next 10-year for adults aged 20 and older without heart disease or diabetes . participants were divided into three groups : low risk ( < 10% risk of developing a chd event over the next 10-year ) , intermediate risk ( 1020% risk ) , and high risk ( > 20% risk ) . however , the number of high - risk group was so small that it included the high - risk group into intermediate - risk group and beyond . smoking status was classified as either current smoker or nonsmoker . for analysis relating to serum ggt within reference interval to the intermediate - risk group and beyond , we constructed adjusted logistic regression analyses that considered bmi , weekly alcohol intake , and ldl cholesterol . the results of group data were expressed as mean standard error ( se ) . data were analyzed using pasw spss version 18.0 ( spss inc . , chicago , il , usa ) . the knhanes has been conducted periodically by the korea centers for disease control and prevention since 1998 and provides comprehensive information on health status , health behavior , nutritional status , and sociodemographics in 600 national districts . the cross - sectional analysis used samples containing serum ggt taken from knhanes data ( knhanes v-1 , 2010 and v-2 , 2011 ) . about 5932 out of 8983 people were excluded due to missing data on frs , smoking , or alcohol history . moreover , approximately 1750 subjects with diabetes mellitus , chd , hepatobiliary disease , positive tests for antibody to hepatitis b surface antigen , antibody to hepatitis b virus core antigen or anti - hepatitis c virus , and patients taking drugs influencing liver function and lipid - lowering drugs were excluded . subjects that had been smoking cigarettes regularly 1-year before the time of survey were considered as current smokers . the weekly alcohol intake was calculated and converted based on grams of ethanol consumed . a reference sample constituted by healthy subjects was obtained according to the following inclusion criteria : no smoking , no heavy alcohol intake ( < 70 g of ethanol per week ) , and absence of cardiovascular disease , hypertension , diabetes , obesity , renal diseases , metabolic syndrome , and dyslipidemia . the final reference serum ggt was drawn from the population , and reference range between 6 and 65 iu / l , which was a statistical interval representing 95% or 2 standard deviations . before collection of blood samples , each subject fasted for more than 10 h. after overnight fasting , a venous blood sample was obtained between 08:00 and 10:00 a.m. to measure ggt and fasting blood glucose ( fbg ) , liver enzymes , total cholesterol , triglycerides , high - density lipoprotein ( hdl ) cholesterol , and low - density lipoprotein ( ldl ) cholesterol . serum ggt was assayed by the standard method recommended by the international federation for clinical chemistry using l - f - glutamyl-3-carboxy-4-nitroanilide as substrate with a toshiba 200fr autoanalyzer . in addition , the fbg , liver enzymes , and lipid levels were assayed using a toshiba-200fr automatic analyzer ( toshiba medical systems , tokyo , japan ) . blood pressure ( bp ) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement , where the average measurement was recorded . hypertension was defined as a systolic bp ( sbp ) 140 mmhg or a diastolic bp 90 mmhg or by the use of antihypertensive medication . body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) . the distribution of ggt values and weekly alcohol consumption were right - skewed ; therefore , a natural log - transformation was applied . after assessment , the general characteristics were presented , and the study subjects were grouped into quartiles according to levels of serum ggt . analysis of variance ( anova ) trend analysis using polynomial contrasts was adapted to perform tests for trends . in order to evaluate the relationship between serum ggt activity within reference range and the individual components of the frs , spearman 's correlation analysis was employed . the frs was calculated from the ncep atp iii algorithm based on six coronary risk factors : age gender , total cholesterol , hdl - cholesterol , sbp , and smoking habit . among these factors , framingham risk equations were used to predict the risk of developing coronary disease events ( myocardial infarction or chd death ) over the next 10-year for adults aged 20 and older without heart disease or diabetes . participants were divided into three groups : low risk ( < 10% risk of developing a chd event over the next 10-year ) , intermediate risk ( 1020% risk ) , and high risk ( > 20% risk ) . however , the number of high - risk group was so small that it included the high - risk group into intermediate - risk group and beyond . smoking status was classified as either current smoker or nonsmoker . for analysis relating to serum ggt within reference interval to the intermediate - risk group and beyond , we constructed adjusted logistic regression analyses that considered bmi , weekly alcohol intake , and ldl cholesterol . the results of group data were expressed as mean standard error ( se ) . data were analyzed using pasw spss version 18.0 ( spss inc . , chicago , il , usa ) . the range of the first - to - fourth quartiles of serum ggt values was 613 , 1418 , 1928 , and 2965 participants in higher ggt quartiles were older and had more general chd risk factors such as hypertension , dyslipidemia , more alcohol consumption , and current smoking history . in addition , higher quartiles of serum ggt concentration were significantly associated with increasing trends in framingham point scores and the risk of 10-year chd prediction ( p for trend < 0.05 ) . the general characteristics of the study subjects according to serum ggt grading data are expressed as mean se after data weighting in complex sample analysis . ggt : gamma - glutamyl transferase ; bw : body weight ; bmi : body mass index ; wc : waist circumference ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; hdl - c : high - density lipoprotein cholesterol ; ldl - c : low - density lipoprotein cholesterol ; tg : total glycerides ; ast : aspartate aminotransferase ; alt : alanine aminotransferase ; chd : coronary heart disease ; se : standard error ; q1 : 1 quartile ; q2 : 2 quartile ; q3 : 3 quartile ; q4 : 4 quartile . the amount alcohol consumption calculation formula : 10 g shots frequency / week . the correlation between log - transformed ggt and 10-year chd risk was r = 0.237 ( p < 0.001 ) [ table 2 ] . log - transformed ggt was also well - correlated with individual risk factor scores including age , smoking , total cholesterol , hdl - cholesterol , and sbp ( p < 0.05 for all ) . the spearman 's rank correlation coefficients relating individual components and total framingham risk score to log - transformed ggt ggt : gamma - glutamyl transferase ; hdl - c : high - density lipoprotein cholesterol ; sbp : systolic blood pressure ; chd : coronary heart disease . table 3 shows the odds ratio ( or ) for intermediate - risk and beyond for chd in relation to quartiles of serum ggt . the 10-year chd risk was significantly associated with increasing quartiles of serum ggt ( p for trend < 0.05 ) . compared with individuals with lowest quartile of serum ggt , the nonadjusted or ( 95% confidence interval [ ci ] ) was 1.18 ( 0.781.78 ) for the second quartile , 1.42 ( 0.94 2.16 ) for the third quartile , and 2.77 ( 1.74 4.40 ) for the highest quartile . after adjustment of bmi , the amount of alcohol intake and ldl cholesterol , the or ( 95% ci ) for intermediate risk and beyond of 10-year chd prediction with lowest quartile of participants was 1.21 ( 0.78 1.87 ) for second quartiles , 1.39 ( 0.88 2.21 ) for third quartiles , and 2.03 ( 1.23 3.34 ) for highest quartiles . in addition , the or for intermediate risk and beyond of 10-year chd prediction showed an increased tendency as the serum ggt quartile gets greater ( p for trend < 0.05 ) . the or of intermediate - risk and beyond for chd ( 10-year risk 10% ) by ggt multivariate logistic regression model was used after data weighting in complex sample analysis . model 1 : unadjusted ; model 2 : after adjustment for bmi , the amount of alcohol intake and ldl - c . ggt : gamma - glutamyl transferase ; chd : coronary heart disease ; or : odds ratio ; ci : confidence interval ; bmi : body mass index ; ldl - c : low - density lipoprotein cholesterol ; q1 : 1 quartile ; q2 : 2 quartile ; q3 : 3 quartile ; q4 : 4 quartile . in the use of serum ggt as a test variable to predict the presence of intermediate risk beyond a 10-year chd prediction , a receiver - operating characteristic ( roc ) curve was prepared [ figure 1 ] . based on the roc curve , the best serum ggt cut - off obtained was 28.5 iu / l that predicted the intermediate risk , beyond a 10-year chd prediction with sensitivity of 58% , specificity of 52% , positive predictive value of 54.7% , and negative predictive value of 55.3% . we investigated the association between the ggt level in normal range and the possible prevalence of chd in 10-year , using frs calculated from the ncep atp iii algorithm in korean men . frs is one of the numbers of scoring systems used to determine an individual 's chances of developing chd . cardiovascular risk scoring systems presents an estimate of the probability that a person will develop chd within a given time - frame , usually 1030 years . in this study , a higher quartile of serum ggt level , even within the reference range , was found to be significantly related to the elevated risk of chd which resulted from the calculation of frs in men . moreover , this association persisted even after adjusting established cardiovascular risk factors such as ldl - cholesterol and bmi , which were not used in the necp atp iii and confounding factors like the amount of alcohol intake . the or of a 10-year chd risk prediction increased in dose - dependent manner with increasing quartiles of serum ggt activity . the best serum ggt cut - off was 28.5 kim et al . previously demonstrated that multivariable - adjusted ors for frs > 20% were significantly increased from the lowest to highest ggt quartiles , compared to the lowest baseline ggt category . however , it was a single center - based study and did not target reference value of serum ggt . in other words , the present study derives its significance in the aspect that the association between serum ggt activity within reference value and a 10-year chd prediction risk was evaluated . the present study also concluded that individuals with higher quartiles of serum ggt were well correlated with individual factors such as higher bmi , waist circumference , sbp , lipid levels , and fbg , which include frs components . , the prevalence rate of metabolic syndrome increased significantly in relation to serum ggt level within normal range in korean men . demonstrated that serum ggt level even within the reference interval was correlated with nonalcoholic fatty liver disease ( nafld ) and the or of ggt activity for nafld was elevated according to elevation of the ggt grading . liu et al . also mentioned that ggt was strongly consistent with cardiovascular and metabolic variables in the cross - sectional study among 616 young healthy participants . this tendency has been proven not only in asian studies but also in previous studies targeting caucasians , africans , and other countries . they indicated that individuals with metabolic syndrome , impaired fasting glucose , and nafld had considerably elevated levels of serum ggt regardless of any ethnicity . increased serum ggt level has been traditionally understood as a marker of alcohol abuse and/or liver damage however , a large number of studies suggested that serum ggt is not only a marker for oxidative stress but also a relative factor of cardiovascular disease and metabolic syndrome . despite the fact that serum ggt activity which reflects the risk of chd is not completely understood , there are several possible mechanisms that support the hypothesis . the first probable mechanism is that ggt is regarded as a biomarker for oxidative stress . in other words , the oxidative stress could be a crucial factor in the pathophysiology of cardiovascular disease and ggt has an important role in maintaining intracellular glutathione transport into most types of cells . elevated ggt could be the expression of subclinical inflammation because serum ggt is highly associated with white blood cell count and some features of low - grade inflammation . furthermore , excess reactive oxygen species and superoxide which is generated by oxidative stress and low - grade inflammation recapitulate not only endothelial dysfunction but also cardiovascular dysfunction . the third acceptable mechanism could be a strong correlation of ggt with various atherosclerotic risk factors . previous studies reported that serum ggt concentrations were related with hypertension , metabolic syndrome , and diabetes . serum ggt level was also found to increase insulin resistance and be positively correlated with risk factors of atherosclerotic chd and inflammation such as male gender , crp , total cholesterol and uric acid level . reversely , as also shown in this study , serum ggt was negatively correlated to hdl - cholesterol level which is a well - known negative risk factor of chd . the origins of ggt in plaques could be through the influx of lipoproteins which carry it into lesions . one of the products of glutathione hydrolysis which were produced by ggt is cysteinyl - glycine . it is also observed in the studies which examined the enzymatic activity of ggt in the coronary and carotid atheroma . furthermore , the data may guarantee that it represent the whole population because it was derived from a random selection . therefore , it is reasonable for the results to be generalized into the korean population . in addition , a 10-year chd risk prediction was computed using the ncep atp iii . however , even though this study showed a positive relationship between serum ggt level within the reference range concentration and a 10-year chd risk prediction , there are several limitations . this study was cross - sectional , and we did not measure oxidative stress directly . additionally , the end point of this study is a mathematical substitute for the presence of chd , even though frs is a generalized tool . furthermore , the statistical analysis to serum ggt concentration in women was not applied because the number of subjects with intermediate - risk and beyond for chd ( frs 10% ) in this gender group was too small . therefore , further longitudinal cohort studies are needed to evaluate the predictive value of biomarkers for the increased risk of chd in the korean population . in conclusion , this study suggests that serum ggt activity , within its reference range , is a useful predictor of a 10-year chd risk using frs calculated by ncep atp iii . therefore , serum ggt within the reference range could be a helpful tool and an additional marker in the prediction of chd risk in korean men .

lichen planus ( lp ) is a chronic mucocutaneous disease widely recognized in adults , but uncommon in children . the purpose of this paper is to report two cases of lp in children . the diagnosis was made based on clinical and histopathological findings . the treatment consisted of antifungal and multivitamin therapy . regression of lesions was observed . the patients are still under follow - up . although lp is uncommon in children , it is necessary to have adequate knowledge about this condition for proper diagnosis and treatment .

lichen planus is an inflammatory mucocutaneous disease ; it is a very common disease in adults but uncommon in children . an autoimmune basis has been proposed ; however , there is evidence suggesting that lp is not a true autoimmune disease but rather a chronic , cell - mediated immune disorder , involving activated lymphocytes and up - regulating cytokine production . several retrospective reviews have estimated that only 1% 16% of lp patients are younger than 15 years . factors responsible for the rarity of juvenile olp include a low incidence of autoimmune diseases , systemic diseases , precipitating factors such as stress and lp related infections in children . the purpose of this paper is to report two cases of childhood olp and review the literature in this regard . an 8 year - old male patient was referred to the department of pedodontics , pms college of dental science and research with the complaint of pain and white discoloration under the tongue . the patient had difficulty tolerating spicy food . the medical history revealed that the boy had been vaccinated against japanese encephalitis one year earlier as part of school immunization program . almost 10 months after this , fluid - filled blisters appeared on both feet and ruptured after 23 days . these were associated with severe itching for which he had taken ayurvedic medicine . on examination , 1 ) , on the ventral tongue mucosa and also on the labial mucosa ( fig . white interlacing striae on the left buccal mucosa striae on the upper labial mucosa ( case 1 ) on palpation , these lesions were non - tender and non - scrapable and the buccal mucosae appeared rough and corrugated . differential diagnoses included olp , lichenoid reaction and leukoplakia and a provisional diagnosis of lp was made by correlating the clinical appearance with the age of the patient . the provisional diagnosis was confirmed by histopathological examination following incisional biopsy from the left buccal mucosa . the biopsy specimen showed typical features of lp including the saw - toothed rete pegs , hyperkeratosis and lymphocytic infiltrate ( fig . 3 ) . pictomicrograph of case 1 showing saw - toothed rete pegs and lymphocytic infiltration as the lesions were symptomatic , topical anti - fungal treatment ( clotrimazole mouth paint twice daily ) was prescribed for the first two weeks . on the recall session , the burning sensation had reduced considerably and the clinical appearance of the lesions had improved with regard to severity . an 8 year - old male patient was referred from a local clinic with burning sensation while eating spicy food . they gave a history of burning sensation since three months earlier after which a local dentist replaced an amalgam restoration with glass ionomer in the left posterior mandible . due to no relief , the patient was referred to a hospital . on examination , the patient had a glass ionomer restoration on tooth # 75 , and all other teeth were caries - free . white interlacing striae were noted on the left buccal mucosa ( unilaterally ) ( fig . 5 ) revealed saw - toothed epithelial rete pegs , basal cell degeneration with subepithelial lymphocytic infiltrates and melanin incontinence , which confirmed the diagnosis of lp . after the biopsy , the lesion and the symptoms subsided ; thus , no treatment was warranted . striae on the left buccal mucosa ( case 2 ) pictomicrograph of case 2 showing saw - toothed rete pegs and lymphocytic infiltration an 8 year - old male patient was referred to the department of pedodontics , pms college of dental science and research with the complaint of pain and white discoloration under the tongue . the patient had difficulty tolerating spicy food . the medical history revealed that the boy had been vaccinated against japanese encephalitis one year earlier as part of school immunization program . almost 10 months after this , fluid - filled blisters appeared on both feet and ruptured after 23 days . these were associated with severe itching for which he had taken ayurvedic medicine . on examination , 1 ) , on the ventral tongue mucosa and also on the labial mucosa ( fig . white interlacing striae on the left buccal mucosa striae on the upper labial mucosa ( case 1 ) on palpation , these lesions were non - tender and non - scrapable and the buccal mucosae appeared rough and corrugated . differential diagnoses included olp , lichenoid reaction and leukoplakia and a provisional diagnosis of lp was made by correlating the clinical appearance with the age of the patient . the provisional diagnosis was confirmed by histopathological examination following incisional biopsy from the left buccal mucosa . the biopsy specimen showed typical features of lp including the saw - toothed rete pegs , hyperkeratosis and lymphocytic infiltrate ( fig . 3 ) . pictomicrograph of case 1 showing saw - toothed rete pegs and lymphocytic infiltration as the lesions were symptomatic , topical anti - fungal treatment ( clotrimazole mouth paint twice daily ) was prescribed for the first two weeks . on the recall session , the burning sensation had reduced considerably and the clinical appearance of the lesions had improved with regard to severity . an 8 year - old male patient was referred from a local clinic with burning sensation while eating spicy food . they gave a history of burning sensation since three months earlier after which a local dentist replaced an amalgam restoration with glass ionomer in the left posterior mandible . due to no relief , the patient was referred to a hospital . on examination , the patient had a glass ionomer restoration on tooth # 75 , and all other teeth were caries - free . white interlacing striae were noted on the left buccal mucosa ( unilaterally ) ( fig . 5 ) revealed saw - toothed epithelial rete pegs , basal cell degeneration with subepithelial lymphocytic infiltrates and melanin incontinence , which confirmed the diagnosis of lp . after the biopsy , the lesion and the symptoms subsided ; thus , no treatment was warranted . striae on the left buccal mucosa ( case 2 ) pictomicrograph of case 2 showing saw - toothed rete pegs and lymphocytic infiltration lichen planus is a chronic inflammatory mucocutaneous disease most frequently seen in the middle aged and the elderly populations with a female to male ratio of approximately 2:1 . childhood lp is common in the tropics , and children of asian origin may be more prone to this condition . this possibly indicates that a specific genetic predisposition ( hla - dependent ) in the asian race , in spite of a negative family history , may be involved in the pathogenesis of olp . the etiology of lp remains obscure , but recently pathogenic mechanisms with an immune - mediated basis have been proposed . causes such as allergy to dental restorative materials , local trauma ( koebner phenomenon ) and several infections ( plaque causing microorganisms ) have also been reported . also , lp has been reported as a complication of hepatitis b vaccination in both children and adults . reactions to measles - mumps - rubella ( mmr ) and diphtheria - tetanus pertussis - polio ( dtap - ipv ) vaccinations can also be related to this disease . moreover , our first case gave a positive medical history of vaccination against japanese encephalitis within the previous year . but this association can not be confirmed conclusively because there is no available literature regarding development of lp following this particular vaccination . this agrees with nnoruka , who reported a relationship between this phenomenon and the occurrence of lp . in our second case , the etiology could be the amalgam restoration but persistence of symptoms even after replacement of amalgam with glass ionomer cement led to the diagnosis of idiopathic lp . some patients report roughness of the lining of the mouth , sensitivity of the oral mucosa to hot or spicy foods , presence of red or white patches or oral ulcerations . but in our second case , unilateral presence of the lesion led us to a differential diagnosis of lichenoid reaction . the only difference is in the site of involvement like the palate , which is atypical of lp . in most cases , the cause for lichenoid reactions can not be identified ; hence the diagnosis by exclusion is idiopathic lp . although the differential diagnoses were considered , histopathological features confirmed the diagnosis of lp in both cases . use of topical corticosteroids is the most widely accepted treatment for olp because it reduces patients pain and inflammation . several therapies , including intra - lesional injection , retinoids , dapsone , tacrolimus , and ultra - violet light have been tested with varying results . clinical improvement with relief of symptoms has been reported following the use of amphotericin - b , nystatin and azole antifungals . miconazole gel is useful as an adjunct to topical steroids . in our first case , since the patient complained of severe burning sensation , immediate treatment with topical antifungals was done and there was marked reduction in the symptoms . while in our second case , since the lesion subsided spontaneously after the incisional biopsy , no treatment was warranted . there are a number of studies on olp with regard to its malignant transformation in the past few decades although olp is considered rare in children , presence of a non - scrapable white lesion should alert the clinicians to consider lp in the list of differential diagnoses . timely diagnosis with proper treatment oriented towards the etiological factor

key clinical messagedorsal agenesis of the pancreas is a rare congenital anomaly . fiftyeight cases were reported from 1913 till 2015 , nine of which were associated with tumors . we present the 10th case , the first to be associated with pancreatic mucinous adenocarcinoma and cystic teratoma , successfully managed by whipple procedure and total pancreatectomy .

pancreatic development is a complex process . however , pancreatic congenital anomalies are a rare entity . among these anomalies , it can be asymptomatic and diagnosed incidentally during investigations carried out for unrelated causes , or symptoms including abdominal pain , diabetes mellitus , jaundice , and weight loss might present initially . other anomalies might be present as well , in association with dorsal agenesis of the pancreas such as polysplenia , ectopic spleen , mesenteric malrotation , bicornuate uterus , and cardiac anomalies 25 . to our knowledge , around 58 cases of dorsal agenesis of the pancreas have been reported from 1913 till 2015 , and around nine cases only were associated with pancreatic tumor . we present a case of dorsal agenesis of the pancreas associated with pancreatic tumor , along with ectopic splenia , mesenteric malrotation , and vascular malformations . this is the case of a 29yearold male patient presenting to the hospital with a 2month history of abdominal pain , worsening progressively then localizing in the right upper abdominal quadrant and radiating to the back over the last 2 weeks . patient admits one episode of abdominal pain a year ago , due to which he underwent an endoscopic gastroduodenoscopy that revealed diffuse mild gastritis and no evidence of helicobacter pylori infection . patient was treated with a 3month course of protonpump inhibitors with clinical improvement in his pain . he has no past medical or surgical history and no allergies , does not consume alcohol and is not a smoker . ultrasound of the abdomen was performed showing a right subhepatic complex cystic lesion 12 7 6 cm containing dense debris inside and a thick wall . magnetic resonance cholangiopancreatography ( mrcp ) was performed showing a loculated mass , 5.6 9.7 cm , located behind the head of the pancreas , displacing it anteriorly and to the right . ( mrcp ) : cuts showing the pancreatic mass ( white arrows ) as it appeared on mrcp in an axial ( a ) and coronal fashion ( b ) . abdominopelvic ct scan was performed showing a multiloculated cystic retroperitoneal lesion at the level of the second portion of the duodenum in continuity with the pancreatic head which is displaced anteriorly and to the right ( fig . 2 ) associated with a mild malrotation of the mesentery and proximal small bowels ( fig . ( ct scan ) : ( a and b ) the multiloculated cystic retroperitoneal lesion ( white arrows ) , at the level of the second portion of the duodenum in continuity with the pancreatic head which is displaced anteriorly and to the right . ( a ) coronal cut from the abdominopelvic ct scan , showing bowels malrotation and an inferiorly located hepatic flexure ( white arrow ) . ( b ) 3d reconstruction of the scan performed with iv contrast showing the vascular variation where the sma branches from the celiac trunk ( thick white arrow ) , giving off the gastroduodenal artery ( thin white arrow ) . ( ct scan ) : ( a ) presence of the ectopic spleen ( white arrow ) ; ( b ) cut showing the absence of pancreatic tissue anterior to the splenic vein , where it is usually located ( white arrows ) . laboratory studies showed the following : hemoglobin 12.8 g / dl , white blood cells 5000/mm , neutrophils 53% , platelets 345,000/mm , pt inr 1.1 , sgpt ( serum glutamicpyruvic transaminase ) 10 /l , sgot ( serum glutamicoxaloacetic transaminase ) 10 /l , direct bilirubin 0.18 mg / dl , total bilirubin 0.45 mg / dl , alkaline phosphatase 101 /l , amylase 77 /l , lipase 55 /l , cea ( carcinoembryonic antigen ) 2.09 ng / ml , ca199 19.95 /ml , and ggt ( gammaglutamyltransferase ) 45 /l . analysis of the aspirate revealed the following : culture : escherichia coli growth , resistant to ampicillin.pathology : necrotic inflammatory smear with no suspicious content , poorly cellular with no ductal cells , no atypical cells.amylase = 982 /l ; lipase = 4498 /l.cea = 700 ng / ml ; ca199 = 10.8 /ml . culture : escherichia coli growth , resistant to ampicillin . pathology : necrotic inflammatory smear with no suspicious content , poorly cellular with no ductal cells , no atypical cells . cea = 700 ng / ml ; ca199 = 10.8 /ml . based on the data collected at that point , the cyst was considered benign and cystojejunostomy was planned . in the operating room , a right subcostal incision was made , and inspection and palpation of the abdominal organs revealed no evidence of metastatic lesions or carcinomatosis . chevron incision was then performed , and the transverse colon was not in its anatomical position , with the hepatic flexure located inferiorly around the right gutter . cyst fluid was suctioned with care taken to avoid any spillage ; then , intraoperative wedge biopsy was performed and sent to the pathology laboratory as frozen . results came back positive for mucinous cystadenocarcinoma , moderately to poorly differentiated cells , ductal in origin . at this point , dissection was started at the hepatic pedicle , identification of the common hepatic duct , portal vein , and right hepatic artery . the anatomical variation at this level was as follows : superior mesenteric artery branching off the celiac trunk , superior mesenteric vein superficially located with a malrotation of the mesentery , and a rightsided ligament of treitz . common hepatic duct was divided and enterectomy was performed , and retroportal pancreatic dissection with ligation of venous tributaries and total pancreatectomy were performed . lymph node dissection around the hepatic artery and celiac trunk was performed , and hepaticojejunostomy and rouxeny gastrojejunostomy were performed as well . macroscopically , the pancreatic piece was 14 7.5 7.5 cm showing on cut section a partly necrotic partly cystic surface , and microscopically , the cyst located in the lateral peripancreatic region is composed of dense mesenchymal tissues ( muscle , fat , cartilage ) admixed with neural elements on a background of dense lymphoid tissue with lymphoid follicles , all lined by mature squamous epithelium . pancreatic head shows numerous cystic dilated ducts lined by flattened epithelium alternating with small cystic spaces lined by tall columnar epithelium exhibiting dysplasia . common bile duct lining epithelium shows marked atypia and is surrounded by numerous clusters and neoplastic glands invading the underlying pancreas ( figs 5 , 6 , 7 , 8) . cyst lined by mature squamous epithelium ( 1 ) continued by either columnar ( 2 ) or at places by cuboidal epithelium . cyst wall composed of mature mesenchymal tissue : cartilage ( 1 ) , muscle ( 2 ) , and clusters of benign salivarytype glands ( 3 ) . major pancreatic duct ( mpd ) surrounded by small irregular neoplastic glands ( adenocarcinoma ) . diagnosis : pancreatic adenocarcinoma , moderately differentiated mucin secreting arising in common bile duct region , infiltrating pancreatic head with absence of body and tail , measuring 6 4 4 cm with free surgical margins . ct scan ( computed tomography scan ) was then performed day 7 postoperation showing no intraabdominal collections , drains were removed , patient was started on liquid diet , and no complications were encountered . patient 's diet was progressed and he was discharged home on insulin replacement therapy and pancreatic enzyme supplementation . being part of the gastrointestinal organs system , the pancreas develops from the endoderm . between the sixth and eighth week of embryonic life , two evaginations , dorsal and ventral , the ventral evagination arises from a liver diverticulum and will eventually form the posterior part of the pancreatic head and the uncinate process . on the opposite site of the foregut , the dorsal evagination develops and will eventually form the tail and body of the pancreas . later on , ducts begin to develop in a treelike fashion , endocrine cells appear , and the islets of langerhans emerge with time 1 , 2 . the pancreas is a retroperitoneal organ , extending from the duodenal arc to the spleen . it harbors two ducts : the main duct of wirsung and the duct of santorini 3 , 4 . with the advancement of imaging techniques , and increasing diagnostic workups made , pancreatic malformations are more described . total pancreatic agenesis has been described in the literature but is an extremely rare entity , usually incompatible with life , and associated mainly with sever intrauterine growth retardation 5 , 6 . other types of congenital anomalies of the pancreas are more frequently encountered such as pancreas divisum , common bile duct syndrome , ectopic pancreas , accessory pancreatic lobe , annular pancreas , and agenesis of the dorsal pancreas ( table 1 ) . pancreatic malformations 7 over the last 100 years , 58 cases were reported with agenesis of the dorsal pancreas , whether partial or complete , with the latter being less frequent , and around nine cases only were associated with pancreatic tumor ( table 2 ) . it has been associated with pancreatic bud primary dysgenesis , ischemic insult to the organ during its development , and an autosomal dominant genetic mode of transmission 1 , 9 , 10 . cases of agenesis of the dorsal pancreas with associated pancreatic tumors found in the literature agenesis of the dorsal pancreas is a rare congenital malformation , and its association with tumors makes it a hard condition to deal with . many studies are trying to identify the reason behind this anomaly , with findings suggestive of genetic inheritance in an autosomal dominant mode or xlinked mode 11 , 19 . other hypotheses state a genomic activation process , notably the sonic and indian hedgehog genes 20 , hnf1 gene 18 , and others . as demonstrated by the literature review done , the cornerstone for curing tumors in association with dorsal agenesis of the pancreas is surgery . most of the time , the lack of sufficient pancreatic tissue makes it hard to achieve safe margins without undergoing a total pancreatectomy . adenocarcinoma associated or arising from previous teratoma tumors is by itself a rare entity 21 , 22 , 23 , 24 . this constellation of presenting tumors with a rare congenital anomaly makes the genetic hypothesis in the frontline of investigations . until today , the proper management of tumors associated with agenesis of the dorsal pancreas consists of surgical intervention for malignant cases and/or symptomatic benign cysts , with or without chemotherapy regimens with respect to the pathology finding . the additional challenge comes afterward involving pancreatic endocrine and exocrine replacement therapies . such cases should be handled in referral center where patient 's monitoring can be achieved optimally on the surgical and medical level . furthermore , associated genes should be evaluated in such patients and on a larger scale as well . the approval of the university of balamand faculty of medicine irb was obtained ( irb / o/03116 ) . the datasets supporting the conclusions of this article are included within the article and its references .

cysticercosis , which has a worldwide distribution is found in man , who is usually infected by eating inadequately cooked pork or other contaminated food . cysticercosis develops most commonly in the muscles and brain . pulmonary involvement is very rare and also difficult to recognize because pulmonary lesions caused by the presence of cysticerci are difficult to discern from pulmonary infiltrates , because other parasitic infestations or tuberculosis , as well as metastatic lesions , produce similar chest x - ray findings and similar clinical symptoms.we experienced a case of pulmonary cysticercosis confirmed at gyeongsang national university hospital by means of an open lung biopsy and treated successfully with praziquantel ( 50 mg / kg per day for 15 days).this case seems to indicate that pulmonary cysticercosis should be considered as a diagnostic possibility in patients with nodular infiltrates in the lungs , especially in endemic areas , until such infiltrates are otherwise explained .

cysticerci , the larval forms of taenia solium , develop in the muscles of the pig , an intermediate host . inadequately heated or raw pork containing adult worms is the main source of human infection . other sources of human infections with c. cellulosae occur by way of 1 ) ingestion of the eggs found on contaminated hands or in food , 2 ) self - contamination by people who have the adult worm in their intestines , and 3 ) internal autoinfection in which the eggs of the adult worm residing in the upper gastrointestinal tract are returned to the stomach by reverse peristalsis . the eggs are hatched in ugi to the embryos which penetrate the intestinal wall and are then carried along blood vessels to all parts of the body , where they soon become cysticerci . cysticerci most frequently invade the intermuscular and subcutaneous tissues ; next , the eye and then the brain . they may also invade the heart , liver , abdominal cavity , kidneys , and adrenals , as well as the lungs . it is so rare that , worldwide , only a few cases have ever been reported , with none in korea . among those few cases reported in other areas , this report , then , together with relevant literature , cites another one of the few documented cases of pulmonary cysticercosis and further cites the only case of pulmonary cysticercosis ever documented or reported in korea . a 65-year - old man was admitted to the hospital because of generalized weakness , coughing , and sputum production . two months before his admission , weakness , sweating and anorexia had developed together with an intermittent cough and whitish sputum production . the patient had eaten raw pork several times 20 years ago , but there was no specific family history . vital signs were as follows : temperature 36 , pulse 98/min , and respiration 20/min . the blood pressure was 110/75 mmhg . on examination the patient appeared weak and chronically ill . an abdominal examination disclosed mild tenderness to deep palpation in the right upper quadrant , without diminution of bowel sounds ; the liver was palpated by four finger breadths . laboratory data on admission were as follows : hemoglobin and hematocrit were 13.9 g / dl and 41.8% on admission ; white cell count was 8700 with 4% eosinophils ; urine and stool specimens gave normal results ; blood urea nitrogen 5 mg / dl ; creatinine 1.3 mg / dl ; total protein 8.1 g / dl ; albumin 4.3 g / dl ; and sgot / gpt 42/32 u / dl . the stain for acid fast bacilli and cytologic examination of sputum did not reveal anything microscopically . the pulmonary function test was within normal range ; the fev1 was 4.1 , the fev1/fvc was 87% . an x - ray film of the chest showed multiple nodular densities throughout both lower lung fields ( fig . 1 ) . a computed tomographic scan of the brain and chest revealed multiple nodular and small calcified densities ( fig . 3 , 4 ) . a biopsy of a subcutaneous nodule was done on the 4th hospital day , and upon microscopic examination , a bladder worm ( fig . 6 ) and parenchymatous portion with spinal canal and separated bladder of the cysticercus ( fig . 7)was observed . on the 12 th hospital day , the presence of cysticercus was confirmed by an open lung biopsy ( fig . afterward , the patient was treated with praziquantel 50 mg / kg / day for 15 days and discharged on the 18 th hospital day , even though he still reported some weakness , cough , and sputum production . an x - ray film of the chest revealed nearly normal conditions in the lungs(fig . taenia solium has been recognized from the time of hippocrates but was never specifically differentiated from the beef tapeworm , t. saginata , until the time of goseze ( 1782 ) . leukart ( 1856 ) first worked out the life cycle and demonstrated that the bladder worm in the tissues of the pig was in the larval stage and infective for man . taenia solium has a world wide distribution but is most commonly found in the soviet union , asia , africa and in central and south america . the eggs of taenia solium and taenia saginata in korea were found in 1.1% of the population , according to a broad parasite study done on 35,018 people in 1981 . in 1986 , the infection rate for the both parasites decreased to 0.27% ( 119 of 43,590 people ) . the eggs of taenia solium are spherical or subspherical in shape , measure 31 to 43 um in diameter and can not be microscopically distinguished from those of t. saginata . cysticerci , the larval forms , are subspherical to ovoid , have milky white bladders with heads invaginated into the bladders , and are approximately 5 mm wide and 8 to 10 mm long . after human ingestion , the eggs develop in the stomach and intestine into embryos , which penetrate the intestinal mucosa , then circulate in the entire body through the venous system of the mesentery and are finally distributed in the muscles and other tissues where development into cysticerci takes place over a period of 60 to 70 days . the fibers of the involved muscles atrophy and their function decreases . in the subcutaneous tissues , however , only a mild tissue reaction occurs , despite the swollen appearance of the protruded skin . the clinical picture of brain cysticercosis varies according to the involved area of the brain cortex and can easily be mistaken for a brain tumor because of those symptoms which involve the central nervous system . if cysticerci develop in the ventricles of the brain , headache , nausea and vomiting occur due to increased intracranial pressure . precysticercus larvae lodged in the brain produce little disturbance during their life spans , but as soon as the larvae begin to lose vitality and parts of them die , dead larvae and their remnants are sensitized as foreign materials and evoke a great variety of brain symptoms , such as allergic reactions . when examples of pulmonary cysticercosis are found , cysticerci may be assumed to have also invaded many other organs like the brain , subcutaneous tissues and muscles of humans or animals as well . the radiological appearance of cysticercosis in the lungs can not be differentiated from other parasitic infections , e.g. , ecchinococcosis , pentastomiasis , paragonimiasis , and histoplasmosis , or other conditions such as tuberculosis , alveolar carcinoma and metastases . this is due to the varying reactions of the lung tissues and to the difference in size of the larvae . the reason for the rarity of lung involvement is probably explained by the life cycle of the taenia solium parasite . humans may serve as intermediate hosts for the adult larvae which favor muscle and brain tissue to complete their life cycle . another reason for the rarity of lung involvement may be that pulmonary lesions are overlooked because of the asymptomatic clinical features and because patients usually present with neurological symptoms . although rare , pulmonary cysticercosis does occur ; therefore it should not be overlooked in differential diagnosis of multiple lung opacities just because they are relatively frequent in korea . like brain cysticercosis , which is treated with praziquantel ( 50 mg / kg / day ) for 15 days , pulmonary cysticercosis , in this case , was treated completely by the same dose , even though no regimen has yet been established .

backgroundpterygium is a proliferative disease with hyperplastic growth of corneoconjunctival fibro vascular tissue onto the cornea . surgical therapy can be used to successfully manage pterygia ; however , recurrence remains a problem . to reduce recurrence , surgical management may include autoconjunctival grafting , lamellar keratoplasty , amniotic membrane transplantation , and intraoperative antimetabolites application.purposeto assess the safety and the efficacy of intraoperative mitomycin c ( mmc ) and 5-fluorouracil ( 5-fu ) application in preventing recurrence of pterygium after excision.patients and methodsthe study design is a prospective , randomized clinical trial . a total of 50 patients with bilateral pterygium were recruited for the study . the first group of patients ( 25 ) underwent surgical excision of the pterygium with bare sclera in one eye and mmc was applied as adjunctive therapy for the other eye . in the second group 5-fu was used instead of mmc . recurrences and postoperative complications were measured in the two groups . the mean follow up period of the patients was 18.8 months . chi square test , odds ratio , and frequency distribution were used to determine significance levels ; p - values < 0.05 were considered statistically significant.resultsin group 1 the recurrence rate was 8% for the mmc treated eyes and 32% for their fellow eyes ( p = 0.03 ) . in group 2 the rate was 18% for the 5-fu treated eyes and 34% for their fellow eyes ( p = 0.07 ) . no serious complications were recorded in either group.conclusionboth mmc and 5-fu reduce the recurrence rate of pterygium after simple surgical excision ; statistically , the effect of the former was significant , but insignificant for the latter . both antimetabolites were safe during the whole study period , but 5-fu recurrent cases showed cosmetically unacceptable appearances with excessive vascularization . mmc , but not 5-fu , is recommended as an adjunctive therapy to prevent recurrence of pterygium after surgical excision .

the word pterygium is derived from the greek word pterygion , meaning wing . it refers to an elevated , superficial , external ocular triangular fibro vascular mass that usually forms over the perilimbal conjunctiva and extends onto the corneal surface.1 pterygia are reported to affect males twice as frequently as females and it is uncommon for patients to present with pterygia prior to the age of 20 years.2 countries nearer the equator have higher rates of pterygia , especially in rural areas . a possible reason for this geographic variation is that ultraviolet light may be a risk factor for the development of pterygia . other potential risk factors include ocular dryness , inflammation , occupational exposure to irritants , and ocular dominance where the dominant eye tends to be kept open more in bright sunlight.3 pterygia may be classified clinically as being active or inactive . the progression of active pterygium onto the cornea can lead to both significant corneal distortion and the development of corneal astigmatism.4 patients with pterygia might present with a variety of complaints , ranging from no symptoms to significant redness , swelling , itching , irritation , and blurring of vision associated with elevated lesions of the conjunctiva and contiguous cornea in one or both eyes.5,6 it is more common for the pterygium to present on the nasal side , although it can present temporally as well as in other locations.7 patients with pterygia can be observed without intervention unless the lesions exhibit growth towards the center of the cornea or if the patient exhibits symptoms of significant redness , discomfort , or alterations in visual function.8 medical treatments include artificial tears to lubricate the ocular surface and anti - inflammatory drops to reduce any inflammation . major indications for surgery include involvement of the visual axis , progressive growth that threatens the visual axis , induced irregular astigmatism that reduces vision , and restriction of ocular motility . it is best to excise a pterygium before it reaches a point where it obscures part of the pupil that is why a pterygium extending 3 mm or more from the limbus is a major indication for surgery.9,10 minor indications for surgery include contact lens intolerance , chronic irritation , and disfigurement . while pterygium surgery is often considered a simple office procedure , the decision to operate , especially for minor indications , must weigh the risk of complications or recurrence against the degree to which the symptoms trouble the patient.10 the major endpoints of pterygium removal relate to recurrence , which may be corneal or conjunctival.11 there are other minor less common endpoints such as ocular motility restriction , cosmetic appearance , or tear outflow.1214 simple excision of pterygium ( bare sclera technique ) is associated with a high rate of recurrence ( 40%80% ) that may be more aggressive than the initial lesion.15,16 other surgical techniques such as conjunctival graft or rotation flap , amniotic membrane graft , thermal cautery , or beta irradiation are associated variably with lower recurrence rates.10,16 the use of medical adjuvant agents capable of stopping pterygium regrowth , such as 5-fluorouracil ( 5-fu ) and mitomycin c ( mmc ) , have had extensive practice worldwide with variable results.10 in this study we evaluated the efficacy and safety of the use of antimetabolites as adjunctive medical therapy after simple excision of primary pterygium . the current prospective , randomized clinical trial study was conducted on 50 patients ( 30 males , 20 females ) with bilateral primary pterygium during a 3-year period . the studied patients fulfilled the following inclusion criteria : age > 20 years , living in rural areas or working in outdoor conditions , and pterygium size of 2.5 mm or more measured from the limbus to the cornea . the patients were grouped into two groups : group 1 ( 25 patients ) underwent excision of pterygia with a bare sclera technique for one eye and mmc was applied intraoperatively for the other eye . in group 2 ( 25 patients ) , 5-fu was used instead of mmc . after applying an eyelid speculum , the pterygium head was lifted off the corneal surface by blunt dissection from the head in a smooth plane to bare sclera at the limbus , then the pterygium base was excised at its nasal end . approximately 3 mm of bare sclera was left in one eye ; in the fellow eye the antimetabolite was applied intraoperatively at the scleral bed after the excision for 3 minutes using a weck - cel sponge ( beaver - visitec international , inc , waltham , ma ) soaked in 0.5 mg / ml mmc solution or 50 mg / ml 5-fu solution . extreme care was taken not to apply the antimetabolite on the cornea and thorough irrigation with at least 30 ml balanced salt solution was used to make sure that all the antimetabolite was washed out . all patients received ciprofloxacin ( antibiotic ) and dexamethasone ( steroid ) eye drops for four weeks postoperatively . chi square test , odds ratio ( or ) , and frequency distribution were used to determine significance levels ; p - values < 0.05 were considered statistically significant . after applying an eyelid speculum , the pterygium head was lifted off the corneal surface by blunt dissection from the head in a smooth plane to bare sclera at the limbus , then the pterygium base was excised at its nasal end . approximately 3 mm of bare sclera was left in one eye ; in the fellow eye the antimetabolite was applied intraoperatively at the scleral bed after the excision for 3 minutes using a weck - cel sponge ( beaver - visitec international , inc , waltham , ma ) soaked in 0.5 mg / ml mmc solution or 50 mg / ml 5-fu solution . extreme care was taken not to apply the antimetabolite on the cornea and thorough irrigation with at least 30 ml balanced salt solution was used to make sure that all the antimetabolite was washed out . all patients received ciprofloxacin ( antibiotic ) and dexamethasone ( steroid ) eye drops for four weeks postoperatively . chi square test , odds ratio ( or ) , and frequency distribution were used to determine significance levels ; p - values < 0.05 were considered statistically significant . age of the patients ranged from 2340 years with a mean age of 36.4 years . this study showed that patients who underwent primary pterygium excision without using antimetabolites as adjunctive agents developed higher recurrence rates than those who received them ( mmc in group 1 , 5-fu in group 2 ) . or = 2.3 in general . use of mmc was superior to 5-fu in prevention of recurrence ( or = 5.4 for mmc and only 1.4 for 5-fu ; table 2 ) . pterygium is a common external ocular problem that is closely related to exposure to uv light , especially in hot , dusty , and dry climates . true estimates of its prevalence in iraq are not well known but having a risky environment makes it a very frequently encountered disease in our daily practice ; furthermore , recurrent cases after surgical excision are expected because of the environmental risks . pterygium is a proliferative disease with hyperplastic growth of the corneo - conjunctival fibro vascular tissue onto the cornea.17 surgical therapy can be used to successfully manage pterygia ; however , recurrence remains a problem.18 recurrence of the pterygium is usually defined as a corneal recurrence that is evidenced by growth of fibrovascular tissue across the limbus onto the cornea . this usually excludes the persistence of deeper corneal vessels and corneal scaring , which may be left even after adequate pterygium removal . 12 bunching of conjunctiva and formation of parallel loops of vessels , which aim almost like an arrowhead at the limbus , usually denotes a conjunctival recurrence ; although , this appearance is sometimes determined by the method of removal . for example , a simple conjunctival closure that may lend itself more to the appearance of a conjunctival recurrence than a broad conjunctival autograft.13,14 cosmetic appearance is another postoperative endpoint ; however , as long as there are no structural changes which affect vision or movement and as long as the patient is asymptomatic with respect to irritation then the issue of cosmetic appearance could not realistically be defined in terms of what can be seen at the slit - lamp magnification , but rather what the patient observes by looking in the mirror or other relatives and friends observe by looking at the patient s eye.19 simple excision leaving the sclera bare is the simplest approach for surgical management of pterygium but with the highest recurrence rate.20 the frustration of watching the rapid , inexorable progression of recurrent pterygium after what seemed to be an adequate surgical excision has provided investigators with strong motivation to find a medical adjunctive treatment capable of preventing pterygium regrowth . two such agents , 5-fu and mmc , are being used extensively worldwide with variable success . 5-fu is a pyrimidine analogue that inhibits dna synthesis and is active on the s phase ( synthesis phase ) of the cell cycle . its effect is most pronounced on rapidly proliferating cells that occur in response to inflammation . it is an alkylating agent rather than an antimetabolite , and selectively inhibits dna replication , mitosis , and protein synthesis . mmc inhibits proliferation of fibroblasts , suppresses vascular ingrowths , and is much more potent than 5-fu.16 fortunately , we did not face any of the expected serious complications secondary to the use of the antimetabolites during the follow - up period . looking at the recurrence rates in the treated groups , simple excision leaving the sclera bare yielded about 33% recurrence rate in all patients . mitomycin was utilized as an ancillary treatment due to its antiproliferative effects ; it reduced the recurrence rate successfully to 8% in our series . the antifibroblastic activity of mmc is known to be more than that of 5-fu ; accordingly , we observed that the recurrence rate with mmc was much less than that of 5-fu ( table 2 ) . variable recurrence rates of pterygia have been reported and surgical results are undoubtedly influenced by several factors , including type of pterygium , size , surgical technique , geographical location , and the surgeon s skill . hameed reported a 20% recurrence rate after simple pterygium excision.20 in the usa , vastine reported up to 60% recurrence rate after simple excision leaving the sclera bare , and zloty was faced with only two recurrent cases after performing 400 pterygium exisions with mmc application in an average follow - up of 3 years ( cited in gans).10 literature reveals 0%10% recurrence rates after using intraoperative mmc adjunctively with surgical excision.16 although the use of 5-fu as an adjunctive medical therapy to reduce the failure rates of trabeculectomy has been widely explored , to our knowledge no sufficient data are available for comparison with regards to the use of 5-fu as an adjuvant therapy after surgical excision of pterygium . optimum concentration and exposure time for mmc is not well known and vary between 0.10.5 mg / ml and 15 minutes . in general , low or intermediate risk of recurrence indicates the use of a low concentration ( 0.2 mg / ml ) , whilst high risk implies the need for a higher concentration ( 0.40.5 mg / ml ) . higher concentrations and extended exposure times are associated with an increased risk of complications.15,21 few data are available to compare regimens , and most surgeons increase concentration or duration based on risk factors for recurrence . gans reported a safe dose of 0.3 mg / ml for 5 minutes , and that zloty applied 0.4 mg / ml for 60 seconds only.10 the concentration we used was 0.5 mg / ml ( 0.05% ) for 3 minutes because all of our patients were defined as having high risks of recurrence , all were young patients with relatively large pterygia , and they share the same exposure risks to environmental factors . this concentration yielded only 8% recurrences and there were no serious side effects or signs of toxicity during the whole follow - up period . the mean follow - up period in the current study was 18.8 months ; all our recurrent cases were reported within the first 48 months postoperatively . table 3 shows a summary of the results of other studies with comparable follow up periods in terms of numbers of eyes with primary pterygium , mmc concentration , intraoperative exposure time , recurrence rates , and mean follow - up periods.22 even when used in correct dosages for brief periods , mitomycin has been associated with prolonged , irreversible stem cell damage with resultant chronic keratopathy and toxic keratoconjunctivitis . it is important to note that any use of topical mmc can be toxic and may cause visually significant complications such as aseptic scleral necrosis and infectious sclerokeratitis , secondary glaucoma , uveitis , cataract , corneal edema , corneal perforation , and endophthalmitis.16,23 moreno et al reported corneal melting 2 weeks after the use of mmc in surgical management of pterygium and mentioned that scleral thinning can occur as early as 1 week postoperatively or even years after the use of mmc ( cited in menghini et al).21 the critical point regarding these complications is that they may occur many months , or even years , after the use of mmc . the statistically significant reduced recurrence rate that we found and the rarity of postoperative complications during a reasonably long follow up period justify the use of mmc as an adjuvant drug to be applied during surgical excision of pterygium . however , it is not advisable to think about using such medication topically postoperatively because of the possible serious complications that might be encountered during long periods of topical administration or because of drug abuse . intraoperative use should be controlled with regards to the dose and time of application , unlike postoperative topical application which would be dependent on patient administration and overdosing might be encountered , increasing the potential of developing side effects secondary to local tissue damage . upon reviewing the characteristics of recurrent cases in our series , the recurrent pterygia after using mmc were atrophic with scanty vascular components ( figure 2a ) and they were hardly noticeable by the patients ( ie , cosmetically insignificant ) . on the other hand , the recurrent cases following application of 5-fu shared a highly vascular mass with variable rate of regrowth ( figure 2b ) , which suggests that the role of such an agent in vascular growth suppression is negligible when compared to the antifibroblastic activity . several studies have recently emphasized the importance of vascular endothelial growth factor in the development and recurrence of pterygium.21 therefore , the timely recognition and treatment of an impending recurrence with subconjunctival anti - vegf drugs injection is crucial in stopping recurrences , especially when unusual vascular growth is noticed . the intraoperative application of antimetabolites is of value in reducing the recurrence rate after simple primary pterygium excision . both mmc and 5-fu were safe during the follow up period but a statistically significant high success rate and more cosmetically acceptable appearance after mmc use justifies recommending its use to be superior to 5-fu as a medical adjuvant in the surgical management of primary pterygium .

artificial tear preparations are important in the management of dry eye syndrome . we present the findings from four recently published studies conducted in russia assessing hylabak ( marketed as hyabak in europe ) , a preservative - free hyaluronic acid preparation , for the treatment of dry eye syndrome . all studies had an open , noncomparative design , but one compared the findings with those from 25 patients treated with tear naturale in previous studies . a total of 134 children and adults were enrolled , and the etiologies of dry eye syndrome included contact lens use , intensive office work , adenovirus eye infection , postmenopausal status , persistent meibomian blepharitis , sjgren s syndrome , phacoemulsification with intraocular lens implantation , and refractive surgery . the patients were treated with hylabak for 2 weeks to 2 months . all studies showed that hylabak resulted in marked improvement as assessed by subjective sensations / complaints , schirmer s test , norn s test , impression cytology and biomicroscopy , staining , and tear osmolarity . greater benefits were also reported compared with tear naturale , including a faster onset of action . hylabak was well tolerated . in conclusion , hylabak provided rapid and safe relief from the signs and symptoms of dry eye syndrome , as well as improvement in objective measures , in a wide range of patients .

symptoms include discomfort or burning sensation , photophobia , blurred or abnormal vision , and eye watering , and the longer - term sequelae can be serious.1 factors contributing to dry eye are numerous , and include aging , autoimmune disorders , infection , abnormalities of the lipid tear layer , menopause , wearing contact lenses , exposure to air conditioning , and use of computers . a modern understanding of dry eye includes etiologic factors such as inflammatory mediators , instability of the tear film , meibomian gland dysfunction , and hyperosmolarity of the tear film.2 the incidence of dry eye has risen considerably in recent years,3,4 partly due to increased computer use in air - conditioned offices . often known as office eye syndrome,5 it results from exposure of the precorneal tear film , corneal epithelium , and conjunctiva to artificial heating and dehumidified air provided by air conditioning and is likely amplified by the reduced eye blink rate and increased tear film evaporation consequent to extended viewing of computer screens . the everyday use of computers may be specifically related to the increased reporting of dry eye syndrome in younger populations,6 as may routine contact lens use , which frequently results in changes in the corneal epithelium and lacrimal film . a variety of treatments are available for dry eye , including surgery , moisture - retaining spectacles , and anti - inflammatory drugs , but artificial tear preparations are the cornerstone of dry eye management across the severity spectrum and almost 20 of these products are currently available in russia.7 they are widely used for the prolonged treatment of patients with early - stage dry eye syndrome and for improving ocular comfort in patients with transient secondary tear film instability ( eg , office workers ) . some such preparations have been developed based on the naturally occurring polysaccharide , hyaluronic acid . this has excellent water - retaining and lubricant properties , as well as viscoelastic effects that aid vision during blinks but maintain hydration and lubrication between blinks.8,9 studies have shown good efficacy in patients with dry eye syndrome.1014 hyperosmolarity has recently been identified as an important factor in the etiology of dry eye ; tear instability and hyperosmolarity are now considered to be interacting and key mechanisms in dry eye and precursors to inflammatory processes and corneal damage.15 indeed , the most recent definition of dry eye by the dry eye workshop ( 2007 ) makes specific mention of tear hyperosmolarity . clinical studies also suggest that hypotonic eye drops have an advantage over isotonic eye drops in the treatment of dry eye.1619 whilst the efficacy of hyaluronate - containing tear substitutes has been well demonstrated , there remain issues over the use of preservatives in such therapies , particularly when the treatment is likely to be long - term . it is now generally recognized that elimination of preservatives such as benzalkonium is important in the long - term safety and tolerability of ocular preparations,20,21 to the extent that some authorities consider the elimination of preservatives from tear substitutes as one of the most critical advances in the treatment of dry eye.22 the preservative - free hyaluronic acid preparation , hylabak ( laboratoires thea , clermont ferrand , france ) , has recently been developed to address this issue . hylabak comprises hyaluronic acid 0.15% and actinoquinol in a hypo - osmolar , preservative - free abak bottle ( laboratoires thea ) . sterility of the open container is assured by the abak system , that consists of a multidose eye drop dispenser closed by an adapted , small - pore sterilizing filter.23 we present here the findings from four recently published studies with broadly comparable methodologies conducted in russia that have assessed hylabak for the treatment of dry eye.2427 patients enrolled in these studies had a range of symptoms and etiologies ranging from contact lens use and visually intensive occupations to persistent meibomian blepharitis , sjgren s syndrome , and the sequelae of ocular surgery . patient characteristics and a methodologic summary of the four individual studies are shown in table 1 . one study,25 however , compared its findings with those from 25 patients treated with tear naturale ( 0.1% dextran 70 , 0.3% hydroxypropyl methylcellulose , preserved with benzylalkonium [ 0.05% ] , alcon laboratories , fort worth , tx , usa ) in previous studies.28 a total of 134 children and adults ( aged 755 years ) were enrolled in the studies . there were various etiologies accounting for the dry eye syndrome , including contact lens use , intensive office work , adenovirus eye infection , postmenopausal status , persistent meibomian blepharitis , sjgren s syndrome , phacoemulsification with intraocular lens implantation , and refractive surgery ( table 2 ) . the patients were treated with hylabak for 2 weeks to 2 months , depending on the study , and assessments were performed at various times during treatment . all studies assessed subjective sensations / complaints using a 04 scale ( 0 , absence of symptoms ; 1 , slight feeling of discomfort ; 2 , evident feeling of discomfort ; 3 , the worst feeling of discomfort ) , tear production ( schirmer s test , without anesthesia ) and tear stability / break - up time ( norn s test);29 other assessments ( impression cytology and biomicroscopy , staining , tear osmolarity ) performed in the individual studies are shown in table 1 . all four studies showed an improvement in dry eye syndrome of varying etiologies with hylabak treatment ( table 2 ) . a brief summary of the results from each of the individual studies is given below . maychuck and yani24 conducted an open study in 40 patients with dry eye syndrome ( adenovirus infection , soft contact lens use , phacoemulsification with intraocular lens implantation , and refractive surgery ) who were treated for 28 days . assessments were performed on days 1 , 7 , 14 , 21 , and 28 , and included subjective complaints , schirmer s test , norn s test , meniscometry , osmometry , and conjunctival xerosis . results for patients divided into those with mild or moderate disease showed that subjective complaints were reduced or had disappeared from the first days of treatment irrespective of symptom severity . complaints were typical of dry eye syndrome and included feelings of a foreign body in the eye , eye reddening , eyelid edema , itching , burning sensation , and variation in visual acuity . . tear production ( schirmer s test ) and precorneal tear break - up time ( norn s test ) were increased at each assessment in patients with both mild and moderate disease ( table 2 ) . similarly , there was an improvement to near normal values in tear osmolarity from 327 0.6 and 336 1.2 mosm / l in the mild and moderate groups , respectively , on day 1 to 286 1.7 and 302 1.5 mosm / l , respectively , on day 28 . in both mild and moderate groups combined , the height of the tear meniscus increased markedly from 0.38 0.2 mm on day 1 to 0.58 0.2 mm on day 28 , and conjunctival xerosis fell from 4.55 0.5 points on day 1 to 0.4 0.4 points on days 28 . the authors concluded that hylabak showed good therapeutic efficacy in the treatment of dry eye syndrome of varying etiologies and severity , and that it was well tolerated during prolonged use . brjesky et al25 conducted an open study in 32 patients with dry eye syndrome ( deficiency of tear film production , persistent meibomian blepharitis , sjgren s syndrome ) who were treated for 28 days , and the results were compared with those from 25 patients treated with tear naturale in previous studies . assessments were performed on days 3 , 7 , 14 , and 28 , and included subjective discomfort , schirmer s test , norn s test , tear meniscus index , and basal and total tear production . subjective discomfort , measured on a four - point scale from 0 ( no symptoms ) to 3 ( worst feelings of discomfort ) , was significantly ( p < 0.05 versus baseline ) reduced by hylabak from day 7 onwards in postmenopausal women ( 2.0 0.2 at baseline versus 1.2 0.1 on day 28 ) and from day 3 onwards in patients with persistent meibomian blepharitis ( 2.1 0.2 at baseline versus 0.6 0.1 on day 28 ) , or sjgren s syndrome ( 2.6 0.2 at baseline versus 1.4 0.2 on day 28 ) ( figure 1 ) . this compares with findings using tear naturale in previous studies in which significant differences were reported from day 7 onwards in postmenopausal women ( 2.0 0.1 at baseline versus 1.4 0.2 on day 28 ) and those with meibomian blepharitis , and from day 14 onwards ( 1.9 0.2 at baseline versus 0.8 0.1 on day 28 ) in those with sjgren s syndrome ( 2.6 0.2 at baseline versus 1.7 0.1 on day 28 ) . objective signs ( four - point scale ) were significantly ( p < 0.05 ) reduced by hylabak from day 14 onwards in postmenopausal women ( 1.4 0.1 at baseline versus 0.3 0.1 on day 28 ) , in patients with sjgren s syndrome ( 2.1 0.2 at baseline versus 1.3 0.1 on day 28 ) , and from day 7 onwards in those with meibomian blepharitis ( 1.1 0.1 at baseline versus 0.6 0.1 on day 28 , figure 2 ) . in contrast , no significant changes were seen with tear naturale in patients with meibomian blepharitis ( 1.2 0.1 at baseline versus 0.7 0.2 on day 28 ) or sjgren s syndrome ( 2.1 0.3 at baseline versus 1.5 0.1 on day 28 ) , and a significant decrease was seen only on day 28 in post - menopausal women ( 1.3 0.2 at baseline versus 0.5 0.1 on day 28 ) . tear film stability ( norn s test ) was significantly increased from day 3 onwards in all groups of patients given hylabak , and in postmenopausal women and patients with sjgren s syndrome given tear naturale ; patients with meibomian blepharitis given tear naturale had a significant improvement only from day 7 onwards . tear meniscus index significantly improved from day 3 onwards in all groups of patients , irrespective of treatment . increases from 1.1 0.1 , 1.9 0.1 , and 1.1 0.1 at baseline to 2.1 0.2 , 2.6 0.1 , and 2.2 0.1 at day 28 were observed with hylabak in postmenopausal women and those with meibomian blepharitis or sjgren s syndrome , respectively . the corresponding increases in patients given tear naturale were from 1.1 0.1 , 1.8 0.2 , and 1.1 0.1 at baseline to 1.9 0.1 , 2.4 0.1 , and 1.9 0.1 on day 28 . basal and total tear production was not significantly changed by either treatment in any group of patients . the results showed that both hylabak and tear naturale were effective in treating the various etiologies of dry eye syndrome , but that benefits with regard to subjective discomfort , objective signs , and tear film stability were more marked with hylabak ( although the differences between the groups did not reach statistical significance ) . in an open study by petrayevsky et al,26 32 women with dry ( office ) eye syndrome due to intensive use of personal computers in an air - conditioned office environment were treated for 2 weeks . assessments performed before and after treatment included recording of subjective complaints , schirmer s test , norn s test , and cytologic analysis of the bulbar conjunctiva . subjective signs and symptoms , measured on a four - point scale from 0 ( no symptoms ) to 3 ( severe ) , were improved after hylabak treatment in patients with mild or moderate symptoms ( figure 3 ) . patients with mild symptoms showed nonspecific objective symptoms ( eg , local edema of the bulbar conjunctiva involving the free edge of the eyelid and mild hyperemia ) , whilst those with moderate symptoms showed both specific ( eg , reduction of tear meniscus at the lid margin ) and nonspecific objective symptoms , all of which were improved or resolved after hylabak treatment . functional tests ( schirmer s test and tear break - up time assessed by norn s test ) were also significantly ( p < 0.01 ) improved after hylabak ( table 2 ) , with the exception of schirmer s test in patients with mild symptoms . cytologic examination of the conjunctiva showed that 50% of patients with mild symptoms had early loss of goblet cells and the remainder had total loss of goblet cells , all without keratinization ( tseng stage 1 and 2 , respectively ) . there was a marked improvement after hylabak treatment , with 28% of patients showing normalization and 61% with tseng stage 1 ( mild dry eye population ) . among the patients with moderate symptoms , tseng stage 1 was seen in 36% and tseng stage 2 in 64% at baseline . the authors concluded that hylabak resulted in subjective and objective improvement , as well as normalization of functional tests and the cytologic profile of the conjunctiva , in a population suffering from office eye syndrome . thirty children and adolescents were enrolled in this open study by nagorsky et al,27 and were assessed according to whether they wore soft contact lenses during the day or orthokeratologic lenses at night . assessments performed before treatment and after 1 and 2 months included subjective sensations , schirmer s test , norn s test , meniscometry and conjunctival xerosis . amongst these 20 patients , there was a marked improvement in subjective complaints after one month of hylabak treatment , that was increased further after 2 months ( figure 4 ) . when asked about the duration of the most comfortable period of wearing their contact lenses this was supported by improvements in objective measures to within the normal range ( table 3 ) . there was also a significant ( p < 0.05 ) increase in the height of the tear meniscus , from 0.62 0.20 mm to 0.95 0.20 mm after 2 months , and a reduction in the intensity of corneal xerotic changes . there was also a reduction in corneal xerotic change intensity with hylabak treatment in the 10 patients wearing orthokeratologic lenses , and patients reported that it was faster , easier , and more comfortable to manipulate their lenses . there were no cases of unpleasant sensation , discomfort , or prolonged blurred vision , and most patients noted improved comfort compared with previous eye drops . the authors concluded that hylabak had good therapeutic efficacy and was easy to use , cost - effective , and well tolerated in this group of young patients . maychuck and yani24 conducted an open study in 40 patients with dry eye syndrome ( adenovirus infection , soft contact lens use , phacoemulsification with intraocular lens implantation , and refractive surgery ) who were treated for 28 days . assessments were performed on days 1 , 7 , 14 , 21 , and 28 , and included subjective complaints , schirmer s test , norn s test , meniscometry , osmometry , and conjunctival xerosis . results for patients divided into those with mild or moderate disease showed that subjective complaints were reduced or had disappeared from the first days of treatment irrespective of symptom severity . complaints were typical of dry eye syndrome and included feelings of a foreign body in the eye , eye reddening , eyelid edema , itching , burning sensation , and variation in visual acuity . this was confirmed by objective measures . tear production ( schirmer s test ) and precorneal tear break - up time ( norn s test ) were increased at each assessment in patients with both mild and moderate disease ( table 2 ) . similarly , there was an improvement to near normal values in tear osmolarity from 327 0.6 and 336 1.2 mosm / l in the mild and moderate groups , respectively , on day 1 to 286 1.7 and 302 1.5 mosm / l , respectively , on day 28 . in both mild and moderate groups combined , the height of the tear meniscus increased markedly from 0.38 0.2 mm on day 1 to 0.58 0.2 mm on day 28 , and conjunctival xerosis fell from 4.55 0.5 points on day 1 to 0.4 0.4 points on days 28 . the authors concluded that hylabak showed good therapeutic efficacy in the treatment of dry eye syndrome of varying etiologies and severity , and that it was well tolerated during prolonged use . brjesky et al25 conducted an open study in 32 patients with dry eye syndrome ( deficiency of tear film production , persistent meibomian blepharitis , sjgren s syndrome ) who were treated for 28 days , and the results were compared with those from 25 patients treated with tear naturale in previous studies . assessments were performed on days 3 , 7 , 14 , and 28 , and included subjective discomfort , schirmer s test , norn s test , tear meniscus index , and basal and total tear production . subjective discomfort , measured on a four - point scale from 0 ( no symptoms ) to 3 ( worst feelings of discomfort ) , was significantly ( p < 0.05 versus baseline ) reduced by hylabak from day 7 onwards in postmenopausal women ( 2.0 0.2 at baseline versus 1.2 0.1 on day 28 ) and from day 3 onwards in patients with persistent meibomian blepharitis ( 2.1 0.2 at baseline versus 0.6 0.1 on day 28 ) , or sjgren s syndrome ( 2.6 0.2 at baseline versus 1.4 0.2 on day 28 ) ( figure 1 ) . this compares with findings using tear naturale in previous studies in which significant differences were reported from day 7 onwards in postmenopausal women ( 2.0 0.1 at baseline versus 1.4 0.2 on day 28 ) and those with meibomian blepharitis , and from day 14 onwards ( 1.9 0.2 at baseline versus 0.8 0.1 on day 28 ) in those with sjgren s syndrome ( 2.6 0.2 at baseline versus 1.7 0.1 on day 28 ) . objective signs ( four - point scale ) were significantly ( p < 0.05 ) reduced by hylabak from day 14 onwards in postmenopausal women ( 1.4 0.1 at baseline versus 0.3 0.1 on day 28 ) , in patients with sjgren s syndrome ( 2.1 0.2 at baseline versus 1.3 0.1 on day 28 ) , and from day 7 onwards in those with meibomian blepharitis ( 1.1 0.1 at baseline versus 0.6 0.1 on day 28 , figure 2 ) . in contrast , no significant changes were seen with tear naturale in patients with meibomian blepharitis ( 1.2 0.1 at baseline versus 0.7 0.2 on day 28 ) or sjgren s syndrome ( 2.1 0.3 at baseline versus 1.5 0.1 on day 28 ) , and a significant decrease was seen only on day 28 in post - menopausal women ( 1.3 0.2 at baseline versus 0.5 0.1 on day 28 ) . tear film stability ( norn s test ) was significantly increased from day 3 onwards in all groups of patients given hylabak , and in postmenopausal women and patients with sjgren s syndrome given tear naturale ; patients with meibomian blepharitis given tear naturale had a significant improvement only from day 7 onwards . tear meniscus index significantly improved from day 3 onwards in all groups of patients , irrespective of treatment . increases from 1.1 0.1 , 1.9 0.1 , and 1.1 0.1 at baseline to 2.1 0.2 , 2.6 0.1 , and 2.2 0.1 at day 28 were observed with hylabak in postmenopausal women and those with meibomian blepharitis or sjgren s syndrome , respectively . the corresponding increases in patients given tear naturale were from 1.1 0.1 , 1.8 0.2 , and 1.1 0.1 at baseline to 1.9 0.1 , 2.4 0.1 , and 1.9 0.1 on day 28 . basal and total tear production was not significantly changed by either treatment in any group of patients . the results showed that both hylabak and tear naturale were effective in treating the various etiologies of dry eye syndrome , but that benefits with regard to subjective discomfort , objective signs , and tear film stability were more marked with hylabak ( although the differences between the groups did not reach statistical significance ) . in an open study by petrayevsky et al,26 32 women with dry ( office ) eye syndrome due to intensive use of personal computers in an air - conditioned office environment were treated for 2 weeks . assessments performed before and after treatment included recording of subjective complaints , schirmer s test , norn s test , and cytologic analysis of the bulbar conjunctiva . subjective signs and symptoms , measured on a four - point scale from 0 ( no symptoms ) to 3 ( severe ) , were improved after hylabak treatment in patients with mild or moderate symptoms ( figure 3 ) . patients with mild symptoms showed nonspecific objective symptoms ( eg , local edema of the bulbar conjunctiva involving the free edge of the eyelid and mild hyperemia ) , whilst those with moderate symptoms showed both specific ( eg , reduction of tear meniscus at the lid margin ) and nonspecific objective symptoms , all of which were improved or resolved after hylabak treatment . functional tests ( schirmer s test and tear break - up time assessed by norn s test ) were also significantly ( p < 0.01 ) improved after hylabak ( table 2 ) , with the exception of schirmer s test in patients with mild symptoms . cytologic examination of the conjunctiva showed that 50% of patients with mild symptoms had early loss of goblet cells and the remainder had total loss of goblet cells , all without keratinization ( tseng stage 1 and 2 , respectively ) . there was a marked improvement after hylabak treatment , with 28% of patients showing normalization and 61% with tseng stage 1 ( mild dry eye population ) . among the patients with moderate symptoms , tseng stage 1 was seen in 36% and tseng stage 2 in 64% at baseline . the authors concluded that hylabak resulted in subjective and objective improvement , as well as normalization of functional tests and the cytologic profile of the conjunctiva , in a population suffering from office eye syndrome . thirty children and adolescents were enrolled in this open study by nagorsky et al,27 and were assessed according to whether they wore soft contact lenses during the day or orthokeratologic lenses at night . assessments performed before treatment and after 1 and 2 months included subjective sensations , schirmer s test , norn s test , meniscometry and conjunctival xerosis . amongst these 20 patients , there was a marked improvement in subjective complaints after one month of hylabak treatment , that was increased further after 2 months ( figure 4 ) . when asked about the duration of the most comfortable period of wearing their contact lenses , the patients reported a more than three - fold increase with hylabak treatment . this was supported by improvements in objective measures to within the normal range ( table 3 ) . there was also a significant ( p < 0.05 ) increase in the height of the tear meniscus , from 0.62 0.20 mm to 0.95 0.20 mm after 2 months , and a reduction in the intensity of corneal xerotic changes . there was also a reduction in corneal xerotic change intensity with hylabak treatment in the 10 patients wearing orthokeratologic lenses , and patients reported that it was faster , easier , and more comfortable to manipulate their lenses . there were no cases of unpleasant sensation , discomfort , or prolonged blurred vision , and most patients noted improved comfort compared with previous eye drops . the authors concluded that hylabak had good therapeutic efficacy and was easy to use , cost - effective , and well tolerated in this group of young patients . all four studies found that hylabak resulted in a marked improvement in patients with dry eye syndrome of heterogeneous etiology . subjective sensations and complaints were reduced , and these findings were supported by results from a wide range of objective measures , including schirmer s test , norn s test , impression cytology and biomicroscopy , staining , and tear osmolarity . complete resolution of signs and symptoms and normalization of functional tests and cytologic profile was reported in some patients . hylabak resulted in rapid relief from symptoms . in one study assessing effects during the first week of treatment , there were statistically significant improvements in subjective discomfort , tear film stability , and tear meniscus index from day 3 onwards , with objective signs improving significantly from day 7 in some patients . in contrast , significant improvements with tear naturale did not occur until later in treatment . in addition , benefits on subjective discomfort , objective signs , and tear film stability were more marked with hylabak than with tear naturale . not all artificial tear preparations are suitable for all types of dry eye syndrome patients . whilst preserved artificial tears may be more convenient for short - term use , for example , a preservative - free formulation would be more appropriate where treatment is likely to continue on a long - term basis . a review of the patients included in the four studies discussed here shows that hylabak was safe and effective in a wide range of dry eye syndrome etiologies including contact lens use , intensive exposure to air - conditioning and computer use , eye infection , postmenopausal status , meibomian blepharitis , sjgren s syndrome , phacoemulsification with intraocular lens implantation , and refractive surgery . there was also a wide age range , with one study enrolling children from 7 years of age . hylabak proved to be well tolerated , effective and easy to use in these young patients , and patients reported that they found contact lens use faster , easier , and more comfortable compared with their previous eye drops . treatment was given daily in all four studies , suggesting that hylabak is suitable for patients with secondary transitional dry eye syndrome , such as office workers who require regularly improved ocular comfort . the findings in this wide - ranging population suggest that hylabak is an ideal choice for first - line treatment of dry eye syndrome , irrespective of the etiology of the disorder . in conclusion , hylabak provided rapid and safe relief from the signs and symptoms of dry eye syndrome , as well as improvement in objective measures , in patients with a wide range of dry eye etiologies .

this study was conducted to investigate the effects of foliar endophytic fungi and arbuscular mycorrhizal fungi ( amf ) on plant community structure in experimental microcosms containing an assemblage of five species of plants ( oenothera odorata , plantago asiatica , trifolium repens , isodon japonicas and aster yomena ) . leaves of sasa borealis , potentilla fragarioides , and viola mandshurica were collected in chungbuk , korea . endophytic fungi were isolated from the surface sterilized leaves and identified to species level using molecular and morphological techniques . four isolates of the endophytic fungi were inoculated to the leaves of host plants in the microcosms . also , three species of amf spores were extracted from pure cultures and the mixture of the three species inoculated to the roots of the plants . after four months of growth in a green house , effects of both symbiotic fungi on plant species diversity , community composition and productivity were examined . the plant species diversity showed significant differences with inoculation of the symbiotic fungi . results indicate that amf significantly affect plant productivity and plant community structure .

healthy leaves of capsicum annuum , sasa borealis , potentilla fragarioides and viola mandshurica collected from an arable site and its surrounding field areas in goesan - gun , chungbuk , korea ( 128 1'e , 36 46'n ) . only green leaves without signs of insect or microbial injury were used for fungal isolation . collected leaves were gently washed with ddh2o and cut with a sterile scalpel into a small piece ( 1 1 cm ) . the pieces were surface - sterilized with 5% sodium hypochlorite ( 5 min ) and 70% ethyl alcohol ( 1 min ) . after surface sterilization , four pieces on 0.5% malt extract agar ( mea ) medium and the medium was incubated in dark at 25 for four weeks . fungal hyphae and structures from slide cultures were observed under a light microscope . the size of condia was measured and the shape was carefully described . for molecular identification , total dna of the fungi was extracted from cultures isolated from each plant using dneasy plant mini kit ( qiagen science , usa ) and was amplified using pcr . the partial internal transcribed spacer ( its ) region including 5.8 s ribosomal dna ( rdna ) was amplified with a universal primer set , its1f and its4 , for fungi ( gardes and bruns , 1993 ) . the thermal cycler was programmed for 1 cycle of 3 min at 94 , and 30 cycles of 1 min at 94 denaturing , 1 min at 55 annealing and 1 min at 72 extension , and finally 1 cycle of 5 min at 72 for hold . nucleotide sequences were determined using abiprism 377 automated sequencer ( perkin - elmer , usa ) . a sequence similarity search of the national center for biotechnology information ( ncbi ) database was conducted using the basic local alignment search tool ( blast ) algorithm . two species of pestalotiopsis ( p. sp1 and p. sp2 ) were identified using morphological and molecular characteristics ( table 1 ) . spores of four species of amf , acaulospora longula , glomus mosseae and archeospora leptotica were extracted from pure cultures using wet - sieving and sucrose density gradient centrifugation methods ( daniels and skipper , 1982 ) . the extracted spores were observed under light microscopes and identified based on their morphological characteristics such as color , shape , surface ornamentation , contents and wall structures of spores ( schenck and perez , 1990 ) . am fungal spores were surface - sterilized for inoculation ( hildebrandt et al . , 2002 ) . three treatments by types of fungal inoculum were designed in this study ; amf only , endophytes only , and combination amf and endophytes . no inoculum on plants was included as control . for root inoculation with amf , plastic pots ( 50(w ) 38(l ) 8(h ) cm ) were filled with the sterilized vermiculites and sands ( 1 : 1 , v / v ) . seeds of five plant species ( oenothera odorata , plantago asiatica , trifolium repens , isodon japonicas and aster yomena ) were obtained from a commercial seed source ( seedkorea co. , ltd . , a potting medium were mixed with four species of amf spores , and 400 spores of each amf species were contained in a pot . for leaf inoculation , newly emerged leaves of healthy plants were inoculated with mycelium of four species of endophytes by brushing without wounds . each treatment was replicated four times at the same time . after four months of growth in a greenhouse , effects of both symbiotic fungi on species diversity , species composition , and productivity of plant community within the microcosm were examined . dry weights of plants were measured after dehydration in a drying oven at 70 for 48 hours . shannon - wiener index of species diversity ( h ' ) was calculated for plant community analysis ( magurran , 1988 ) . all data was analyzed with one - way analysis of variance ( anova ) using statistical package spss - win . the mean values were compared by fisher 's least significant difference test ( lsd , p < 0.05 ) . healthy leaves of capsicum annuum , sasa borealis , potentilla fragarioides and viola mandshurica collected from an arable site and its surrounding field areas in goesan - gun , chungbuk , korea ( 128 1'e , 36 46'n ) . only green leaves without signs of insect or microbial injury were used for fungal isolation . collected leaves were gently washed with ddh2o and cut with a sterile scalpel into a small piece ( 1 1 cm ) . the pieces were surface - sterilized with 5% sodium hypochlorite ( 5 min ) and 70% ethyl alcohol ( 1 min ) . after surface sterilization , four pieces on 0.5% malt extract agar ( mea ) medium and the medium was incubated in dark at 25 for four weeks . fungal hyphae and structures from slide cultures were observed under a light microscope . the size of condia was measured and the shape was carefully described . for molecular identification , total dna of the fungi was extracted from cultures isolated from each plant using dneasy plant mini kit ( qiagen science , usa ) and was amplified using pcr . the partial internal transcribed spacer ( its ) region including 5.8 s ribosomal dna ( rdna ) was amplified with a universal primer set , its1f and its4 , for fungi ( gardes and bruns , 1993 ) . the thermal cycler was programmed for 1 cycle of 3 min at 94 , and 30 cycles of 1 min at 94 denaturing , 1 min at 55 annealing and 1 min at 72 extension , and finally 1 cycle of 5 min at 72 for hold . nucleotide sequences were determined using abiprism 377 automated sequencer ( perkin - elmer , usa ) . a sequence similarity search of the national center for biotechnology information ( ncbi ) database was conducted using the basic local alignment search tool ( blast ) algorithm . two species of pestalotiopsis ( p. sp1 and p. sp2 ) were identified using morphological and molecular characteristics ( table 1 ) . spores of four species of amf , acaulospora longula , glomus mosseae and archeospora leptotica were extracted from pure cultures using wet - sieving and sucrose density gradient centrifugation methods ( daniels and skipper , 1982 ) . the extracted spores were observed under light microscopes and identified based on their morphological characteristics such as color , shape , surface ornamentation , contents and wall structures of spores ( schenck and perez , 1990 ) . am fungal spores were surface - sterilized for inoculation ( hildebrandt et al . , 2002 ) . three treatments by types of fungal inoculum were designed in this study ; amf only , endophytes only , and combination amf and endophytes . no inoculum on plants was included as control . for root inoculation with amf , plastic pots ( 50(w ) 38(l ) 8(h ) cm ) were filled with the sterilized vermiculites and sands ( 1 : 1 , v / v ) . seeds of five plant species ( oenothera odorata , plantago asiatica , trifolium repens , isodon japonicas and aster yomena ) were obtained from a commercial seed source ( seedkorea co. , ltd . , a potting medium were mixed with four species of amf spores , and 400 spores of each amf species were contained in a pot . for leaf inoculation , newly emerged leaves of healthy plants were inoculated with mycelium of four species of endophytes by brushing without wounds . each treatment was replicated four times at the same time . after four months of growth in a greenhouse , effects of both symbiotic fungi on species diversity , species composition , and productivity of plant community within the microcosm were examined . dry weights of plants were measured after dehydration in a drying oven at 70 for 48 hours . shannon - wiener index of species diversity ( h ' ) was calculated for plant community analysis ( magurran , 1988 ) . all data was analyzed with one - way analysis of variance ( anova ) using statistical package spss - win . the mean values were compared by fisher 's least significant difference test ( lsd , p < 0.05 ) . experimental microcosms contained an assemblage of five species of plants treated with amf and endophytes were set up in a greenhouse . after four months of growth , effects of the fungi on plant productivity were examined . the responses of plant growth to each treatment showed variation among plant species ( table 2 ) . growth of o. odorata , p. asiatica , i. japonicus and a. yomena in the community inoculated with amf was significantly increased compared to controls , while no significant growth effect of amf was detected in t. repens . also , microsms inoculated with both symbiotic fungi , the growth of o. odorata , p. asiatica , i. japonicus and a. yomena was significantly increased compared to controls . however , in the communities inoculated with endophytes , a significant increase of dry weights was observed only in p. asiatica and i. japonicas . in plants with amf , mean biomass of all 5 plant species were significantly higher than biomass of control for each plant species , indicating positive mycorrhizal responsive ( mr , table 3 ) . mr showed significant variation among plant species ; highest in p. asiatica and lowest in a. yomena . the total biomass of plants in a microcosm was significantly higher in communities with amf or with both symbiotic fungi than control ( table 2 ) . however , a significant increase of growth was not observed in the microcosms inoculated with endophytes . the species diversity index ( h ' ) was significantly increased in the plant community inoculated with symbiotic fungi ( fig . the number of species in the microcosm was not changed during the period of this study , but relative abundances of each species consisting of a community were changed ( table 2 ) . there was a positive correlation between species diversity and total dry weight of plants per microcosm ( fig . however , influence of amf and endophytes to host plants is not fully understood yet . in this study , the effects of foliar endophytes and amf on plant community in experimental microcosms containing an assemblage of five species of plants were investigated . the results in this study showed that amf and endophytes affect growth of host plants and plant community structure . for example , the nutrition and inter - specific relationships of plants were affected by microbes of root systems ( stanton , 1988 ) and therefore composition of the plant community was affected by mycorrhizas ( allen and allen , 1984 ; bever , 1994 ) . nutrients flowing through the hyphal connection plant to plant affect both growth and competition ability of plants resulted in influence of the species composition in plant community . also , studies were reported that host plant species differed in their dependency on amf ( hetrick et al . 1998 ) as in this study ( table 3 ) showing variation among the plant species in mycorrhizal response of each plant species . although amf increased the growth of all plants in the community , they could influence relative abundance of each species in the community due to the interspecific variation of response to the mycorrhizas . as a result , in this study , amf would contribute to raise the species evenness of plant community . mycorrhizal symbiosis can strongly influence the patterns and intensity of both intraspecific density effects and interspecific competition of plants ( grime et al . mycorrhizas were played a vital role in the maintenance of biological diversity on the plant community . a significant increase was not observed in the productivity of the community with endophytes in this study . however , relative abundance of plant species in the communities inoculated with endophytes was changed and the species evenness of plant community was increased with inoculation of endophytes , indicating endophytes increased species diversity of plant community through increasing species evenness . in this study , endophytes changed the relative abundance by increasing growth of two species , p. asiatica and i. japonicus , in the community and the endophytes infected to these plants could promote resistance of the host plants to various environmental stresses ( west et al . , 1993 ) . it has been observed that plants infected with the endophytic fungi had greater resistance to the drought and high temperatures ( marks and clay , 1996 ) . the increased resistance to the environmental stresses with infection of endophytes could raise inter - specific competitive ability of the plants and these factors could influence the composition of plant community ( hill et al . the community treated with both symbiotic fungi showed significant differences in growth responses of plant in the community from the community with only amf and endophytes , respectively . the biomass of p. asiatica in the community treated with both symbiotic fungi was lower than when treating only amf , suggesting that the endophytes could negatively influence amf of p. asiatica . if plants were infected with endophytes , the movement of photosynthetic products toward a root could be limited , and it would be possible to affect growth of amf ( clay , 1992 ) . also , toxic metabolites produced by endophytes may inhibit colonization of mycorrhizal fungi . in this study , the plant species diversity and the total dry weight of plants showed a significant positive correlation . these results were supported by preceding studies showing that the productivity in the plant community was increased with the increased plant diversity ( van der heijden et al . , 1998 ) . results of this study indicate that both symbiotic fungi may significantly affect the plant community structure . the interactions between the symbiotic fungi and host plants might increase the species diversity and plant productivity . the result of study would increase the scope of understanding with respect to the role of symbiotic fungi on the plant community .

pulmonary hypertension ( ph ) contributes to the mortality of patients with lung and heart diseases . however , the underlying mechanism has not been completely elucidated . accumulating evidence suggests that inflammatory response may be involved in the pathogenesis of ph . macrophage migration inhibitory factor ( mif ) is a critical upstream inflammatory mediator which promotes a broad range of pathophysiological processes . the aim of the study was to investigate the role of mif in the pulmonary vascular remodeling of hypoxia - induced ph . we found that mif mrna and protein expression was increased in the lung tissues from hypoxic pulmonary hypertensive rats . intensive immunoreactivity for mif was observed in smooth muscle cells of large pulmonary arteries ( pas ) , endothelial cells of small pas , and inflammatory cells of hypoxic lungs . mif participated in the hypoxia - induced pasmcs proliferation , and it could directly stimulate proliferation of these cells . mif - induced enhanced growth of pasmcs was attenuated by mek and jnk inhibitor . besides , mif antagonist iso-1 suppressed the erk1/2 and jnk phosphorylation induced by mif . in conclusion , the current finding suggested that mif may act on the proliferation of pasmcs through the activation of the erk1/2 and jnk pathways , which contributes to hypoxic pulmonary hypertension .

chronic pulmonary hypertension ( ph ) is a disease characterized by a sustained pulmonary arterial pressure with increases in pulmonary vascular resistance . the pathogenesis of ph has been ascribed to two mechanisms , the initial event of vasoconstriction followed by remodeling of small- and medium - sized pulmonary arteries which is a hallmark of severe and advanced pulmonary hypertension . the main pathological change related to vascular remodeling is an abnormal pulmonary artery smooth muscle cells ( pasmcs ) hypertrophy and proliferation resulting in obstruction of small pulmonary arteries [ 2 , 3 ] . recently , more attention has been given to the facts that pulmonary inflammation could contribute to hypoxic vasoconstriction and remodeling . it has been reported that inflammatory cell infiltrates in the areas of plexiform lesions in human severe chronic pulmonary arterial hypertension . circulating inflammatory and/or progenitor cells contribute to hypoxia - induced pulmonary vascular remodeling [ 5 , 6 ] . in addition , accumulating evidence confirms that chronic hypoxia results in the increased expression of lung inflammatory cytokines and chemokines , including interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , tumor - necrosis - factor- ( tnf- ) , and fractalkine , which may potentiate the development of ph [ 710 ] . for instance , il-6 promotes the development and progression of pulmonary vascular remodeling and ph through proproliferative antiapoptotic mechanisms . therefore , there is a growing interest in inflammatory mediators in the pulmonary hypertensive process . macrophage migration inhibitory factor ( mif ) was originally identified as a t - cell - derived cytokine that inhibits the random migration of macrophages . currently , mif is proved to be an important proinflammatory cytokine , secreted by most of the cells including t cells , macrophages / monocytes , endothelial cells , and smooth muscle cells and induce the production of a large number of inflammatory mediators , such as tnf- , il-1 , il-6 , and il-8 [ 12 , 13 ] . thus , many studies revealed mif to be involved in the pathogenesis of inflammatory diseases , such as atherosclerosis , rheumatoid arthritis , sepsis , asthma , and acute respiratory distress syndrome [ 1418 ] . besides , mif may act beyond inflammatory cytokine , as suggested by its proliferative effect on vascular smooth muscle cells demonstrated in atherosclerosis . the evidence provides an idea that mif might play a role in the vascular disease . however , there is no report that mif participates in the pulmonary vascular remodeling during exposure to chronic hypoxia . in the present study for that purpose , the expression of mif was examined in the lungs of hypoxic pulmonary hypertensive ( hph ) rats . then we investigated the effect of mif on hypoxia - induced pasmcs proliferation and the underlying mechanism . all of the experimental procedures were approved by the animal use and care committee for research and education of the fourth military medical university . recombinant mouse mif was purchased from r&d systems ( minneapolis , mn , usa ) . mtt , pd 98059 , sb 203580 , and sp 600125 were from sigma ( st . louis , mo , usa ) . chuan - min hu , the third military medical university , chong qing , china . antibodies to erk1/2 and phospho - erk1/2 , jnk and phospho - jnk were from cell signaling ( beverly , ma , usa ) . the final amount of dmso in the bath solution was less than 0.1% . according to the previous reports , rats were housed intermittently in a chamber containing 10% oxygen , for exposure to continuous hypobaric hypoxia . the intermittent regime consisted of 10 hours in the hypoxic chamber followed by 14 hours in room air ( 21% oxygen ) . the normoxic control rats were housed continuously in room air . at the end of hypoxia exposure , measurement of the right ventricle systolic pressure ( rvsp ) and the ratios of right ventricle/[left ventricle + septum ] ( rv/[lv+s ] ) weight were determined . briefly , as soon as median sternotomy was performed , lungs were removed with hearts in fresh pbs . under a dissecting microscope , the 2nd-3rd - division ( external diameter < 300 m ) pulmonary arteries were isolated carefully . after the adventitial layers together with the surrounding tissue and endothelium were removed , the pulmonary arteries were dissected into small pieces and cultured in dmem supplemented with 100 u / ml penicillin , 0.1 mg / ml streptomycin , 2 mm l - glutamine , and 10% fbs and grown in humidified incubators at 37c in 95% o2 and 5% co2 . cells were used for experiments between passages 3 and 6 . in the hypoxic groups , pasmcs were transferred into a hypoxic chamber containing 2% o2 , 5% co2 , and 93% n2 for 24 hours . before exposure to hypoxia or treatment with different agents , lungs were homogenized , and total rna was extracted from the lung tissues by using the rneasy total rna isolation kit ( qiagen , valencia , ca ) . the primers for the rat mif gene were sense , 5-tctccgccaccatgcctatg-3 , and antisense , 5-gggtcgctcgtgccactaaa-3 , and for the housekeeping gene -actin were sense , 5-atcatgtttgagaccttcaaca-3 , and antisense , 5-catctcttgctcgaagtcca-3. pcr reaction was carried out under the following conditions : 30 cycles of denaturation at 94c for 30 s , annealing at 56c for 30 s , and extension at 72c for 30 s. a final extension was performed at 72c for 1 min . lung homogenates were prepared in ripa lysis buffer , containing 50 mm tris ( ph 7.4 ) , 150 mm nacl , 1% triton x-100 , 1% sodium deoxycholate , 0.1% sds , 2 mm naf , 5 mm edta ( ph 8.0 ) , and 1 mm sodium orthovanadate ( beyotime inc , jiangsu , china ) . the protease inhibitor of phenylmethylsulfonyl fluoride ( pmsf , 1 mm ) was added to the ripa buffer in advance . equivalent amounts of protein ( 30 g ) from each sample were separated on 12% sds - polyacrylamide gels and then transferred onto 0.22 m nitrocellulose filter membranes ( millipore , bedford , usa ) . the membrane was blocked and incubated with primary antibodies for mif , or phosphospecific erk1/2 , erk1/2 , phospho - jnk , and jnk . the levels of proteins and phosphoproteins were detected with enhanced chemiluminescent substrate ( pierce , rockford , il , usa ) . mif ( 50 , 100 , 200 ng / ml ) or pd 98059 ( 20 m ) , sb 203580 ( 20 m ) and sp 600125 ( 20 m ) and iso-1 ( 10 , 50 , 100 m ) were added , respectively . after being cultured for 24 hours under normoxic condition or hypoxia exposure , solution mtt was added into each well with a 5 mg / ml concentration . cells were cultured for another 4 hours , and then dimethyl sulfoxide ( dmso ) was added in . after vibrating for 10 minutes , the optical density values were detected at 490 nm wavelength by using a spectrophotometer ( bio - tek power wave xs , usa ) . cells were stimulated with mif or inhibitors as described above , respectively , and after 24 hours they were harvested by mild trypsinization and counted with a hemocytometer . statistical analysis was processed by using one - way anova , followed by lsd test for post hoc multiple comparisons ( spss for windows version 16.0 , chicago , usa ) . rats exposed to hypoxia for 28 days developed pulmonary hypertension ( table 1 ) , as demonstrated by an increase in rvsp ( 48 2.8 mmhg in hypoxic rats versus 23 0.5 mmhg in control rats ) ( n = 8 , p < 0.05 ) and the ratios of rv/(lv+s ) weight ( 0.41 0.05 in hypoxic rats versus 0.26 0.02 in control rats ) ( n = 10 , p < 0.05 ) . lung homogenates from hypoxic rats showed increases in both mif mrna ( figure 1(a ) ) and protein ( figure 1(b ) ) compared with control rats . as the immunohistochemistry results show , there was mif staining in bronchial epithelial cells ( figure 2(b ) ) , but no positive immunoreactivity for mif in the pulmonary vasculature in control rats ( figures 2(a ) and 2(b ) ) , while intense mif staining of smooth muscle cells of large pulmonary arteries ( diameter > 100 m ) ( figure 2(d ) ) , endothelial cells of smaller pa ( diameter < 100 m ) , and bronchial epithelial cells ( figure 2(e ) ) , as well as the inflammatory cells around the alveoli ( figure 2(f ) ) , was observed in hypoxic lungs . it is known that hypoxia exposure significantly increases the pasmcs proliferation , but we found that this proliferation was obviously inhibited by three various concentrations of mif antagonist iso-1 ( figure 3 , p < 0.05 ) . iso-1 had no effect on normoxic control pasmcs proliferation . since we have established that hypoxia enhanced mif expression and mif contributed to the hypoxia - induced proliferation of pasmcs , we further tested the direct effect of mif on pasmcs proliferation . both mtt assay and cell counting showed that higher concentration ( 100 , 200 ng / ml ) of mif could directly stimulate pasmcs proliferation ( figure 4 ) . as shown in figure 5(a ) , 100 ng / ml mif - induced pasmcs proliferation was significantly blocked by specific mek inhibitor pd 98059 and jnk inhibitor sp 600125 ( p the similar results were found as evaluated by cell counting ( figure 5(b ) ) . 100 ng / ml mif - induced erk1/2 activation was increased in a rapid time - dependent manner with maximal at 30 min and followed by a downregulation of erk1/2 phosphorylation after 60 min ( figure 6(a ) ) . mif could also activate phosphorylation of jnk with peaks at 60 min ( figure 6(b ) ) . treatment with iso-1 ( 50 m ) , mif - induced both erk1/2 and jnk activation was attenuated markedly ( figure 7 ) . chronic hypoxic exposure induces changes in the structure of pulmonary artery which is associated with increased pulmonary vascular resistance , pulmonary hypertension , and right heart failure . the present study provides evidence that mif is upregulated in the lungs of hph rats and stimulates rats pasmcs proliferation , indicating a possible role for this cytokine in the pathogenesis of ph . investigations revealed that chronic hypoxia induced upregulation of gene expression of a wide spectrum of proinflammatory mediators , including chemokines and their receptors , cytokines , growth and differentiation factors , and adhesion and fibrosis - associated molecules . it is increasingly appreciated that inflammatory mediators could directly contribute to the pulmonary vascular remodeling . they have significant effects on the local vascular wall cells , including increases in proliferation and matrix protein production . it is reported that il-6 may affect pulmonary vascular remodeling via direct stimulation of vascular smooth muscle cell ( smc ) migration or by indirect effects on vascular smc proliferation . in addition , increased serum level of il-1 was observed in the serum of patients with severe primary pulmonary hypertension . these findings suggest that inflammatory mediators are closely associated with pulmonary hypertensive process . as a critical proinflammatory cytokine , mif is constitutively expressed in a variety of immune and nonimmune cells and also effectively secreted from various phenotype cells into the circulation . upon secretion , mif exhibits broad regulatory properties , including stimulation of the growth of a number of cell lines , except for a key mediator in a number of immune and inflammatory diseases . yang detected that mif was a potent human - endothelial - cell - growth - promoting agent . neutralizing mif bioactivity in atherosclerosis - susceptible mice reduces vascular smc proliferation and neointimal thickening . additionally , fu et al . found that mif mediated the hypoxia response of vascular smc , including cell migration and proliferation . in human , it is reported that patients with ph showed higher mif levels than patients without these manifestations in systemic sclerosis . however , no studies have yet addressed the role of mif in pasmcs proliferation in the pulmonary circulation exposed to hypoxia . therefore , it will be of interest to explore whether mif affects pasmcs proliferation during exposure to chronic hypoxia and contributes to the pulmonary vascular remodeling . in the current study , we found that both mif mrna and protein expression were increased in the lung tissues from hph rats . furthermore , the increased mif mostly located in smooth muscle cells of large pulmonary arteries and endothelial cells of smaller pa , as well as the inflammatory cells and bronchial epithelial cells . these results indicated that mif overexpression under hypoxia exposure condition might influence the cells in the vascular walls . due to the key role of pasmcs in hph as the results show , the isolated pasmcs proliferated in response to hypoxia , which was consistent with previous reports [ 29 , 30 ] . iso-1 is a small molecule inhibitor targeting mif which inhibits the catalytic site of mif and leads to a marked reduction in the biological function of mif [ 31 , 32 ] . next , to observe the direct stimulatory effect of mif on pasmcs growth , recombinant mif was administrated to cells however , there is evidence showing that proliferation of pasmcs was not affected by exogenous mif . . we also added 1% fbs to the culture medium which may contribute to the stimulated effect of mif . thus , our findings add mif to the list of proliferative agents which may contribute to pasmcs proliferation in ph . although mif is a weak stimulator of proliferation , it might work in combination with something else such as other inflammatory mediators , or its receptor expression is altered during hypoxia exposure , which needs to be further studied . mitogen - activated protein kinases ( mapks ) , including extracellular signal - regulated kinase ( erk ) , c - jun nh2-terminal kinase ( jnk ) , and p38 map kinase ( p38 ) , play the critical role in cell proliferation , survival , or apoptosis [ 34 , 35 ] . studies have implied that mif - mediated signaling is associated with a sustained phosphorylation and activation of the p44/p42 erk1/2 subfamily of mapk [ 36 , 37 ] . on the other hand , mif could mediate phosphorylation of jnk in septic shock and utilizes the jnk pathway in t cells and fibroblasts . also , mif treatment strongly activated erk1/2 and p38 mapk in endometriotic cells . showed that mif mediated adhesion molecule expression via the promotion of p38 mapk activation in human endothelial cells . although mif has been reported to activate mapk signaling in several phenotype cells , there is no available information on the role of mapk pathway in mif - induced pasmcs response . here , we demonstrated that mek and jnk inhibitors block mif - stimulated pasmcs growth , respectively . thus , our findings support the idea that erk1/2 and jnk signaling participate in the mif proliferative pathway . but our findings also show that p38 mapk did not appear to contribute to mif - promoted pasmcs proliferation . the previous findings of amin reported that mif - induced migration of human dermal microvascular endothelial cells was not blocked by inhibitors of src and p38 mapk . it indicated that mif mediated the various effects through different signal transduction pathways in different cell types . in summary , the current study showed that mif expression was increased in the lungs from hph rats . this cytokine may act as a growth factor for pasmcs partially through erk1/2 and jnk pathway without the involvement of p38 mapk . we conclude that mif contributes to the hypoxic pulmonary hypertension , but further studies will be needed to extend these findings , with the aim of identifying new therapeutic targets for treatment of pulmonary hypertension .

angiomyolipoma of the liver ( aml ) is an infrequent neoplasm composed of three tissues ( adipose , muscle and vessels ) . in spite of advances in radiology , preoperative correct diagnosis is difficult . clasically , a conservative management strategy was adopted in patients with asymptomatic tumors less than 5 cm with undoubtful diagnosis . but after publishing some few cases of malignant angiomyolipoma a more radical has been advocated . laparoscopic resection of liver tumors is becoming a excellent approach for operating on benign liver tumors . usually is performed using five trocars but in some cases a less invasive technique with three trocars could be used . we present a laparoscopic resection of liver angiomyolipoma in a 65 year - old male using only three trocars and also discuss the optimal management of aml and technical tips of three - trocar technique .

the angiomyolipoma of the liver ( aml ) is a very infrequent benign tumor characterized by three components : adipose , vascular , and muscular [ 17 ] . progresses achieved in imaging techniques have substantially increased the number of correct preoperative diagnoses of aml . the publications in the medical literature of some cases that confirm a malign transformation of aml have made the surgical indications for this pathology a controversial issue [ 3 , 4 , 6 ] . laparoscopic approach , when feasible , is a technique of choice that is gaining ground and which is commonly performed with the use of 4 or 5 trocars [ 8 , 9 ] . we describe here a new case concerning a laparoscopic resection with only three trocars ; we have revised the literature and discussed surgical indications . a 65-year - old male patient presented with epigastric pain , with no medical records of interest , and was not affected by tuberous sclerosis . ultrasonography and abdominal ct ( figure 1 ) revealed an adipose - looking lesion of around 4 cm located on the left lateral hepatic sector . the patient was placed in the french position : the surgeon between his legs and the assistant on the left side of the patient . three trocars were placed : a 10 mm trocar in umbilical position for the 30 camera , a 5 mm trocar placed in the right hypochondrium , and another 10 mm trocar ( operator 's trocar ) in the left hypochondrium ( see figure 2 ) . pringle 's maneuver was prepared passing a nylon loop , although no vascular control was applied . a partial left sectionectomy was carried out , sealing the parenchyma with ligasure 5 mm ( figure 3 ) . the piece was removed in a bag through the umbilical trocar , widening the incision up to 2 cm . the procedure lasted 40 minutes , and the loss of blood amounted to 75 cc . macroscopically , it is a well - delimited 4 cm lesion , although not encapsulated . the histological study revealed a mesenchymal lesion with a muscular , adipose , and vascular component . immunohistochemical study was negative for pankeratin , ae1-ae3 , and cd117 but positive for s-100 with reference to the adipose tissue ; hmb45 was positive within the cytoplasm of spindle - shaped and epithelioid cells and also for actin and desmin in the vascular component of the thick wall ( figure 4 ) , so the final diagnosis confirmed a benign mixed angiomyolipoma . the radiological control carried out 12 months later was normal and did not show any relapse , and the patient is now free of symptoms . regularly , the hepatic angiomyolipoma ( aml ) is a solitary mesenchymal tumor , not encapsulated and of a variable size ( 1 to 36 cm ) [ 1 , 5 , 6 ] that is composed of three variable tissues : muscular cells , thick - wall vessels , and mature adipose tissue [ 17 ] . it occurs more frequently in women , with no age preference , and has a low incidence . it was described for the first time by ishak in 1976 , and there are only 300 cases published ever since [ 16 ] . commonly , it is a sporadic tumor , but it is associated with tuberous sclerosis in 6% of patients [ 1 , 2 ] . this predisposition suggests that genes involved in the tuberous sclerosis ( tsc1 and tsc2 ) may be instrumental in aml pathogenesis . aml muscular cells are called pec ( perivascular epithelioid cell ) , while identical cells are seen in other tumors ( lung lymphangiomatosis , lung or pancreas clear cells , cardiac rhabdomyoma , etc . ) , so it has been suggested to include these tumors in a family called pecomas , although this idea is not widely accepted [ 1 , 2 ] . there are 3 types of aml , histologically speaking , depending on the amount of fat they contain , which are called lipomatous tumors ( > 70% of fat ) , myomatous ( < 10% ) , and angiomatous and mixed variants [ 1 , 2 , 5 ] . the immunohistochemical study of aml is always positive for hmb45 and frequent for s100 and actin [ 2 , 4 , 6 ] . the aml has always been regarded as benign tumor , with a slow growth and with no chances of a malign transformation [ 3 , 4 ] , but four works published from 2000 to now report the occurrence of 4 malignant aml cases that have or can develop the capacity to relapse and lead to vascular invasion [ 24 ] . the combined use of abdominal ultrasonography , ct , and mri has increased preoperative diagnostic certainty of aml , especially in those cases when a fat component and some central and prominent abnormal vessels can clearly be detected ; however the correct preoperative diagnosis does not exceed 50% [ 25 ] . the ultrasonography reveals a heterogeneous hyperechoic mass that sometimes is difficult to distinguish from a hemangioma [ 2 , 4 , 5 ] . the ct shows an aml with two parts : a peripheral angiomatous component and a lipomatous one with a low attenuation [ 1 , 6 ] . the mri of the aml reveals an intense signal in t1 and t2 , and it seems that a somewhat higher specificity is obtained when compared with other imaging systems [ 16 ] . a differential diagnosis is suggested in the event of hepatocarcinoma and other liver tumors that may include a variable fat content ( adenoma , lipoma , liposarcoma , sarcoma , gist , metastasis , etc . ) [ 13 , 6 ] . the fnap technique may yield a low rate of correct diagnoses , so its utility is reduced [ 1 , 4 ] . aml is generally asymptomatic , but big - sized tumors may produce compressive symptoms , such as abdominal pain in superior hemiabdomen , plenitude sensation after intake of food , palpable mass , and other symptoms that include weight loss or fever [ 14 , 6 ] . there seems to be a correlation between a higher size than 5 cm and the occurrence of symptoms . general indications for the resection of benign liver tumor ( blt ) include diagnostic doubt , occurrence of symptoms , or onset of complications [ 8 , 10 ] . there is currently a tendency to perform these resections by laparoscopy when it is possible , although this laparoscopic approach should not increase the amount of surgery indications for blt [ 10 , 11 ] . accepted indications for resection of aml include symptomatic patients , those cases where malignancy can not be excluded , rapid growth tumors , and lesions with an exophytic component , as this last increases the risk of rupture . some authors recommend the resection of all amls larger than 5 cm [ 3 , 4 ] . the most accepted criteria for a preserving management comprise aml cases that are smaller than 5 cm , asymptomatic , whenever their histology has been tested through fnap , and uninfected by hepatotropic viruses , as it could lead to an erroneous diagnosis of hepatocarcinoma [ 3 , 4 ] . the above - mentioned existence of malignant cases has reopened the debate on the need to approach all aml cases . the only medical available treatment for aml although no randomized trial has been carried out yet is based on sirolimus , as it seems to reduce the aml size by inhibiting mtorc1 . our opinion is that asymptomatic aml , less than 5 cm , in a noncirrhotic liver could be observed but after explaining to the patient the very low ( 3% ) but possible incidence of cancer . the rest of the aml , symptomatic , quick growing , bigger than 5 cm , not of clear diagnosis , should be resected . the first laparoscopic resection of the liver ( lrl ) was performed in 1992 , but it had no exponential growth until the last five years when it coincided with a better technology that did not exist previously [ 8 , 12 ] . the more frequent lrl ( 65% ) comprises minor hepatectomies ( left lateral sectionectomy and atypical resections ) , and segments ii to vi are regarded as the ideal ones for lrl [ 8 , 9 , 12 ] . a progressive experience on lrl has made possible a higher number of resections on segments that initially were more complex and major hepatectomies [ 8 , 9 , 12 ] . rlr on blt is an excellent indication since it rules out the risk of tumor dissemination , presents the benefits of the laparoscopic surgery for usually young patients , and reduces the mean in - hospital stay and recovery time [ 8 , 9 , 12 ] . the potential disadvantages of lrl include slow progress of the learning curve , bleeding , inaccurate assessment of lesions , which may go unnoticed , and the risk of air embolism . in 2009 , an exhaustive worldwide revision was published on 2801 patients who had undergone lrl , out of which 44.7% ( n = 1253 ) of lrl procedures dealt with blt cases . there are , though , few publications devoted exclusively to the implementation of lrl on blt cases [ 8 , 9 ] ; the two most numerous series on this issue totalized 70 patients and only two of them belonged to the aml group [ 8 , 9 ] . the percentage of complications concerning lrl on blt varies between 10 and 20% , while mortality is 0% , a fact that proves that lrl is feasible and safe [ 8 , 9 ] the classical lrl technique employs 5 trocars ( two of 12 mm , one of 10 mm , and two of 5 mm ) and systematic portal clamping [ 8 , 9 ] . however , the published series reveal that in a percentage of patients , which varies between 12.5% and 46% , the technique was performed without pringle 's maneuver . our posture is that in peripheral resections concerning small tumors , or in segmentectomies , it is not so strictly necessary to perform the portal clamping , although it is advisable to be prepared for any contingency . there is only one publication that mentions the use of three trocars in 9 lrl cases . all patients had malign tumors ( 8 hepatocarcinomas and one hepatic metastasis ) , superficially located from segments ii to vi and viii and exhibited a mean size of 3 cm . the mean time of procedures was 2 hours , pringle 's maneuver was not used , blood loss amounted to 75 ml , and morbidity - mortality was negative . our opinion is that in selected patients those peripheral lesions smaller than 5 cm can be resected with only three trocars instead of the classical 5 ones . the main problems for liver sils surgery are new learning curve , loss of instrumental triangulation , vision problems because camera and instruments are parallel , expensive costs and incisional hernias [ 1419 ] . after an extensive bibliographical search , only 9 liver sils surgeries have been found , two laparoscopic fenestration of simple cyst and 7 liver resections . lesions were always located in left lateral segment [ 1419 ] . in every case , the surgeon 's opinion is that liver sils surgery is complex , technically demanding , and not always feasible . besides , in some cases additional ports are required [ 18 , 19 ] . the advantages of 3 trocars technique versus sils are conventional devices , more anatomical vision , nonlearning curve , and costs . so , three - port laparoscopic resection is safe and feasible in some liver lesions . aml is a rare neoplasm of the liver with not well - defined malignant potential so surgical resection in suitable patients is indicated .

to investigate the effects of collagen cross - linking using glyceraldehyde on the biomechanical properties of the sclera and the axial elongation of form - deprived myopia in the guinea pig . thirty - six guinea pigs were randomly assigned to four groups : fdm ( form - deprived myopia ) ; fdmg ( form - deprived myopia treated with glyceraldehyde ) ; fdms ( form - deprived myopia treated with 0.9% isotonic sodium chloride ) ; and normal control ( free of form - deprivation ) . fdm was achieved in the right eye using a latex facemask . the right eye in fdmg was treated with a posterior subtenon injection of 0.5 m glyceraldehyde ; 0.9% isotonic sodium chloride was administered to the right eye in fdms group using the same method . axial length , refraction , and stress - strain of the sclera were measured at scheduled time points . the treated eyes were also examined histologically by light microscopy . it was found that glyceraldehyde treatment significantly increased the stiffness of the sclera in the fdm eyes and abnormalities have not been observed in the retina and optic nerve of the treated eyes . but the development of myopia was not affected .

the incidence of myopia in the usa and europe is reported to be around 30% , and in asian countries it affects around 60% of the general population [ 1 , 2 ] . pathologic myopia of more than 6.0 d can be found in 1215% of all myopic patients . low to moderate degrees of myopia can be easily corrected using optical or refractive surgical means . pathologic myopia , while also correctable using these optical approaches , is of major concern because of sight - threatening consequences such as retinal detachment , macular schisis , and macular degeneration . all these complications are associated with progressive axial elongation which can not be treated by refractive means . the pharmacological treatment currently in use is topical atropine , which is accompanied by numerous side effects and offers little benefit for already highly myopic eyes [ 4 , 5 ] . surgical approaches include scleral reinforcement surgery in which donor sclera or synthetic bands are placed around the back of the globe and sutured to the sclera to provide scleral support or injection of a polymeric composition forming a gel under tenon 's capsule and inducing scar tissue to prevent axial elongation [ 68 ] . however , the outcomes of all of these surgical therapies are controversial and surgical trauma also should be taken into consideration . there is strong evidence from clinical and experimental studies indicating that the biochemical and biomechanical properties of the sclera play a major role in the progression of myopia . thinning of the sclera and weakened biomechanical properties , particularly at the posterior pole of the eye , have long been known to be an important feature in the development of high myopia in human and mammalian models . thus , the sclera has been considered to be a prime target for therapeutic manipulation regarding myopia progression . as mentioned above surgical treatments based on sclera reinforcement because of the controversial results and complications , the various surgical approaches have not been widely applied in clinical practice . animal studies involving tree shrews have shown that impaired collagen cross - linking is an important factor in the weakening process of the myopic sclera . indeed , collagen cross - linking has been successful in treating progressive keratoconus in which the cornea undergoes a thinning process and exhibits weakened biomechanical properties , as in the myopic sclera [ 11 , 12 ] . several studies have attempted to increase the rigidity of the sclera through collagen cross - linking . collagen cross - linking induced by the photosensitizer riboflavin and ultraviolet a ( uva ) has been shown to lead to a significant increase in young 's modulus for treated porcine , rabbit , and human sclera [ 13 , 14 ] . however , scleral cross - linking using riboflavin and uva requires an operation entailing surgical exposure of the posterior sclera and has a potential cytotoxic risk for the retina . an alternative method of chemical cross - linking using glyceraldehyde has been shown to significantly increase young modulus in porcine sclera in vivo and in rabbit sclera in vitro [ 13 , 15 ] . another study involving the rabbit demonstrated that the efficacy of glyceraldehyde in increasing scleral biomechanical strength can extend over a period of 8 months . glyceraldehyde is used for tissue engineering in the pharmaceutical and food industry and is generally considered nontoxic [ 15 , 17 ] . the safety of glyceraldehyde treatment on eyes has also been studied by wollensak and iomdina . light microscopy examination revealed that there were no abnormalities in the optic nerve and retina , and only some moderate infiltration of neutrophils and bleeding adjacent to the injection site were found in several animals . unlike riboflavin cross - linking and sclera reinforcement surgery , glyceraldehyde can be easily administered by means of sequential parabulbar injections and have large treatment area . consequently , scleral cross - linking with glyceraldehyde may be a promising method for the treatment of myopia . although glyceraldehydes could improve sclera rigidity in normal animal eyes , its effect on progressive myopic eyes has not yet been studied . the aim of the present study was to investigate the effectiveness of collagen cross - linking using glyceraldehyde in increasing scleral rigidity and retarding the axial elongation in form - deprived myopia ( fdm ) of guinea pig . this study was approved by the animal care and ethics committee at tianjin medical university ( tianjin , china ) . the treatment and care of animals were conducted according to the arvo statement for the use of animals in ophthalmic and vision research . thirty - six pigmented guinea pigs ( cavia porcellus ; approximately 3 weeks old ) were obtained from the animal breeding unit at tianjin medical college and randomly assigned to the following four groups with nine animals per group : fdm ( form - deprived myopia ) ; fdmg ( form - deprived myopia treated with glyceraldehyde ) ; fdms ( form - deprived myopia treated with 0.9% isotonic sodium chloride ) ; and normal control ( free of form deprivation ) . all animals underwent biometric measurement ( refraction and axial length ) prior to the experiment . the right eye in the fdmg group was treated using a sub - tenon injection of 0.5 m glyceraldehyde ; 0.9% isotonic sodium chloride was administered to the right eye in the fdms group using the same method . biometric measurement was undertaken at four time points ( 0 , 2 , and 4 weeks ) . following the final ocular measurements , the right eyes were enucleated after the animal had received an overdose of anesthesia . the stress - strain of the sclera was measured and histological examination was undertaken with light microscopy . fdm was achieved using a latex facemask ( oujie , suzhou , china ) covering the right eye . the left eye , nose , and both ears remained exposed , as described by lu et al . the facemasks were examined once daily to ensure that they were in place and fitted well . after topical anesthesia using 0.5% proparacaine hydrochloride ( alcon , purrs , belgium ) , sub - tenon injections of 0.05 ml of 0.5 m glyceraldehydes ( sigma - aldrich , steinheim , germany ) dissolved in physiologic saline solution were administered as a depot with the injection site 3.0 mm behind the limbus in the superonasal and inferotemporal quadrant , respectively ; then the injection site was transferred to the other two quadrants for every other injection ; a 1.0 ml tuberculin syringe with a sharp 25-gauge injection needle was used . the first injection was given at day 1 just before achieving fdm and the injections were given twice a week . 0.05 ml of 0.9% isotonic sodium chloride solution ( dazhong , tianjin , china ) was administered in the same way . ofloxacin eyedrops ( santen , osaka , japan ) were applied four times a day . retinoscopy for all animals was performed by the same optometrist in a dark room using a streak retinoscope . before examination , 1% cyclopentolate hydrochloride ( alcon ) was topically administered to the eye every 5 min for four repetitions to achieve a completely dilated pupil . the refraction was recorded as the mean value of the horizontal and vertical meridian [ 19 , 21 , 22 ] . after topical anesthesia with 0.5% proparacaine hydrochloride ( alcon ) , ocular axial length was measured using an a - scan ultrasonography ( cinescan a / b , quantel medical , clermont ferrand , france ) . sound velocities were assumed to be 1557.5 m / s for the anterior segment , 1723.3 m / s for the lens , and 1540 m / s for the vitreous humor [ 19 , 23 , 24 ] . the animals were sacrificed by overdose anesthesia with 160 mg / kg pentobarbital sodium ( sigma - aldrich , steinheim , germany ) . after making a complete circular incision located at a distance 1 mm anterior the limbus , two scleral strips of width 2 mm were dissected sagittally using a double - shade shaver , from the nasal and temporal margin of the optic nerve to the anterior end almost at 1 and 11 o'clock . care was taken to insure that all strips were cut in a similar orientation to minimize any differences resulting from possible anisotropy . scleral strips with a width of 2 mm were clamped horizontally with a distance of 5 mm between the jaws of the microcomputer - controlled biomaterial tester ( shanghai university ; shanghai ; china ) . strain was increased linearly with a velocity of 1 mm / min and stress was measured up until tissue rupture ( figure 2 ) . the strain - stress curve was recorded and the parameters stress ( mpa ) and young 's elastic modulus ( mpa ) from 2% to 14% of the strain of the samples were used for analysis . all the eyes of fdms and fdmg group perform histological examination . after the scleral strips used for biomechanical test were removed carefully , the left eye cups were fixed in 10% neutral buffered formalin for at least 24 hours for light microscopy . the specimens were embedded with paraffin . 4 mm thin sections were cut within and adjacent to the treatment area and stained with hematoxylin - eosin . the slides were examined using a light microscope ( leica dm4000b ; germany ) at 401000 magnification . the refraction and axial length of the right eye were statistically compared to the left eye within the same group prior to the experiment using the paired sample t - test . the biometric and stress - strain results for the right eye were compared between the different groups using one - way analysis of variance ( anova ) with bonferroni correction . prior to the form deprivation ( 0 time - point ; table 1 ) , there was no significant difference between the right eye and the left eye of the animals regarding refraction and axial length within each individual group ( p > 0.05 ; paired sample t - test ) . the difference in refraction and axial length of the right eyes was not significant among all the four groups ( refraction , p = 0.685 ; axial length , p = 0.949 ; one - way anova with bonferroni correction ) . all of the right eyes of animals in the fdmg , fdm , and fdms groups developed significant myopia in 4 weeks . similar to the fdm group , the biometric parameters of the right eyes of animals in the fdmg and fdms groups kept developing towards myopia during the 4-week observation period . data regarding the development of refraction and axial length in the four groups is detailed in table 1 . at all time points the development of refraction and axial length in the deprived eyes of animals in the fdm , fdmg , and fdms groups was similar but was significantly faster than the normal control group ; the results were presented in tables 1 and 2 . when compared with the normal control eyes , statistically significant changes occurred in the biomechanical parameters of the deprived eyes of animals in the fdmg , fdm , and fdms groups ( table 3 ) . the stress - strain curves and young 's elastic modulus - strain curves for all of the four groups are presented in figures 3 and 4 , respectively . for each group the stress - strain curve approximated to a straight line ; however , when the thickness of the sclera was considered , there was not a single young 's elastic modulus at different strain levels . the elastic modulus increased from 2% strain and reached a value that was almost stable at 6% strain ; the difference between the groups also reached almost stable value at 6% strain . the elasticity of the sclera in the eyes of animals in the fdm group increased ; the stress in the deprived eyes of animals in the fdm group was significantly lower than normal control eyes , with the exception of the 2% and 4% strain levels . in contrast with the stress results , although young 's elastic modulus for the deprived eyes of animals in the fdm group was lower than in the normal control eyes , the difference was not significant at all strain levels . saline injection could improve the biomechanical properties of the sclera in the form - deprived eyes . the stress and young 's elastic modulus for the right eyes of animals in the fdms group were both significantly higher than in the fdm group , with the exception of 2% and 4% strain ( stress : fdms versus fdm , 4% strain , p = 0.052 , 6% strain p = 0.01 , and 8% strain , p = 0.009 ; young 's elastic modulus : fdms versus fdm , 4% strain , p = 0.072 , 6% strain , p = 0.014 , and 8% strain , p = 0.042 ; one - way anova with bonferroni correction ) . however , when compared to normal control eyes , there were no significant differences between the groups at all strain levels ( table 3 ) . for the right eyes of animals in the fdmg group , the stress and young 's elastic modulus at all strain levels were significantly higher than for the right eyes of animals in all of the other groups ( p < 0.01 ; one - way anova with bonferroni correction ) . the stress in the right eyes in animals in the fdmg group was 1.69 times greater and young 's elastic modulus was 1.08 times greater than in the right eyes of animals in the fdm group at 6% strain . the comparison of stress and young 's elastic modulus between fdmg and normal control group was showed in table 3 . light microscopy examination showed that , in 7 eyes of fdmg group and 6 eyes in fdms group , mild inflammatory infiltration or hemorrhage was seen in the episcleral tissue in the injection area . it adhered to the sclera tightly , but no scar has been observed ( figure 5 ) . the optic nerve and retina in all samples were without abnormalities ( figure 6 ) . in the present study , it was confirmed that form deprivation can cause significant myopia ; in the eyes of guinea pigs with form - deprived myopia the sclera had weakened biomechanical properties . the stress in the eyes of animals in the fdm group was lower than that in the eyes of animals in the normal control group at all strain levels , and the difference was significant at 6% strain . young 's elastic modulus for the sclera was lower in the eyes of animals in the fdm group than in the normal control group , but the difference was not significant . these results were similar to those reported by phillips and mcbrien . in their study , the sclera extended over a distance that was 25% greater than in the controls at a load corresponding to 20 mmhg intraocular pressure ; however , when the thickness of the sclera was taken into consideration , young 's elastic modulus was similar between the myopic and normal eyes under physiological pressure . consequently , it was concluded that the alterations in elasticity were mainly the result of a thinner sclera in the eyes with myopia . it is believed that scleral thinning in myopia is not the result of passive stretch of the sclera but is caused by active tissue remodeling . decrease in the amount of collagen present and fibril diameter are two critical events that lead to scleral thinning and weakening . in an eye with a weakened sclera , physiological intraocular pressures may be sufficient to induce progressive ocular enlargement given sufficient time . consequently , treatment targeting scleral collagen may be effective in retarding the progression of myopia . experiments both in vivo and in vitro have demonstrated that the biomechanical parameters of sclera in normal eyes could be improved by treatment with glyceraldehyde [ 13 , 16 , 18 ] . wollensak and iomdina have demonstrated in vivo in rabbit sclera after glyceraldehyde treatment for 14 days that the ultimate stress increased by 409.7% and young 's modulus increased by 1027% ; the ultimate strain decreased by 48.2% . in vitro after treatment with glyceraldehyde , the stress of treated porcine and human sclera increased by 487% and 34% , respectively . in our study , it was found that in form - deprived eyes the biomechanical rigidity of the sclera could also be increased by treatment with glyceraldehyde . the stress and elastic modulus in the eyes of animals treated with glyceraldehydes was significant higher than in the eyes of animals in the normal control and fdm groups at all strain levels . this indicates that injected glyceraldehydes could overwhelm the scleral biomechanical changes caused by form deprivation . through the maillard reaction cascade , glyceraldehyde can be added to the ends of protein molecules and can be further transformed to more stable molecules called advanced glycation end products ; this results in covalent collagen cross - links that can promote increased tissue stiffness and resistance to enzymatic degradation [ 2628 ] . it had been demonstrated by ultrasound sub - tenon injections can reach posterior sclera immediately and then disperse into surrounding tissue . in this study repeated injections in four quadrants , respectively , facilitated glyceraldehydes reaching the whole posterior sclera which play most important role in the axial elongation . abnormalities of the retina and optic never have not been observed in our study ; the same results have also been reported by wollensak and iomdina . the tenon capsule of the fdmg eyes became compact and adhered to the sclera tightly that may be caused by injections and glyceraldehydes . the result of these changes has not been studied furtherly , but there was no scar observed and the injections have been done well in this study . in the current study , although the injection of glyceraldehydes increased scleral rigidity , the development of myopia was not retarded in experimental eyes relative to the normal control eyes . all of the biometric parameters regarding the eyes of animals in the fdmg group were not significantly different from those in the fdm group at all time points . the results of other studies aimed at evaluating the efficiency of sclera reinforcement have been controversial . scleral strengthening by means of polymer injection to slow ocular elongation has been studied in animal models and humans ; the results were reciprocal . su et al . reported that polymeric hydrogels , either implanted or injected adjacent to the outer scleral surface , could not slow ocular elongation in chicken eyes , although the sclera thickness was significantly increased . the authors considered that a possible reason for this result relates to the bilayered structure of the chick sclera ; the stiffer cartilage component of the chick sclera may determine the rate of elongation . in mammalian and primate eyes that have monolayered fibrous scleras , the addition of the fibrous capsule is likely to have a greater impact on scleral biomechanical properties and could possibly slow ocular relongation . indeed , it has been reported by avetisov et al . that repeated injections of liquid polymeric composition could promote collagen formation in 146 rabbit eyes and slow myopia progression in 240 human eyes . this finding is at odds with our observations regarding form - deprived myopia in the guinea pig . the axial elongation could not have been retarded by increased scleral stiffness in our study . one possible reason may be that myopia was progressive and more pronounced in the former study . in addition to this , a reduction in collagen fibril diameter has been reported to occur at the posterior pole . in experimental myopia , the collagen fibril diameter has been found to decrease only after a long period of myopia ( 3 months in the tree shrew ) . therefore , collagen change may not be the most important factor that contributes to the progression of short - term experimental myopia . the improvement in scleral rigidity based on collagen cross - linking may be more effective in long - term progressive myopia . on the other hand , the results of our study may indicate that enhancement of scleral biomechanical properties does not alone guarantee slowed eye growth in the fdm guinea pig eye model , consistent with suggestions by mcbrien and norton . they found that form - deprived myopia could be increased by administration of aminopropionitrile , which could prevent collagen cross - linking ; at the same time it was also found that eyes treated with aminopropionitrile without form deprivation were no different from normal untreated eyes . it appears that focused images falling on the retina could control ocular development in a growing eye despite the presence of structural changes involving collagen . several factors are involved in the progression of myopia , a predisposition to myopia or other error signals combined with abnormal collagen structure , and work together to produce myopia . it is reasonable to assume that if the reinforcement of sclera reaches a sufficiently high level it can retard myopia by overwhelming all of the factors leading to its progression . but to make the sclera more rigidity may be combined with more side effects . in summary , the results of the present study represent proof that the scleral biomechanical properties of form - deprived eyes can be improved using sub - tenon injection of glyceraldehydes ; however , the rate of ocular elongation was not affected . because of differences in high and moderate myopia , a follow - up study involving progressive high and long - term myopia in a mammalian animal models will be necessary to establish their suitability for myopia control with collagen cross - linking .

objective . we aimed to assess peripheral vascular endothelial function in open - angle glaucoma ( poag ) by measuring flow - mediated dilatation ( fmd ) . materials and methods . the study included 20 cases with poag ( group 1 , mean age 58.68 13.3 years ) and 30 healthy individuals ( group 2 , mean age 58.68 13.6 years ) . in all cases , responses of endothelial function were assessed by a cardiologist through measurement of fmd following brachial artery occlusion . results . mean percent of fmd , an indicator of endothelial function , was found to be 11.9 4.2% in group 1 and 12.3 4.4% in group 2 ( p = 0.86 ) . conclusion . no impairment in systemic vascular function of cases with poag suggests that poag could be a local disorder rather than being a component of systemic disease .

glaucoma is an irreversible optic neuropathy characterized by specific optic disc changes and corresponding visual field alterations which are often associated with elevated intraocular pressure ( iop ) . mechanistic theory proposes that increased iop is the most important risk factor in glaucomatous optic neuropathy ; however , optic neuropathy can be worsened in patients with well - controlled primary open - angled glaucoma and low iop . vascular dysregulation resulting from vascular endotheliopathy is the most important factor in decreased ocular blood flow [ 24 ] . in addition to responses to several vasoactive agents and hormones , endothelium itself also releases substances relaxing or constricting vascular smooth muscles such as nitric oxide ( no ) or endothelin-1 ( et-1 ) , respectively . it was found that level of et-1 was increased in the aqueous fluid samples of patients with glaucoma [ 6 , 7 ] . endothelium - dependent fmd by occlusion at brachial artery is a noninvasive method which is previously used in the measurement of endothelial function in patients with risk factors ( dm , hypertension , hypercholesterolemia , homocysteinemia , etc . ) for coronary artery disease . the aim of the present study using vascular endothelial function measurement to assess fmd was to evaluate relationship between cases with primary open - angled glaucoma and fmd . the study included 20 patients with poag and 30 age- and sex - matched healthy controls . the diagnostic criteria for paog included iop > 22 mmhg without treatment , open angle at gonioscopy , glaucomatous optic disc changes ( cup / disc > 0.7 and thinning or pitting at rim ) , and characteristic visual field defects corresponding to optic disc . the cases with poor sita reliability indices ( false negative errors , false positive errors , and fixation loss exceeding 20% ) were excluded . also , patients with previous ocular trauma or surgery , those with history of steroid use , or those having an eye disorder other than glaucoma were excluded . in addition , patients with systemic diseases such as hypertension , congestive cardiac failure , hypercholesterolemia , diabetes mellitus , cerebrovascular event , or autoimmune diseases were excluded . control group included healthy individuals without history of medication who presented for routine ophthalmological visits . the study was approved by the local ethics committee and was conducted in accordance with the declaration of helsinki , good clinical practice guidelines . the participants were informed of the nature of the study , and informed consent was obtained from each patient . in all subjects , blood samples were drawn for measurements of fasting blood glucose , lipid profile , sedimentation rate , and c - reactive protein ( crp ) . fmd measurements in brachial artery were performed by a cardiologist blinded to clinical characteristics of the patients with 2-dimensional high - resolution ultrasound device ( 7.013.0 mhz , siemens medical sol . ten minutes resting at supine position was provided before measurements and ecg monitoring was applied throughout measurements . radial artery measurement was performed at a point 35 cm superior to antecubital fossa . transition zone was set at a depth between area at 3 cm depth and closer wall . by magnifying image , intermediate zone between tunica media and adventitia on pulse doppler sonography , blood flow velocity and volume were assessed through two - dimensional images generated by signal obtained from center of artery with a sampling angle of 6570 and sampling distance of 1 mm . a 5-minute occlusion was obtained by inflating cuff up to 250 mmhg at the level of brachial artery . radial artery measurements were recorded during r wave on ecg ( end - diastole ) . response of brachial artery diameter to hyperemia was calculated as percent increase by using the following formula : 100x [ postocclusion diameter ( mm ) baseline diameter ( mm)/baseline diameter ] . statistical analysis was performed by using spss for windows version 17.0 ( statistical package for social science , chicago , il , usa ) . student 's t - test was used to assess differences in mean values between groups . table 1 summarizes demographic characteristics and blood measurements of poag and control groups . there were no significant differences between the groups regarding age , sex , and biochemical measurements . percent of fmd increases was 11.9 4.2% and 12.3 4.4% in poag and control groups , respectively ( p = 0.86 ) . glaucoma represents a group of eye disorders in which many risk factors play a role in its development and progression . iop is the best known risk factor and reducing iop is the only available method in the treatment of glaucoma . thus , it is needed to investigate ocular dynamic in patients with glaucoma to further improve our understanding of ocular dynamics . previous studies have reported the role of vascular factors in the development and progression of glaucomatous optic neuropathy in paog [ 9 , 10 ] . these previous works focused on ocular blood blow . in the present study , it was revealed that there is no impairment in systemic vascular endothelial function in patients with poag when compared to controls by using brachial artery fmd measurements . currently , fmd is the gold standard used in the measurement of arterial endothelial function in clinical trials . fmd is high - frequency sonographic visualization of brachial artery that shows endothelial - dependent flow - mediated vasodilation . hyperemia - induced transient ischemia leads to increased stress in brachial artery wall and it causes vasodilation by promoting endothelial no release . however , previous studies showed that inter- and intraobserved differences are minimal without significant effect on results [ 8 , 12 ] . in our study , measurements were performed by an experienced cardiologist . fmd measurement is considered an important marker in establishing prognosis and treatment response in coronary artery disease . , et-1 causes decreased retinal , choroidal , and optic nerve head blood flow and vasoconstriction at posterior ciliary artery . increased et-1 levels can play a role in the astrocyte proliferation seen in glaucomatous optic neuropathy . increasing et-1 levels in the aqueous humor of patients with poag reduces outflow of aqueous fluid by causing contraction in trabecular network . in studies on normotensive glaucoma ( ntg ) , it was found that et-1 levels were increased in both serum and aqueous humor , suggesting both ocular and systemic vascular dysfunction in ntg [ 1618 ] . in studies on patients with paog , et-1 levels were found to be increased in aqueous humor , while no change was observed in serum et-1 levels [ 7 , 16 , 1922 ] . this suggests that the role of et-1 in vascular dysfunction in poag is localized to tissue . reported increased serum et-1 levels in selected cases with disease progression despite well - controlled iop . in the previous 3 studies using fmd , it was reported that fmd was decreased in patients with poag [ 1 , 23 , 24 ] . however , there are inconsistencies among these studies . in the study by su et al . , in which patients with ntg and poag were compared , it was reported that mean percent of fmd increase in patients with ntg was lower than that in patients with poag . found no significant difference between patients with paog and ntg ; even authors reported lower percent of fmd increase in poag group when compared to ntg group . in the study by cellini et al patient group consisted of those with progressive visual field defect despite well - controlled iop ; thus , they emphasized vascular theory . in our study , it was demonstrated that there is no impairment in systemic vascular function of cases with poag . we think that , in contrast to ntg with systemic component , poag is a disease specific to the eye . valuable information could be obtained about systemic vascular function in patients with poag by designing studies with larger sample size which demonstrate indirect vascular endothelial function such as et-1 level and number of endothelial progenitor cells .

congenital trcheo - oesophageal fistula ( tof ) without oesophageal atresia is usually diagnosed and managed in the neonatal period . its presentation in adulthood is a rarity . traditional treatment of a tof in adults involves its repair via a thoracotomy . we report the case of a 23-year - old man diagnosed with an h - type tof during workup undertaken for his symptoms of gastro - oesophageal reflux . this fistula located at the level of third thoracic vertebra was repaired successfully using a thoracoscopic approach .

oesophageal atresia ( oa ) is a congenital condition of oesophageal discontinuity that results in proximal oesophageal obstruction and a tracheo - oesophageal fistula ( tof ) is an abnormal communication between the oesophagus and the trachea . oa and tof can occur alone or in combination . congenital tof without oa ( h - type tof ) account for < 4% of congenital oesophageal anomalies the case presented here is distinctive because of ( a ) the rarity of an h - type tof in adults and ( b ) the utilization of a thoracoscopic approach to repair it . a 23-year - old man was investigated for complaints of persistent night - time regurgitation for 8 years without any dysphagia . an upper gastrointestinal ( ugi ) endoscopy revealed a fistulous opening in the oesophagus 20 cm from the incisors [ figure 1a ] . a computed tomography ( ct ) scan with reconstruction confirmed an h - type tof in the mid - oesophagus at t3 level [ figure 1c ] and ruled out any extrinsic mediastinal pathology . a virtual bronchoscopy [ figure 1d ] showed the fistula to be 3 cm above the carina . oesophageal manometry demonstrated a lower oesophageal sphincter pressure to be 4.9 cm of h2 o and ineffective peristalsis in 30% of the wet swallows . the patient was counselled regarding a thoracoscopic repair of the tof in the first instance followed by reassessment of his reflux symptoms after surgery . ( b ) view of the carina obtained upon passing the endoscope through the fistulous tract . ( d ) virtual bronchoscopic image demonstrating the tracheal opening ( arrow ) the surgery was performed under double - lumen endotracheal anaesthesia with the patient placed in the left lateral decubitus position . a 10 mm port was established in the 6 intercostal space in the midclavicular line , and two 5 mm ports were placed on either side in the 5 and 7 intercostal space . the parietal pleura overlying the oesophagus above the level of the azygous vein was incised widely , and a pleural flap was harvested for later use . the trachea was identified , and the fistulous tract extending from the oesophagus was dissected . vascular slings were passed to encircle the broad fistulous tract and the oesophagus distal to it [ figure 2a ] . the 10 mm port was exchanged for a 12 mm endopath xcel port ( ethicon endosurgery , mumbai , india ) . the nasogastric tube was withdrawn , an endoscopic stapler with a blue cartridge ( 3.5 mm staple depth ) was locked on to the fistula , and the sling isolating the fistula was removed . the oesophageal staple line was oversewn with a 3 - 0 polyglycolic acid ( vicryl , johnson and johnson , mumbai , india ) suture . the pleural flap was interposed between the two staple lines and sutured to the trachea in a similar manner . a 32f intercostal drain was placed through the 12 mm port site , and the incisions were closed . ( a ) sling around the fistula ( f ) between the oesophagus ( o ) and trachea ( t ) . ( b ) arrow points to the tracheal end of the staple - divided fistula post - operatively the patient was prescribed parenteral analgesics for the first 48 h , and he was kept nil by mouth for 4 days . an esophagogram obtained on the 5 day showed smooth passage of contrast into the stomach without any leakage . it is proposed to assess his reflux symptoms a year after surgery to determine whether he would merit long - term proton - pump inhibitor therapy or anti - reflux surgery . benign , acquired tofs are rare in adults and may result as a complication of inflammatory disorders , foreign body ingestion , trauma or prolonged tracheal intubation . congenital h - type tofs presenting in adulthood are even rarer and only around 16 cases have been documented in the surgical literature . the minimal symptoms in many patients may be explained on the basis of the oblique passage ( an n rather than h configuration ) of the fistulous tract leading to a valve - like action that results in the occlusion of its lumen during the passage of the bolus of food or peristaltic wave . presence of a membrane covering the opening or contraction of the of the muscle wall of the fistulous tract are considered to be the other likely explanations for the delay in onset of symptoms until adulthood . a high index of suspicion is needed to diagnose a tof in adults , in whom the respiratory symptoms are far commoner than the gastrointestinal symptoms . the commonest complaint is cough , while eating or drinking ( ohno 's sign ) followed by recurrent bouts of respiratory tract infection . development of tof in utero disrupts the normal development of the oesophageal myenteric plexuses resulting in dysmotility . this may lead to gastroesophageal reflux ( as seen in our patient ) and esophagitis . the fistulous opening is often apparent during a ugi endoscopy , but may be missed if covered with a membrane or located on the anterior wall of the upper third of the oesophagus . similarly , the lower positioning of the oesophageal opening in relation to the one in the trachea may preclude outlining of the tract on a contrast swallow . unless the fistula is wide ( as was the case in our patient ) coronal sections of the ct scan may fail to identify it . sagittal views along with a three - dimensional reconstruction are more likely to demonstrate the fistula . bronchoscopy is useful for identifying the fistulas not seen on ugi endoscopy and ruling out intrinsic trachea - bronchial pathology . as in neonates , surgery is the only definitive treatment for tof presenting in adults . traditionally , either a cervical route ( for a fistula above the t2 level ) or thoracotomy ( for fistulas at or below t3 level ) is employed for the repair . the repair involves ligation of the fistula or its division , followed by repair of the trachea and oesophagus . a muscle or pleural flap is usually interposed between the divided ends . over the past decade , the feasibility and safety of thoracoscopic repair of the congenital tofs in neonates has been well documented . a recent meta - analysis comparing open and thoracoscopic surgery in the treatment of oa and tof in neonates indicated that the outcomes of the two approaches were comparable . in the only previously reported case of thoracoscopic repair of tof in an adult , staple - divided a mid - thoracic level tof in a 79-year - old female . thoracoscopic approach affords excellent magnification for safe visualization , dissection and division of the tof , even when fistula is a narrow one . this complex surgery is best - undertaken either by a thoracic surgeon well versed with thoracoscopy or ( as was the case here ) by an experienced minimal access surgeon teamed up with a paediatric surgeon . thoracoscopic repair of a tof may not necessarily shorten the hospital stay , as it is customary to commence oral intake around the 5 day after confirming integrity of the staple / suture line with a contrast study . however , these patients do enjoy the other well - proven benefits of thoracoscopic surgery such as reduced pain , speedy recovery and reduction in wound - related and pulmonary morbidity . thoracoscopic repair appears to be a feasible and safe option for the treatment of tof presenting in adulthood , which is an extremely rare clinical entity .

introductionisolated endometriosis of the intestine causing large bowel obstruction is rare.presentation of casewe present a case of endometriosis presenting as large bowel obstruction in a woman of childbearing age . she had no previous symptoms to suggest endometriosis and on presentation urgent surgery was required . the diagnosis of endometriosis was made only after pathological evaluation of the specimen.discussionno cases of endometriosis confined to this sigmoid colon without pelvic involvement were noted in the literature.conclusionthe diagnosis of endometriosis should be entertained when women of childbearing age presents with large bowel obstruction , whether or not the patient has other evidence of the disease .

we present a case of endometriosis presenting as large bowel obstruction in a woman of childbearing age . she had no previous symptoms to suggest endometriosis and on presentation urgent surgery was required . no cases of endometriosis confined to this sigmoid colon without pelvic involvement were noted in the literature . when women of childbearing age seek medical attention for signs and symptoms of intestinal obstruction , and there is no obvious etiology , intestinal endometriosis should be considered as a differential diagnosis . a 37 year - old housewife and mother of a 10 year - old by caesarian section , presented with a history of generalized , colicky abdominal pain and constipation for 6 days , with bilious vomiting and distention for 2 days . she had normal , regular menses with no history of dyspareunia , dysmenorrhea , abdominal pain , constipation , diarrhea or rectal bleeding . on examination , she was in painful distress , ill looking and mildly dehydrated , with a tachycardia of 108/min . the abdomen was distended and tympanitic with generalized mild tenderness but no peritonism or palpable mass . ct - scan showed grossly distended large intestine from the caecum to the sigmoid colon , with no air in the rectum ( see fig . the complete blood count , liver function test and carcino - embryonic antigen were all within normal limits . at surgery , the large intestine was grossly distended from the caecum to sigmoid colon where there was a palpable solid tumor in the wall of the bowel . the pelvis , ovaries , tubes and uterus were grossly normal and there were no other intra - abdominal abnormalities . with an operative diagnosis of carcinoma , sigmoid colectomy with primary anastomosis the cut specimen revealed an obstructing tumor on the mesenteric border , within the wall of the sigmoid colon without involvement of the mucosa ( see fig . she was subsequently referred to the gynecologist for treatment and remains asymptomatic 15 months later . intestinal involvement by endometriosis occurs in 5% of premenopausal women . of these , 70% present with large bowel obstruction . however , the vast majority of these patients reported are known cases of endometriosis , having complaints of pelvic pain , dyspareunia and/or dysmenorrhea . many of them also have a history of infertility , for which they were subjected to investigation , such as laparoscopy , hence , presenting with a previous diagnosis of endometriosis . our patient had no history to suggest the condition with no visible endometrioma , ovarian or pelvic abnormality indicative of endometriosis . the pathophysiology of endometriosis has been explained by various theories ; direct myometrial extension , coelomic metaplasia , lymphatic and hematogenous metastasis , reverse menstruation and implantation during salpingography or due to operative spillage . isolated endometriosis within the muscular layer of the bowel with no involvement of mucosa or any pelvic organ remains unclear . although seat belt trauma had , in one case , been suggested as an etiologic factor , no clear evidence was provided for this . in our patient when the initial presentation of endometriosis is intestinal obstruction with no previous history and no suspicious findings at surgery , the diagnosis is unlikely to be made preoperatively . in most reported cases , the patients had a known history of endometriosis or the surgical findings were very suggestive of it . both these observations were absent in our patient . in the non - obstructed case , colonoscopy , endoscopic we believe that one should always maintain a high level of suspicion of endometriosis , when a woman of childbearing age presents with intestinal obstruction and there is no other obvious cause . if the diagnosis is made preoperatively , surgery may be avoided in the non - obstructed case and only a limited resection done in the event of obstruction . when a woman of childbearing age presents with large bowel obstruction , one should always entertain a possible diagnosis of endometriosis whether or not the patient has other evidence of the disease . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . all the authors of this article contributed to the study design , data collection , data analysis and writing .

influenza a virus exerts a large health burden during both yearly epidemics and global pandemics . however , designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly . therefore , it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets . we have previously measured host protein responses in continuously cultured a549 cells infected with mouse - adapted virus strain a / pr/8/34(h1n1 ; pr8 ) . we here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial ( hbae ) cells . a total of 3740 cytosolic hbae proteins were identified by 2d lc ms / ms , of which 52 were up - regulated 2-fold and 41 were down - regulated 2-fold after pr8 infection . up - regulated hbae proteins clustered primarily into interferon signaling , other host defense processes , and molecular transport , whereas down - regulated proteins were associated with cell death signaling pathways , cell adhesion and motility , and lipid metabolism . comparison to influenza - infected a549 cells indicated some common influenza - induced host cell alterations , including defense response , molecular transport proteins , and cell adhesion . however , hbae - specific alterations consisted of interferon and cell death signaling . these data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells .

influenza a virus ( iav ) , a member of the orthomyxoviridae family , is an enveloped , single - stranded rna virus with a genome of 8 negative - sense segments , with most encoding a single gene . these eight segments give rise to 10 or 11 distinct proteins depending on the strain ( na , ha , ns1 , ns2/nep , m1 , m2 , np , pa , pb1 , pb1-f2 , and pb2 ) , and these have been shown to interact extensively with each other and with host cell proteins throughout the virus lifecycle . some interactions elicit alterations in the host proteome , as exemplified by the virus s ability to both induce and evade a host immune response , to influence autophagy and apoptosis , and to increase viral protein synthesis while shutting down host protein synthesis . numerous host proteins may also be important for influenza replication , as recently shown in whole genome sirna screens that identified 100 genes in drosophila , 120 genes in u2os cells , as well as 295 genes and 287 genes in the a549 human carcinoma lung cell line . current anti - influenza therapies consist of only two classes of treatments , and these each target a viral protein : zanamivir and oseltamivir are neuraminidase inhibitors , while amantidine and rimantidine target the m2 protein . recent and currently circulating strains from the 20102011 season have remained susceptible to m2 inhibitors ; however , resistance to neuraminidase inhibitors is emerging ( www.cdc.gov/flu/weekly/ ) . thus , new targets for which resistance will not quickly be developed are needed , and host cell proteins essential for viral replication represent one option . host interactions is increasingly reliant on quantitative proteomic techniques such as 2d - dige , isotope - encoded affinity tag ( icat ) , and stable isotope labeling of amino acids in cell culture ( silac ) . for example , silac profiling was used to determine differences in expression between hiv- and mock - infected t - cells and the response of several host cell types to hepatitis c infection . quantitative proteomics has also been applied to influenza host interactions , including several studies probing primary macrophages with itraq and 2dige - ms / ms and multiple studies using similar techniques in continuous epithelial cell lines such as ags , mdck , a549 , and calu-3 cells . several genomic and proteomic studies have also been carried out in influenza - infected macaques . a novel aspect of the current study is the use of more relevant human primary bronchial epithelial cells , a model that more closely mimics in vivo infection conditions . it has been shown that the response to influenza depends not only on the viral strain but also the host species , cell type and location in the lung , and the state of cell differentiation . while the effect of these different parameters have been characterized in part by measuring secreted cytokines and the kinetics of viral replication , very little is known about differences in host similarly , few studies have compared the effect of iav infection in continuous and primary cell lines of specific cell types . therefore , it is important to extend our previous silac studies using continuous human lung a549 cells , a type ii alveolar epithelial cell , to primary human tracheobronchial airway epithelial ( hbae ) cells to compare pathways affected by influenza replication , and identify pathophysiologically relevant responses . influenza virus strain a / pr/8/34 ( h1n1 ; pr8 ) , an attenuated mouse - adapted strain , was grown in embryonated hens eggs from laboratory stocks , after which chorioallantoic fluid was harvested , aliquoted , and titered in mdck cells by standard procedures . primary normal human broncho - tracheal epithelial cells were obtained from healthy donors by lonza inc . and were certified as mycoplasma- , hiv- , hbv- , and hcv - negative . notably , these cells were capable of > 10 cell doublings , which made the six doublings needed for silac labeling possible . cells were cultured in bronchial epithelial growth media ( begm , lonza inc . ) , which consists of bronchial epithelial basal media ( bebm , lonza inc . ) supplemented with singlequots ( lonza inc . ) containing bovine pituitary extract , hydrocortisone , hegf , epinephrine , transferrin , insulin , retinoic acid , triiodothyronine , and gentamicin-1000 . cells were maintained as monolayers in 10% co2 at 37 c and passaged by trypsinization at 8090% confluence . for silac labeling , cells were grown in silac media that consisted of arginine- and lysine - free begm supplemented with either c12 ( light ) or c13 ( heavy ) lysine ( 29 mg / l ) ( mass difference of 6.0 da ) and c12/n14 ( light ) or c13/n15 ( heavy ) arginine ( 348 mg / l ) ( mass difference of 10.0 da ) . light amino acid containing media were prepared from regular stock powder ( sigma , st , louis mo ) , and heavy amino acids were obtained from the silac phosphoprotein identification and quantification kit ( invitrogen canada inc . ; burlington , ontario ) . separate silac labeling and infection experiments were performed four times . for silac experiments , all cells were grown in a pair of t75 flasks in silac media for six cell doublings ; after the sixth cell doubling , cells were allowed to reach near confluency . in three replicates c12/n14-light cells were infected with egg - grown pr8 virus that was diluted in gel saline to achieve a multiplicity of infection ( moi ) of 7 plaque forming units ( pfu ) per cell . an equivalent number of c13/n15-heavy cells were mock infected as a control using only gel saline . previous studies indicate the small amount of egg protein makes no significant measurable contribution to results or conclusions . label swapping was performed for the fourth replicate ; the c13/n15 heavy cells were infected , and the c12/n14 light cells were mock infected . for all other infections , cells were grown to near confluency and infected at various mois as indicated . in order to synchronize infections , virus- and mock - infected cells were placed at 4 c for the 1 h virus adsorption , after which inoculum was removed and cells were overlaid with appropriate prewarmed light or heavy silac media . infected and mock - infected cell cultures were then kept at 37 c until analysis of viral replication and host protein expression . efficiency of infection was always confirmed by a standard plaque assay as described previously . twenty - four hours postinfection , cells grown in c12/n14-light or c13/n15-heavy begm were collected by brief trypsinization and counted , and then equivalent numbers ( roughly 3 10 cells ) of each group were mixed together . to verify infection status of each culture , aliquots of all separate cultures were saved for virus titration by plaque assay as mentioned above . mixed cells were washed 3 times in > 50 volumes of ice - cold phosphate - buffered saline ( pbs ) and lysed for cytosolic proteins by adding np-40 buffer ( 10 mm trishcl ph 7.4 , 3 mm cacl2 , 2 mm mgcl2 , 0.5% np-40 , 1.1 m pepstatin a ) to the cells and incubating them for 30 min on ice . nuclei were pelleted at 5000 g for 10 min and the supernatant was saved as cytosol . protein content in the cytosolic fractions were measured using a protein assay kit ( biorad ) and bovine serum albumin standards . three hundred micrograms of each was reduced , to which 6 the volume of 100 mm ammonium bicarbonate was added . then 100 mm dithiothreitol ( dtt ) in 100 mm ammonium bicarbonate was added to the peptide mixture , and samples were incubated for 45 min at 60 c . iodoacetic acid ( 500 mm in 100 mm ammonium bicarbonate ) was added to each tube for alkylation , and the tubes were incubated for a further 30 min ( room temperature , in the dark ) . samples were digested overnight at 37 c with 6 g of sequencing grade trypsin ( promega , madison , wi ) and then stored at 80 c until further processing . peptide fractionation was carried out using a 2d rp ( reversed - phase ) high ph rp low ph peptide system as described previously . in short , lyophilized tryptic digests were dissolved in 200 l of 20 mm ammonium formate ph 10 ( buffer a ) , injected onto a 1 100 mm xterra ( waters , milford , ma ) column and fractionated using a 0.67% acetonitrile per minute linear gradient ( agilent 1100 series hplc system , agilent technologies , wilmington , de ) at a 150 l / min flow rate . sixty 1-min fractions were collected and concatenated using procedures described elsewhere ; the last 30 fractions were combined with the first 30 fractions in sequential order ( i.e. , no . combined fractions were vacuum - dried and redissolved for the second dimension rp separation ( 0.1% formic acid in water ) . the second dimension was run on a splitless nanoflow tempo lc system ( eksigent , dublin , ca ) with 20 l sample injection via a 300 m 5 mm pepmap100 precolumn ( dionex , sunnyvale , ca ) and a 100 m 200 mm analytical column packed with 5 m luna c18(2 ) ( phenomenex , torrance , ca ) . both eluents a ( water ) and b ( acetonitrile ) contained 0.1% formic acid as an ion - pairing modifier . a 0.33% acetonitrile per minute linear gradient ( 030% b ) was used for peptide elution , providing a total 2-h run time per fraction in the second dimension . a qstar elite mass spectrometer ( applied biosystems , foster city , ca ) was used in a data - dependent ms / ms acquisition mode . one - second survey ms spectra were collected ( m / z 4001500 ) followed by ms / ms measurements on the 3 most intense parent ions ( 80 counts / s threshold , + 2 to + 4 charge state , m / z 1001500 mass range for ms / ms ) , using the manufacturer s smart exit previously targeted parent ions were excluded from repetitive ms / ms acquisition for 60 s ( 50 mda mass tolerance ) . files from analyst were submitted simultaneously to protein pilot 3.0 ( applied biosystems ) for relative quantification and protein identification using the paragon algorithm as the search engine . each ms / ms spectrum was searched against a database of human protein sequences ( ncbinr , released march 2008 , downloaded from ftp://ftp.ncbi.nih.gov/genomes/h_sapiens/protein/ ) . the search parameters allowed for cysteine modification by iodoacetic acid and biological modifications programmed in the algorithm ( i.e. , phosphorylations , amidations , semitryptic fragments , etc . ) . the threshold for detecting proteins ( unused protscore ( confidence ) ) in the software was set to 2.0 to achieve 99% confidence , and identified proteins were grouped by the progroup algorithm ( applied biosystems , foster city , ca ) to minimize redundancy . the protein sequence coverage was calculated using peptides identified with > 95% confidence . a decoy database search strategy ( ncbinr homo sapiens with all protein sequences reversed ) was also used to estimate the false discovery rate ( fdr ) , defined as the percentage of reverse proteins identified against the total protein identification . for our data , the estimated fdr was 0.56% , which is low compared to other studies and indicates a very high reliability of the proteins identified . for relative quantitation , only peptides unique for a given protein were considered , thus excluding those common to other isoforms or proteins of the same family . proteins were identified on the basis of having at least one peptide with an ion score above 99% confidence . among the identified peptides , some of them were excluded from the quantitative analysis for one of the following reasons : ( a ) the peaks corresponding to the silac labels were not detected . ( b ) the peptides were identified with low identification confidence ( < 1.0% ) . ( c ) either the same peptide sequence was claimed by more than one protein or more than one peptide was fragmented at the same time because of shared ms / ms spectra . ( d ) the sum of the signal - to - noise ratio for all of the peak pairs was 6 for the peptide ratios . in order to compare multiple biological replicates , protein ratios within each replicate were converted to a z - score that allowed protein ratios to be normalized to the mean and standard deviation of its individual experiment . thus , a protein with a z - score > 1.960 indicates that protein s differential expression lies outside the 95% confidence level , 2.576 indicates 99% confidence , and 3.291 indicates 99.9% confidence ; z - scores > 1.960 were considered significant . the weighted average z - score was then calculated for each protein found in multiple replicates . gi numbers of all significantly regulated proteins were submitted to and analyzed by the david bioinformatic suite at the niaid , version 6.7 , and gene ontologies were examined with the fat and panther databases . the gi numbers were also submitted to , and pathways constructed with , ingenuity pathway analysis software ( ipa ) . mock- and influenza - infected hbae cells were scraped into cold pbs at 24 hpi , pelleted at 5000 g for 5 min , and lysed with lysis buffer ( 20 mm tris ph 7.5 , 100 mm nacl , 0.5% np-40 , 0.5 mm edta , 1 antiprotease cocktail ( pierce ) , 1 phosphatase inhibitor cocktail ( pierce ) ) . forty micrograms of protein was loaded per lane into sds - page gels , separated , and transferred to nitrocellulose membranes . membranes were probed with primary antibodies for viral np ( in - house antibody ) , viral ns-1 ( in - house antibody ) , samd9 ( sigma ) , ifit1 ( epitomics ) , e - cadherin ( cell signaling ) , glg1 ( sigma ) , isg15 ( rockland ) , mxb ( santa cruz ) , rsad2 ( abcam ) , stat1 ( cell signaling ) , -tubulin ( cell signaling ) , ppia ( epitomics ) , oasl ( epitomics ) , -actin ( sigma ) , and gapdh ( santa cruz ) and rabbit hrp - conjugated secondary antibodies . bands were detected with ecl ( amersham ) and the alphainnotech fluorchemq multiimage iii instrument , quantitated using alphaease software and virus - to - mock ratios reported without normalization . hbae cells were grown to 80% confluence on 25 mm coverslips and infected or mock - infected at moi = 7 . mock and 0 , 12 , and 24 h infected cells were fixed in 3% paraformaldehyde for 15 min , permeabilized with 0.3% triton - x100 in 3% paraformaldehyde for 15 min , and blocked with 1% bsa in pbs . cells were treated with a primary antibody for np ( made in - house ) and a cy3-conjugated rabbit secondary antibody ( jackson immuno research ) ; all antibodies were diluted in 1% bsa and pbs . coverslips were then mounted onto slides using dapi - prolong gold antifade , dried , and sealed . fluorescent images were obtained using a computer - controlled olympus ix70 microscope ( 20x objective ) equipped with ccd camera and nis - element software . actively proliferating primary epithelial cell cultures are not commonly used for influenza studies ; thus , we initially determined optimal conditions for influenza replication in nondifferentiated hbae . a low ( 0.01 ) and a high ( 7 ) moi were initially chosen to measure the amount of infectious progeny virus released into the supernatant over time . at the low moi , viral replication was most efficient in mdck cells , as peak titers reached almost 10 pfu / ml by 48 hpi , whereas maximum titers of 10 pfu / ml were attained by 48 hpi in a549 cells and no detectable virus was recovered from hbae cells ( figure 1a ) . at moi = 7 , mdck and a549 cells reached similar peak titers but at earlier time points , 24 and 31 hpi , respectively ; hbae produced detectable virus by 12 hpi and reached a maximum titer of 10 pfu / ml by 24 hpi ( figure 1b ) . influenza replication in hbae at moi = 7 was further confirmed by western blotting for the viral non - structural-1 protein ( ns1 ) , which is expressed only during active viral replication . ns1 was clearly expressed in hbae cells by 6 hpi and increased in abundance at both 12 and 24 hpi ( figure 1b ) . additionally , we found that an moi = 7 was sufficient to demonstrate active and productive infection in approximately 50% of cells by 12 hpi , while > 95% of cells demonstrated productive infection by 24 hpi ( figure 1c ) . for silac experiments we therefore chose to study cells 24 h after infection to maximize the amount of time for cellular proteomic changes to occur while retaining active virus replication and minimizing any cytopathic effect ( figure 1d ) . in addition , to verify that hbae cells could be labeled , we analyzed only heavy - labeled cells using lc , we determined that 91% of all peptides contained the c13-label modification ( supplementary figure 1 ) . the c13-containing peptides were highly enriched for high h : l ratios ; 66% of these peptides had an h : l ratio > 9 ( > 90% incorporation ) , and 92% had an h : l ratio > 2 ( > 67% incorporation ) . in contrast , the peptides that did not contain a c13 label were enriched for peptides with low h : l ratios , e.g. , 75% of these peptides had an h : l ratio < 1 ( < 50% incorporation ) , and virtually all unlabeled peptides belonged to keratins . therefore , we concluded that the six hbae cell doublings were sufficient to label the cell proteome . ( a ) hbae , a549 , and mdck cells were infected at moi = 0.01 ( left ) and 7 ( right ) pfu / cell to monitor and compare efficiency of infectious progeny virus production . supernatants were collected and titered for progeny virus production by standard plaque assay at indicated time points . ( b ) hbae cells were infected with influenza a / pr/8/34 at moi = 7 pfu / cell , and protein lysates were assayed for accumulation of viral ns-1 protein at 6 , 12 , and 24 h post infection by western blotting . ( c ) hbae cells were infected for 0 , 12 , and 24 h at moi = 7 pfu / cell . after fixation , the percentage of cells infected by virus was shown using immunocytochemistry for the viral nucleoprotein . ( d ) phase - contrast images of hbae cells at 24 and 48 h post infection and mock infection . we performed four separate replicate experiments using primary human bronchial epithelial cell cultures from the same donor in which the light - labeled culture was infected in three replicates and the heavy - labeled culture was infected for the fourth replicate . this allowed us to demonstrate that the heavy isotopes had no effect on the virus replication . several proteins were found significantly up - regulated in the light - labeled infections but significantly down - regulated in the heavy - labeled infection . these proteins , which therefore probably represent contaminants , include several keratin species ( 1 , 2 , 9 ) , dermcidin , transferrin , thyroid hormone receptor associate protein 3 , and nipsnap2 and were excluded from subsequent analysis . overall , a total of 3740 unique cytoplasmic proteins were detected and quantified ; 2282 ( 61% ) of these proteins were detected in 2 or more replicates , 1453 ( 39% ) were found in 3 or more replicates , and 621 ( 17% ) were common to all four replicates ( figure 2a ) . as these proteins were identified using a human database and rely upon the presence of these proteins in both cultures , no viral proteins were identified during mass spectrometry . to confirm that the samples were infected , supernatants were titered for progeny virus using a standard plaque assay on mdck cells ( figure 2b ) . distributions of total , up - regulated , and down - regulated proteins identified . ( a ) a total of 3740 proteins were identified from four separate biological replicates ; 30% of all proteins were found in every run , 53% were found in two or more runs , and 46% were unique to each run . ( b ) up- and down - regulated proteins were determined statistically using a frequency distribution curve . in order to compare the four replicate runs , infected : mock ratios for each run were normalized by converting them to z - scores , a measure of deviation from the average . therefore , positive values represent proteins that are up - regulated in virus - infected cells , and negative values represent down - regulated proteins . to determine which proteins are significantly altered in abundance we used different confidence intervals as indicated . most proteins were not significantly altered ( z - score between 1 and 1 ) ; however , a small subset of proteins were strongly up- or down - regulated ( z - score > 1.96 and ( c ) after silac - labeled hbae cells were infected for 24 hpi , the supernatant was collected and titered by plaque assay to demonstrate that the cell monolayer had been infected and had productively produced progeny virus . ( d ) the number of up- and down - regulated protein pairs identified in each trial at a confidence level of 95% ( z - score > 1.96 ) . specific peptides identified , measured , and used to measure up - regulated and down - regulated proteins are listed in supplementary table 3 . comparing the abundance of proteins in infected cultures to uninfected cultures revealed that 97% of all proteins were present in approximately equal amounts ( near a 1:1 ratio ) , thus indicating that iav infection does not alter the expression level of most proteins in host cells . to determine cutoff values for identifying significantly up- and down - regulated proteins , ratios for infected : uninfected in each trial were first converted to z - scores to normalize each run to its own mean and standard deviation , thus allowing us to compare multiple runs . by converting an average z - score back into an average protein ratio , we determined that a z - score of 1.96 ( 95% confidence interval ) corresponded to a 2-fold change . similarly , z - scores of 2.58 ( 99% confidence interval ) corresponded to a protein ratio change of 2.5-fold and z - scores of 3.29 ( 99.9% confidence interval ) corresponded to a 3.0-fold change in protein ratio ( figure 2c ) . further bioinformatic analyses were primarily performed with the 95% confidence data set but were also compared to more restricted data sets based on 99% and 99.9% confidence intervals ( not shown ) . proteins that were identified with only a single peptide were excluded from our data set if they were detected in only a single run ; however , any protein identified in multiple trials was included . we also excluded proteins from further analysis if they were not consistently up- or down - regulated between replicate runs . some proteins identified in the current study were unaltered in at least one replicate and up- or down - regulated in other replicate(s ) . for these scenarios we grouped ratios into three groups : ( a ) z - scores 0.09 to 0.9 ( not altered ) , ( b ) 1.0 to 1.96 ( moderately altered ) , or ( c ) greater than 1.96 ( highly altered ) . we then included only those proteins that had significant values ( group c ) in at least two runs or one of two runs ; additionally , any trial with a nonsignificant value had to have that value still fall within the 80% confidence range ( group b ) to be considered . outliers could be found at both the peptide and the protein level such that peptides and proteins that were found only in the virus or mock sample were labeled 9999 or 0000 , respectively . we did not remove any outliers at the peptide level but found that they made up a very small percentage of the peptides in our lists of differentially regulated proteins ( the details may be found in supplementary table 2 ) and removing them had minimal impact on the overall protein ratio . for proteins that had a ratio of 9999 and 0 we arbitrarily assigned a z - score of 50 and 50 , respectively . these outliers were not included in the statistical analysis but were reincorporated into data sets for further bioinformatic and biological analyses . in summary , we used stringent parameters to generate a data set of proteins of interest based on 99% confidence in protein identification ; protein identification based on 2 or more peptide pairs ; 95% confidence interval for altered regulation ; and the necessity for proteins to show consistent infected : unifected ratios . in this manner we identified 52 up - regulated proteins and 41 down - regulated proteins from pr8-infected primary hbae cells ( figure 2c and listed in tables 1 and 2 ) . the identities , associated z - scores and protein percent coverage of the 52 up - regulated and 41 down - regulated proteins are listed in supplementary table 1 , and the identities , confidences and other statistical parameters of the specific peptides used for quantitation are listed in supplementary table 2 . several of the up - regulated and nonregulated proteins that were identified in the silac analysis were confirmed by western blotting ( figure 3 ) , and most western blot results confirmed silac - determined regulation status . only one protein , ppia , that was shown to be up - regulated in silac , was not validated using western blotting . this may be caused by inherent differences in sampling ( partially degraded proteins would not be measured by western blot but their peptides would be detected by ms ) or by inherent differences in the different methods ' levels of sensitivity . hbae cells were harvested and lysed with 0.5% np-40 detergent , nuclei were removed , and cytosolic fractions were dissolved in sds electrophoresis sample buffer , resolved in 8% , 10% , or 15% mini bands were visualized and intensities were measured with an alpha innotech fluorchemq multiimage iii instrument . molecular weight standards are indicated at left and ratios of each protein ( infected divided by mock - infected ) are indicated for each protein at right , along with silac - measured ratios ( far right ) . western blot ratios for host proteins that were detected only in infected cells ( undetectable in mock ) are designated n.a . . silac ratios are an average of the four replicate runs performed , and western blotting ratios are from a single experiment in a different donor than was used for silac . bands from western blotting were quantitated using alphaease software and are reported without normalization . * : no viral proteins measured by silac because not present in mock - infected samples . gene ontology analyses using go and panther classification terms were determined for proteins that were up - regulated in response to influenza infection and compared at three different confidence intervals : 95% ( z - score > 1.96 ) , 99% ( z - score > 2.58 ) and 99.9% ( z - score > 3.29 ) ( figure 4a ) . additionally , canonical pathways and networks that were represented by influenza - altered proteins were constructed with ipa at the 95% , 99% , and 99.9% confidence intervals . approximately half of the proteins up - regulated in response to influenza infection in hbae could be attributed to interferon and other known host cell defense responses . these proteins were identified by several gene ontology categories including defense response , immune response , response to virus and defense response to virus ( figure 4a ) . collectively , these categories included proteins isg15 , rsad2 , mx1 , mx2 , ifit1 , ifit2 , ifit3/4 , ifitm1 , samd9 , samhd1 , oasl , oas3 , tlr2 , stat1 , gbp-1 , and hla - b / c . pathway analyses also identified immune systems and interferon signaling ( figure 5 , supplementary figure 2a ) . proteins belonging to endocytic and intracellular protein trafficking pathways were also indicated as up - regulated ( figure 4a ) and included -actin , mx1 , mx2 , and ppia . related canonical pathways that were identified by ipa include caveolar - mediated endocytosis , viral entry via endocytic pathways , and mechanisms of viral exit from host cells ( supplementary figure 3b ) . a third category that was identified among influenza - up - regulated proteins was the presence of gtpase activity , which included proteins mx1 , mx2 , and gbp1 ( figure 4a ) . lists of ( a ) up- and ( b ) down - regulated protein ids were uploaded into david separately and analyzed for enrichment of categories belonging to biological processes , cellular components , and molecular functions . additionally , lists of proteins determined at different confidence intervals ( 95% , 99% , 99.9% ) were compared . interactions between up- and down - regulated proteins . protein ids and ratios from tables 1 and 2 ( 95% confidence interval ) were combined and imported into the ingenuity pathways analysis ( ipa ) tool from which interacting pathways were constructed . up- and down - regulated proteins are denoted in red and green , respectively ; gray proteins indicate that they were detected in our study but not regulated ; white proteins interact with many proteins in the network but were not detected in this study . any known direct connections between these proteins are indicated by solid lines ; indirect interactions are not shown here . networks are titled ( a ) infection , gene expression , antimicrobial processe ; ( b ) cancer , dermatological conditions , cellular development ; and ( c ) connective tissues , genetics , cv system development and function . many biological processes were represented among proteins down - regulated at the 95% confidence level . notably , many of these are not cytosolic proteins but were mainly transmembrane ( fads2 , odz2 , fat1 , f3 , glg1 , tnfrsf10a , jag1 , fdft1 , slc16a2 , tnfrsf10b ) and extracellular matrix - related proteins ( col12a1 , lama3 , lama5 , fn1 , adamts1 , bigh3 ) ( figure 4b ) . cellular functions that were attributed to these proteins included cell adhesion ( col12a1 , fat1 , fn1 , glg1 , lama3 , lama5 ) ( figure 4b ) as well as signal transduction ( odz2 , collagen , fat1 , fn , f3 , bigh3 , tnfrsf10a , tnfrsf10b , adamts1 ) ( figure 4a ) . trail signaling , a process involved in inducing cell death , was also prominently identified ( tnfrsf10a , tnfrsf10b ) ( figures 4b and 5b , supplementary figures 2a and 3d ) . third , several enzymes involved in lipid metabolic processes were consistently down - regulated including sterol metabolism ( fdft1 ) and fatty acid metabolism ( fads2 , fads3 ) ( figures 4b and 5b , supplementary figures 2b and 3d ) . our lab has previously used silac to study a549 alveolar epithelial cell host pathways that are altered in response to infection by influenza virus a / pr/8/34 , a highly attenuated mouse - adapted strain of influenza virus . we have now extended this work to include primary human airway epithelial cells , a model that is more closely related to the in vivo situation and that offers opportunity to assess the global relevance of proteomics data obtained using transformed human cell lines . using a human database , we identified 3740 cytosolic hbae protein pairs , of which only a small fraction was altered in response to a / pr/8/34 influenza infection . proteins that were up- and down - regulated were determined statistically using different confidence intervals for which the 95% confidence interval corresponded to a 2-fold or more difference , the 99% limit to at least a 2.5-fold difference , and 99.9% confidence to a 3-fold or greater change . since many previous studies have used either a 2-fold or 1.5-fold change in protein abundance to create data sets of infection - altered proteins , the cutoff values used here are comparable . as viral proteins are not detected by human database searches , we titered the amount of virus secreted by our silac labeled cells at 24 hpi to ensure that viral infection and replication had been successful . it is worth noting that the maximum virus titers produced by hbae vary from donor to donor . for example , the particular cells used in figure 1a produced the highest titers we have observed in hbae , whereas the cells used for the silac experiments ( figure 2c ) produced considerably lower titers . however , despite these differences , we find robust viral protein expression in these cells from 6 to 72 hpi and can find comparable proteomic expression patterns among hbae from different donors ( data not shown ) . the current study consists of the cytoplasmic cellular fraction , which includes all np40 soluble components such as proteins of the cell membrane , cytoplasm , and cytoplasmic organelles , thereby excluding mainly nuclear proteins . we recognize that the nuclear sample is also an important component of the cell to analyze , particularly because influenza virus replicates within the nucleus , and anticipate analyzing these fractions in future studies . however , the primary purpose of the present study was to compare hbae results to a549 results , and the later study had only been performed with the np-40-soluble fraction . it is well documented that in many cell types interferon expression is a central cellular response to viral infection . this is thought to be key in developing an antiviral state in both infected and neighboring noninfected cells to limit viral replication . consistent with this paradigm , in our current study up - regulated proteins in hbae were chiefly enriched for host cell defense responses such as interferon ( inf ) and the jak / stat pathway ( figures 4a and 5a , supplementary figures 2a and 3a ) . of note , influenza a can also antagonize interferon signaling via the viral ns-1 protein through multiple mechanisms including blocking host translation and sequestering viral rna in order to prevent its detection by host pathogen recognition processes . however , the extent of this inhibitory effect is strain - dependent , and since the ns1 protein produced by the a / pr/8/34 influenza strain is generally thought to be a less potent inhibitor of interferon production than other strains ns1 protein , this correlates well with our data . some of these findings were also unique to hbae compared to our previous a549 study . interestingly , neither go nor ipa identified interferon signaling in pr8-infected a549 cells , whereas it was highly significant in hbae cells ( p - value = 0.001 ) . in addition , while a few interferon - induced proteins ( rsad2 , mx1 , mx2 , and isg15 ) were highly up - regulated in both cell types , several ( stat1 , rig - i , samd9 , samhd1 , ifit1 , ifit2 , ifit3 ) were highly up - regulated in hbae but unaltered or undetected in a549 . indeed , we were able to confirm with western blotting that cytosolic stat1 abundance was increased in hbae after influenza infection , whereas levels in a549 remained relatively unchanged . samd9 , a molecule downstream of stat1 , was more highly induced in hbae than a549 after infection , whereas ifit1 was detected only in hbae after infection ( figure 6 ) . this suggests that less interferon is produced by a549 than hbae cells and that this difference is dependent either on the cell type ( a549 are alveolar epithelial in origin whereas hbae are from airways ) or properties of the cell line ( i.e. , primary versus transformed ) rather than the virus strain . notably , a difference in interferon signaling and production could explain the ability of a549 , but not hbae , to support replication of the pr8 virus when infected at a low moi ( 0.01 pfu / cell ) . these observations provide rationale for future studies assessing the precise role(s ) of interferon - associated proteins in preventing viral replication in primary epithelia and mechanisms that suppress this effect in transformed or alveolae - derived epithelial cells . comparison of jak / stat signaling molecule abundance in a549 and hbae cells after influenza infection . cells were harvested and lysed with 0.5% np-40 detergent , nuclei were removed , and cytosolic fractions were dissolved in sds electrophoresis sample buffer , resolved in 10% or 12% mini sds - page , transferred to pvdf , and probed with indicated antibodies . bands were visualized and intensities were measured with an alpha innotech fluorchemq multiimage iii instrument . molecular weight standards are indicated at left , and ratios of each protein ( infected divided by mock - infected ) are indicated for each protein at right . protein ratios are an average of three experiments each from a549 cells and a single hbae donor and are reported after normalization to gapdh . the interferon - induced proteins commonly up - regulated in both a549 and hbae ( rsad2 , mx1 , mx2 , and isg15 ) were also identified in numerous other influenza studies . for example , myxovirus resistance host proteins mx1 and mx2 are up - regulated in a variety of cell models including mdck and a549 cells , as well as in vivo in studies with macaques . isg15 and rsad2 , interferon - induced proteins with anti - influenza activity , were also up - regulated in a549 cells and primary macrophages . these proteins have also been identified in proteomics studies of human host cell responses to infection with other viruses such as rsv , adenovirus , and sindbis virus and hsv . interferon - induced tetratricopeptide repeat proteins ( ifit13 ) have also been found in multiple proteomic studies of influenza involving primary macrophages , the polarized calu-3 airway epithelial cell line and animal models . interestingly , these three ifit proteins have been discovered to form an antiviral complex against 5-triphosphate rna , which is thought to be present during an influenza infection . it is likely that these similar responses to different types of infection reflect the induction of a nonspecific innate immune response against rna viruses by the host cells . we found 19 proteins that were consistently and strongly down - regulated in primary hbae cells in response to influenza infection , and these were mainly linked to cell adhesion , death receptor signaling , and lipid metabolism . cellular adhesion is necessary for the survival of primary cells , and previous studies have found that a reduction in adhesion molecules precedes and may possibly be instrumental in inducing cell death . in epithelial cells adherens junction proteins are essential for cell adhesion and these are largely composed of integrin , cadherin , catenin , and actin complexes . many proteins belonging to these complexes and downstream signaling pathways were detected in our silac data among which integrin 4 was down - regulated 2.4-fold , glg1 2.5-fold , and thbs1 2.6-fold . other adhesion - related proteins such as fibronectin , laminin , tgfbi , and tnc were down - regulated > 2.0-fold . however , cell death processes were not identified by any of our analyses , except for the down - regulation of two trail receptors , tnfsfr10a and tnfsfr10b ( figures 4b and 5b ) . tnfrsf10a , tnfrsf10b and f3 are additionally associated with caspase activity induction , which was similarly indicated as down - regulated ( figure 4b ) . in contrast , tnfsfr10d was found up - regulated 1.4-fold in pr8-infected a549 cells at the same time point . given that trail signaling is important in clearing influenza - infected epithelial cells , it would be interesting to further investigate the mechanism by which trail - receptors are down - regulated in hbae . as cholesterol and lipids are key to maintaining the integrity of the cell membrane and the organization of lipid rafts , it was interesting to find that several proteins related to lipid biosynthesis were down - regulated in response to influenza . for example , a previous study indicated that rsad2 interferes with cholesterol synthesis and subsequent lipid raft integrity by binding and inhibiting fpps , an enzyme in the cholesterol biosynthetic pathway . our silac / hbae data found rsad2 highly up - regulated ( > 3-fold ) and fpps moderately up - regulated ( 1.5-fold ) ( supplementary figure 1c ) . importantly , fpps is involved not only in the production of cholesterol but also other intermediates , such as isoprenoids fpp and ggpp , which serve as essential activators of gtpase proteins . our silac data identified a number of proteins from this pathway , of which fdft1 was most significantly altered ( 2.9-fold down - regulated ) . fatty acid synthesis was also affected through a strong downregulation of fads2 ( 3.45-fold ) . a second study has also found that interferon signaling can suppress sterol biosynthesis in macrophages , suggesting that this may be a protective response induced by the host cell rather than by the virus . while none of fpps , fdft1 , nor fads2 were altered in a549 cells at 24 hpi , ipa indicated other lipid - related pathways as down - regulated in a549 cells including sphingolipid metabolism and vdr / rxr signaling . collectively , these data suggest that lipid metabolism may be inhibited in epithelial cells during influenza infection . overall this proteomics study provides a broad and unbiased profile of cytoplasmic host cell proteins that are up- and down - regulated in response to an h1n1 mouse - adapted influenza virus in primary human airway epithelial cells . while some of our findings correlate with previous proteomic studies involving influenza or other viruses , we have identified many proteins that have not been previously studied with respect to influenza . thus , our work has expanded the knowledge base for understanding host cell response to influenza infection . of note , we performed our studies using relevant primary human bronchial epithelial cells , whereas many previous studies have been conducted using transformed cell lines such as alveolar a549 cells or nonlung hek293 cells . a comparison of functional categories and pathway alterations between our studies with a549 and hbae identified some common characteristics such as downregulation of cell adhesion and lipid metabolism , as well as upregulation of host defense responses against influenza infection . further validation and investigation into the functions of the candidate proteins we have identified will contribute to understanding novel virus - host interactions and permit integration of this information with previous large - scale analyses of influenza to identify new directions for developing anti - influenza therapies .

often regarded as benign , plasmodium vivax infections lay in the shadows of the much more virulent p. falciparum infections . however , about 1.98 billion people are at risk of both parasites worldwide , stressing the need to understand the epidemiology of plasmodium vivax , particularly under the scope of decreasing p. falciparum prevalence and ecological interactions between both species . two epidemiological observations put the dynamics of both species into perspective : ( 1 ) act campaigns have had a greater impact on p. falciparum prevalence . ( 2 ) complete clinical immunity is attained at younger ages for p. vivax , under similar infection rates . we systematically compared two mathematical models of transmission for both plasmodium species . simulations suggest that an act therapy combined with a hypnozoite killing drug would eliminate both species . however , p. vivax elimination is predicted to be unstable . differences in age profiles of clinical malaria can be explained solely by p. vivax 's ability to relapse , which accelerates the acquisition of clinical immunity and serves as an immunity boosting mechanism . p. vivax transmission can subsist in areas of low mosquito abundance and is robust to drug administration initiatives due to relapse , making it an inconvenient and cumbersome , yet less lethal alternative to p. falciparum .

plasmodium falciparum has traditionally been the main focus of malaria control programs worldwide , mainly because this parasite is the major cause of severe morbidity and mortality in tropical africa . however , at a time when global eradication is advocated as the ultimate goal of malaria control strategies worldwide , p. vivax needs to be given much more attention from researchers , policy makers , and funding agencies . p. vivax is a major public health challenge in central and south america , the middle east , central , south , and southeast asia , oceania , and east africa , where 2.85 billion people are currently at risk of infection and as many as 250 million infections may be due to this species each year . the emergence of drug - resistant strains and severe ( sometimes fatal ) disease challenges the traditional view of vivax malaria as a benign infection . although cytoadhesion of p. vivax - infected erythrocytes to endothelial cells has been recently demonstrated and might contribute to the pathogenesis of severe vivax malaria , this phenomenon is likely to be much less common than with p. falciparum infections . compared to p. falciparum , p. vivax has a slightly longer incubation period ( 12 days to several months ) and a similarly phased erythrocytic cycle ( 4248 hours ) that yields fewer merozoites per schizont . however , the distinct ability of p. vivax to stay dormant in host 's liver cells and cause relapses weeks or months after the primary infection is the most striking difference between p. vivax and p. falciparum . although the molecular mechanism of relapse remains undisclosed , progression of the parasite through its life cycle is fairly well described [ 5 , 6 ] . a proportion of sporozoites remain dormant in the liver , as hypnozoites , for prolonged periods of time before developing and causing recurrent infection . the ability to relapse in fact , in areas where both species are present , act ( artemisinin - based combination therapy ) campaigns have had a greater impact on p. falciparum than on p. vivax prevalence [ 7 , 8 ] . we demonstrate how this is evident when contemplating elimination scenarios , with p. vivax elimination being extremely difficult to achieve by mass drug administration . epidemiological studies have accumulated evidence that clinical ( antidisease ) and antiparasite immunity is attained at younger ages for p. vivax , when compared to p. falciparum , under similar infection rates [ 911 ] . we put forward an alternative hypothesis arguing that differences between the observed age profiles lie in the characteristic life cycles of both parasites . explicitly , we propose that the ability of p. vivax to relapse can accelerate the piecemeal acquisition of clinical immunity . we developed a model representing the transmission dynamics of p. vivax by adding new elements to the foundation laid by previous work in p. falciparum . we have thus a model structure for p. vivax transmission , represented by figure 1(a ) , which contains the topology representing p. falciparum as a submodel ( figure 1(b ) ) . the falciparum dynamics are retrieved by equating p1 to 1 in the p. vivax model . natural history of p. vivax infection is generally similar to that of p. falciparum , with a few but crucial idiosyncrasies . susceptible individuals ( s ) are subject to a certain rate of infection ( here represented by the force of infection ) , which depends on local environmental and socioeconomic factors . in p. vivax , after a mosquito infectious bite , an indeterminate proportion of the inoculated sporozoites remains dormant in the liver , whilst the remaining develops into erythrocyte invading merozoites . the model describing the transmission dynamics of p. vivax must then include a latent class , representing those individuals who , after recovering from infection , keep a remnant of dormant liver forms , called hypnozoites ( subject to reactivation at rate ) rather than clearing all parasites while acquiring clinical immunity . reactivation is still a rather cryptic process , and most relapses seem to result from activation of heterologous hypnozoites , which suggests that genotype - specific immunity somehow modulates the occurrence of relapses , much to the resemblance of how the clinical outcome of a given infection is determined . here , parameter p1 accounts for episodes not followed by a relapse , either because no hypnozoites were formed or because the remaining hypnozoites do not reactivate during their lifespan . for illustration purposes , throughout the paper we keep p1 = 0.25 for p. vivax ( and p1 = 1 for p. falciparum ) . it is generally accepted that the severity of malaria episodes decreases as the host accumulates exposures to the parasite . we implement this aspect of malaria immunity by discretizing the malaria clinical spectrum into two compartments : i1 represents the severe end of the spectrum , and is labeled clinical malaria , " while i2 represents the less severe part and is labeled asymptomatic malaria " . naturally , there is a degree of arbitrariness in this compartmentalization , and the results should be interpreted in this context . we consider that immunologically nave individuals will display clinical symptoms when infected ( i1 ) . although p. vivax infections are generally not as severe as those caused by p. falciparum , they are far from benign . community studies have revealed that the proportion of p. vivax infections presenting with fever is similar to the one registered for p. falciparum . the probability of clinical outcome upon relapse in individuals that kept hypnozoites ( l1 ) from a previous clinical infection is determined by parameter p2 . latent individuals carrying hypnozoites are subject to reinfection at rate , with the resultant infection phenotype being determined by parameter p2 as well . individuals who have just recovered from a clinical malaria episode are said to have acquired temporary clinical immunity ( r ) . this means that they do not display clinical symptoms upon reinfection and that , unless they are challenged again within a given time frame , they will lose that clinical protection . in fact these infections are crucial in boosting acquired clinical immunity , and the interplay between the rate of infection in clinically immune individuals and the rate of clinical immunity loss is fulcrum in determining the number of expected clinical malaria cases during one 's lifespan . as such , those recovering from an asymptomatic infection are also subject to loss of immunity at rate . these include individuals that either clear all parasite forms and return to r ( a proportion p1 ) or keep a remnant of hypnozoites and go to the l2 class . we consider that , in the latter case , subsequent infections and relapse will give rise to asymptomatic malaria . the described dynamics can be written as the following system of differential equations : ( 1)st+sa=r((a)+)s,i1t+i1a=(a)s+p2(+(a))l1(1+)i1,rt+ra = p11i1+p12i2(+(a)+)r,i2t+i2a=(a)r+(+(a))l2 + ( 1p2)(+(a))l2(2+)i2,l1t+l1a=(1p1)1i1+l2(+(a)+)l1,l2t+l2a=(1p1)2i2(+(a)++)l2 , with boundary conditions at age a = 0 : s(t , 0 ) = and ii(t , 0 ) = li(t , 0 ) = r(t , 0 ) = 0 for i = 1,2 . the p. falciparum transmission dynamics are retrieved by making p1 = 1 : ( 2)st+sa=r((a)+)s,i1t+i1a=(a)s(1+)i1,rt+ra=1i1+2i2((a)++)r,i2t+i2a=(a)r(2+)i2 . the force of infection was constructed as an age - dependent parameter ( 3)(a)=0(1ceka ) . the function is strictly increasing with age , with a minimum 0(1 r ) ( at age zero ) converging asymptotically to 0 as age increases . parameter k determines how steeply the force of infection increases with age , and r controls the magnitude of that increase . a summary measure of transmission is obtained by integrating the force of infection over age as ( 4)=(a)p(a)da , where p(a ) = e is the total population distributed over age and is the birth and death rate . adopting standard assumptions , is proportional to the frequency of infectious individuals , the proportionality constant being the transmission coefficient , ( 5)=i1+i2 . this standard assumption allows us to analyze how the equilibrium behavior of the system depends on , which is a critical transmission parameter , representing the sylvatic portion of the classical macdonald formulation of the basic reproduction number for vector - borne diseases . the basic reproduction number for the p. vivax model presented here assumes the form ( 6)r0=(p2n1+1p1n2+2p1n3+(+)n4+1n5)2d1+2d2+1d3+12p1d4+2d5+3d6 , where ( 7)n1=1(1p1)()+2,n2=p2,n3=++p2,n4=(+)++2,n5=+(p1+p2)+,d1=1+2p11p2,d2=(p2+p1p1p2)+1(1+p1p2+p1p2),d3=(+2p2+p2p1)+2p1,d4=2p22+p2+p22p1,d5=(1+)(+2)+2(+p1 ) + 1(2p2p2p1+2),d6=+2++1 + 2. epidemiological changes are often attributed to thresholds in transmission , and these are detected through longitudinal trends and comparative studies across multiple communities . we consider the transmission coefficient , ( or the basic reproduction number , r0 ) as a control parameter and describe the significance of these indices of transmission on selected epidemiological variables . to do so , we calculated the endemic equilibria for systems ( 1 ) and ( 2 ) , without age dependence . age profiles were obtained by following a cohort under the pressure of an age - dependent force of infection defined by ( 3 ) , for 20 years . the common variables between systems ( 1 ) and ( 2 ) have the same boundary conditions . we used the escalator boxcar train ( ebt ) technique to simulate the dynamics in our age - structured population . to assert the benefits of specific interventions we first simulate our age - structured model in equilibrium conditions to obtain the age profile of clinical disease prevalence without intervention . we use that age profile as the initial condition for the simulation in which the drug administration trial is in vigor . in such a setup , asymptomatic malaria is treated as effectively as clinical malaria , and thus the recovery rate from an asymptomatic infection , 2 , takes the same value as the recovery rate from a clinical infection , 1 . the expected prevalence of malaria cases of each plasmodium species is highly dependent on local environmental and socioeconomic factors that can be summarized into some transmission index . in figures 2(a ) and 2(c ) , infectious proportions are plotted in terms of a transmission coefficient , , which encapsulates information on contact rates and infectivity . comparing the model outputs for the p. falciparum ( red ) and p. vivax ( blue ) systems , we verify that , if a significant proportion of individuals recovers from infection with dormant forms of the parasite which reactivate later on , it is easier for the parasite to be transmitted in a sustainable manner ( figure 2(a ) ) . latency then generates a mechanism by which the parasite population can be maintained in scenarios where vectors are not very abundant or where human - mosquito contacts are sparse . strikingly , for any value of transmission coefficient that sustains both species , the proportion of individuals with a clinical episode due to p. vivax is lower than that due to p. falciparum ( figure 2(a ) ) , while the overall parasite prevalence ( measured as proportion of individuals in the population which carry parasites in the blood stream and are thus potentially infectious ) is expected to be higher for p. vivax ( figure 2(c ) ) . figures 2(b ) and 2(d ) display how equilibrium solutions depend on the basic reproduction number , r0 , as a proxy for transmission . figure 3 simulates the introduction of malaria control measures in an area supporting p. vivax transmission versus in an area supporting p. falciparum transmission , with the same parasite prevalence . the intervention consists of applying an mda ( mass drug administration ) campaign using act aimed at reducing the infectious period of asymptomatic infections , making all infections , regardless of clinical outcome , last the same . figure 3 portrays that by treating asymptomatic infections equally for both species one can reduce falciparum prevalence ( red lines ) to a much greater extent than vivax prevalence ( blue lines ) within the same time frame ( dot - dashed lines ) . this intervention alone leads to sustained elimination of p. falciparum if implemented for 274 days ( time for the system to enter the basin on attraction of the disease - free equilibrium ) . in the case of p. vivax , elimination is only possible through a stochastic event , and even then the risk of reemergence will be high as the disease - free equilibrium is unstable for this system . if a drug that can eliminate the dormant forms of the parasite ( primaquine ) is included in the mda strategy , then the p. . however , once the intervention is halted the risk for reemergence would still be much higher for p. vivax due to instability of the disease - free equilibrium . in figure 4 , we simulate the age profiles for p. vivax and p. falciparum for an equal risk of infection ( 0 ) . the predicted age profiles for p. vivax ( blue ) display a higher value for clinical malaria cases at the peak , when compared with p. falciparum ( red ) , and reveal a decrease in the average age at infection . this means that the risk of a p. vivax episode relative to the risk of having a p. falciparum episode is greater in very young children and lower throughout childhood . this is consistent in the two transmission settings chosen for this illustration as well as for the entire transmission spectrum . the true impact of p. vivax transmission on human populations stands in the shadow of the overwhelming mortality and morbidity burden exerted by p. falciparum worldwide . p. vivax importance has been increasingly recognized over the years , and new estimates of the global malaria burden revealed that there are slightly more people at risk of having a p. vivax infection than a p. falciparum infection . however , probably more interesting is to consider the importance and impact of p. vivax under the scope of its ecological interactions with p. falciparum , especially considering that coinfection with these species might somehow modulate the clinical outcome of infection [ 2123 ] , and that there might be cross - specific immunity [ 2427 ] . understanding the transmission dynamics of p. vivax is crucial to understand the current epidemiological scenario and the potential long - term impact of control interventions . we have previously developed a mathematical model to represent the dynamics of p. falciparum transmission in human populations . the model was calibrated on hospitalization data from 8 endemic regions in sub - saharan africa , estimating a fundamental difference between the duration of clinical and asymptomatic infections . this result led to the identification of a deterministic elimination threshold for p. falciparum malaria in areas of low to moderate transmission . we adopted the same generic model for p. vivax while adding latency classes to represent those individuals who recover from infection with a remnant of dormant parasites , called hypnozoites . model outputs were generated and compared with and without latent classes , to mimic vivax and falciparum , respectively , while all other features were unchanged . the expected levels of clinical episodes of both p. vivax and p. falciparum for the same levels of the transmission coefficient , , indicate that p. vivax transmission can be sustained for much lower values of this parameter , when compared with p. falciparum ( figure 2(a ) ) . this becomes intuitive in light of p. vivax 's ability to relapse , which can transform a single infectious bite into more than one malaria episode leading to higher parasite prevalence in the p. vivax system ( figure 2(c ) ) . these relationships are inverted , however , when parasite prevalence is represented against the basic reproduction number , r0 , ( figures 2(b ) and 2(d ) ) attesting the importance of standardizing transmission indices . more importantly the deterministic elimination threshold described for the p. falciparum system is no longer present under the conditions simulated for p. vivax . the parameter regime sustaining the bistability phenomenon that gives rise to the elimination threshold is contracted when latency comes into play . a major advance in the search for effective malaria drug treatment , following the demise of most known drugs in the battle against resistant parasites , came in the form of artemisinin , a very potent and effective drug against chloroquine and sulphadoxine - pyrimethamine - resistant infections , which can clear parasites and resolve fever faster than any other licensed antimalarial . however , artemisinin derivatives have a very short half - life , translating into substantial treatment failures when used as monotherapy , which motivated the combination of artemisinin with longer - lasting partner drugs in the so - called artemisinin combination therapies ( acts ) , assuring that there is substantial antimalarial pressure to deal with the residual parasite biomass that may persist when the artemisinin derivatives have fallen below therapeutic levels . curiously , empirical studies have revealed that the deployment of act as a control strategy affects p. falciparum transmission much more than it does p. vivax transmission [ 8 , 9 ] . furthermore , unexpected resurgences of p. vivax malaria in areas where elimination attempts were thought to have been successful question to what extent p. vivax control is sustainable [ 32 , 33 ] . our simulations suggest that , by treating all infections ( equally for both species ) with an act therapy , one can reduce falciparum prevalence to a much greater extent than vivax prevalence ( in the same time scale ) . this is intrinsically associated with act 's inability to kill the dormant forms of p. vivax , which escape drug action , subsist , and can cause a relapse later on , thus sustaining the parasite pool . in such scenario one should invoke the use of drugs targeting hypnozoites ( primaquine is the only approved and available drug at the moment ) as a means of counteracting the parasite 's ability to relapse . if everyone was to receive a dose of primaquine to kill the hypnozoite parasite forms , the dynamics would be similar to that of p. falciparum . a remaining difference , however , is that p. vivax elimination is predicted to be unstable , meaning that any perturbation in the system ( introduction of infectious individuals from neighboring populations for instance ) would drive it back to the endemic equilibrium . another epidemiological observation that deserves careful consideration is the differential speed at which complete clinical immunity is attained when comparing p. vivax to p. falciparum for scenarios of equal risk of infection [ 911 ] . while others have interpreted this phenomenon as an evidence of there being different mechanisms by which immunity is acquired , our results suggest that the difference in age profiles of clinical malaria can be accounted for by the intrinsic transmission dynamics inherent to the natural history of infection of each species . in figure 4 , we can clearly see that the model topologies we used to describe the p. vivax and p. falciparum transmission dynamics can account for differences in the age profiles of clinical malaria , specifically for low levels of p1 and p2 . we then propose that differences in age profiles of clinical malaria can be explained solely by p. vivax 's ability to relapse , which converts a single infectious mosquito bite into one or more malaria infections , thus accelerating the acquisition of clinical immunity . relapse also serves as an immunity boosting mechanism that prevents onsets of malaria episodes in older ages . on a final note , we recall that all parameters other than those governing hypnozoite formation and relapse were unchanged between the two scenarios . p. vivax and p. falciparum transmission dynamics might , however , be modulated by a number of biological characteristics such as gametocyte production and antigenic variation . although our model was not designed to explore these processes , some analogies can be made as detailed in the supplementary material available online at doi:10.1155/2012/921715 . first , it should be acknowledged that p. vivax produces gametocytes earlier than p. falciparum during a human infection and these gametocytes are shorter lived in the bloodstream . as a consequence , vivax patients are expected to have transmitted more by the time malaria infection is confirmed and treatment is provided . second , the acquisition of immunity to p. vivax relies on expectations regarding strain specificity of natural immunity and antigenic similarity between primary infections and relapses that deserve further attention [ 35 , 36 ] .

purpose . to investigate whether patients with age - related macular degeneration ( amd ) run a potentially higher risk of developing visual , musculoskeletal , and balance complaints than age - matched controls with normal vision . methods . visual assessments , self - rated visual function , self - rated visual , musculoskeletal , and balance complaints , and perceived general health were obtained in 37 amd patients and 18 controls , at baseline and after an average of 3.8 years later . results . at follow - up both groups reported decreased visual acuity ( va ) and visual function , but only amd patients reported significantly increased visual , musculoskeletal , and balance complaints . decreased va , need for larger font size when reading , need for larger magnification , and decreased self - rated visual function were identified as risk markers for increased complaints in amd patients . these complaints were also identified as risk markers for decreased health . for controls , decreased va and self - reported visual function were associated with increased visual and balance complaints . conclusions . visual deterioration was a risk marker for increased visual , musculoskeletal , balance , and health complaints in amd patients . specifically , magnifying visual aids , such as cctv , were a risk marker for increased complaints in amd patients . this calls for early and coordinated actions to treat and prevent visual , musculoskeletal , balance , and health complaints in amd patients .

age - related macular degeneration ( amd ) is one of the most common reasons of visual impairment in the western world [ 1 , 2 ] . the prevalence of amd is approximately 2% at the age of 40 , with an increase to about 17%35% at the age of 80 [ 3 , 4 ] . patients with amd experience irreversible and progressive visual loss , leading to reduced quality of life [ 57 ] , reduced health , and increased risk of mortality [ 8 , 9 ] . in step with visual deterioration amd patients report additional complaints , such as loss of postural stability [ 1013 ] , fall accidents , and neck / scapular area complaints [ 1517 ] . in primary health care these symptoms are often downplayed as part of the aging processes and not typically associated with visual impairments [ 6 , 19 ] . however , research indicates that people suffering from amd may pose a higher risk of developing visual , musculoskeletal , and balance complaints . genetics , lifestyle , body mass index , and smoking have all been found to increase the risk of developing amd [ 2 , 3 ] . amd is identified by retinal changes beyond what are age - normal [ 1 , 3 , 18 ] . early stages are associated with minor visual disturbance , followed by increased retinal changes during the intermediate stages and the development of pronounced visual deterioration at the late stages [ 1 , 2 ] . amd follows no particular schedule , but studies show that approximately 20% of cases escalate from intermediate to late stage within 6 years [ 1 , 2 , 21 ] . as vision deteriorates the visual system this may adversely affect visual performance and further distort visual feedback to other systems that are normally supported by visual inputs such as the musculoskeletal and the postural systems . as a consequence there are a number of enhancing devices to help amd patients adjust to their deteriorated vision . although magnifying aids are aimed at facilitating everyday life , they come with some drawbacks such as reduced field of view or the need to use both hands or adopt awkward postures [ 22 , 23 ] . straining conditions such as orbicularis squinting ( to increase effective focus imaging ) or unfavorable gaze - angles ( to locate the best viewing visual field or facilitate convergence movements ) may also occur as the person struggles to acquire an acceptable image , which may in turn impose increased strain in the muscles used for positioning the head [ 16 , 17 , 24 , 25 ] . visual deterioration also negatively influences postural control as this is largely based on adequate visual feedback [ 1214 ] . hence , visual deterioration and increased use of visual aids may increase the risk of musculoskeletal and balance complaints [ 12 , 13 , 1517 , 26 ] . however , our knowledge of what influence visual loss in amd may pose on musculoskeletal and balance complaints is limited . previous research addressing the impact of visual loss in amd patients on visual , musculoskeletal , and balance complaints is limited or has used cross - sectional design . this research has identified concurrent relationships ; however , less is known about the influences of visual decline in amd on visual , musculoskeletal , and balance complaints across time . by using a longitudinal cohort design these issues can be addressed . therefore studies using longitudinal design are warranted in order to investigate to what extent visual deterioration influences visual , musculoskeletal , and balance complaints . the present study aims to identify risk markers for increased visual , musculoskeletal , and balance complaints and perceived general health in patients with amd by using a longitudinal cohort design . based on the previous research described above , we hypothesized that visual , musculoskeletal , and balance complaints shouldincrease more in amd patients than in age - matched individuals with age appropriate vision , increase with increased visual deterioration , negatively affect perceived general health . increase more in amd patients than in age - matched individuals with age appropriate vision , increase with increased visual deterioration , negatively affect perceived general health . this study has a prospective longitudinal case / control design , in which a group of amd patients and an age- and sex - matched reference group with normal vision were assessed twice . at baseline , amd patients were recruited in consecutive order from the queue system of the low vision centre at region rebro county , sweden . inclusion criteria were being diagnosed with late- or intermediate - stage amd according to an ophthalmologic examination at a hospital eye clinic with best corrected visual acuity ( bcva ) worse than 0.5 logmar and no additional eye disease . the patient should have had the amd diagnosis for at least one year in order to adapt to the visual impairment and become accustomed with use of magnifying visual aids . individuals in the reference group were recruited from relatives and companions of the amd patients visiting the clinic . inclusion criteria included age - normal bcva defined as better than 0.10 logmar with correction if needed and without any known eye disease . actual refraction and bcva were tested in both groups in an eye - examination conducted by an optometrist at the low vision clinic . individuals in either group were excluded at baseline or at follow - up if they were medically diagnosed with musculoskeletal or balance disorders , such as whiplash , arthritis , myalgia , or parkinson 's disease . the study included 88 individuals , 64 cases ( 43 women/21 men , mean age 78.6 years , sd = 5.81 , and range 61.885.9 years ) , and 24 referents ( 11 women/13 men , mean age 73.9 years , sd = 6.08 , and range 64.983.0 years ) . in 2012 , a mean of 3.8 years and sd = 0.46 years later , the former participants were contacted by telephone to schedule the follow - up appointment . at follow - up 55 individuals remained , including 37 cases ( 28 women/9 men , mean age 81.1 , sd = 5.43 , and range 67.289.2 years ) and 18 referents ( 11 women/7 men , mean age 77.6 , sd = 5.60 , and range 69.078.1 years ) . none of the amd patients were diagnosed with wet neovascular amd at baseline ; however , six amd patients have had ranibizumab injections prior to baseline . twenty amd patients ( 31% ) had reached late amd at baseline according to clinical classification of age - related macular degeneration . two participants ( one amd patient and one referent ) have had monocular cataract surgery with a replaced intraocular lens . among the 27 cases not participating in the follow - up 16 were diseased , 5 were infirm or suffered from dementia and were not able to participate , 2 had moved , and 4 declined without giving any specific reason . among the 6 referents not participating in the follow - up , one was deceased , one had moved , one had acquired amd , and 3 declined without giving any specific reason . consort flow chart is shown in figure 1 . informed consent was collected from all participants at baseline and at follow - up . best corrected visual acuity ( bcva ) using habitual visual aids ( ordinary spectacles or contact lenses ) was assessed under monocular and binocular viewing conditions using the early treatment diabetic retinopathy study ( etdrs ) test chart . if va was very low , the bailey - lovie letter - by - letter chart was used to capture va beyond 1.0 logmar . critical print size was assessed by the smallest font size that could be read fluently / best acceptable reading pace , with the use of the participants ' normal visual aids . cps was measured in points ( p ) , where 1 p = 1/72 of an inch . a font size of 8 p is commonly used in newspapers and is equivalent to a snellen notation of n8 or 1 m . cps was assessed as participants read the appropriate printed texts , ranging from 4 p to 64 p. reading distance was assessed by measuring the distance in cm between the text and the eyes when the participant was reading the near charts with assistance of their normal visual aids . visual aids contained head - worn visual aids ( single vision reading glasses , bifocals , or progressives ) used alone or combined with handheld magnifiers or closed circuit television ( cctv ) . the magnification used while reading was estimated by summing up the dioptric power ( d ) of those visual aids that normally were used simultaneously . the amount of dioptres was then transformed into units of magnification by dividing the sum of d by 4 based on a simplified commonly used nominal magnification transformation formula ( m = f/4 ) . this calculation does not give a perfect value of the provided enlargement [ 23 , 27 , 28 ] , as it is not based on the equivalent viewing distance . for example , if reading glasses of + 8 d were used , this refers to a magnification of 8/4 = 2x , but combined with a handheld magnifying aid of + 20 d , this refers to a magnification of 8/4 + 20/4 = 7x . this assumes a reading distance of less than 20 cm , whereof at least half the distance refers to the distance between the specs and the magnifying aid . in clinical practice , most of the elderly have difficulties accomplishing a shorter reading distance than what is provided by reading glasses above + 8.0 , which is the reason why the additive handheld magnifying aid is often combined with their best reading glasses to overbridge effects from the continuous visual deterioration and does not shorten the reading distance any further [ 22 , 23 ] . controls also used near visual aids , in order to compensate for the distance , which was estimated in the same manner . near visual function was assessed on the near activities subscale of the national eye institute - visual function questionnaire 25 ( nei - vfq 25 ) . the visual function questionnaire - near activities subscale ( vfq - nas ) consists of six questions and has shown excellent internal consistency and reliability ( cronbach alpha 0.91 ) as well as convergent validity with bcva and health - related quality of life among patients with amd [ 30 , 31 ] . the first five alternatives describe the quality of visual function , ranging from 0 to 100 at equal steps ( i.e. , 0 , 25 , 50 , 75 , and 100 ) . the sixth alternative , stopped doing this for other reasons or not interested in doing this , is not related to the quality of visual function and therefore does not contribute to the total vfq - nas score . the sum score from the six questions was divided by the number of contributing questions to form a total near activity visual function score . generally , a total score above 80 indicates minor visual problems and a score of 70 or less is considered clinically significant . visual , musculoskeletal , and balance complaints were measured on the visual , musculoskeletal , and balance complaints questionnaire ( vmb ) . it consists of 15 questions , with five questions each in visual ( vmb - v ) , musculoskeletal ( vmb - m ) , and balance ( vmb - b ) domains . the questions are rated on visual analogue scales , ranging from 0 ( no problem at all ) to 10 ( problems all the time ) , with verbal anchors at 3 ( occasionally ) and 7 ( quite often ) . the sum of the five scores in each domain is then calculated , with scores ranging from 0 to 50 . the present study was performed with the use of the original vmb - scale , which was used at baseline and then at follow - up allowing for detection of individual changes using gee . the participant could choose from two alternatives , feeling healthy ( 1 ) or not feeling healthy ( 0 ) . data analyses were performed using ibm spss statistics version 22 ( ibm corp , armonk , ny ) . most of the data were positively skewed , which does not necessarily indicate a problem with the scales but reflects the underlying nature of the construct in focus . wilcoxon signed rank test was used to compare differences across time and mann whitney u test was used to compare differences between groups . generalized estimation equation ( gee ) was used to obtain robust parameter estimates and standard errors and to further estimate the correlation of multiple observations for each subject over time . the gee is an extension of generalized linear models , which facilitate regression analyses of dependent variables that are not normally distributed . by using marginal models the analysis gives an average response for observations sharing the same covariates as a function of the covariates ; that is , for every one - unit increase in a covariate across the population in focus , gee tells the user how much the average response should change ; gee can thus account for correlations in repeated measures , and the interpretation of the estimates is much the same as that in ordinary least squares regression when the dependent variable is normally distributed . in the present study , each predictor variable was regressed separately on each of the vmb to identify significant risk markers for visual , musculoskeletal , and balance complaints . since the vmb variables were positively skewed , the gee regression models were specified with a log link function . the influences of predictor variables on participants ' perceived health ( dichotomous data ) were evaluated using gee logistic models , which were expressed in odds ratios ( or ) and 95% confidence intervals ( 95% ci ) . at baseline there was no significant difference in sex ratios between cases and referents , but there was a trend for a difference at follow - up ( = 5.33 , p = 0.069 ) . this difference was mainly due to a larger number of dropouts among men with amd ( 57% ) than among women with amd ( 35% ) at follow - up . there was also a difference in age between groups at baseline ( u = 445 , p = 0.002 ) but not at follow - up ( u = 256 , p = 0.10 ) . as shown in table 1 , cases had worse bcva at baseline than referents , needed larger font size when reading , used shorter reading distances , and needed greater magnification . fewer cases than referents used progressives , but several more of them used a combination of different near visual aids . cases also reported lowered near visual function , lowered perceived general health , and more visual and balance complaints , but not significantly more musculoskeletal complaints than referents . at follow - up , within group analyses revealed that both groups showed deteriorated bcva and visual function ( table 2 ) . cases needed larger font size when reading and more magnification , but referents did not . however , referents needed significantly shorter reading distances at follow - up , which was not found for cases , although cases still needed shorter reading distances than referents ( u = 49.5 , p < 0.001 ) . at follow - up cases no longer used bifocals or progressives as a basic solution , and none of them reported that they solely relied on these visual aids . instead cases reported significantly more visual , musculoskeletal , and balance complaints at follow - up than at baseline , but no change in the level of complaints was found for the referents ( figure 2 ) . complaints increased among cases from 3 times ( vmb - m ) to 10 times ( vmb - v ) as much as among referents . general health decreased in cases , where fewer reported good general health at follow - up than at baseline , whereas there was no significant deterioration of perceived general health found in referents . in regard to the first aim , gee analysis of complaints showed significant group influences , indicating that the increase of visual , musculoskeletal , and balance complaints from baseline to follow - up was larger in cases than in referents ( b = 15.177 , 95% ci : 11.467 ; 18.888 , p < 0.001 , b = 4.180 , 95% ci : 0.018 ; 8.342 , p = 0.049 , and b = 18.862 , 95% ci : 14.373 ; 23.351 , p < 0.001 , resp . ) . in support for the second aim , and as shown in table 3 , visual and balance complaints increased in step with deteriorating va in cases , along with the need for larger critical print size . the analyses also revealed that all three complaints increased with increased use of magnification and deteriorating visual function . also in referents , visual complaints increased with declining va , resulting in more visual and balance complaints as visual function deteriorated ( measured by vfq - nas ) , but there were hardly no changes in magnification and thereby no association between magnification and any of the three vmb complaints . to further evaluate influences from use of visual aids , types of aid were regressed on visual , musculoskeletal , and balance complaints , serving as dependent variables . as shown in table 3 , these analyses showed that in cases reduced use of bifocals and progressives was associated with decreased visual complaints , while increased use of cctv was associated with increased visual , musculoskeletal , and balance complaints . in referents , the use of reading glasses was associated with decreased visual complaints and use of bifocals was associated with increased visual and balance complaints . in support for the third aim , gee analyses showed that cases perceived poorer health with increasing visual and musculoskeletal complaints ( table 4 ) . the need for larger font size was also a marker for poorer perceived health in cases . this study followed a group of amd patients and age - matched referents responding to reported change in perceived complaints during visual decline over a period of four years . that is , amd patients were more at risk of increased visual , musculoskeletal , and balance complaint than similarly aged individuals with age - normal vision . additionally , visual deterioration was a risk marker for increased visual and balance complaints both in cases and in referents . in referents this decrease must be considered consistent with the age - normal decline . finally , increased visual , musculoskeletal , and balance complaints constituted risk factors for decreased perceived general health . at baseline amd patients had significantly worse conditions in all assessed areas compared to referents , except for reported musculoskeletal complaints . most of these group differences remained or increased at follow - up , which supports the hypothesis that having amd entails a greater risk of poorer health [ 79 ] . it can be hypothesized that , in pace with visual function decline and increased need for support and assistance , low vision patients may successively abandon physical and social activities resulting in a less satisfactory quality of life [ 5 , 6 , 8 , 9 , 34 ] . visual deterioration , such as worsening bcva , need of larger print size , larger magnification when reading , and worsened near visual functioning were risk markers for increased visual and balance complaints among amd patients . interestingly , the referents showed a similar pattern to amd patients , in regard to bcva and near visual functioning . thus , visual deterioration seems to be a risk marker for increasing visual and balance complaints regardless of initial vision status ( normal or impaired ) . these results are of course consistent with general deterioration of sensory function with age ; however , the increased magnitude of complaints is more profound among amd patients , which may put them at higher risk of developing lower health and lower quality of life than those with same age but with age - normal vision ( table 3 ) . reduced near vision function and increased need of magnification were the most prominent risk markers for musculoskeletal complaints among amd patients . this result is consistent with previous research showing that during near work tasks an increased use of optical enlargement in visual enhancing devices may adversely result in restricted postures [ 22 , 26 , 27 , 35 ] that subsequently can lead to increased musculoskeletal complaints as well as balance complaints [ 15 , 17 ] . magnifying aids facilitate everyday life but may have side effects such as limited and restricted field of view and the need to adopt awkward ( nonneutral ) head and body postures to see as well as possible . as a consequence , magnifying aids may lead to increased strain on the visual and musculoskeletal systems , subsequently resulting in increased complaints . when the portable visual aids are not sufficient any longer , stationary visual aids as cctv may be needed , on the cost of increasing strain on the visual and musculoskeletal systems . in the present study , the increased use of visual enhancing devices such as cctv was related to increased visual and musculoskeletal complaints in amd patients . thus , as vision deteriorates increased visual inputs from use of specialized visual enhancing aids , such as hyperoculars , magnifiers , and cctv , are needed , where amd patients need to adopt certain postures to adjust the eye to the best position for viewing . these adopted postures can be compared in many ways to those found in people using visual display units ( vdu ) at work , where visual ergonomics are monitored carefully in order to prevent neck / scapular complaints [ 3638 ] . however , visual ergonomic guidelines might be overlooked when providing cctv , especially as they are situated in old patients ' homes ( or nursing homes ) with limited ability for adequate adjusting . one should , however , bear in mind that cctv is typically prescribed in situations when near vision is severely impaired and when the enlargement gained by ordinary optical enhancing devices is insufficient . cctv is thus often one of a very few remaining solutions when extreme magnification is needed . normally a two times increase in dioptric power represents three line improvements on the near reading test chart but may not be applicable as the distance between the eye and the magnifier may vary . our estimates of magnification at use do not intend to estimate angular enlargement but give a hint of the increasing limits of high powered visual aids . at baseline , fewer cases , proportionately , than referents perceived themselves as healthy . at follow - up , the number of cases perceiving themselves as unhealthy had increased , while there was no significant change among referents . visual function ( need for a larger font size ) , visual complaints , and musculoskeletal complaints constituted risk markers for perceived unhealthiness in amd patients , and visual and balance complaints were significant risk markers among referents . thus , aspects of visual loss affect perceived health as people grow older , irrespective of whether they have visual impairment . however , the magnitude of complaints is larger among amd patients , thus increasing the risk of developing lower health . our results are in line with studies showing that visual functional loss is associated with depression [ 58 ] and that loss of vision is one of the most - feared disabilities . this is in line with findings in a previous study describing decreased health and increased need of health care in people with neck or back pain . also the association of increased balance complaints and unhealthiness is consistent with previous research [ 5 , 9 , 11 , 12 , 40 ] . this result was somewhat unexpected since both groups showed increased balance complaints and decreased general health over time . the lack of association found between balance complaints and health in amd patients may be statistical in nature and due to ceiling effect . that is , because amd patients already had a greater level of balance problems than referents ( by four times ) the increase of balance complaints from an already high level may have less effect on perceived health than a similar magnitude of increase from much lower levels , as in the control group . the overall dropout rate was 38% , with a larger dropout rate in the amd group . given that this study was conducted in a group of elderly participants who have an increased risk of illness and mortality , this rate was to be expected . the larger proportion of dropouts due to mortality in amd patients is consistent with research showing that among the elderly late - stage amd is associated with increased risk of all - cause mortality [ 8 , 9 ] . since the mortality rate is probably larger among those with the most complaints and the poorest health , the effect of this attrition bias would probably decrease any differences between amd patients and referents ; thus , differences between the groups may be larger than those reported here and observed differences are probably underrated . it should be noted that six amd patients had ranibizumab injections at the low vision clinic prior to registration and two participants ( one amd patient and one referent ) had been treated with monocular cataract surgery between registration and follow - up . this may have slowed down their visual deterioration and thus any potential influences on visual , musculoskeletal , or balance complaints in the amd group . therefore , the observed level of complaints may underestimate the level in an amd population not being treated with eye surgery or receiving ranibizumab injections . we hypothesized that magnifying visual aids may give rise to suboptimal ergonomic head postures that affect the muscles in the neck scapular area , resulting in subsequent musculoskeletal complaints and increasing the risk of more balance complaints . at the same time , we can not neglect the fact that in late - stage amd visual performance may not be sufficient for adequate visuomotor support . research in this area is limited and future research is warranted . it should be noted that our estimates of magnification do not reflect the exact equivalent viewing power ( evp ) as we did not collect all required distances for this estimate , that is , the distances between the naked eye / reading glass and the magnifying aid compared to evp . the magnification estimate can therefore be somewhat overestimated . however , the magnification estimate reflects the use of higher dioptric power under conditions of deteriorating visual function . in pace with need of higher amounts of magnification , the reading distance variation gets more limited and the focal depth gets more specific resulting in the need for adopting a more constricted and static posture [ 22 , 26 , 28 ] . it was not possible to separate the influences of visual aids from the influences of loss of central vision in the present data because these factors are intertwined . that is , amd patients start increasing the use of visual aids when they start losing their central vision . future research measuring the amount of time in visual aid use might shed light on the magnitude of influence from visual aids and from loss of central vision this study had a longitudinal design where temporal order of causality was met . however , the result that visual decline evoked increased visual , musculoskeletal , and balance complaints does not reveal the mechanisms behind these findings or whether there are other variables involved in this process , such as personality , depression , or socioeconomic status . prevalence and intervention studies could shed light on how best to prevent increased complaints in people with amd and on treatments to decrease complaints among those affected . the present study demonstrated that visual , musculoskeletal , and balance complaints increase with visual decline and increase more among people with amd than among people with normal vision . increased visual , musculoskeletal , and balance complaints were also found to negatively affect general health . the study showed that visual decline and increased use of greater magnification in visual aids are important risk factors for increased complaints . the results from this study show that amd patients ' use of magnifying visual aids has side effects that optometrists and low vision staff must be aware of when prescribing visual aids . as a preventive measure , optometrists may suggest alternative use of both optical and technical visual enhancing aids as well as using devices with text - to - speech function ( e.g. , electronic readers ) when applicable . visual aids are important , especially in elderly people with severe amd , as these aids enable users to continue daily activities and maintain their quality of life . optometrist may also refer patients to a physiotherapist for investigation , treatment , and preventive training [ 4042 ] . this calls for coordinated actions at an early stage in order to prevent visual - related musculoskeletal and balance complaints in amd patients to minimize the further risk of more serious complaints .

backgroundhuman serum has the potential to become the most informative source of novel biomarkers , but its study is very difficult due to the incredible complexity of its molecular composition . we describe a novel tool based on biodegradable nanoporous nanoparticles ( npnps ) that allows the harvesting of low - molecular - weight fractions of crude human serum or other biofluids . npnps with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon . when incubated with a solution , the npnps harvest only the molecules small enough to be absorbed into the nanopores . then they can be recovered by centrifugation and dissolved in water , making the harvested molecules available for further analyses.resultsfluorescence microscopy , gel electrophoresis , and mass spectrometry were used to show the enrichment of low - molecular - weight fraction of serum under physiological conditions , with a cut - off of 13 kda and an enrichment factor > 50.conclusionfrom these findings , we conclude that ability to tune pore size , combined with the availability of hundreds of biomolecule cross - linkers , opens up new perspectives on complex biofluid analysis , discovery of biomarkers , and in situ drug delivery .

the search for novel tools for early diagnosis is one of the major issues in medical research . the discovery of biomarkers in biological fluids and blood is especially challenging due to the tremendous number of biomolecular species , which differ by many orders of magnitude in their relative abundance.1,2 of the several approaches proposed in the last few years , the study of the proteome seems to hold the greatest potential.2,3 proteomics is a quickly developing area of biochemical investigation . the basic aim of proteomic analysis is the identification of specific protein patterns from cells , tissues , and biological fluids related to physiological or pathological condition.2,4 it provides a different view from that of gene expression profiling , which does not evaluate post - transcriptional and post - translational modifications , or protein compartmentalization and half - life changes ( eg , ubiquitination and proteosome - driven degradation ) . all these characteristics make the protein profile much more complex but more informative than gene expression profiling . several approaches can be used to perform proteomic analysis ; among these , the most common are methods based on 2d - polyacrylamide gel electrophoresis ( 2d - page ) and mass spectrometry ( ms).59 it is now well accepted that the low - molecular - weight ( lmw ) , low - abundance fraction of biological fluids might contain the most informative source of novel biomarkers . the conventional analytical methods mentioned above do not seem to reach a sufficient degree of resolution and sensitivity to reliably detect and identify lmw and low - abundance peptides . therefore , research has recently focused on the development of innovative devices that are able to enrich this fraction of body fluid proteome , making it available for use in common analytical tools . harvesting and enrichment of candidate biomarkers from complex protein mixtures can be pursued in different ways . usually , in the plasma or serum , in which albumin and immunoglobulin alone account for > 90% of total protein content , these species are conventionally removed prior to 2d - page and ms by immunoaffinity depletion columns . however , it must be emphasized that many potentially informative biomarkers , represented by very small peptides noncovalently associated with the carrier protein albumin , are lost following this procedure . many commercial albumin removal kits based on different methods are available , but it has been demonstrated that they can cause loss of several low - abundance proteins , including albuminome.1012 moreover , most of these sieving / filtering systems do not allow sufficient flexibility of the whole process . therefore , nanotechnology methods appear to offer a promising and powerful strategy for overcoming these limitations.1316 discovered over 40 years ago , porous silicon ( psi ) has attracted increasing attention in many fields of research for its interesting features . in particular , the demonstration of its biodegradability in physiological environments has opened up new perspectives for biomedical application.17 the desired dissolution rate can be obtained through the accurate control of the morphology , pore size , and ph . the typical dissolution rate in alkaline conditions ranges from a few minutes to up to a few days . moreover , the well - known functionalization processes of the porous surface provide further control of bioreactivity and hydrophobicity.1821 various surface derivatization has been reported in the literature and hundreds of different cross - linking agents are now available to selectively bind the target molecules.22 in this paper , we present a direct approach to harvesting the lmw fraction of a complex solution that relies on 3 important properties of silicon nanoporous nanoparticles ( npnps ) : ( a ) they can act as nanosponges and absorb small molecules depending on nanopore size ; ( b ) they can be separated from solution through efficient centrifugation ( the nanoparticle density is higher than that of the solvent ) ; ( c ) they can be dissolved in water . this last property is relevant because the filtration process can be carried out in physiological solution , without the need for introducing particular solvents , which can contaminate , denaturate , or degrade the potential biomarkers . the whole process is depicted in figure 1 ( figures 1a c fabrication process and figures 1c h harvesting process ) . the starting solution is incubated with npnps which , bcause of size - exclusion , can absorb only the lmw fraction into the nanopore . afterwards , npnps can easily be recovered from solution by means of centrifugation , and resuspended in water , or other solvents in which they can be dissolved . the harvested molecules are then available , in their native state , for further analyses . this easy , cheap , and fast process enables the harvesting of peptides < 13 kda from raw serum . modern biology demands not only fast and easy techniques but also full compatibility with existing protocols . the present approach can be mixed and matched with the majority of current investigation protocols . npnps were fabricated by ultrasonication of a thin film of nanoporous silicon.1324 psi was obtained by anodization of a boron - doped silicon wafer ( resistivity 510 cm ) of crystal orientation , using an electrolyte binary mixture of hydrofluoric acid ( 25% ) , water ( 25% ) , and ethanol ( 50% ) . the psi film was oxidized in an oven at 200c for 2 hours . in order to obtain npnps , the psi film was sonicated in dimethylformamide for about 60 minutes and then , after washes in ethanol , ultrasonicated ( 5 w ) in water for 10 minutes at a constant temperature of 4c , and finally filtered to eliminate impurities > 500 nm . an ad hoc protein mixture was prepared by mixing 50% ( v / v ) human serum albumin ( mw 66,000 da ; sigma - aldrich , st . louis , missouri , usa ) ; 30% ( v / v ) bovine plasma gamma globulin ( heavy chain mw 45,000 da ; light chain mw 30,000 da ; biorad , berkeley , california , usa ) ; and 20% ( v / v ) aprotinin ( 6,500 da ; sigma - aldrich ) . all proteins were dissolved at a concentration of 1 mg/ ml in 100 mm sodium phosphate buffer and 9% ( w / v ) sodium chloride ph 7.4 ( pbc ) to reproduce physiological conditions . human serum was obtained from a healthy anonymous male donor and collected in accordance with human proteome organization ( hupo ) plasma proteome project guidelines.25 approximately 8 ml of blood were drawn by venipuncture and collected in tubes without additive and allowed to clot at room temperature for 40 minutes . the sample was centrifuged within 2 hours of collection at 1300 g for 10 minutes , aliquoted into silicon tubes , and stored at 80c . the fabricated npnps were deposited onto a glass substrate and characterized using scanning electron microscopy ( sem ) and fluorescence microscopy . the pores of the particles are too small to be shown by sem ( figures 2 a and b ) , but the typical emission spectrum peak at around 620 nm ( figures 2c and d ) indicates a pore size of about 23 nm ( excitation wavelength 408 nm ) . the nanoparticle diameter is about 200 nm , and it can be adjusted by changing the power and duration of the sonication process . we studied the interactions of the nanoparticles with complex biological fluids in different environmental conditions . here we report 3 experiments with fluids of increasing complexity in order to show the splitting capability of the npnps : experiment 1 . interaction of a complex mixture of proteins with a wide range of mws simulating a biological fluid . npnps were incubated with 2 solutions of fluorescent polymer of different mws ( 6 kda and 14 kda dextran - fluorescein isothiocyanate [ fitc ] 10 mg , npnp 5 mg , water 10 ml , 1 hour ) . after incubation , the npnps were separated from supernatant ( centrifugation ) , dropped on a slide , dried , and analyzed with fluorescence microscopy . optical and fluorescence images collected on the npnps incubated with polymers of 14 and 6 kda are reported in upper and lower panels , respectively ; for higher mw polymer there is no trace of absorption , and only a weak blue fluorescence coming from salt residue is visible . in contrast , green fluorescence emitted by npnps incubated with the lower mw polymer indicates good absorption . after incubation and centrifugation , the recovered npnps can be dissolved in water at a rate depending on the temperature and acidity of the medium . at ph 8 and 90c , the dissolution takes a few minutes , but it can be also carried out at room temperature in a few hours , if nondenaturing conditions are needed . at ph < 5 , the npnps do not dissolve , allowing their long - term storage in water at or above room temperature . in experiment 1 , the amount of harvested fraction of dextran - fitc ( mw 6 kda ) can be evaluated by comparing the total fluorescence intensity of 2 solutions ( harvested vs supernatant ) . the 2 solutions show very similar fluorescence intensity values , indicating that , under the experimental conditions described above , 50% of the molecules were harvested , whereas the remaining 50% were left in solution . this result shows that the loading capacity is very high , about 1 mg of harvested molecules for each mg of npnps . in the second experiment , the ability of npnps to enrich the lmw fraction of a complex mixture was tested with an ad hoc protein mixture ( see materials and methods ) . for this purpose , the effects of several parameters such as ph , osmolarity , temperature , and incubation time were studied , and conditions were optimized , and , finally , a volume of 200 ml of protein mixture was incubated with 5 mg of npnps ( about 10 particles ) at room temperature for 1 hour . npnps were subsequently separated from supernatant by centrifugation , and washed once with pbc buffer . the sodium dodecyl sulfate - page ( sds - page ) in figure 4 ( panel a ) clearly shows that nanoparticles selectively retain small molecules as aprotinin ( mw 6,500 da ) , whereas proteins with higher mw are completely excluded from the nanopores . we noted that during the incubation a small fraction of the nanoparticles dissolve releasing silicic acid into the solution ( about 8% of npnp volume under our conditions).26 this amount can be decreased by lowering the incubation temperature , ph , or incubation time when possible . in the third experiment , the same protocol for experiment 2 was applied to raw human serum to demonstrate that npnps are able to selectively enrich lmw serum proteome ( lmwp ) . after 1 hour of incubation ( 100 ml of serum sample diluted 1:2 with pbc buffer , 5 mg of npnps ) , the lmw fraction was recovered by dissolving the nanoparticles in pbc buffer for 12 hours at 37c . we noted that the dissolution process can be hastened by heating the solution and adding a base ( to increase ph ) or other solvent . an aliquot of serum , before and after incubation , was analyzed by sds - page using a 16.5% ready prepared tris - tricine / peptide gel . the efficiency of npnps to selectively enrich the lmwp is clearly demonstrated in the sds - page analysis shown in figure 4 ( panel b ) : in lines 2 and 3 , crude human serum and supernatant are represented . in line 4 , the lmw fraction of human serum extracted from npnps is visible : no molecules > 12 kda are still present , except for a very small trace of albumin . from densitometric analysis of lane 4 , it was estimated that > 50% of total pixel volume was from lmw species , whereas in lane 2 ( unprocessed serum ) the same area represented < 1% of the total pixel volume . the enrichment factor was thus estimated to be > 50 . in order to characterize the lmw protein species extracted by incubation with npnps , gel bands from sds - page - separated extracts ( figure 4b , lane 4 ) were cut and processed for tryptic digestion according to the protocol shown in the supplementary information , section 1 . based on mw marker information , the 3 processed gel bands corresponded to mw intervals of 1520 kda , 1015 kda and < 10 kda . tryptic peptides were analyzed by nanoscale liquid chromatography interfaced with tandem ms ( nanolc - ms / ms , supplementary information , section 2).27,28 in an alternative approach , proteins extracted by incubation with npnps were directly digested in solution by trypsin and analyzed by nanolc - ms / ms . validated protein identifications are reported in 2 tables ( supplementary information , section 3 ) . besides abundant lmw serum proteins , such as apolipoprotein a - ii and transthyretin , a database search identified a number of lmw serum proteins that are not considered abundant , such as tetranectin , platelet basic protein , and dermcidin . furthermore , lc - ms / ms analysis identified several high - mw proteins in the in - gel - digested sds - page protein bands . such identifications confirm that the lmw proteome is also populated with fragments of abundant high - mw serum proteins.29 a comparison of the results of these experiments with those reported in the literature shows many remarkable characteristics of the npnps : easy and cheap production . easy recovery by centrifugation , long - term storage wet ( water solution of ph < 5 ) or dry . we note that such properties are very attractive also for drug delivery applications , for which the use of nanocarriers is attracting a lot of interest . we report a straightforward tool relying on water - soluble silicon npnps used to harvest the lmw molecules in their native state from a complex fluid . the method is based on the porosity of the nanoparticles , which act as a molecular sieve , and their solubility in a physiological environment . the proposed approach can be mixed and matched with currently available techniques and protocols , and does not require high temperature , denaturing solvents , or other contaminants . a cut - off of about 13 kda was demonstrated for crude human serum . the ability to tune pore size , combined with the availability of hundreds of biomolecule cross - linkers , opens up new perspectives on complex biofluid analysis , discovery of biomarkers , and in situ drug delivery . gel bands below 25 kda were excised and digested by trypsin incubation ; selected bands were punched out manually and placed in a silicon eppendorf tube . gel pieces were washed once with 150 ml of deionized water and then destained by 3 washes of 150 ml 35% acetonitrile in 25 mm nh4hco3 buffer . after destaining , trypsin digestion was performed overnight at 37c with modified trypsin ( sigma - aldrich , st louis , mo , usa ) 0.2 mg / ml . the resulting tryptic peptides were acidified , purified by ziptips c18 ( millipore , billerica , ma ) according to the manufacturer s procedure and eluted with 2 l of a 1:1 mixture of acetonitrile and 0.1% trifluoro acetic acid ( v / v ) . 28 l of loading pump solvent ( see below ) were added , and 10 l were injected for nanolc - ms / ms analysis . 500 ng of sequencing grade modified trypsin ( sigma - aldrich ) were added , and digestion was allowed to proceed for 16 h. the resulting tryptic peptides were fractionated by off - gel eletrophoresis before nanoscale lc - ms / ms analysis , in order to achieve a 2-dimensional fractionation of the peptide mixture . an agilent 3100 off - gel fractionator ( agilent technologies , santa clara , ca ) was used . the peptide mixture was diluted with carrier ampholyte mixture ( 3.6 ml final volume , 10% carrier ampholyte concentration ) . the sample was then loaded on separate immobiline drystrip , linear ph range 3.010 , 18 cm long , purchased from ge healthcare ( chalfont st . peptides were focused at a constant temperature of 20c , and at constant current intensity of 50 a . after focusing was complete , fractions were collected . in order to improve peptide recovery , sample wells were washed with 100 l of a water / methanol / formic acid mixture , 49:50:1 ( v / v / v ) . the wash solution was added to each well and allowed to incubate for 90 minutes before being collected and pooled with the corresponding fraction supernatant . pooled supernatants were reduced to a volume of approximately 10 l in a vacuum centrifuge . a 1:4 mixture of concentrated eluates and loading pump solvent of the nanolc - ms / ms system ( see below ) was injected for nanolc - ms / ms analysis . considering the injection volume of 10 l , approximately 1/5 of each oge fractions was injected for nanolc - ms / ms . chromatography was performed on an ultimate nanolc system from dionex ( sunnyvale , ca , usa ) , using a valveless setup.1,2 the peptide extracts were redissolved in 30 l of loading pump solvent ( see below ) and 10 l were loaded onto an in - house packed 100 m i.d . , integra frit ( new objective , cambridge , ma ) trapping column ( packing bed length 1.5 cm ) at 10 l / min of loading pump solvent , consisting of h2o / acetonitrile / trifluoroacetic acid ( tfa ) 97.95:2:0.05 ( v / v / v ) . after 4 minutes of column washing , the trapping column was switched on - line to the analytical column : an in - house packed 50 m i.d . , pico frit column ( new objective ) , filled with the same stationary phase used for the trapping column packing : 3 m c18 silica particles ( dr maisch , entringen , germany ) . mobile phase a was h2o / acetonitrile / formic acid / tfa 97.9:2:0.09:0.01 ( v / v / v / v ) ; mobile phase b was h2o / acetonitrile / formic acid / tfa 29.9:70:0.09:0.01 ( v / v / v / v ) . after 10 minutes at 95% b , the column was re - equilibrated at 5% b for 20 minutes before the following injection . ms detection was performed on a qstar xl hybrid lc - ms / ms from applied biosystems ( foster city , ca , usa ) operating in positive ion mode , with nesi potential at 1300 v , curtain gas at 15 units , cad gas at 3 units . information - dependent acquisition ( ida ) was performed by selecting the 2 most abundant peaks for ms / ms analysis after a full tof - ms scan from 400 to 1600 m / z lasting 4 seconds . both ms / ms analyses were performed in enhanced mode ( 3 seconds / scan ) . ms / ms data were converted to mascot generic format ( mgf ) by the analyst software 1.1 ( applied biosystems ) . data were searched on the mascot search engine ( www.matrixscience.com ) , version 1.9 , against the international protein index database ( ipi version 3_38 ) using the following parameters : ms tolerance 30 ppm ; ms/ ms tolerance 0.2 da ; variable modifications methionine oxidized ; enzyme trypsin ; max . missed cleavages 1 . ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained ( 4% false discovery rate ) . proteins identified with a minimum of 2 peptides were retained ( protein probability score > 0.9 ) . proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes : in gray , protein identification obtained with a single hit . those hits were validated by visual inspection . ms / ms data are reported in supplementary information . mascot score ( * ) is reported for identifications which have not passed tpp validation , but were above mascot threshold of 29 and for which manual validation was undertaken . additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes : in gray , protein identification obtained with a single hit . gel bands below 25 kda were excised and digested by trypsin incubation ; selected bands were punched out manually and placed in a silicon eppendorf tube . gel pieces were washed once with 150 ml of deionized water and then destained by 3 washes of 150 ml 35% acetonitrile in 25 mm nh4hco3 buffer . after destaining , trypsin digestion was performed overnight at 37c with modified trypsin ( sigma - aldrich , st louis , mo , usa ) 0.2 mg / ml . the resulting tryptic peptides were acidified , purified by ziptips c18 ( millipore , billerica , ma ) according to the manufacturer s procedure and eluted with 2 l of a 1:1 mixture of acetonitrile and 0.1% trifluoro acetic acid ( v / v ) . 28 l of loading pump solvent ( see below ) were added , and 10 l were injected for nanolc - ms / ms analysis . 500 ng of sequencing grade modified trypsin ( sigma - aldrich ) were added , and digestion was allowed to proceed for 16 h. the resulting tryptic peptides were fractionated by off - gel eletrophoresis before nanoscale lc - ms / ms analysis , in order to achieve a 2-dimensional fractionation of the peptide mixture . an agilent 3100 off - gel fractionator ( agilent technologies , santa clara , ca ) was used . the peptide mixture was diluted with carrier ampholyte mixture ( 3.6 ml final volume , 10% carrier ampholyte concentration ) . the sample was then loaded on separate immobiline drystrip , linear ph range 3.010 , 18 cm long , purchased from ge healthcare ( chalfont st . peptides were focused at a constant temperature of 20c , and at constant current intensity of 50 a . after focusing was complete , fractions were collected . in order to improve peptide recovery , sample wells were washed with 100 l of a water / methanol / formic acid mixture , 49:50:1 ( v / v / v ) . the wash solution was added to each well and allowed to incubate for 90 minutes before being collected and pooled with the corresponding fraction supernatant . pooled supernatants were reduced to a volume of approximately 10 l in a vacuum centrifuge . a 1:4 mixture of concentrated eluates and loading pump solvent of the nanolc - ms / ms system ( see below ) was injected for nanolc - ms / ms analysis . considering the injection volume of 10 l , approximately 1/5 of each oge fractions was injected for nanolc - ms / ms . chromatography was performed on an ultimate nanolc system from dionex ( sunnyvale , ca , usa ) , using a valveless setup.1,2 the peptide extracts were redissolved in 30 l of loading pump solvent ( see below ) and 10 l were loaded onto an in - house packed 100 m i.d . , integra frit ( new objective , cambridge , ma ) trapping column ( packing bed length 1.5 cm ) at 10 l / min of loading pump solvent , consisting of h2o / acetonitrile / trifluoroacetic acid ( tfa ) 97.95:2:0.05 ( v / v / v ) . after 4 minutes of column washing , the trapping column was switched on - line to the analytical column : an in - house packed 50 m i.d . , pico frit column ( new objective ) , filled with the same stationary phase used for the trapping column packing : 3 m c18 silica particles ( dr maisch , entringen , germany ) . mobile phase a was h2o / acetonitrile / formic acid / tfa 97.9:2:0.09:0.01 ( v / v / v / v ) ; mobile phase b was h2o / acetonitrile / formic acid / tfa 29.9:70:0.09:0.01 ( v / v / v / v ) . after 10 minutes at 95% b , the column was re - equilibrated at 5% b for 20 minutes before the following injection . ms detection was performed on a qstar xl hybrid lc - ms / ms from applied biosystems ( foster city , ca , usa ) operating in positive ion mode , with nesi potential at 1300 v , curtain gas at 15 units , cad gas at 3 units . information - dependent acquisition ( ida ) was performed by selecting the 2 most abundant peaks for ms / ms analysis after a full tof - ms scan from 400 to 1600 m / z lasting 4 seconds . both ms / ms analyses were performed in enhanced mode ( 3 seconds / scan ) . ms / ms data were converted to mascot generic format ( mgf ) by the analyst software 1.1 ( applied biosystems ) . data were searched on the mascot search engine ( www.matrixscience.com ) , version 1.9 , against the international protein index database ( ipi version 3_38 ) using the following parameters : ms tolerance 30 ppm ; ms/ ms tolerance 0.2 da ; variable modifications methionine oxidized ; enzyme trypsin ; max . missed cleavages 1 . ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained ( 4% false discovery rate ) . proteins identified with a minimum of 2 peptides were retained ( protein probability score > 0.9 ) . proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes : in gray , protein identification obtained with a single hit . those hits were validated by visual inspection . ms / ms data are reported in supplementary information . mascot score ( * ) is reported for identifications which have not passed tpp validation , but were above mascot threshold of 29 and for which manual validation was undertaken . additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes : in gray , protein identification obtained with a single hit . ms / ms data were converted to mascot generic format ( mgf ) by the analyst software 1.1 ( applied biosystems ) . data were searched on the mascot search engine ( www.matrixscience.com ) , version 1.9 , against the international protein index database ( ipi version 3_38 ) using the following parameters : ms tolerance 30 ppm ; ms/ ms tolerance 0.2 da ; variable modifications methionine oxidized ; enzyme trypsin ; max . missed cleavages 1 . ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained ( 4% false discovery rate ) . proteins identified with a minimum of 2 peptides were retained ( protein probability score > 0.9 ) . proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes : in gray , protein identification obtained with a single hit . those hits were validated by visual inspection . ms / ms data are reported in supplementary information . mascot score ( * ) is reported for identifications which have not passed tpp validation , but were above mascot threshold of 29 and for which manual validation was undertaken . additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes : in gray , protein identification obtained with a single hit .

backgroundthe purpose of this study was to examine the effects of carvedilol therapy on autonomic control of the heart and qt - interval dispersion ( qtd ) among children with idiopathic dilated cardiomyopathy ( dcm ) whose symptoms were not adequately controlled with standard congestive heart failure therapy.material/methodspatients with dcm who were treated with carvedilol were enrolled in the study . all patients had undergone carvedilol therapy in addition to standard therapy for at least 6 months . clinical , echocardiographic , and electrocardiographic parameters , and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment.resultsa total 34 patients ( mean age : 7.44.3 years ) with dcm were analyzed in the study . the median follow - up period was 9.5 months . after the 6 months of carvedilol therapy the clinical score significantly improved , left ventricular ejection fraction ( lvef ) and fractional shortening ( lvfs ) significantly increased , and left ventricle end - diastolic dimensions and end - systolic dimensions significantly decreased . there were statistically significant increases in mean sdnn , sdann , rmssd , and pnn50 ( p=0.002 , p=0.001 , p=0.008 , and p=0.026 , respectively ) . after the carvedilol therapy , sdnn was correlated with the clinical score , heart rate , lvef , lvfs , and total premature ventricular contractions ( pvcs ) . in addition , rmssd and pnn50 were correlated with heart rate , lvef and lvfs . a significant reduction was observed in qtc - minimum , qtc - maximum , and qtd values ( 434.940.7 vs. 416.136.5 , 497.843.6 vs. 456.341.7 , 58.617.1 vs. 49.315.6 ; p<0.001 , p=0.001 , and p=0.008 , respectively ) . qtd was significantly related to pvcs ( r=0.62 , p=0.02).conclusionswe conclude that the addition of carvedilol to standard therapy can improve clinical symptoms and heart rate variability , and reduce in arrhythmia markers in children with dcm .

dilated cardiomyopathy ( dcm ) is a rare disease in children , with an annual incidence of 0.34/100 000 persons . recent studies have suggested that multiple neuroendocrine factors , including the reduction in parasympathetic activity and activation of sympathetic nervous and renin - angiotensin - aldosterone ( raa ) system , play an important role in the genesis and progression of congestive heart failure ( chf ) [ 24 ] . such observations have led to the use of neurohormonal antagonists such as aldosterone antagonists , angiotensin - converting enzyme inhibitors ( acei ) , and -adrenergic blockers for the treatment of chf [ 1,79 ] . reduced measures of heart rate variability ( hrv ) , considered a marker of tonic sympathetic and vagal outflow , several investigators have demonstrated that measurements of hrv can be used to evaluate the effect of acei and -adrenergic receptor antagonists on cardiac autonomic activity . dysregulation of autonomic nervous system function and impaired homogeneity of myocardial repolarization are 2 major mechanisms for the genesis of ventricular arrhythmias . increased qt - interval dispersion ( qtd ) and decreased hrv have been reported in adult patients with chf , and are considered as potential markers for arrhythmogenicity and for use in predicting mortality [ 1214 ] . it decreases the chronic adrenergic overstimulation of the myocardium and improves myocardial function , and has been shown to improve survival , decrease morbidity , and improve quality of life in adults with chf . although some studies have suggested that carvedilol therapy decreased qtd and had a beneficial effect on hrv parameters in adult patients , the effects of carvedilol therapy on inhomogeneity of ventricular repolarization and autonomic nervous regulation are not clear . to the best of our knowledge , there have been few published studies about the effect of carvedilol on arrhythmias in dilated cardiomyopathy in pediatric patients , and there are no published data available about the effects of carvedilol therapy on heart rate dynamics and arrhythmia markers in children . therefore , the aim of this study was to examine the effects of carvedilol therapy on autonomic control of the heart and qt - interval dispersion as an arrhythmia marker among children with dcm whose symptoms were not adequately controlled with standard chf therapy . after obtaining approval from the institutional review board of our institution , we retrospectively reviewed the hospital records of the 34 patients who were followed - up in our pediatric cardiology clinic with dcm and treated with carvedilol in addition to standard therapy of digoxin , diuretics , and acei . data collected included : age at diagnosis , therapy prior to carvedilol , time between diagnosis and initiation of carvedilol , age , weight , symptoms at carvedilol initiation and last follow - up , dosage , and adverse effects of the drug . all patients who received carvedilol in addition to standard therapy were followed - up for at least 6 months . the diagnosis of dilated cardiomyopathy was defined as a child having both left ventricular ( lv ) contractility 2 sd below the normal mean and lv end - diastolic dimension 2 standard deviations above the normal mean , which was not caused by dysrhythmia or any other structural heart disease . we used a modified scoring system of chf signs and symptoms described by ross and reithmann et al . ( table 1 ) . each sign or symptom was graded on a scale of 0 , 1 , or 2 points according to the severity . the sum of points formed the clinical score ( range 012 points ) , with a higher score corresponding to more severe heart failure . the inclusion criteria were : patients with dcm whose symptoms were not adequately controlled with standard chf therapy ( clinical score of 5 or more ) , and whose systemic ventricle is morphologic left ventricle and left ventricular ejection fraction ( lvef ) 0.40 . the exclusion criteria were : congenital heart defect , atrial fibrillation , sustained or symptomatic ventricular dysrhythmias , sinus or av node dysfunction , bradycardia , acute myocarditis , bronchial asthma , obstructive or severe regurgitative valvular disease , significant renal , hepatic , gastrointestinal disease , endocrine disorders , taking any drugs influencing qt dispersion , and use of antiarrhythmic drugs . patient characteristics including age , sex , weight , height and concomitant medications were collected for all patients . clinical , echocardiographic , electrocardiographic parameters , and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment . echocardiographic data obtained before and after carvedilol therapy were retrospectively reviewed from patient medical records . for estimates of left ventricular dimension , function and shortening fraction were reported with the standard method of 2-dimensionally directed m - mode measurements . standard 12-lead electrocardiography was obtained simultaneously using a recorder set at 50 mm / s paper speed and calibration of 1milivolt / centimeter , in a comfortable supine position . qt intervals ( qtc = qt/rr , maximum and minimum qtc intervals ) were also measured . qt dispersions were manually measured in all electrocardiograms ( ecg ) by the same investigator . all measurements were repeated by a second investigator who was blinded to the demographic information and therapy . qt intervals were measured from the beginning of the qrs complex to the end of the t wave , which was defined as return to baseline in each ecg lead . when u waves were present , the qt interval was measured to the nadir of the curve between the t and u waves . for each lead , 2 or more consecutive cycles were measured and the arithmetic mean of the qt interval for that lead was used in all calculations for qtd . qtd was calculated as the difference between the longest and shortest qt interval measured in each individual ecg lead . premature ventricular contractions ( pvcs ) were characterized by the following : ectopic , premature , and bizarrely shaped qrs complexes , usually wider than 120 msec ; absence of p waves preceding a qrs complex ; the t wave is usually large , and its direction is opposite the major deflection of the qrs . couplets were characterized by 2 consecutive premature ventricular contractions . nonsustained ventricular tachycardia ( vt ) was characterized by 3 or more consecutive beats lasting less than 30 seconds , at a rate > 100/min . all records of the 24-holter monitoring before and after carvedilol therapy were obtained from the computer - based electronic holter archive of our institution . holter studies were considered adequate for interpretation if there was greater than 16 hours of analyzable data for the 24-hour recording . the tapes were manually reviewed by an independent observer who was blinded to the patient s identity and study treatment . total numbers of pvc and number of episodes of vt were calculated . we also performed hrv analysis by using 24-hour holter ecg monitoring ( dms 300 holter recorder ; dms inc . abnormal beats and areas of artifact were automatically and manually identified and excluded from the analysis . we analyzed hrv in the time domain by the following 5 standard 24-hour time - domain measures : sdnn ( standard deviation of all normal sinus r - r intervals during 24 hours ) , sdnni ( mean of the standard deviation of all normal sinus r - r intervals for all 5-minute segments ) , sdann ( standard deviation of the average normal sinus r - r intervals for all 5-minute segments ) , rmssd ( root mean square of the successive normal sinus r - r interval difference ) , and pnn50 ( percentage of successive normal sinus r - r intervals longer than 50 ms ) . changes from pre- to post - carvedilol therapy were assessed using paired t - tests for continuous variables when the differences were approximately normally distributed , and by using wilcoxon signed rank tests for differences with skewed distributions . the relationship among variables was evaluated by pearson s correlation coefficient . a p - value < 0.05 was considered to be significant . analyses were performed with the software package spss 11.0 ( spss , inc , chicago , il , usa ) . after obtaining approval from the institutional review board of our institution , we retrospectively reviewed the hospital records of the 34 patients who were followed - up in our pediatric cardiology clinic with dcm and treated with carvedilol in addition to standard therapy of digoxin , diuretics , and acei . data collected included : age at diagnosis , therapy prior to carvedilol , time between diagnosis and initiation of carvedilol , age , weight , symptoms at carvedilol initiation and last follow - up , dosage , and adverse effects of the drug . all patients who received carvedilol in addition to standard therapy were followed - up for at least 6 months . the diagnosis of dilated cardiomyopathy was defined as a child having both left ventricular ( lv ) contractility 2 sd below the normal mean and lv end - diastolic dimension 2 standard deviations above the normal mean , which was not caused by dysrhythmia or any other structural heart disease . we used a modified scoring system of chf signs and symptoms described by ross and reithmann et al . was graded on a scale of 0 , 1 , or 2 points according to the severity . the sum of points formed the clinical score ( range 012 points ) , with a higher score corresponding to more severe heart failure . the inclusion criteria were : patients with dcm whose symptoms were not adequately controlled with standard chf therapy ( clinical score of 5 or more ) , and whose systemic ventricle is morphologic left ventricle and left ventricular ejection fraction ( lvef ) 0.40 . the exclusion criteria were : congenital heart defect , atrial fibrillation , sustained or symptomatic ventricular dysrhythmias , sinus or av node dysfunction , bradycardia , acute myocarditis , bronchial asthma , obstructive or severe regurgitative valvular disease , significant renal , hepatic , gastrointestinal disease , endocrine disorders , taking any drugs influencing qt dispersion , and use of antiarrhythmic drugs . patient characteristics including age , sex , weight , height and concomitant medications were collected for all patients . clinical , echocardiographic , electrocardiographic parameters , and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment . echocardiographic data obtained before and after carvedilol therapy were retrospectively reviewed from patient medical records . for estimates of left ventricular dimension , function and shortening fraction were reported with the standard method of 2-dimensionally directed m - mode measurements . standard 12-lead electrocardiography was obtained simultaneously using a recorder set at 50 mm / s paper speed and calibration of 1milivolt / centimeter , in a comfortable supine position . qt intervals ( qtc = qt/rr , maximum and minimum qtc intervals ) were also measured . qt dispersions were manually measured in all electrocardiograms ( ecg ) by the same investigator . all measurements were repeated by a second investigator who was blinded to the demographic information and therapy . qt intervals were measured from the beginning of the qrs complex to the end of the t wave , which was defined as return to baseline in each ecg lead . when u waves were present , the qt interval was measured to the nadir of the curve between the t and u waves . for each lead , 2 or more consecutive cycles were measured and the arithmetic mean of the qt interval for that lead was used in all calculations for qtd . qtd was calculated as the difference between the longest and shortest qt interval measured in each individual ecg lead . premature ventricular contractions ( pvcs ) were characterized by the following : ectopic , premature , and bizarrely shaped qrs complexes , usually wider than 120 msec ; absence of p waves preceding a qrs complex ; the t wave is usually large , and its direction is opposite the major deflection of the qrs . nonsustained ventricular tachycardia ( vt ) was characterized by 3 or more consecutive beats lasting less than 30 seconds , at a rate > 100/min . all records of the 24-holter monitoring before and after carvedilol therapy were obtained from the computer - based electronic holter archive of our institution . holter studies were considered adequate for interpretation if there was greater than 16 hours of analyzable data for the 24-hour recording . the tapes were manually reviewed by an independent observer who was blinded to the patient s identity and study treatment . we also performed hrv analysis by using 24-hour holter ecg monitoring ( dms 300 holter recorder ; dms inc . abnormal beats and areas of artifact were automatically and manually identified and excluded from the analysis . we analyzed hrv in the time domain by the following 5 standard 24-hour time - domain measures : sdnn ( standard deviation of all normal sinus r - r intervals during 24 hours ) , sdnni ( mean of the standard deviation of all normal sinus r - r intervals for all 5-minute segments ) , sdann ( standard deviation of the average normal sinus r - r intervals for all 5-minute segments ) , rmssd ( root mean square of the successive normal sinus r - r interval difference ) , and pnn50 ( percentage of successive normal sinus r - r intervals longer than 50 ms ) . changes from pre- to post - carvedilol therapy were assessed using paired t - tests for continuous variables when the differences were approximately normally distributed , and by using wilcoxon signed rank tests for differences with skewed distributions . the relationship among variables was evaluated by pearson s correlation coefficient . a p - value < 0.05 was considered to be significant . analyses were performed with the software package spss 11.0 ( spss , inc , chicago , il , usa ) . a total 34 patients ( 18 male , 16 female , mean age : 7.44.3 years , range 32 months to 14 years ) with dcm were analyzed in the study . all patients had undergone carvedilol therapy in addition to standard therapy for at least 6 months . the median follow - up period was 9.5 months ( range 6.413.7 months ) after the initiation of carvedilol therapy . the initial mean carvedilol dose was 0.140.07 mg / kg / day , and 0.460.28 mg / kg / day at 6 months . as standard treatment , 34/34 ( 100% ) were on digoxin , 32/34 ( 94.1% ) were on furosemide , and 32/34 ( 94.1% ) were on acei . the average heart rate was significantly reduced after carvedilol treatment ( 11423 vs. 8921 beat / min , p=0.008 ) . systolic blood pressure tended to decrease after carvedilol therapy , but did not reach statistical significance . after 6 months of carvedilol treatment , the ross clinical score significantly improved from 6 to 3 ( p=0.03 ) ( table 3 ) . the most common adverse events were dizziness ( 22% ) , vomiting ( 13% ) , hypotension ( 8% ) , and headache ( 5% ) . lvef significantly increased from 34.77.6% ( range 2240% ) to 45.29.6% ( range 2961% ) following carvedilol treatment ( p=0.002 ) . after carvedilol treatment , the left ventricular fractional shortening ( lvfs ) significantly increased from 16.49.7% ( range 1033% ) to 23.97.7% ( range 1637% ) ( p=0.016 ) , left ventricle end - diastolic dimensions ( lvedd ) significantly decreased from 45.78.1 mm ( range 3160 mm ) to 41.46.5 mm ( range 2658 mm ) ( p=0.026 ) and the left ventricle end - systolic dimensions ( lveds ) significantly decreased from 39.46.9 mm ( range 2447 mm ) to 34.26.0 mm ( range 2043 mm ) ( p=0.047 ) ( table 3 ) . there were significant increases in mean sdnn , sdann , rmssd , and pnn50 after carvedilol therapy ( p=0.002 , p=0.001 , p=0.008 , and p=0.026 , respectively ) . a trend toward an increase in sdnni after carvedilol therapy , sdnn was correlated with the clinical score of chf , heart rate , lvef , lvsf , and total pvcs . in addition , rmssd and pnn50 were correlated with heart rate , lvef , and lvsf after carvedilol therapy . the comparison of clinical , hemodynamic , heart rate variability , and ventricular arrhythmia parameters of patients receiving carvedilol therapy at baseline and after the treatment are shown in table 3 , and the correlation between changes in hrv in the time domains and changes in hemodynamic parameters are presented in table 4 . intra- and interobserver variability were assessed in 18 randomly chosen patients ; all intra- and interobserver variability for ecg parameters ranged from 3.1 to 4.8% . a significant reduction was observed in maximum and minimum qtc interval , qtc , and qtd values after carvedilol treatment . qtd was slightly higher in patients with a lower clinical score than in those with a higher clinical score , but the difference was not statistically significant . qtd was significantly related to total pvcs , but qtd was not related to age , sex , clinical score of chf , heart rate , lvef , lvsf , or lvedd ( table 4 ) . although 8 patients ( 23.5% ) had pvcs before treatment , they disappeared in 4 patients and pvc decreased in 2 patients after treatment . sustained ventricular tachycardia was not observed in any patients . before carvedilol therapy , 5 patients ( 14.7% ) had ventricular couplets and 2 patients ( 5.8% ) had nonsustained ventricular tachycardia . a trend toward a decrease in ventricular couplets and nonsustained ventricular tachycardia did not reach statistical significance after 6 months . patients with dcm who had arrhythmic events during follow - up had significantly greater qtd than those without arrhythmic events ( 59.716.8 vs. 51.415.6 , p=0.026 ) . the initial mean carvedilol dose was 0.140.07 mg / kg / day , and 0.460.28 mg / kg / day at 6 months . as standard treatment , 34/34 ( 100% ) were on digoxin , 32/34 ( 94.1% ) were on furosemide , and 32/34 ( 94.1% ) were on acei . the average heart rate was significantly reduced after carvedilol treatment ( 11423 vs. 8921 beat / min , p=0.008 ) . systolic blood pressure tended to decrease after carvedilol therapy , but did not reach statistical significance . after 6 months of carvedilol treatment , the ross clinical score significantly improved from 6 to 3 ( p=0.03 ) ( table 3 ) . the most common adverse events were dizziness ( 22% ) , vomiting ( 13% ) , hypotension ( 8% ) , and headache ( 5% ) . lvef significantly increased from 34.77.6% ( range 2240% ) to 45.29.6% ( range 2961% ) following carvedilol treatment ( p=0.002 ) . after carvedilol treatment , the left ventricular fractional shortening ( lvfs ) significantly increased from 16.49.7% ( range 1033% ) to 23.97.7% ( range 1637% ) ( p=0.016 ) , left ventricle end - diastolic dimensions ( lvedd ) significantly decreased from 45.78.1 mm ( range 3160 mm ) to 41.46.5 mm ( range 2658 mm ) ( p=0.026 ) and the left ventricle end - systolic dimensions ( lveds ) significantly decreased from 39.46.9 mm ( range 2447 mm ) to 34.26.0 mm ( range 2043 mm ) ( p=0.047 ) ( table 3 ) . there were significant increases in mean sdnn , sdann , rmssd , and pnn50 after carvedilol therapy ( p=0.002 , p=0.001 , p=0.008 , and p=0.026 , respectively ) . a trend toward an increase in sdnni did not achieve statistical significance after 6 months . after carvedilol therapy , sdnn was correlated with the clinical score of chf , heart rate , lvef , lvsf , and total pvcs . in addition , rmssd and pnn50 were correlated with heart rate , lvef , and lvsf after carvedilol therapy . the comparison of clinical , hemodynamic , heart rate variability , and ventricular arrhythmia parameters of patients receiving carvedilol therapy at baseline and after the treatment are shown in table 3 , and the correlation between changes in hrv in the time domains and changes in hemodynamic parameters are presented in table 4 . intra- and interobserver variability were assessed in 18 randomly chosen patients ; all intra- and interobserver variability for ecg parameters ranged from 3.1 to 4.8% . a significant reduction was observed in maximum and minimum qtc interval , qtc , and qtd values after carvedilol treatment . qtd was slightly higher in patients with a lower clinical score than in those with a higher clinical score , but the difference was not statistically significant . qtd was significantly related to total pvcs , but qtd was not related to age , sex , clinical score of chf , heart rate , lvef , lvsf , or lvedd ( table 4 ) . although 8 patients ( 23.5% ) had pvcs before treatment , they disappeared in 4 patients and pvc decreased in 2 patients after treatment . sustained ventricular tachycardia was not observed in any patients . before carvedilol therapy , 5 patients ( 14.7% ) a trend toward a decrease in ventricular couplets and nonsustained ventricular tachycardia did not reach statistical significance after 6 months . patients with dcm who had arrhythmic events during follow - up had significantly greater qtd than those without arrhythmic events ( 59.716.8 vs. 51.415.6 , p=0.026 ) . the neurohumoral mechanisms of chf involve activation of the sympathetic nervous system and the raa system , leading to intrinsic myocardial dysfunction , apoptosis , and remodeling . stimulation of -receptors increases oxygen consumption of the myocardium by increasing the afterload , which causes peripheral and coronary vasoconstriction that results in accumulation of calcium in the myocyte , leading to cell death , and contributes to remodeling of the heart with fibrosis and hypertrophy . carvedilol is a third - generation -blocking agent that at therapeutic target doses blocks all 3 adrenergic receptors that decrease the chronic adrenergic overstimulation of the myocardium and improve myocardial function , and it has been shown to inhibit free radical induced cardiac contractile dysfunction . therefore , it is important to examine the clinical effect of a -adrenergic blocker therapy on dcm ( e.g. , carvedilol ) to verify its efficacy in children . we have shown that oral carvedilol added to standard drug therapy improved ventricular function and clinical symptom scores in children with dcm , and we also found a significant correlation between changes in lvef and time domain parameters of hrv , including sdnn , rmssd , and pnn50 . in accordance with the results obtained from adults , the improvement of the autonomic function seen after the initiation of carvedilol therapy is likely to play an important role in children with dcm . recently , some authors have observed a statistically significant association between sudden cardiac death and depressed sdnn in patients with dcm . however , bilchick et al . demonstrated that sdnn has a strong and independent association with mortality in patients with moderate - to - severe chf . in this context , the uk - heart prospective study has recently demonstrated that reduced sdnn was the best noninvasive independent predictor of cardiac death in patients with chf . in our study , sdnn and sdann were found to be increased after the addition of carvedilol to standard medical therapy ; these results may have important clinical implications . the pnn50 and rmssd predominantly reflect parasympathetic activity and are independent of long - term trends . in the present study , improvements in rmssd and pnn50 were noted , and pnn50 and rmssd correlated to improvement in lvef . our findings confirm that carvedilol treatment has a beneficial effect on the mechanisms that sustain the harmful hyperadrenergic state and may improve prognosis in children with dcm . qtd has been found to be a significant , noninvasive prognostic marker of inhomogeneity of myocardial repolarization in several disease settings , and increased qtd may predispose to arrhythmic events . in a retrospective study of adult patients with chf , fu et al . found a larger qtd in patients who died suddenly or had spontaneous ventricular tachycardia than in survivors . however , only limited data is available regarding the effects of carvedilol on qtd in children with dcm . one retrospective study reported significant increases in qtd values in patients with lv systolic dysfunction . our study showed that carvedilol therapy decreased qt dispersion and qtc parameters , and improved ventricular repolarization characteristics in children with dcm after 6 months of follow - up . a randomized trial in adult patients with dcm showed a significant effect of carvedilol in reducing ventricular arrhythmias . our study results suggest that the increase in sdnn and reduction in qtd are related with decrease in total pvcs . the increase in sdnn and reduction in qtd under carvedilol treatment may be partly due to an adrenergic blocking effect . moreover , antiapoptotic effects and inhibition of chronic remodeling of the myocardium may indirectly contribute to the observed homogenization of the ventricular repolarization process and prevention of induction of arrhythmia in patients with chf . we observed that ventricular ectopic beats disappeared in 4 patients and decreased in 2 patients with carvedilol treatment . reduced heart rate at rest may lead to better oxygen supply , lower the risk for life - threatening arrhythmia , and slow the myocardial remodeling process ; thus , carvedilol may have also been effective in the control of ventricular ectopic beats . our data support the evidence that the increase in heart rate variability reflects improved autonomic regulation of heart rate , and show a significant correlation between heart rate variability changes and hemodynamic improvement with carvedilol therapy in children with dcm . we conclude that the addition of carvedilol to standard medical regimens can improve clinical symptoms and heart rate variability in association with improved left ventricular function , and reduce arrhythmia markers in children with dcm . a randomized , controlled , prospective trial is required to determine the true efficacy of carvedilol on the progression of congestive heart failure and to more clearly define its role in the cardiac autonomic dysfunction and rhythm disorders of children with dcm .

drawing from multiple sources of evidence , this paper updates previous descriptions ( iom , 2002 ) of measurement strategies and teaching techniques to promote four theoretically derived abilities thought to be necessary conditions for the responsible conduct of research . data from three samples ( exemplary professionals , professionals disciplined by a licensing board , and graduates who completed an ethics program designed to promote the four interrelated abilities ) suggest that development of a moral identity that is consistent with the norms and values of a profession is the driving force that gives rise to ethical sensitivity , ethical reasoning , and ethical implementation . evidence from the cited studies supports the usefulness of the theoretical model to ( a ) deconstruct summary judgments about character and see them as abilities that can be assessed and developed ; ( b ) guide the design assessments that are sensitive to the effects of interventions ; and ( c ) augment previous iom recommendations for the development of meaningful learning activities .

in chapter 5 of integrity in scientific research ( 19 ) , the institute of medicine ( iom ) committee described a theory - driven , evidence - based approach to designing instruction in the responsible conduct of research that would maximize the likelihood that education would influence individuals and institutions rather than merely satisfy an item on a check - off list for that institution . the recommended model for education included these principles : the educational program should be built around the development of abilities that give rise to responsible conduct . these include the ability to : 1 ) identify the ethical dimensions of situations that arise in the research setting and the laws , regulations , and guidelines governing one s field that apply ( ethical sensitivity ) ; 2 ) develop defensible rationales for a choice of action ( ethical reasoning ) ; 3 ) integrate the values of one s professional discipline with one s own personal values ( identity formation ) and appropriately prioritize professional values over personal ones ( showing moral motivation and commitment ) ; and 4 ) perform with integrity the complex tasks ( e.g. , communicate ideas and results , obtain funding , teach , and supervise ) that are essential to one s career ( survival skills).the program should be designed in accordance with basic principles of adult learning.the instruction should be provided as much as possible by faculty who are actively engaged in research related to that of the trainees . the educational program should be built around the development of abilities that give rise to responsible conduct . these include the ability to : 1 ) identify the ethical dimensions of situations that arise in the research setting and the laws , regulations , and guidelines governing one s field that apply ( ethical sensitivity ) ; 2 ) develop defensible rationales for a choice of action ( ethical reasoning ) ; 3 ) integrate the values of one s professional discipline with one s own personal values ( identity formation ) and appropriately prioritize professional values over personal ones ( showing moral motivation and commitment ) ; and 4 ) perform with integrity the complex tasks ( e.g. , communicate ideas and results , obtain funding , teach , and supervise ) that are essential to one s career ( survival skills ) . the instruction should be provided as much as possible by faculty who are actively engaged in research related to that of the trainees . in chapter 5 ( 19 ) , the four abilities drawn from rest s ( 20 ) four component model of morality ( fcm)are operationally defined . each is seen as a mix of cognitive and affective processes that contribute to the component s primary function . following the definitions , research conducted in professional education settings is summarized , and teaching strategies , assessment methods , and guidelines for designing cases to promote development of the abilities in the research setting are described . since the iom report also addressed the institutional culture that either enables or impedes researchers ability to act at the leading edge of their ethical competence , a separate appendix ( 18 ) described outcome measures that could be used or adapted ( a ) to study organizational culture and ( b ) to study the ethical competences of individuals . potential outcome measures referenced in chapter 5 are fully described in appendix b ( 18 ) . following publication of the iom report , michael zigmond and i prepared a document ( appendix 1 ) to illustrate how the guidelines for designing cases to promote the four abilities could be applied to a set of circumstances that might arise in the research environment . since publication of the iom report ( 17 ) , new findings have emerged that have implications for the structuring of a professional ethics curriculum . early studies cited in the iom report had shown that the abilities were independent of one another , as rest ( 20 ) had predicted . in other words , competence in one did not predict competence in another , and a shortcoming in a single ability could account for a moral failing . this paper provides summaries of findings for each ability as well as new evidence as to their interconnectedness . following implications for education , the summary and conclusion section expands on earlier recommendations for structuring education and assessment to promote the responsible conduct of research . studies using well validated measures of ethical sensitivity illustrate that competence in the ability to interpret the moral dimension of professional problems is distinct from the ability to apply professional norms and values to determine what ought to be done . what is clear from the research is that if one fails to see the moral issue in a professional problem , competence in reasoning and problem solving , even if well - developed , are not brought to bear ( 3 , 5 , 26 ) . further , just as students and professionals show remarkable variability in their level of moral reasoning development ( see next section ) , they also vary greatly in their ability to interpret patient / client characteristics and professional responsibilities embedded in ethical sensitivity tests . finally , ethical sensitivity can be influenced by educational interventions ( 3 , 5 ) , and , in some settings ( 26 ) , researchers have observed small , though statistically significant gender difference favoring women . in a meta - analysis of ethical sensitivity research ( 26 ) , researchers identified 37 studies in which 23 measures were described to assess ethical sensitivity in professional settings ( e.g. , dentistry , medicine , nursing , professional psychology , business , and science ) . after classifying the measures along several dimensions , including the extent to which the construct was elicited by the stimulus materials , they concluded that only seven of the measures met criteria , and most had not been extensively validated . examples of validated measures that elicit the process include the dental ethical sensitivity test ( dest ) designed for dentistry ( 6 , 11 ) and the racial ethical sensitivity test ( rest ) designed for counseling psychology ( 13 , 22 ) . what distinguishes measures like the rest and dest is the extent to which the stimulus presents clues to one or more moral problems without ever signaling either the moral issues at stake or the particular professional responsibilities called for . by way of contrast , some test designers seem to conceptualize ethical sensitivity as the ability to simply name the moral issue when a condensed synopsis of a moral problem is presented . examples in appendix 1 show how distinctly different the stimulus for assessing ethical sensitivity is from the stimulus for assessing moral reasoning development . the iom report ( 18 ) described the usefulness of the defining issues test ( dit ) for assessing students capacity for reasoning about moral issues . subsequent reviews ( 2 , 9 ) update that literature and describe use of the dit to assess ( a ) the reasoning development across and within professions , and ( b ) the effectiveness of interventions to promote moral judgment development . as described below , the measure is used in remediation programs to identify shortcomings in ethical decision making of practicing dentistry ( 4 ) and medical ( 7 ) students . strategies for assessing students and giving individualized feedback on reasoning development are included in chapter appendices ( 7 ) . in addition to life - span measures of moral judgment development like the dit , researchers have designed measures of ethical reasoning for specific educational contexts . referred to as intermediate concept measures ( icms ) , because they assess ethical concepts in the intermediate zone between broad moral ideals and profession - specific codes of conduct ( e.g. , professional autonomy , informed consent ) , icms present discipline - specific problems and assess agreement between respondents and experts as to the appropriateness or inappropriateness of particular action choices and justifications . it is important to note that test developers engage respondents from the discipline to generate items . an analysis of results from multiple icm measures across populations ( adolescents , young adults , and professionals ) ( 24 ) revealed that irrespective of the sample , age group , or icm concept assessed , respondents had more difficulty identifying the bad choices / justifications than the good items . in addition , the obtained mean differences suggested different levels of performance , as there was little to suggest that these findings were due to methodological issues . further , it appeared that identifying inappropriate and appropriate choices or justifications were not poles on a single dimension , as participants seemed not to apply information and experience gained on one set of considerations to the other . perhaps instruction focuses on what one ought to do and what are appropriate choices , and individuals must then infer what is inappropriate and poorly conceived . if so , ethics education may benefit by expanding its focus on inappropriate choices in order to provide more guidance in developing an experiential base to support more optimal moral decisions . when rest proposed his fcm of moral functioning in the early 1980s , moral motivation was featured , though less well articulated than the other three components . rest ( 20 ) thought that moral motivation influenced moral action directly and in interaction with the other components of the moral system . in the last decade , considerable attention has been given to the study and measurement of lifespan identity formation particularly in the professions . in an edited volume on moral motivation , researchers ( 23 ) summarize early and more recent efforts to operationally define and measure moral motivation . measures include the professional role orientation inventory ( proi ) described in the iom report ( 18 ) , and the theoretical importance of a newer measure ( the professional identity essay [ pie ] ) used to assess life - span identity formation in dentistry ( 10 ) and law ( 16 ) and recently adapted for ethics remediation in medical education ( 7 ) . note : in addition to providing guidance on the use of the dit , chapter appendices ( 7 ) include the pie , adapted for medicine , and criteria for assessing and strategies promoting identity formation . in the same edited volume , researchers ( 8) summarize evidence from dentistry , medicine , law , and the military that supports constructivists theoretical understanding of a developmental continuum of moral motivation and commitment ( rest s component 3 ) . the continuum proceeds from self - interest and concreteness of thought characteristic of entering professionals including entrants into doctoral education programs ( 1 ) to more other - oriented and abstract ways of making sense of the self in relation to others . at advanced levels of moral motivation , the exemplary individual s personal and professional moral values ( 14 , 21 ) are fully integrated , as evidenced by behavior that is consistent across contexts and situations . for example , the exemplary dental professionals studied by rule and bebeau ( 21 ) were able to articulate the public duties of their profession , integrate them with personal value frameworks , and regularly and consistently engage in socially responsible actions . the identity of such exemplary professionals was contrasted with the identities of entering students and entering professionals across several professions ( reviewed by bebeau and monson ) and with professionals who have been disciplined by a licensing board ( 5 ) . a consistent shortcoming of all but two of 41 professionals referred for an ethics assessment because they violated laws governing professional practice was in the ability to articulate professional duties and responsibilities . whereas referrals demonstrated a great deal of variability on measures of ethical sensitivity , moral reasoning , and ethical implementation , they consistently demonstrated only vague notions of their responsibilities to patients , their profession , their community , and society . the importance of professional identity formation to professional practice is further supported by a reexamination of data from a study of gender differences ( 25 ) on measures of the four ethical abilities ( ethical sensitivity , reasoning , role concept , and ethical implementation ) for five cohorts who participated in a well - validated four - year dental ethics education program . the researchers ( bebeau and thoma , in preparation ) noted that those graduates who had developed a mature professional identity by graduation also had developed high levels of competence on measures of the other three components ( ethical sensitivity , reasoning , and implementation ) . taken together , evidence from the three samples ( exemplary professionals , disciplined professionals , and recent graduates who completed an ethics curriculum ) strongly suggests that development of a moral identity that is consistent with the norms and values of the profession is the driving force that gives rise to the development of other abilities that account for responsible professional conduct . whether ethical sensitivity , reasoning , and competence in implementing ethical solutions develop in tandem with the development of a moral identity is a question for further research . as noted in the iom report ( 19 ) , developing self - regulation , self - efficacy and implementation abilities is necessary for effective and responsible professional practice what fisher and zigmond ( 15 ) describe as survival skills in the responsible conduct of research . a wide variety of assessment strategies is possible . in the dental ethics curriculum ( 25 ) , competence in ethical implementation was based on performance score for eight complex cases presented during the last two years of the dental ethics curriculum . stimulus for assessment was similar to dest cases in that students needed to : 1 ) interpret the facts that must be addressed if the problem is to be resolved efficiently ; 2 ) design an action plan ; and 3 ) create a dialogue to illustrate effective plan implementation . all responses were assessed by the same rater , and students could challenge the assessment , revise , and resubmit . like other assessments used in the curriculum , an unexpected finding ( 25 ) was an observed gender difference in ethical implementation for the 60 men and 60 women randomly selected from five cohorts ( 25 ) to explore previously observed gender differences ( favoring women ) in ethical sensitivity and moral reasoning . no gender difference was observed on ethical sensitivity , and pretest and posttest differences on moral reasoning were trivial . whereas men and women had similar scores on the responsibility dimension of moral motivation ( proi scores ) at entrance to professional school , at graduation , an effect size of 0.75 for women versus 0.5 for men indicated greater change for women . a statistically significant gender difference , favoring women , was also evident on the measure of moral implementation , though pretest data were not collected to judge the role of general implementation abilities ( e.g. , interpersonal effectiveness , problem solving , etc . ) . an effect size of 0.57 suggested that the women s ability to implement more effective action plans was not a trivial difference . recall that a reanalysis of this data set ( bebeau and thoma , in preparation ) suggests that those students with a highly developed sense of professional identity also scored high on the measure of ethical sensitivity , reasoning , and implementation . given that the observed gender differences were not anticipated , replication with other cohorts and measures is indicated . an ethics education program of moderate duration ( 9 , 25 ) can facilitate development of the four abilities described by the iom report , provided attention is directed to the educational principles the report outlines . in addition to the effect sizes reported for the undergraduate dental ethics curriculum ( 25 ) , enhanced ethical competence was also achieved for professionals referred for assessment / instruction as a condition for license renewal . of the 41 dentists referred over a 20-year period ( 5 , 4 ) , two were exempt from instruc tion based on pretest performance on the five well - validated measures of the fcm ( the dest , dit , derjt , proi , and role concept essays [ rce ] ) and 38 completed an individualized course designed to remediate deficiencies in ethical abil ities identified at the pretest . statistically significant pre- to posttest changes ( effect sizes ranging from 0.55 to 5.0 ) were reported ( 5 ) for ethical sensitivity ( dest scores ) , moral reasoning ( dit scores ) , and role concept ( rce essays and proi scores ) . analysis of the relationships between ability deficiencies and disciplinary actions supported the explanatory power of rest s fcm . of particular interest was the way the model helped the referrals to deconstruct a summary judgment about their character ( as unethical or unprofessional ) and to see the self as lacking in particular capacities or abilities that could be further developed . finally , though time - consuming to implement , the individualized remediation programs were highly effective in promoting ethical competencies , in reducing recidivism , and in influencing practitioner perceptions of the programs value . an examination of the extensive practitioner self - assessment data ( 5 ) provides guidance for structuring instruction . beginning the instructional process with a discussion of the distinguishing features of a profession and the expectations that follow further , the use of cases to assess and facilitate ethical sensitivity and reasoning was viewed as relevant to professional practice . of particular interest instead of focusing on what is happening ? and what ought to be done ? as is typical of much ethics instruction , the courses emphasized how to implement an action plan , including what to say and how to say it . with the exception of fisher and zigmond s ( 15 ) work on survival skills , this appears to be an often neglected area of ethics instruction . for students and practitioners alike , there is a clear hunger for help with strategies and language to deal with human interaction problems that have clear ethical implications . for further discussion of curriculum and resources to promote ethical implementation , newly designed resources for assessing and promoting identity formation are provided in a chapter ( 7 ) , in remediation in medical education . the authors coach educators to use measures of moral reasoning and professional identity formation to provide a diagnostic assessment of a student s strengths and shortcomings in their understanding of the ethical and moral dimensions of professionalism . next , the authors describe a remediation curriculum developed for a group of students who violated professional norms . this program has also been used to address individual transgressions ( e.g. , cheating , subpar behavior in practice ) . lastly , the authors highlight strategies they have found effective in therapeutic interactions with individual students who present particular challenges . studies using well validated measures of ethical sensitivity illustrate that competence in the ability to interpret the moral dimension of professional problems is distinct from the ability to apply professional norms and values to determine what ought to be done . what is clear from the research is that if one fails to see the moral issue in a professional problem , competence in reasoning and problem solving , even if well - developed , are not brought to bear ( 3 , 5 , 26 ) . further , just as students and professionals show remarkable variability in their level of moral reasoning development ( see next section ) , they also vary greatly in their ability to interpret patient / client characteristics and professional responsibilities embedded in ethical sensitivity tests . finally , ethical sensitivity can be influenced by educational interventions ( 3 , 5 ) , and , in some settings ( 26 ) , researchers have observed small , though statistically significant gender difference favoring women . in a meta - analysis of ethical sensitivity research ( 26 ) , researchers identified 37 studies in which 23 measures were described to assess ethical sensitivity in professional settings ( e.g. , dentistry , medicine , nursing , professional psychology , business , and science ) . after classifying the measures along several dimensions , including the extent to which the construct was elicited by the stimulus materials , they concluded that only seven of the measures met criteria , and most had not been extensively validated . examples of validated measures that elicit the process include the dental ethical sensitivity test ( dest ) designed for dentistry ( 6 , 11 ) and the racial ethical sensitivity test ( rest ) designed for counseling psychology ( 13 , 22 ) . what distinguishes measures like the rest and dest is the extent to which the stimulus presents clues to one or more moral problems without ever signaling either the moral issues at stake or the particular professional responsibilities called for . by way of contrast , some test designers seem to conceptualize ethical sensitivity as the ability to simply name the moral issue when a condensed synopsis of a moral problem is presented . examples in appendix 1 show how distinctly different the stimulus for assessing ethical sensitivity is from the stimulus for assessing moral reasoning development . the iom report ( 18 ) described the usefulness of the defining issues test ( dit ) for assessing students capacity for reasoning about moral issues . subsequent reviews ( 2 , 9 ) update that literature and describe use of the dit to assess ( a ) the reasoning development across and within professions , and ( b ) the effectiveness of interventions to promote moral judgment development . as described below , the measure is used in remediation programs to identify shortcomings in ethical decision making of practicing dentistry ( 4 ) and medical ( 7 ) students . strategies for assessing students and giving individualized feedback on reasoning development are included in chapter appendices ( 7 ) . in addition to life - span measures of moral judgment development like the dit , researchers have designed measures of ethical reasoning for specific educational contexts . referred to as intermediate concept measures ( icms ) , because they assess ethical concepts in the intermediate zone between broad moral ideals and profession - specific codes of conduct ( e.g. , professional autonomy , informed consent ) , icms present discipline - specific problems and assess agreement between respondents and experts as to the appropriateness or inappropriateness of particular action choices and justifications . it is important to note that test developers engage respondents from the discipline to generate items . an analysis of results from multiple icm measures across populations ( adolescents , young adults , and professionals ) ( 24 ) revealed that irrespective of the sample , age group , or icm concept assessed , respondents had more difficulty identifying the bad choices / justifications than the good items . in addition , the obtained mean differences suggested different levels of performance , as there was little to suggest that these findings were due to methodological issues . further , it appeared that identifying inappropriate and appropriate choices or justifications were not poles on a single dimension , as participants seemed not to apply information and experience gained on one set of considerations to the other . perhaps instruction focuses on what one ought to do and what are appropriate choices , and individuals must then infer what is inappropriate and poorly conceived . if so , ethics education may benefit by expanding its focus on inappropriate choices in order to provide more guidance in developing an experiential base to support more optimal moral decisions . when rest proposed his fcm of moral functioning in the early 1980s , moral motivation was featured , though less well articulated than the other three components . rest ( 20 ) thought that moral motivation influenced moral action directly and in interaction with the other components of the moral system . in the last decade , considerable attention has been given to the study and measurement of lifespan identity formation particularly in the professions . in an edited volume on moral motivation , researchers ( 23 ) measures include the professional role orientation inventory ( proi ) described in the iom report ( 18 ) , and the theoretical importance of a newer measure ( the professional identity essay [ pie ] ) used to assess life - span identity formation in dentistry ( 10 ) and law ( 16 ) and recently adapted for ethics remediation in medical education ( 7 ) . note : in addition to providing guidance on the use of the dit , chapter appendices ( 7 ) include the pie , adapted for medicine , and criteria for assessing and strategies promoting identity formation . in the same edited volume , researchers ( 8) summarize evidence from dentistry , medicine , law , and the military that supports constructivists theoretical understanding of a developmental continuum of moral motivation and commitment ( rest s component 3 ) . the continuum proceeds from self - interest and concreteness of thought characteristic of entering professionals including entrants into doctoral education programs ( 1 ) to more other - oriented and abstract ways of making sense of the self in relation to others . at advanced levels of moral motivation , the exemplary individual s personal and professional moral values ( 14 , 21 ) are fully integrated , as evidenced by behavior that is consistent across contexts and situations . for example , the exemplary dental professionals studied by rule and bebeau ( 21 ) were able to articulate the public duties of their profession , integrate them with personal value frameworks , and regularly and consistently engage in socially responsible actions . the identity of such exemplary professionals was contrasted with the identities of entering students and entering professionals across several professions ( reviewed by bebeau and monson ) and with professionals who have been disciplined by a licensing board ( 5 ) . a consistent shortcoming of all but two of 41 professionals referred for an ethics assessment because they violated laws governing professional practice was in the ability to articulate professional duties and responsibilities . whereas referrals demonstrated a great deal of variability on measures of ethical sensitivity , moral reasoning , and ethical implementation , they consistently demonstrated only vague notions of their responsibilities to patients , their profession , their community , and society . the importance of professional identity formation to professional practice is further supported by a reexamination of data from a study of gender differences ( 25 ) on measures of the four ethical abilities ( ethical sensitivity , reasoning , role concept , and ethical implementation ) for five cohorts who participated in a well - validated four - year dental ethics education program . the researchers ( bebeau and thoma , in preparation ) noted that those graduates who had developed a mature professional identity by graduation also had developed high levels of competence on measures of the other three components ( ethical sensitivity , reasoning , and implementation ) . taken together , evidence from the three samples ( exemplary professionals , disciplined professionals , and recent graduates who completed an ethics curriculum ) strongly suggests that development of a moral identity that is consistent with the norms and values of the profession is the driving force that gives rise to the development of other abilities that account for responsible professional conduct . whether ethical sensitivity , reasoning , and competence in implementing ethical solutions develop in tandem with the development of a moral identity is a question for further research . as noted in the iom report ( 19 ) , developing self - regulation , self - efficacy and implementation abilities is necessary for effective and responsible professional practice what fisher and zigmond ( 15 ) describe as survival skills in the responsible conduct of research . a wide variety of assessment strategies is possible . in the dental ethics curriculum ( 25 ) , competence in ethical implementation was based on performance score for eight complex cases presented during the last two years of the dental ethics curriculum . stimulus for assessment was similar to dest cases in that students needed to : 1 ) interpret the facts that must be addressed if the problem is to be resolved efficiently ; 2 ) design an action plan ; and 3 ) create a dialogue to illustrate effective plan implementation . all responses were assessed by the same rater , and students could challenge the assessment , revise , and resubmit . like other assessments used in the curriculum , an unexpected finding ( 25 ) was an observed gender difference in ethical implementation for the 60 men and 60 women randomly selected from five cohorts ( 25 ) to explore previously observed gender differences ( favoring women ) in ethical sensitivity and moral reasoning . no gender difference was observed on ethical sensitivity , and pretest and posttest differences on moral reasoning were trivial . whereas men and women had similar scores on the responsibility dimension of moral motivation ( proi scores ) at entrance to professional school , at graduation , an effect size of 0.75 for women versus 0.5 for men indicated greater change for women . a statistically significant gender difference , favoring women , was also evident on the measure of moral implementation , though pretest data were not collected to judge the role of general implementation abilities ( e.g. , interpersonal effectiveness , problem solving , etc . ) . an effect size of 0.57 suggested that the women s ability to implement more effective action plans was not a trivial difference . recall that a reanalysis of this data set ( bebeau and thoma , in preparation ) suggests that those students with a highly developed sense of professional identity also scored high on the measure of ethical sensitivity , reasoning , and implementation . given that the observed gender differences were not anticipated , replication with other cohorts and measures is indicated . an ethics education program of moderate duration ( 9 , 25 ) can facilitate development of the four abilities described by the iom report , provided attention is directed to the educational principles the report outlines . in addition to the effect sizes reported for the undergraduate dental ethics curriculum ( 25 ) , enhanced ethical competence was also achieved for professionals referred for assessment / instruction as a condition for license renewal . of the 41 dentists referred over a 20-year period ( 5 , 4 ) , two were exempt from instruc tion based on pretest performance on the five well - validated measures of the fcm ( the dest , dit , derjt , proi , and role concept essays [ rce ] ) and 38 completed an individualized course designed to remediate deficiencies in ethical abil ities identified at the pretest . statistically significant pre- to posttest changes ( effect sizes ranging from 0.55 to 5.0 ) were reported ( 5 ) for ethical sensitivity ( dest scores ) , moral reasoning ( dit scores ) , and role concept ( rce essays and proi scores ) . analysis of the relationships between ability deficiencies and disciplinary actions supported the explanatory power of rest s fcm . of particular interest was the way the model helped the referrals to deconstruct a summary judgment about their character ( as unethical or unprofessional ) and to see the self as lacking in particular capacities or abilities that could be further developed . finally , though time - consuming to implement , the individualized remediation programs were highly effective in promoting ethical competencies , in reducing recidivism , and in influencing practitioner perceptions of the programs value . an examination of the extensive practitioner self - assessment data ( 5 ) provides guidance for structuring instruction . beginning the instructional process with a discussion of the distinguishing features of a profession and the expectations that follow further , the use of cases to assess and facilitate ethical sensitivity and reasoning was viewed as relevant to professional practice . of particular interest was the value practitioners placed on the curriculum s emphasis on ethical implementation . instead of focusing on what is happening ? and what ought to be done ? as is typical of much ethics instruction , the courses emphasized how to implement an action plan , including what to say and how to say it . with the exception of fisher and zigmond s ( 15 ) work on survival skills , this appears to be an often neglected area of ethics instruction . for students and practitioners alike , there is a clear hunger for help with strategies and language to deal with human interaction problems that have clear ethical implications . for further discussion of curriculum and resources to promote ethical implementation , newly designed resources for assessing and promoting identity formation are provided in a chapter ( 7 ) , in remediation in medical education . the authors coach educators to use measures of moral reasoning and professional identity formation to provide a diagnostic assessment of a student s strengths and shortcomings in their understanding of the ethical and moral dimensions of professionalism . next , the authors describe a remediation curriculum developed for a group of students who violated professional norms . this program has also been used to address individual transgressions ( e.g. , cheating , subpar behavior in practice ) . lastly , the authors highlight strategies they have found effective in therapeutic interactions with individual students who present particular challenges . evidence from the cited studies adds weight to earlier recommendations ( 17 ) for structuring educational programs that use active learning strategies to promote the development of competent , thoughtful , and responsible scientists . it is natural to assume that students who apply for graduate education are mature individuals of good character , and our intention is not to undermine confidence in students personal integrity . yet the evidence ( 10 ) shows that entering students , across professions , have rather vague notions of the norms and values of their chosen profession and seem not to pick them up from role models during the course of their education . more importantly , recent studies ( 5 , 25 ) exploring competence on each of the four ethical abilities defined by rest s four component model of morality illustrate the critical role of professional identity formation . because the formation of a professional identity seems to be the primary driver behind responsible conduct , a first step in designing an educational program in responsible research conduct is to begin by addressing the expectations of a scientist . ask students to respond to open - ended questions in writing which they can reflect upon as they learn about the values and normative practices that guide the research enterprise . by asking students to express the concepts in their own words , and in writing , misperceptions can be identified and addressed before they become an issue . educators may wish to modify the pie applied in medicine ( 7 ) and adapt the reflective learning activities for rcr education . as argued in the iom report , to develop thoughtful and responsible scientists who act with integrity and have a broad understanding of their role and a commitment to integrity in science , educators must do more than teach the rules and policies that apply to the conduct of research . once the expectations of the scientist have been clarified , it is important to engage students in active learning ( using cases , if possible ) to facilitate the abilities that are necessary conditions in addition to a professional identity ethical sensitivity , reasoning , and problem solving for effective moral action . when selecting or designing case materials , the materials must be carefully structured to elicit the process of concern . as argued above , too often cases are written and participants are asked : what should the protagonist do ? such a question focuses on problem solving rather than problem identification or moral reasoning . whereas a skilled facilitator may be able to redirect focus to reasoning or problem identification , it is sometimes more difficult . for this reason , we provided an example ( appendix 1 ) to show how to design stimulus materials that focus on the particular skill needed for effective problem identification , reasoning , or implementation . with rather carefully targeted courses of moderate duration , it is possible to show gains in each of the abilities that give rise to responsible conduct . our goal is not to develop the more advanced skills in ethical reasoning that might result from courses in moral philosophy . yet evidence shows that problem - based practice ( using cases ) can be especially effective in helping students recognize and subsequently avoid personal interest arguments while strengthening awareness and adherence to the rules of responsible research conduct .

background : earlier forms of food frequency questionnaire ( ffq ) used in iran have extensive lists of foods , traditional categories and food - based design , mostly with the interviewer - administered approach . the aim of the current paper is to describe the development of a dish - based , machine - readable , semi - quantitative food frequency questionnaire ( dfq).methods : within the framework of the study on the epidemiology of psychological , alimentary health and nutrition project , we created a novel ffq using harvard ffq as a model.results:the following steps were taken to develop the questionnaire : construction of a list of commonly consumed iranian foods , definition of portion sizes , design of response options for consumption frequency of each food item and finally a pilot test of the preliminary dfq . from a comprehensive list of foods and mixed dishes , we included those that were nutrient - rich , consumed reasonably often or contributed to between - person variations . we focused on mixed dishes , rather than their ingredients , along with foods . to shorten the list , the related food items or mixed dishes were categorized together in one food group . these exclusions resulted in a list of 106 foods or dishes in the questionnaire . the portion sizes used in the ffq were obtained from our earlier studies that used dietary recalls and food records . the frequency response options for the food list varied from 6 - 9 choices from never or less than once a month to 12 or more times per day.conclusions:the dfq could be a reasonable dietary assessment tool for future epidemiological studies in the country . validation studies are required to assess the validity and reliability of this newly developed questionnaire .

the global alarming increase in the prevalence of chronic diseases , as the most leading cause of death , has attracted considerable attention over the past decades . a strikingly high prevalence of chronic diseases and their risk factors has been indicated among iranian adults . findings from numerous studies have confirmed the crucial role of life - style factors , in particular dietary intakes , on the etiology and management of chronic diseases ; however , the optimal assessment of dietary intakes as the principal challenge in this regard still remains unresolved . so far , various methods such as dietary recalls , food records , diet histories and food frequency questionnaires ( ffqs ) have been proposed for dietary intake assessment . recall bias , embarrassment of participants , requiring well - trained interviewers and food coding in the dietary recalls might affect its validity and utilization . despite the accuracy of food records , some weaknesses such as the possibility of change in the usual eating habits and the necessity of substantial training of participants can not be excluded . most importantly , dietary recalls and records do not reflect participants usual intakes . to obtain long - term dietary intakes , the potential bias due to an un - standardized interview approach , time - consuming , lengthy and open - ended food lists in the diet histories , have considerably limited the application of this method . now - a - days , ffqs are the most widely used dietary assessment tool in large - scale epidemiological studies . the acceptability of ffqs is due to several important advantages of this method , such as low cost , ease of administration , computer processing , considering within - person variation and reflecting long - term intakes . some characteristics of ffqs , such as their length and complexity , which is mainly due to asking for detailed information on a long list of food items , portion sizes and food preparation methods could affect the accuracy of collected information . on the other hand , poorly designed ffqs without comprehensive food lists may lead to incorrect information and misclassification , which in turn could result in masking diet - disease relations . thus , the development of a comprehensive and appropriate ffq is the cornerstone of nutritional epidemiological studies . so far all previously used ffqs in iranian settings have mainly been consisted of the extensive list of foods and followed a food - based design . however , the limitations and pitfalls in the accuracy of information that is provided using such ffqs have been described before . worldwide , many epidemiological studies , including korean , japanese , brazilian , swiss , bangladeshi and zimbabwean studies have used dish - based ffqs . typical iranian dietary pattern is characterized by various mixed dishes , which contains several ingredients prepared in different ways . with no doubt , it is very difficult to estimate usual intake of single ingredients from various mixed iranian dishes . to the best of our knowledge , no earlier study in iran has used a dish - based ffq or a ffq with multiple - choice frequency response options . furthermore , all previous studies in the country have used the interviewer - administered ffqs . in the framework of the study on the epidemiology of psychological , alimentary health and nutrition ( sepahan ) project , we designed a dish - based machine - readable semi - quantitative food frequency questionnaire ( dfq ) with multiple choice frequency response options for the first time in iran . the aim of the present paper is to describe how this dietary assessment tool was developed . to develop the dfq , we used the harvard ffq as a model , which assesses the dietary intake of a person in the preceding 12 months . the following steps were taken to develop the questionnaire : construction of a list of commonly consumed iranian foods , definition of portion sizes , determining the frequency response options for each food item and finally testing the face validity of the questionnaire in a pilot setting to check for the comprehension and feasibility . as the initial step , a comprehensive list of foods was prepared based on the information provided by local experienced nutritionists , considering all commonly consumed iranian foods and mixed dishes . to avoid missing major food items , we listed foods commonly consumed by iranians as meal ( breakfast , lunch and dinner ) or snack . from the comprehensive list of foods and mixed dishes , we selected those that were nutrient - rich ( such as liver ) , consumed reasonably often ( such as different types of bread ) , or contributed to between - person variations ( such as sausage ) . owing to the high consumption of mixed dishes among iranian population , it is difficult for people to estimate usual intake of ingredients of those dishes . in contrast to previously designed ffqs in the country , we focused on foods and mixed dishes rather than single ingredients . for example , iranians consume potato through several mixed dishes such broth , olviyeh ( potato salad ) , cutlet , different stews , etc . , certainly , it is too hard to estimate total potato intake of a person by asking participants to remember the frequency consumption of potato in the preceding 12 months . participants may get confused while trying to add their usual potato intake that comes from different sources and it is highly possible that some potato - containing dishes be forgotten or ignored . however , it would be much easier for them to make an estimate of the amount and frequency of consuming every single dish . it is generally assumed that questionnaire length has a significant effect on the survey response rate as respondents get tired , bored and/or distracted by external factors . furthermore , a lengthy questionnaire is less likely to be completed and returned . as we attempted to design a self - administered questionnaire , we pilot tested the comprehensive list of food items / dishes among a group of individuals ( n = 35 ) in order to exclude some foods that were consumed rarely or never . finally , these exclusions resulted in a list of 106 food items or dishes in the questionnaire . to simplify the completion of the questionnaire , we categorized food items into five major groups : ( 1 ) mixed dishes ( cooked or canned : 29 items ) ; ( 2 ) grains ( different types of bread , cakes , biscuits and potato : 10 items ) ; ( 3 ) dairy products ( dairies , butter and cream : 9 items ) ; ( 4 ) fruits and vegetables ( 22 items ) ; and ( 5 ) miscellaneous food items and beverages ( including sweets , fast foods , nuts , desserts and beverages : 36 items ) . although portion sizes in ffqs are generally poorly estimated and do not contribute significantly to the between - person variations in dietary intakes , their quantification in a questionnaire will help better ranking of individuals in terms of their dietary intakes . in the dfq , portion sizes for food items and mixed dishes were defined based on the most commonly consumed portion size for each item among iranian general population . to increase precision and accuracy of estimates , we attempted to provide the portion sizes of foods and mixed dishes as a unit with the same perception for all people . to reach these portion sizes , we used reported portion sizes in dietary recalls and food records in our earlier studies . furthermore , we pilot tested the preliminary ffq by administering it in a group of adults ( n = 35 ) to determine the most appropriate portion size for every single food item . a group of nutrition experts also discussed about the portion sizes to finalize the most suitable choice for each food item . loaf for breads , medium - sized plate for cooked rice , tablespoon for raisins and bag for potato chips . similar to the harvard ffq , we used nine multiple - choice options ( for most food items ) varying from never or less than once per month to 12 or more times per day to estimate average frequency of different food intakes . the frequency response options for each food item were defined separately in a row against the food list . this is in contrast to the harvard ffq in which the frequency response options are indicated in the top of a column for all food items . for all frequency response options , we also mentioned the portion sizes repeatedly to simplify responding [ figure 1 ] . the number of frequency response options was not constant for all foods . for foods consumed infrequently , we omitted the high - frequency options while for highly consumed foods , the number of multiple - choice options were increased . for instance , the frequency response for tuna consumption included six options , as follows : never or less than once per month , 1 - 3 times per month , 1 time per week , 2 - 4 times per week , 5 - 6 times per week and 1 - 2 times per day . however , for tea which is consumed more frequently , the frequency response included nine options , as follows : never or less than 1 cup per month , 1 - 3 cups per month , 1 - 3 cups per week , 4 - 6 cups per week , 1 cup per day , 2 - 4 cups per day , 5 - 7 cups per day , 8 - 11 cups per day and 12 cups per day ) . the pilot study on 35 subjects , which was conducted to test the face validity of the dfq , assisted us to select proper number of response options for each food item in the list . sample of questions and layout of the harvard food frequency questionnaire ( a ) in comparison with the dfq ( b ) . different number of response options for each food item , including portion sizes that are more familiar to iranians , and including the response options of consumption frequencies for each food item are among the major features that have been considered in designing the dfq using the dfq , a daily value for each item can be calculated based on food composition , specified portion size and the average of reported frequency . for example , if a person reports to consume macaroni 2 - 4 plates per week , we can compute the daily intake of ground beef from this item according to this formula : 0.43 ( 3 ( average number of plates per week ) divided by 7 ( number of days per week ) ) multiplying 50 ( grams of ground beef in each plate of macaroni ) . moreover , other mixed dishes in this ffq contain ground beef such as barbecue , cutlet , etc . , therefore , total ground beef intake for a participant will be calculated by the sum of his / her daily ground beef intake from different food items in the list . the food composition of mixed dishes was determined based on common recipes consumed in the country . in addition , a group of five nutrition experts collected 15 home or restaurant recipes for each iranian mixed dish that was included the dfq . finally , mean values of different ingredients of a mixed dish was considered as the final food composition details of that dish . given the seasonal variation in the dietary intakes of some fruits and vegetables , in the dfq participants are asked to mention the consumption frequency of such items during the months that those products are widely available in the market . in the calculation of fruit and vegetables , as the initial step , a comprehensive list of foods was prepared based on the information provided by local experienced nutritionists , considering all commonly consumed iranian foods and mixed dishes . to avoid missing major food items , we listed foods commonly consumed by iranians as meal ( breakfast , lunch and dinner ) or snack . from the comprehensive list of foods and mixed dishes , we selected those that were nutrient - rich ( such as liver ) , consumed reasonably often ( such as different types of bread ) , or contributed to between - person variations ( such as sausage ) . owing to the high consumption of mixed dishes among iranian population , it is difficult for people to estimate usual intake of ingredients of those dishes . in contrast to previously designed ffqs in the country , we focused on foods and mixed dishes rather than single ingredients . for example , iranians consume potato through several mixed dishes such broth , olviyeh ( potato salad ) , cutlet , different stews , etc . , certainly , it is too hard to estimate total potato intake of a person by asking participants to remember the frequency consumption of potato in the preceding 12 months . participants may get confused while trying to add their usual potato intake that comes from different sources and it is highly possible that some potato - containing dishes be forgotten or ignored . however , it would be much easier for them to make an estimate of the amount and frequency of consuming every single dish . it is generally assumed that questionnaire length has a significant effect on the survey response rate as respondents get tired , bored and/or distracted by external factors . furthermore , a lengthy questionnaire is less likely to be completed and returned . as we attempted to design a self - administered questionnaire , we pilot tested the comprehensive list of food items / dishes among a group of individuals ( n = 35 ) in order to exclude some foods that were consumed rarely or never . finally , these exclusions resulted in a list of 106 food items or dishes in the questionnaire . to simplify the completion of the questionnaire , we categorized food items into five major groups : ( 1 ) mixed dishes ( cooked or canned : 29 items ) ; ( 2 ) grains ( different types of bread , cakes , biscuits and potato : 10 items ) ; ( 3 ) dairy products ( dairies , butter and cream : 9 items ) ; ( 4 ) fruits and vegetables ( 22 items ) ; and ( 5 ) miscellaneous food items and beverages ( including sweets , fast foods , nuts , desserts and beverages : 36 items ) . although portion sizes in ffqs are generally poorly estimated and do not contribute significantly to the between - person variations in dietary intakes , their quantification in a questionnaire will help better ranking of individuals in terms of their dietary intakes . in the dfq , portion sizes for food items and mixed dishes were defined based on the most commonly consumed portion size for each item among iranian general population . to increase precision and accuracy of estimates , we attempted to provide the portion sizes of foods and mixed dishes as a unit with the same perception for all people . to reach these portion sizes , we used reported portion sizes in dietary recalls and food records in our earlier studies . furthermore , we pilot tested the preliminary ffq by administering it in a group of adults ( n = 35 ) to determine the most appropriate portion size for every single food item . a group of nutrition experts also discussed about the portion sizes to finalize the most suitable choice for each food item . loaf for breads , medium - sized plate for cooked rice , tablespoon for raisins and bag for potato chips . similar to the harvard ffq , we used nine multiple - choice options ( for most food items ) varying from never or less than once per month to 12 or more times per day to estimate average frequency of different food intakes . the frequency response options for each food item were defined separately in a row against the food list . this is in contrast to the harvard ffq in which the frequency response options are indicated in the top of a column for all food items . for all frequency response options , we also mentioned the portion sizes repeatedly to simplify responding [ figure 1 ] . the number of frequency response options was not constant for all foods . for foods consumed infrequently , we omitted the high - frequency options while for highly consumed foods , the number of multiple - choice options were increased . for instance , the frequency response for tuna consumption included six options , as follows : never or less than once per month , 1 - 3 times per month , 1 time per week , 2 - 4 times per week , 5 - 6 times per week and 1 - 2 times per day . however , for tea which is consumed more frequently , the frequency response included nine options , as follows : never or less than 1 cup per month , 1 - 3 cups per month , 1 - 3 cups per week , 4 - 6 cups per week , 1 cup per day , 2 - 4 cups per day , 5 - 7 cups per day , 8 - 11 cups per day and 12 cups per day ) . the pilot study on 35 subjects , which was conducted to test the face validity of the dfq , assisted us to select proper number of response options for each food item in the list . sample of questions and layout of the harvard food frequency questionnaire ( a ) in comparison with the dfq ( b ) . different number of response options for each food item , including portion sizes that are more familiar to iranians , and including the response options of consumption frequencies for each food item are among the major features that have been considered in designing the dfq using the dfq , a daily value for each item can be calculated based on food composition , specified portion size and the average of reported frequency . for example , if a person reports to consume macaroni 2 - 4 plates per week , we can compute the daily intake of ground beef from this item according to this formula : 0.43 ( 3 ( average number of plates per week ) divided by 7 ( number of days per week ) ) multiplying 50 ( grams of ground beef in each plate of macaroni ) . moreover , other mixed dishes in this ffq contain ground beef such as barbecue , cutlet , etc . , therefore , total ground beef intake for a participant will be calculated by the sum of his / her daily ground beef intake from different food items in the list . the food composition of mixed dishes was determined based on common recipes consumed in the country . in addition , a group of five nutrition experts collected 15 home or restaurant recipes for each iranian mixed dish that was included the dfq . finally , mean values of different ingredients of a mixed dish was considered as the final food composition details of that dish . given the seasonal variation in the dietary intakes of some fruits and vegetables , in the dfq participants are asked to mention the consumption frequency of such items during the months that those products are widely available in the market . in the calculation of fruit and vegetables , this factor has also been taken into account . in the present article , we described the development and design of a dish - based , machine - readable , semi - quantitative ffq ( called dfq ) for the iranian adult population . to the best of our knowledge , this is the first time that such a dietary assessment tool has been created in the country . in general , ffqs have been proposed as the optimal instruments for dietary intake assessments in large epidemiological studies . data from ffqs can be used to elucidate diet - disease relations through ranking participants based on their usual dietary intakes . as shown by earlier investigations , along with all dietary assessment methods , some potential disadvantages could also be noted about ffqs including recall bias , overestimation of dietary intakes particularly for rarely - consumed and healthy - perceived foods ( e.g. , fruit and vegetables ) , bias of current intake , misclassification and bias of pre - established food listing . in the dfq , we focused on compiling a comprehensive list of foods to capture total energy intake . as discussed by well - known researchers in the field of nutritional epidemiology , total energy intake must be controlled for in almost all epidemiological studies . moreover , the main objective of the current study was to develop an easy - to - use ffq for future epidemiological studies in iran . given that longer questionnaires may cause respondent fatigue and poorer quality of gathered information , we focused on nutrient - rich frequently consumed foods and dishes . we also included those foods that could explain the between - person variations in diet . earlier ffqs that have been used in iranian epidemiological studies were developed for the tehran lipid and glucose study ( tlgs ) , the isfahan health heart program ( ihhp ) and the golestan cohort study . the difference in food items could be explained by the inclusion of food items in earlier questionnaires but dishes in the dfq . compared with previous iranian ffqs , the principal discriminatory feature of dfq was incorporating iranian mixed dishes instead of including their ingredients . since typical iranian dishes usually consist of various ingredients , it is difficult for respondents to estimate their total intake of one ingredient which may be used in several mixed dishes . inclusion of mixed dishes instead of their ingredients in a ffq will not only facilitate participants responding , but also will reduce the length of the questionnaire . using the current approach of including mixed dishes instead of their ingredients could shorten the list of food items in the dfq , shorten the time required to fill the questionnaire , decrease participants boredom and increase accuracy of dietary intake assessment . furthermore , as cooking and other food processing methods affect the nutritional value of foods , including mixed dishes instead of food ingredients can better elucidate the relationship between diet and diseases . some investigators believe that shortening of food list and using a unique recipe for all participants may lead to decreased accuracy and failure to estimate the total energy intake . although open - ended frequency responses would lead to enhanced precision and continuous variables instead of categorical ones , it has been shown that using multiple - choice frequency response options increases clarity and reduces errors . however , it must be kept in mind that frequency options must be designed in a manner that covers all possible choices of consumption frequency responses in participants . the number of options should range between 5 and 10 ; broadened response options with less than five choices would significantly limit the discrimination capacity , while excessive options may be confusing . between - persons variation for frequently and rarely consumed foods is distinguished at the high and low end of the scale , respectively . the dfq is the first ffq in the country with multiple - choice frequency response options . we believe that the multiple - choice frequency response options will facilitate the completion of the dfq as it can be filled by participants without the help of an interviewer ( i.e. , self - administered questionnaire ) . the frequency response choices in the dfq are different from those in the harvard ffq . as consumption frequency of food items may vary in a broad range , we decided to include a unique set of frequency response options for each food item in the dfq . as generally portion sizes are poorly estimated , the inclusion of portion sizes in ffqs is still controversial . it seems that the large percentage of between - persons variation could be explained by consumption frequency , rather than portion sizes . estimating portion size of foods is difficult for most participants . however , it is important to recognize that calculation of absolute nutrient intake is impossible when information on portion sizes is not available . some investigators have suggested to use the commonly consumed portion sizes for calculating nutrient intakes in case of missing portion sizes in a ffq . for example , the correlations of nutrient intake obtained from ffqs with and without portion sizes were over 0.9 . presumably , inclusion of pre - specified portion sizes make questionnaire easier to complete by respondents . it has been proposed to include the most frequently consumed or traditional portion sizes in ffqs . although earlier ffqs in iran have included portion sizes , this information was mostly based on serving sizes recommended by the u.s . department of agriculture food guide pyramid , not on the most frequently locally used portion sizes . as previously mentioned , the dfq is a machine - readable questionnaire . for this purpose , we developed an optical mark recognition ( omr ) system that can distinguish the selected answer for each food item in the scanned pictures of questionnaires . furthermore to convert selected consumption frequency responses option to exact intake of a food item , appropriate software was developed . results from the pilot study showed the very high accuracy of the developed computerized systems . for large epidemiological studies , using this system will have a crucial role in reducing the expenses ( e.g. , staff , time ) and errors that are inevitable while extracting data manually . various approaches have been used to assess the performance of ffqs including classic validation study and evaluation of the ability to predict expected diet - diseases correlation . although , a classic validation study has not been conducted for the dfq so far , we have recently indicated several established relationships between dietary factors and diseases using this tool . the dfq could be an appropriate dietary assessment tool for future epidemiological studies in iran . however , before its application in large epidemiological studies , the validity and reliability of this newly developed ffq should be assessed among different iranian populations .

renin angiotensin system ( ras ) activation plays a key role in the development of hypertension and cardiovascular disease . drugs that antagonize the ras ( angiotensin - converting enzyme [ ace ] inhibitors and angiotensin receptor blockers [ arbs ] ) have proven clinical efficacy in reducing blood pressure values and cardiovascular morbidity and mortality . ace inhibitors partially inhibit plasma ace , and angiotensin ii generation . thus , arbs , which block selectively type 1 angiotensin ii receptor ( at1r ) , have been developed and used in the clinical management of hypertension and cardiovascular disease . experimental and clinical trials with arbs indicate that this class of drug represents an effective , safe and well tolerated therapeutic option for the prevention and care of hypertension , even though there is no proven superiority as compared to ace inhibitors except for the better tolerability . most arbs may not completely inhibit the at1r at the approved clinical doses . azilsartan medoxomil is a newly approved arb for the management of hypertension . this arb induces a potent and long - lasting antihypertensive effect and may have cardioprotective properties . this article reviews the current evidence on the clinical effectiveness of azilsartan in hypertension .

improved control of blood pressure in patients with hypertension is a key requirement to reduce cardiovascular and renal morbidity and mortality.1,2 despite the effectiveness of the currently available antihypertensive agents , hypertension remains inadequately controlled , with slightly less than half of patients who receive treatment successfully achieving the goals for systolic and diastolic blood pressure.3,4 the renin angiotensin system ( ras ) plays a central role in the pathophysiology of hypertension , cardiovascular and renal disease.5,6 it contributes to the increase of blood volume and arterial pressure , to the alterations of endothelial function , vascular reactivity , fibrosis , tissue remodeling , oxidative stress and inflammation which may predispose to the development of cardiovascular disease.57 while there are many drug classes available to reduce blood pressure , pharmacological agents that modulate the ras are more commonly chosen as the first drug or in combination therapy because of their efficacy and the lowest side effect profile among the antihypertensive agents . the development of angiotensin - converting enzyme ( ace ) inhibitors has represented a cornerstone for the treatment of various pathological cardiovascular conditions , including hypertension . however ace inhibitors only partially inhibit the formation of angiotensin ii ( ang ii ) . from this point of view , angiotensin - receptor blockers ( arbs ) provide a more rational review tool to inhibit ras activity since they block selectivity the coupling of ang ii to type 1 angiotensin ii receptor ( at1r ) . for this reason they have been developed and increasingly used in the clinical management of hypertension and cardiovascular diseases . among the compounds that antagonize the ras , arbs ( which include several molecules such as losartan , candesartan cilexetil , valsartan , irbesartan , telmisartan , eprosartan and olmesartan medoxomil , and the newest azilsartan medoxomil ) represent today the best - tolerated class among antihypertensive agents.8 the clinical efficacy of arbs has been established in hypertensive patients , particularly in terms of reduction in cardiovascular morbidity and mortality , prevention and regression of end organ damage and the slow progression of nephropathy . ras blockers provide independent actions on end - organ protection , beyond their blood pressure lowering effect.9 this has been shown with both ace inhibitors and arbs . a large body of evidence indicate that ace inhibitors are effective for the treatment of various pathological conditions including arterial hypertension , diabetes , and cardiovascular or cerebrovascular diseases.1013 however ace inhibitors antagonize the ras only in part . indeed , these drugs partially inhibit plasma ace and ang ii generation , even at high dosages.14 nevertheless , ang ii can be produced by alternative pathways ( chymases , caspases , elastases ) in the cardiovascular system.15,16 therefore , the selective blockers of the at1r , have been developed and progressively used in the clinical management of hypertension and cardiovascular diseases . moreover , some of the molecules may have ancillary effects such as the increased urinary uric acid excretion ( losartan in particular ) and may activate peroxisome proliferator - activated receptor ( ppar)- which may contribute to a favorable metabolic profile . this latter effect has been described for the arbs telmisartan , irbesartan , candesartan , and losartan.17 randomized clinical trials have proven benefits for the therapy with arbs in primary and secondary prevention in several pathological conditions including hypertension , coronary artery disease , congestive heart failure , and renal disease . arbs have shown cardioprotective effects in patients with hypertension and additional risk factors . in the losartan intervention for endpoint reduction ( life)18 the arb losartan was compared to atenolol in over 9000 patients with moderate - to - severe hypertension and left - ventricular hypertrophy . losartan improved the primary composite endpoint , which included cardiovascular mortality , stroke , and myocardial infarction , independently by blood pressure reduction . the losartan treatment was also associated with a 25% lower incidence of new - onset diabetes . this was observed also in the placebo - controlled kyoto heart study19 in which valsartan used as add - on therapy reduced the incidence of new onset of diabetes as well as cardiovascular outcomes in about 3000 hypertensive patients with additional risk factors . a recent meta - analysis has also proven the effectiveness of ras blockers in reducing the occurrence of new - onset diabetes . hence the ace inhibitors or arbs should be preferred in patients with clinical conditions that may increase the risk of developing diabetes.20 both hypertension and diabetes may induce renal damage in patients at risk for cardiovascular diseases . in turn , renal disease may increase the cardiovascular risk even at a preclinical stage ( ie , presence of microalbuminuria ) . furthermore , diabetic nephropathy is responsible for the majority of end - stage renal disease in most countries . in particular , agents that block the ras can delay or prevent the diabetic nephropathy even at the initial stages as well as reduce the degree of albuminuria and the progression to advanced renal disease . in the randomised olmesartan and diabetes microalbuminuria prevention ( roadmap ) study , the arb olmesartan was associated with a delayed onset of microalbuminuria , although the drug did not reduce the cardiovascular complications associated with the diabetic state and the number of cardiovascular events.21 the arb irbesartan has been shown to delay the progression from microalbuminuria to overt proteinuria and to restore normoalbuminuria in a significant proportion of patients with hypertension and type 2 diabetes.22 moreover , irbesartan protected against advanced nephropathy in hypertensive patients with type 2 diabetes , independently by blood pressure control.23 ventricular dysfunction and heart failure may develop in the progression of the cardiovascular disease in high cardiovascular risk patients , which are generally older than the patients at the early stages of the cardiovascular continuum and have often several comorbidities.24 arbs were equal to ace inhibitors in reducing all - cause mortality in patients with left - ventricular dysfunction or heart failure postmyocardial infarction2426 as well as in chronic heart failure patients.27,28 in patients at higher cardiovascular risk , arbs have been shown to induce similar cardiovascular protective effects . telmisartan was proven to have cardioprotective effects in high cardiovascular risk patients who were intolerant to ace inhibitors.29 in over 25,000 patients with coronary , peripheral or cerebrovascular disease and diabetes with end organ damage the ongoing telmisartan alone and in combination with ramipril global endpoint trial ( ontarget)30 has shown that telmisartan had similar effect to ramipril on the primary composite end point ( including cardiovascular death , myocardial infarction , stroke or hospitalization for heart failure ) and death from any cause . this was the first trial to compare an arb with the ace inhibitor ramipril , which has been proven to improve cardiovascular outcomes in the hope trial.31 in hypertensive patients with a history of a cerebral event within the previous 24 months eprosartan showed to be superior to the calcium - channel blocker nitrendipine for the secondary prevention of morbidity and mortality after stroke.32 however , valsartan was comparable to amlodipine on the primary composite end - point in the valsartan antihypertensive long - term use evaluation ( value ) trial which included over 15,000 patients with hypertension and additional risk factors ( coronary heart disease , diabetes , high cholesterol).33 thus the clinical experience with arbs consistently indicates that this class of drugs represents an effective , safe and well tolerated therapeutic alternative for the prevention and care of cardiovascular disease , even though there is no proven superiority as compared to ace inhibitors except for the well documented better tolerability . caution should be used in considering arbs interchangeably , although a class effect can be advocated for the clinical effectiveness of arbs . therefore , in the clinical practice it is preferable to choose the arb that is proven effective , based on specific evidence derived from clinical studies . while arbs are effective in clinical practice and well tolerated , the extent to which they can reduce blood pressure is eventually considered insufficient and a combination therapy is required in a significant percentage of hypertensive patients.34 moreover most arbs may not completely inhibit the at1r at the approved clinical doses . it is a prodrug that is quickly hydrolyzed to the active moiety azilsartan , a potent and highly selective arb with estimated bioavailability of 60% and elimination half - life of 12 hours.35 findings from pharmacokinetic and dose - ranging studies have assessed that the effective therapeutic antihypertensive dosages of azilsartan medoxomil in humans vary from 40 to 80 mg once daily.35 experimental and clinical studies have shown that this new arb induces a potent and long - lasting antihypertensive effect . in conscious spontaneously hypertensive rats ( shrs ) and renal hypertensive dogs , azilsartan medoxomil induced more potent and longerlasting antihypertensive effects than olmesartan medoxomil , which is the newest to the market and has been reported to be most effective among arbs in terms of blood pressure reduction . the persistent durability of the antihypertensive effects of azilsartan medoxomil may reduce the variations in blood pressure during the day . this may contribute potential protective effects on cardiovascular consequences.36 clinical trials have compared azilsartan medoxomil with other arbs in the class , by studying patients with primary hypertension in randomized , double - blind , multicenter studies using ambulatory ( abpm ) and clinic blood pressure measurements ( table 1 ) . intraclass differences in blood pressure - lowering effects within the arb class can not be recognized using clinic blood pressure measurements and may be better assessed with abpm.37 moreover , mean 24-hour systolic blood pressure has been shown to correlate with cardiovascular morbidity in patients with hypertension.38 phase iii studies evaluated and compared the efficacy of 24-hour mean systolic blood pressure and the safety of azilsartan ( 80 mg once daily ) with placebo and the maximal , approved doses of olmesartan medoxomil ( 40 mg once daily ) and valsartan ( 320 mg once daily ) in hypertensive patients ( stage 12 of hypertension ) using ambulatory and automatically measured clinic blood pressure monitoring.39 azilsartan medoxomil at a dose of 80 mg once daily for 6 weeks showed superior efficacy to the top approved doses for hypertension of both valsartan and olmesartan . moreover , also at the dosage of 40 mg azilsartan medoxomil lowered clinic systolic bp to a greater extent than the other arbs , suggesting that this novel agent has a greater potency than these other molecules at the dosages used in the study . moreover , azilsartan was well tolerated and there was no increase in adverse events during this short - term trial . across the effective dose range azilsartan showed superior efficacy compared to the arb valsartan at its maximal recommended dose40 without any significant increase in adverse events . in particular , the antihypertensive effects , safety and tolerability of azilsartan medoxomil were compared to those of valsartan in patients with stage 1 or 2 hypertension in a 24-week randomized , double - blind , parallel - group , multicenter trial.40 on the basis of either abpm or automatically measured clinic blood pressure measurements azilsartan medoxomil at a dose of 40 mg or 80 mg once daily showed greater efficacy ( about 10% in absolute rate ) than a 320 mg dose of valsartan , the highest approved dose for this drug.40 these findings suggest that azilsartan medoxomil can lower 24-hour blood pressure more effectively than maximally recommended doses of other arbs . this suggests that there may be a measurable hierarchal response in the arb class , as far as the blood pressure levels are considered . azilsartan medoxomil is expected to be able to control the blood pressure for a 24-hour period , which may contribute to the prevention of cardiovascular events . indeed , elevations in blood pressure around midnight and early morning are important predictors of central nervous system and cardiovascular outcomes in hypertensive patients.41,42 azilsartan medoxomil is highly potent in inhibiting the specific binding of 125i - sar1-ile8-ang ii to human at1r , and it is a slowly dissociating ang ii receptor blocker . indeed the inhibitory effect of azilsartan medoxomil persisted after washout of the free compound when compared to other arbs ( including olmesartan , telmisartan , valsartan , and irbesartan ) which presented attenuated inhibitory effects with washout . in this regard , the inhibitory effects of azilsartan on ang ii - induced contractile response persisted after washout in vascular strips and chinese hamster ovary ( cho ) cells which overexpress the human at1r.43 thus azilsartan medoxomil may prove to provide a more complete antagonism against endogenous ang ii . this may explain at least in part the greater blood pressure reduction associated with azilsartan . however , these in vitro determinations are yet to be supported in the whole animal or in human studies . hypertension is often associated with insulin resistance which predisposes to the development of metabolic syndrome and/or diabetes . blockade of ras / at1r signaling has been shown to improve the metabolic syndrome in clinical and experimental studies.44 some arbs including losartan , irbesartan , and telmisartan have been shown to improve insulin sensitivity in rodents and humans,45,46 suggesting the possible involvement of the excess of ang ii in the development of insulin resistance . olmesartan medoxomil produced dose - related improvements in the insulin sensitivity of shrs.36 candesartan cilexetil improved the insulin sensitivity of essential hypertensive patients.47 most recently , azilsartan medoxomil has been proven to improve insulin sensitivity in hypertensive rats.36 interestingly , it has been shown that azilsartan medoxomil is more effective than candesartan in reducing plasma concentrations of glucose and fatty acids in normotensive mice . furthermore this novel arb decreases adipose tissue weight and adipocyte size and increases adipose expression of ppar- and its target gene adiponectin , independently of its effects on blood pressure and plasma insulin concentrations.48 it has also been shown that azilsartan medoxomil induces insulinsensitizing effects in obese koletsky rats , independently of decreases in food intake and body weight increase or of the activation of adipose ppar- , the master regulator of adipogenesis . 49 in particular azilsartan treatment decreased the hyperinsulinemia , improved the homeostasis model assessment ( homa - ir ) index and suppressed the over - increase in plasma glucose and insulin concentrations during oral glucose tolerance tests in obese koletsky rats . in the same rat model , it reduced the basal plasma concentrations of glucose , triglyceride , and nonesterified fatty acids ( nefa ) . it has also been reported that azilsartan medoxomil improved insulin sensitivity in shrs and reduced urinary protein excretion more potently than olmesartan medoxomil.37 taken together , this evidence suggests the possible usefulness of azilsartan in the treatment of insulin resistance / metabolic syndrome , and its potential contribution to reduce the cardiovascular risk linked to glucose and lipid metabolism abnormalities in high risk individuals . indeed , azilsartan medoxomil modulates other metabolic functions which can be involved in the atherosclerotic process . in cultured preadipocytes , azilsartan enhanced adipogenesis and induced the expression of adipokines , including leptin , adipsin , and adiponectin , and enhanced the expression of ppar- and - , at a greater extent than valsartan.50 hypertensive and/or diabetic patients often present microalbuminuria or overt proteinuria which are considered major risk factors for progression to end - stage renal disease and the development of cardiovascular disease.51 reduction and normalization of proteinuria by drug treatment including the arbs is associated with decreased risk for adverse renal outcomes,52 as previously discussed . evidence from experimental studies suggest that similarly to other arbs , azilsartan medoxomil may induce urinary albumin and protein excretion levels . this may possibly occur through the activation of several candidate mechanisms , including normalization of glomerular capillary pressure , inhibition of podocyte injury , inhibition of the proliferation of mesangial cells and inhibition epithelial although the exact mechanisms are unknown.36 azilsartan medoxomil may modulate the cell growth , as it is indicated by the observation that it blocked the ang ii -induced activation of mitogen - activated protein kinases in vascular smooth muscle cells . furthermore azilsartan medoxomil is a potent inhibitor of vascular cell proliferation even at low dosages . this effect is also evident in cells lacking at1r.50 increased expression of plasminogen activator inhibitor type - i ( pai-1 ) in the vessel wall seems to accelerate atherosclerosis and it is involved in increasing the instability of atherosclerotic plaque.53 azilsartan medoxomil reduced the expression of pai-1 in the aortic wall of transgenic mice which overexpressed pai-1 in vsmcs and were prone to atherosclerosis secondary to genetically determined apoe deficiency.54 this was associated with a more stable atherosclerotic plaque . in the same mouse model azilsartan medoxomil reduced the pai-1 levels also in the heart . therefore it may contribute to reduce this profibrotic factor that is associated with the negative left - ventricular remodeling and the development of heart failure after myocardial infarction.55 these evidence suggest that azilsartan could exert pleiotropic cardioprotective effects beyond the expected beneficial effects of the potent and sustained blood pressure - lowering action . azilsartan medoxomil is a new compound proposed for the treatment of stage 12 hypertension , with its potent blood pressure - lowering ability associated with considerably better rates of hypertension control compared with other antihypertensive drugs including arbs at standard doses . in fact even a reduction of systolic blood pressure of 2 mmhg to 3 mmhg or more is associated with greater cardiovascular risk reduction as supported by in epidemiologic reports and interventional trials.56 the pharmacodynamic and pharmacokinetic properties suggest that azilsartan medoxomil should be used as an alternative agent for mild - to - moderate hypertension , particularly when other antihypertensive agents are not well tolerated or as an adjunctive drug in hypertensive patients not controlled with other antihypertensive agents . evidence from experimental studies suggests that azilsartan medoxomil may have cardioprotective properties , through a number of other actions which are independent of effects on blood pressure . at present , there are no data available on the effects of azilsartan on cardiovascular morbidity and mortality as well as on key intermediate endpoints or disease markers .

anomalous aortic origin of the left subclavian artery ( lsca ) from the left pulmonary artery ( lpa ) is a rare congenital cardiac malformation . we describe a case of lsca from the lpa via ductus arteriosus in association with a double - outlet right ventricle , which never has been reported previously in korea .

fetal echocardiography performed at 28 weeks of gestational age in a full - term 2,780-g male neonate showed a double - outlet right ventricle ( dorv ) . the patient was born through a cesarean section with apgar scores of 7 and 9 at 1 and 5 minutes , respectively . the heart rate was 170 beats / min , and the respiratory rate was 54 beats / min . the blood pressure was 71/42 and 43/21 mmhg in the right and the left upper extremities , respectively . the left radial pulse was weak , but the right radial and femoral pulses were normal . two - dimensional echocardiography demonstrated the right aortic arch , anterior malalignment ventricular septal defect , overriding aorta ( 60% ) , and pulmonary and infundibular stenosis . patent ductus arteriosus ( pda ) arose from a tortuous abnormal artery with bidirectional shunting . the left subclavian artery ( lsca ) was not seen arising from the aorta . however , three - dimensional 64-row multidetector computed tomography ( mdct ) showed the lsca arising from the left pulmonary artery via ductus arteriosus ( fig . associated non - cardiac anomalies were also determined and included inguinal hernia , polydactyly , and syndactyly . the fluorescent in situ hybridization test was positive for the digeorge syndrome ( 22q11 deletion ) . the patient 's age at the time of surgery was 28 days , and his weight was 3.8 kg . the aberrant lsca was divided , and its pulmonary stump was oversewn . after appropriate trimming , the lsca was directly reimplanted into the left common carotid artery ( lcca ) with 8 - 0 polypropylene sutures ( surgipro ; tyco healthcare ussc , norwalk , ct , usa ) . the left cerebral oxygen saturation was maintained above 80% of the baseline value during the clamping of the lcca . fifteen months later , the patient underwent intraventricular tunnel repair of the dorv at 16 months of age . three - dimensional 64-row mdct at the time revealed the lsca arising from the lcca without stenosis ( fig . the patient is now 18 months old and has normal left arm function and growth . the right aortic arch with isolation of the lsca is an uncommon arch anomaly in which the lsca arises exclusively from the pulmonary artery via ductus arteriosus ( da ) or ligamentum arteriosum without communication with the aorta . the development of the aortic arch and its branches takes place during the third week of gestation . a common arterial trunk arises from the primitive heart and divides into six paired aortic arches that fuse and form bilateral dorsal aorta which , in turn , fuse caudally into the descending aorta . the persistence or regression of these arches may lead to various arch anomalies [ 1 - 3 ] . edwards ' embryologic model of aortic arch malformation explains this lsca isolation by the interruption of the left aortic arch at two locations : 1 ) between the left carotid and subclavian arteries , and 2 ) between the left ductus arteriosus and the left dorsal aortic root . the isolation of the lsca is commonly associated with congenital heart disease ( chd ) and 22q11 deletion . luetmer et al . reported associated chd in 23 of the 39 cases ( 59% ) of isolated lsca . further , the tetralogy of fallot was the most common ( 14 of the 23 cases ) . double - outlet right ventricle and d - transposition of the great artery have also been reported in a few cases [ 4 - 6 ] . the clinical presentations of patients with isolated lsca depend on the patency of the da . the isolation of the lsca is usually asymptomatic and is usually discovered during the evaluations of the associated cardiac anomalies or when reduced blood pressure is detected in the left arm . in these patients , if the direction of blood flow is from the left vertebrobasilar artery to the pulmonary artery ( pulmonary steal syndrome ) or the subclavian artery ( subclavian steal syndrome ) , the patient has vertebrobasilar insufficiency . symptoms may include the disturbance of vision , faintness , syncope , and headache that can be exacerbated by exercising the left upper limb , resulting in increased left arm circulation . ischemic symptoms of the left arm , including pain , weakness , coldness , and a reduced limb length , may be present [ 3 - 7 ] . in a review of the 39 cases described by luetmer and miller , we found that 5 patients had ischemic symptoms of the left arm and 5 had vertebrobasilar insufficiency . they reported that the ages of the symptomatic patients ranged from 22 to 53 years , and the symptom duration before diagnosis ranged from less than 1 year to 11 years . the authors suggested that initially asymptomatic patients may lose the ability to compensate for the steal phenomenon and that eventually symptoms may develop with age . simple ligation of the lsca , surgical reimplantation , device occlusion of the pda , and follow - up have been described as therapeutic options . however , persistent isolated lsca , which was not surgically reimplanted into the aorta , provides an anatomic substrate for a subclavian steal syndrome [ 4 - 7 ] . although this anomaly may be clinically asymptomatic , we recommend surgical correction before significant vertebrobasilar insufficiency or arm ischemia occurs . in our case , the lsca was directly anastomosed with the lcca to prevent symptomatic subclavian or pulmonary steal . we think that very low operative mortality rates and high success rates justify a surgical approach in patients with isolated lsca . further , surgical reimplantation provides complete abolition of the anatomic substrate for the subclavian steal syndrome .

sturge weber syndrome ( sws ) is a rare , sporadically occurring neurocutaneous disorder with a frequency of approximately 1 per 50,000 . the hallmark is an intracranial leptomeningeal vascular angioma in association with a port wine nevus , usually involving ophthalmic or maxillary distribution of trigeminal nerve . other clinical findings associated with sws are seizures , glaucoma , hemiparesis and mental retardation . the radiological hallmark is tram - line or gyri - form calcification . 25 to 56% of patients experience recurrent episodes of paroxysmal focal neurological deficits in form of transient hemiparesis , which may be due to vascular ischemia or postictal in origin . eeg helps to differentiate the exact etiology , as it is normal in former . aspirin prophylaxis in those , due to ischemia decreases their recurrences and improves overall neurological prognosis . we report a 25-month - old child of sws with recurrent episodes of transient hemiparesis and atypical midline location of facial vascular nevus .

sturge weber syndrome , an encephalo - trigeminal angiomatosis is a rare , congenital neurocutaneous syndrome characterized by unilateral facial cutaneous vascular malformation ( port wine stain ) associated with ipsilateral leptomeningeal angiomatosis involving parietal or occipital regions . it presents with focal seizures and recurrent episodes of transient hemiparesis either due to acute vascular events or of postictal in origin . we here report a 25-month - old male child born by full - term vaginal delivery presented to us with sudden onset of left - sided hemiparesis lasting 2 hours , not preceded by constitutional symptoms or seizure activity . he had similar episodes of transient hemiparesis on either side in past which relieved spontaneously without any neurological deficit . frequency of seizures increased and child was started on antiepileptic drugs . since age of 11 months , child had recurrent episodes of transient hemiparesis on either side with or without associated seizures , lasting 2 - 3 days and relieved by itself . there was history of developmental delay noticed after the age of 4 months and presently he could only walk with support corresponding to developmental age of 11 months . on examination he was afebrile with normal vitals . there was port - wine stain of 4 1 cm in an atypical site involving midline of forehead extending over nose [ figure 1 ] . his modified gcs was 13/15 , had umn type left facial nerve palsy with left - sided hemiparesis . contrast ct scan of brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy [ figures 2 and 3 ] . clinical photograph of patient showing port - wine stain of 4 1 cm in an atypical site involving midline of forehead extending over face axial contrast ct brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy coronal contrast ct brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy eeg done urgently revealed no ictal activity , excluding postictal hemiparesis . child was started on aspirin to prevent further recurrences of these thrombotic events . on 3 day of admission , child developed right - sided tonic clonic seizures with facial twitching lasting 15 minutes , and loss of consciousness followed by right - sided hemiparesis . a concurrent eeg revealed epileptic activity with focus on left side , suggesting hemiparesis of postictal origin . child remained seizure - free thereafter until discharge and his residual deficits resolved within 24 hours . on 6 day of admission , child was discharged on anticonvulsants and aspirin prophylaxis . on follow - up up to 1 year features suggestive of sturge weber syndrome in this child were facial port - wine stain , focal seizures , hemiparesis or paroxysmal vascular events and neuroimaging findings . sws is a phakomatosis characterized by port - wine stain over trigeminal nerve area , leptomeningeal angiomatosis and ocular abnormalities caused by abnormal persistence of embryonal vascular system , which is localized around the cephalic portion of neural tube . normally this vascular plexus forms in sixth week and regresses around ninth week of gestation . failure of normal regression results in residual vascular tissue , which forms angiomata of face , ipsilateral eye and leptomeninges . neurological dysfunction results from secondary effects on surrounding tissue , including hypoxia , ischemia , venous occlusion , thrombosis , infarction and vasomotor phenomenon . type i ( classic ) is the common form with both facial and leptomeningeal angiomas and glaucoma may be present , type ii with facial angioma and glaucoma but without evidence of intracranial disease and type iii with leptomeningeal angiomas without facial nevus with usually no ocular manifestation . and gomez reported that venous occlusion might actually cause the initial neurological event , either a seizure , transient hemiparesis or both , thereby beginning the process of vascular steal phenomenon developing around the angioma , resulting in cortical ischemia . recurrent seizures , status epilepticus , intractable seizures and recurrent vascular events may aggravate this steal further with increase in cortical ischemia , resulting in progressive calcification , gliosis and atrophy , which in turn increase the chance of seizures and neurological deterioration . neurological outcome in sws ranges from minimal or no neurological signs to devastating impairment with uncontrolled seizures , hemiparesis , visual field defects and progressive mental retardation . seizure is a common feature , often occurs during first year of life and results from cortical irritability caused by angioma through the mechanism of hypoxia , ischemia and gliosis . about 80% of affected persons have focal seizures involving the contralateral side of the port wine stain . developmental delay and mental retardation as postictal event may also present as hemiparesis , it is important to differentiate the two . pet and spect scan can also guide to differentiate both , which can reveal decreased perfusion and metabolism during vascular event . calcification is unusual before 2 years of age , usually involves high parietal or occipital lobes . mri demonstrates thickened cortex , decreased convolutions , abnormal white matter and gadolinium enhancement of leptomeningeal angioma . aspirin prophylaxis reduces these vascular phenomenon , thereby such episodes of transient hemiparesis and overall neurological deterioration . any child with a facial nevus and hemiparesis with or without seizures and/or glaucoma should arouse a suspicion of sws .

maraviroc is the first and , so far , the only licensed representative of the class of chemokine receptor type 5 ( ccr5 ) inhibitors used for the treatment of human immunodeficiency virus ( hiv ) infection . its safety and efficacy were demonstrated in several clinical trials , and its use was approved in 2007 by the responsible authorities . some specific issues are correlated with maraviroc and its use . it is the only drug in the antiretroviral armamentarium , which does not interact with the viral enzymes but with a human receptor . hence , it is able to be long - term effective only if the infecting virus uses , exclusively , the ccr5 receptor . occurrence and detection of the ccr5 tropism are some of the great challenges of maraviroc use in treatment - experienced patients . although up to 80% of naive patients harbor ccr5-tropic virus , the occurrence of cxcr4 or other tropisms increases with the duration of hiv infection and treatment . nonetheless , maraviroc is a potent medication for eligible patients and helps to improve the outcome of antiretroviral treatment ( art ) of hiv infection .

since the discovery of the human immunodeficiency virus-1 ( hiv-1 ) in 1983 and the beginning of the battle against acquired immunodeficiency syndrome , the development of antiretroviral medication has succeeded substantially . the development and use of antiretroviral therapy ( arv ) started in the mid to end of the 1980s with the first class of substances , the nucleoside reverse transcriptase inhibitors ( nrtis).1 in the following years , additional substances and classes have been introduced into clinical practice . until now , 5 classes of arv with more than 20 licensed drugs have been established for therapeutic use . most of the proven substances inhibit the reverse transcriptase ( rt ) either as nucleoside antagonists or as non - nrtis ; others work as protease inhibitors ( pis ) or integrase inhibitors by modifying the viral enzymatic activity . however , these substances are underlying a risk to select resistant viral mutants due to the variability of the virus under pharmacological pressure . the only antiretroviral class not interfering with the enzymes of the virus is the group of entry inhibitors . different substances , each with a specific mode of action , are represented within this class . the potency of this fusion inhibitor is based on its ability to block the conformational change of gp41 and to avert the approach of virus and cell membrane.2 despite the proven efficacy , enfuvirtide was not established as a routinely used drug because of its application mode as subcutaneous injections twice daily and the resultant side effects predominantly present as cutaneous irritations and painful indurations . lacking the disadvantages of enfuvirtide , maraviroc was the first , and is still the only , approved entry inhibitor in oral formulation that showed potency and sustained efficacy to suppress hiv-1 viral load . the mechanism of action of maraviroc is based on the need of hiv-1 to use a main receptor ( cd4 ) and a coreceptor ( ccr5 or cxcr4 ) on the surface of the target cells . this is followed by a conformational change of gp120 and the coreceptor binding , which allows for the next step , the fusion of virus and cell membrane mediated by gp41.3,4 in 1996 , huang et al5 demonstrated that a homozygous , 32-base pair mutation in the gene for ccr5 ( 32 ) protects against hiv infection by coding for a dysfunctional protein , which is not expressed on the cell surface . the later genetic studies demonstrated relatively high rates of 15%20% of heterozygous 32 mutations in caucasian populations , but rare cases in other populations . in 1996 , liu et al6 estimated that approximately 1% of the caucasian population appeared to be homozygous for this 32 mutation . in fact , some of these individuals remained hiv - negative despite repeated exposure to hiv . based on this knowledge , it was found that maraviroc inhibits the attachment of hiv to its target cell via an allosteric modification of the ccr5 on the surface of the cd4 cells . it works as a small molecular ccr5 inhibitor through a binding in a cavity in the transmembrane ccr5 receptor . this fixation of maraviroc changes the geometry of the transmembrane protein , which is originally needed for the binding of gp120 and ccr5.710 although hiv-1 predominantly uses the ccr5 coreceptor for cell infection , it is not the only possibility to operate . some viral strains have the ability to use a second coreceptor called cxcr4 to infect the target cells , and some viruses may use both ccr5 and cxcr4 receptors . epidemiological studies revealed that hiv-1 strains in arv - naive patients predominantly exhibit ccr5 tropism ( r5 viral variants).1113 more than 80% of treatment - naive hiv-1-infected individuals carry r5 viruses , whereas approximately 20% show dual - tropic or mixed - tropic viruses ( r5/x4 variants ) . only a minority ( < 1% ) of viral strains are capable of using cxcr4 coreceptors exclusively ( x4 variants ) . in treatment - experienced populations , r5 variants still account for 48%62% of isolates and r5/x4 variants are found in 34%50% of these patients , whereas viruses that exclusively use the cxcr4 coreceptors for cell entry are seen in only 2%4% of the pretreated population.1416 the clinical relevance of ccr5 receptor variants , especially the homozygous one , is still unclear . most recent studies suggest an association of 32 mutation and elevated mortality in cases of west nile virus infections.17,18 in contrast , earlier studies suggested protection against chronic hepatitis b infection and rheumatoid arthritis or prolonged survival of renal transplant individuals.1921 additionally , an evaluation date back to the end of the last millennium discussed prevention of yersinia infections ( bubonic plague).22 in fact , many issues have been discussed but a final conclusion of the individual relevance of the ccr5 has not been established . as maraviroc can not prevent cell infection in x4 or r5/x4 variants , it is necessary to detect viral tropism carefully in each individual before using it for arv . as there are different methods to determine the tropism of the virus , it is still unclear which test is the most appropriate for routine clinical use . phenotypic assays are based on the transfection of the virus into the cell culture , to mark the cell with a luciferase reporter gene , and on the determination of lyzed ccr5 or cxcr4 positive cell , in which a single viral cycle has been completed . finally , the luciferase activity is measured by relative light units.23,24 despite being considered to be the gold standard for the measurement of hiv-1 coreceptor usage , the phenotypic method has some disadvantages that should be kept in mind : the method is technically complex , expensive , and laborious . in fact , performance of a phenotypic tropism test takes up to 4 weeks from blood drawing and costs between $ 750 and $ 1000 . in contrast , genotypic assays may be performed within some days , and the cost is restricted to the performance of a gene amplification , mostly of the v3 region . subsequently , the amplification products are analyzed in a sequencer , and the generated sequences are correlated with several standard sequences . for interpretation , some of the algorithm systems predicting coreceptor usage are available online . these systems , such as webcat , webpssm , and geno2pheno[coreceptor ] , are accessible via the internet . established laboratories use these systems to determine the coreceptor usage . a restriction of these systems is the relatively low sensitivity and specificity , but combining 2 or 3 systems enhances the recall ratio.2527 because of the importance of coreceptors for viral entry and the knowledge of research in other entry inhibitors , the pfizer global research and development discovered maraviroc ( uk-427,857 ) as a highly promising substance to block the ccr5 receptor effectively . clinical studies started with dose - finding and safety issues as short - term monotherapy administration in treatment - naive patients and in antiretroviral treatment ( art)-experienced patients , who had to be on treatment for at least 8 weeks ( a4001007 and a4001015 ) . participation was restricted to individuals with confirmed r5 viral variants , viral load > 5,000 hiv-1 copies / ml , and moderate immunodeficiency with still more than 250 cd4 cells / mm . results of these trials demonstrated efficacy of maraviroc in both naive and experienced patients with r5 but not with x4 viruses.28 following trials ( motivate-1 and motivate-2 ) evaluated the efficacy of maraviroc in treatment - experienced patients harboring r5-tropic variants . both trials were double - blind , placebo - controlled , multicenter phase 2b/3 studies , investigating maraviroc plus optimized background therapy ( obt ) vs placebo plus obt in viremic patients ( viral load > 5000 copies / ml ) carrying ccr5-tropic virus . motivate-1 was conducted in the united states and canada , whereas the identically designed motivate-2 trial enrolled patients in europe , australia , and north america . the primary end point of both studies was viral load change in hiv-1 rna from baseline to week 48 . according to the inclusion criteria , all patients had to be triple - class experienced . the results showed a significant advantage for patients in the maraviroc groups : viral load declined by 1.66 log10 copies / ml and 1.82 log10 copies / ml for once - daily and twice - daily administration of drug , respectively , whereas in the placebo arm , it declined only by 0.80 log10 copies / ml in motivate-1 . nearly the same results could be found in motivate-2 : 1.72 and 1.87 log10 reduction vs 0.76 log10 reduction , respectively . the study data also showed a superior response of cd4 cells in the treatment arms : the mean increase of cd4 cells in motivate-1 was 113 and 122 cells/l for once - daily and twice - daily administration , respectively , vs 54 cells/l in the placebo arm . in motivate-2 , the cd4 cells increased by 122 and 128 cells/l for once - daily and twice - daily administration , respectively , ( verum ) and 69 cells/l ( placebo ) . the pooled safety analysis of both studies demonstrated no statistical significant differences in treatment - related adverse events , which indicates a good compatibility of maraviroc.2931 the results of the motivate studies led to the approval of maraviroc , with the restriction for use in pretreated patients harboring only r5-tropic viral strains . a further study called merit compared maraviroc plus zidovudine / lamivudine with efavirenz plus the same backbone in arv - naive patients . the original study missed its goal to demonstrate noninferiority of maraviroc correlated to efavirenz according to the rate of viral load < 50 copies/l after 48 weeks . however , it demonstrated the very good compatibility of the ccr5-inhibitor : patients following the efavirenz - containing regimen discontinued their therapy thrice , more often than maraviroc recipients did . later analyses verified that the original goal of noninferiority was missed because of the lacking sensitivity of the primarily used test to detect viral tropism . a retrospective analysis , with an exclusion of the additionally found x4-tropic virus in patients , by using an advanced version of the tropism test demonstrated the expected noninferiority of maraviroc . additionally , patients in the maraviroc arm demonstrated a significantly higher cd4 cell increase compared with the efavirenz arm . nonetheless , one of the maraviroc arms was conducted as once - daily administration of study drug . this arm was discontinued prematurely due to a lack of efficacy.32 a compilation of maraviroc - associated data of the motivate and merit trials is shown in table 1 . one of the most problematic issues in battling hiv is the development of resistances to antiretroviral drugs . regularly , selection of resistant mutants develops under pharmaceutical pressure , while viral replication occurs due to the irregular intake of medication and/or suboptimal drug levels . in the rt , the protease , and the integrase , occurrence of mutations may be strongly associated with the drug used : eg , lamivudine regularly causes a m184 v mutation in the rt gene locus , which is associated with a resistance to lamivudine but reduces viral fitness significantly.33 on the other hand , some protease - associated mutations , such as l90 m , may be initiated by 1 drug ( eg , saquinavir ) , but may have impact on other drugs of the same class ( eg , atazanavir and nelfinavir).34 these mechanisms of direct or cross - resistance are characteristic for drugs interfering with viral enzymes . in ccr5 inhibitors , there are more escape mechanisms for the virus ; primarily , the use of the above - mentioned coreceptor may change : a r5-using virus might be successfully defeated but the virus may switch to use cxcr4 as coreceptor . in an in vitro study recently conducted , it has been shown that the presence of maraviroc in a cell culture does not lead to a switch of r5 viral strains to x4 variants . thus , it appears not to be common for a virus to switch from its origin to another tropism , it seems to be rather unusual . therefore , another possibility appears to be more reliable with respect to the development of resistance : a preexisting x4 virus minority may get selected due to the r5 suppression , and as result may lead to resistance against ccr5 inhibitors . however , there are also other mechanisms forcing resistance to maraviroc : as maraviroc directly interacts with the human ccr5 , the viral correlate , the env gene with its gp120 and gp41 , may mutate . studies showed the changes of the v3 loop of gp120 lead to resistance , but mutations have also been reported in other regions of gp120 and gp41 due to the enormous variability of the env gene . additionally , the development of resistance in the presence of ccr5 inhibitors does not select always the same mutations.3539 consequently , the prediction of resistance in ccr5 inhibitors may be more complicated and more difficult to interpret than it is with the rt or pis . a third possibility for the virus to attain infectiveness of its combatant maraviroc or other ccr5 inhibitors is the ability to bind to the coreceptor despite specific drug is bound , whether through a competitive replacement due to higher affinity than the drug or because of the possibility to bind despite a drug - bound receptor.40,41 taking the above - mentioned facts into account , the consequences of therapeutic use still remain elusive . nearly , 80% of all antiretroviral naive patients harbor r5-tropic virus , and therefore , would be eligible for using this drug . however , until 2009 , the worldwide approvals for maraviroc indicate its use only for pretreated patients with r5-tropic hiv-1 , partly with this requirement alone or partly with the necessity of resistances to other antiretroviral drug . since 2009 , maraviroc is licensed in the united states under a statement , for combination antiretroviral treatment of adults infected with only ccr5-tropic hiv-1 ( us food and drug administration approval november 20 , 2009 ) , which means that it can be used also in naive patients . however , this statement is directly followed by a special note : more subjects treated with selzentry experienced virologic failure and developed lamivudine resistance compared to efavirenz , which puts the indication into perspective . hence , the use of maraviroc in treatment - naive individuals would be according to the applicable label , but may induce a higher risk for the patients to develop virological failure and acquiring resistances . therefore , maraviroc should be the first choice in treatment - naive patients only under special conditions and thorough surveillance . in treatment - experienced subjects , the motivate studies demonstrated a benefit of maraviroc compared with the placebo arms in virological and immunological responses . however , 955 individuals ( 29.4% ) of 3,244 screened patients were directly excluded from the studies due to the occurrence of x4 or dual / mixed tropism . overall , in only 61% of the initially tested patients , an r5 tropism could be clearly demonstrated.29 these results were generated with a later revised phenotypic assay ( trofile , monogram bioscience , south san fransico , california , usa ) . unfortunately , the possible subsequently generated data obtained using the enhanced trofile assay , which is more sensitive , are not applicable . nonetheless , the virological activity of maraviroc and the good compatibility could be demonstrated in the studies and in clinical practice . however , before starting arv , it needs to be demonstrated that the patient is harboring r5-tropic virus , exclusively . the mechanism of action of maraviroc is based on the need of hiv-1 to use a main receptor ( cd4 ) and a coreceptor ( ccr5 or cxcr4 ) on the surface of the target cells . this is followed by a conformational change of gp120 and the coreceptor binding , which allows for the next step , the fusion of virus and cell membrane mediated by gp41.3,4 in 1996 , huang et al5 demonstrated that a homozygous , 32-base pair mutation in the gene for ccr5 ( 32 ) protects against hiv infection by coding for a dysfunctional protein , which is not expressed on the cell surface . the later genetic studies demonstrated relatively high rates of 15%20% of heterozygous 32 mutations in caucasian populations , but rare cases in other populations . in 1996 , liu et al6 estimated that approximately 1% of the caucasian population appeared to be homozygous for this 32 mutation . in fact , some of these individuals remained hiv - negative despite repeated exposure to hiv . based on this knowledge , it was found that maraviroc inhibits the attachment of hiv to its target cell via an allosteric modification of the ccr5 on the surface of the cd4 cells . it works as a small molecular ccr5 inhibitor through a binding in a cavity in the transmembrane ccr5 receptor . this fixation of maraviroc changes the geometry of the transmembrane protein , which is originally needed for the binding of gp120 and ccr5.710 although hiv-1 predominantly uses the ccr5 coreceptor for cell infection , it is not the only possibility to operate . some viral strains have the ability to use a second coreceptor called cxcr4 to infect the target cells , and some viruses may use both ccr5 and cxcr4 receptors . epidemiological studies revealed that hiv-1 strains in arv - naive patients predominantly exhibit ccr5 tropism ( r5 viral variants).1113 more than 80% of treatment - naive hiv-1-infected individuals carry r5 viruses , whereas approximately 20% show dual - tropic or mixed - tropic viruses ( r5/x4 variants ) . only a minority ( < 1% ) of viral strains are capable of using cxcr4 coreceptors exclusively ( x4 variants ) . in treatment - experienced populations , r5 variants still account for 48%62% of isolates and r5/x4 variants are found in 34%50% of these patients , whereas viruses that exclusively use the cxcr4 coreceptors for cell entry are seen in only 2%4% of the pretreated population.1416 the clinical relevance of ccr5 receptor variants , especially the homozygous one , is still unclear . most recent studies suggest an association of 32 mutation and elevated mortality in cases of west nile virus infections.17,18 in contrast , earlier studies suggested protection against chronic hepatitis b infection and rheumatoid arthritis or prolonged survival of renal transplant individuals.1921 additionally , an evaluation date back to the end of the last millennium discussed prevention of yersinia infections ( bubonic plague).22 in fact , many issues have been discussed but a final conclusion of the individual relevance of the ccr5 has not been established . as maraviroc can not prevent cell infection in x4 or r5/x4 variants , it is necessary to detect viral tropism carefully in each individual before using it for arv . as there are different methods to determine the tropism of the virus , it is still unclear which test is the most appropriate for routine clinical use . phenotypic assays are based on the transfection of the virus into the cell culture , to mark the cell with a luciferase reporter gene , and on the determination of lyzed ccr5 or cxcr4 positive cell , in which a single viral cycle has been completed . finally , the luciferase activity is measured by relative light units.23,24 despite being considered to be the gold standard for the measurement of hiv-1 coreceptor usage , the phenotypic method has some disadvantages that should be kept in mind : the method is technically complex , expensive , and laborious . in fact , performance of a phenotypic tropism test takes up to 4 weeks from blood drawing and costs between $ 750 and $ 1000 . in contrast , genotypic assays may be performed within some days , and the cost is restricted to the performance of a gene amplification , mostly of the v3 region . subsequently , the amplification products are analyzed in a sequencer , and the generated sequences are correlated with several standard sequences . for interpretation , some of the algorithm systems predicting coreceptor usage are available online . these systems , such as webcat , webpssm , and geno2pheno[coreceptor ] , are accessible via the internet . established laboratories use these systems to determine the coreceptor usage . a restriction of these systems is the relatively low sensitivity and specificity , but combining 2 or 3 systems enhances the recall ratio.2527 because of the importance of coreceptors for viral entry and the knowledge of research in other entry inhibitors , the pfizer global research and development discovered maraviroc ( uk-427,857 ) as a highly promising substance to block the ccr5 receptor effectively . clinical studies started with dose - finding and safety issues as short - term monotherapy administration in treatment - naive patients and in antiretroviral treatment ( art)-experienced patients , who had to be on treatment for at least 8 weeks ( a4001007 and a4001015 ) . participation was restricted to individuals with confirmed r5 viral variants , viral load > 5,000 hiv-1 copies / ml , and moderate immunodeficiency with still more than 250 cd4 cells / mm . results of these trials demonstrated efficacy of maraviroc in both naive and experienced patients with r5 but not with x4 viruses.28 following trials ( motivate-1 and motivate-2 ) evaluated the efficacy of maraviroc in treatment - experienced patients harboring r5-tropic variants . both trials were double - blind , placebo - controlled , multicenter phase 2b/3 studies , investigating maraviroc plus optimized background therapy ( obt ) vs placebo plus obt in viremic patients ( viral load > 5000 copies / ml ) carrying ccr5-tropic virus . motivate-1 was conducted in the united states and canada , whereas the identically designed motivate-2 trial enrolled patients in europe , australia , and north america . the primary end point of both studies was viral load change in hiv-1 rna from baseline to week 48 . according to the inclusion criteria , all patients had to be triple - class experienced . the results showed a significant advantage for patients in the maraviroc groups : viral load declined by 1.66 log10 copies / ml and 1.82 log10 copies / ml for once - daily and twice - daily administration of drug , respectively , whereas in the placebo arm , it declined only by 0.80 log10 copies / ml in motivate-1 . nearly the same results could be found in motivate-2 : 1.72 and 1.87 log10 reduction vs 0.76 log10 reduction , respectively . the study data also showed a superior response of cd4 cells in the treatment arms : the mean increase of cd4 cells in motivate-1 was 113 and 122 cells/l for once - daily and twice - daily administration , respectively , vs 54 cells/l in the placebo arm . in motivate-2 , the cd4 cells increased by 122 and 128 cells/l for once - daily and twice - daily administration , respectively , ( verum ) and 69 cells/l ( placebo ) . the pooled safety analysis of both studies demonstrated no statistical significant differences in treatment - related adverse events , which indicates a good compatibility of maraviroc.2931 the results of the motivate studies led to the approval of maraviroc , with the restriction for use in pretreated patients harboring only r5-tropic viral strains . a further study called merit compared maraviroc plus zidovudine / lamivudine with efavirenz plus the same backbone in arv - naive patients . the original study missed its goal to demonstrate noninferiority of maraviroc correlated to efavirenz according to the rate of viral load < 50 copies/l after 48 weeks . however , it demonstrated the very good compatibility of the ccr5-inhibitor : patients following the efavirenz - containing regimen discontinued their therapy thrice , more often than maraviroc recipients did . later analyses verified that the original goal of noninferiority was missed because of the lacking sensitivity of the primarily used test to detect viral tropism . a retrospective analysis , with an exclusion of the additionally found x4-tropic virus in patients , by using an advanced version of the tropism test demonstrated the expected noninferiority of maraviroc . additionally , patients in the maraviroc arm demonstrated a significantly higher cd4 cell increase compared with the efavirenz arm . nonetheless , one of the maraviroc arms was conducted as once - daily administration of study drug . this arm was discontinued prematurely due to a lack of efficacy.32 a compilation of maraviroc - associated data of the motivate and merit trials is shown in table 1 . one of the most problematic issues in battling hiv is the development of resistances to antiretroviral drugs . regularly , selection of resistant mutants develops under pharmaceutical pressure , while viral replication occurs due to the irregular intake of medication and/or suboptimal drug levels . in the rt , the protease , and the integrase , occurrence of mutations may be strongly associated with the drug used : eg , lamivudine regularly causes a m184 v mutation in the rt gene locus , which is associated with a resistance to lamivudine but reduces viral fitness significantly.33 on the other hand , some protease - associated mutations , such as l90 m , may be initiated by 1 drug ( eg , saquinavir ) , but may have impact on other drugs of the same class ( eg , atazanavir and nelfinavir).34 these mechanisms of direct or cross - resistance are characteristic for drugs interfering with viral enzymes . in ccr5 inhibitors , there are more escape mechanisms for the virus ; primarily , the use of the above - mentioned coreceptor may change : a r5-using virus might be successfully defeated but the virus may switch to use cxcr4 as coreceptor . in an in vitro study recently conducted , it has been shown that the presence of maraviroc in a cell culture does not lead to a switch of r5 viral strains to x4 variants . thus , it appears not to be common for a virus to switch from its origin to another tropism , it seems to be rather unusual . therefore , another possibility appears to be more reliable with respect to the development of resistance : a preexisting x4 virus minority may get selected due to the r5 suppression , and as result may lead to resistance against ccr5 inhibitors . however , there are also other mechanisms forcing resistance to maraviroc : as maraviroc directly interacts with the human ccr5 , the viral correlate , the env gene with its gp120 and gp41 , may mutate . studies showed the changes of the v3 loop of gp120 lead to resistance , but mutations have also been reported in other regions of gp120 and gp41 due to the enormous variability of the env gene . additionally , the development of resistance in the presence of ccr5 inhibitors does not select always the same mutations.3539 consequently , the prediction of resistance in ccr5 inhibitors may be more complicated and more difficult to interpret than it is with the rt or pis . a third possibility for the virus to attain infectiveness of its combatant maraviroc or other ccr5 inhibitors is the ability to bind to the coreceptor despite specific drug is bound , whether through a competitive replacement due to higher affinity than the drug or because of the possibility to bind despite a drug - bound receptor.40,41 taking the above - mentioned facts into account , the consequences of therapeutic use still remain elusive . nearly , 80% of all antiretroviral naive patients harbor r5-tropic virus , and therefore , would be eligible for using this drug . however , until 2009 , the worldwide approvals for maraviroc indicate its use only for pretreated patients with r5-tropic hiv-1 , partly with this requirement alone or partly with the necessity of resistances to other antiretroviral drug . since 2009 , maraviroc is licensed in the united states under a statement , for combination antiretroviral treatment of adults infected with only ccr5-tropic hiv-1 ( us food and drug administration approval november 20 , 2009 ) , which means that it can be used also in naive patients . however , this statement is directly followed by a special note : more subjects treated with selzentry experienced virologic failure and developed lamivudine resistance compared to efavirenz , which puts the indication into perspective . hence , the use of maraviroc in treatment - naive individuals would be according to the applicable label , but may induce a higher risk for the patients to develop virological failure and acquiring resistances . therefore , maraviroc should be the first choice in treatment - naive patients only under special conditions and thorough surveillance . in treatment - experienced subjects , the motivate studies demonstrated a benefit of maraviroc compared with the placebo arms in virological and immunological responses . however , 955 individuals ( 29.4% ) of 3,244 screened patients were directly excluded from the studies due to the occurrence of x4 or dual / mixed tropism . overall , in only 61% of the initially tested patients , an r5 tropism could be clearly demonstrated.29 these results were generated with a later revised phenotypic assay ( trofile , monogram bioscience , south san fransico , california , usa ) . unfortunately , the possible subsequently generated data obtained using the enhanced trofile assay , which is more sensitive , are not applicable . nonetheless , the virological activity of maraviroc and the good compatibility could be demonstrated in the studies and in clinical practice . however , before starting arv , it needs to be demonstrated that the patient is harboring r5-tropic virus , exclusively .

rectal cancer is a distinct subset of colorectal cancer where specialized disease - specific management of the primary tumor is required . there have been significant developments in rectal cancer surgery at all stages of disease in particular the introduction of local excision strategies for preinvasive and early cancers , standardized total mesorectal excision for resectable cancers incorporating preoperative short- or long - course chemoradiation to the multimodality sequencing of treatment . laparoscopic surgery is also increasingly being adopted as the standard rectal cancer surgery approach following expertise of colorectal surgeons in minimally invasive surgery gained from laparoscopic colon resections . in locally advanced and metastatic disease , combining chemoradiation with radical surgery may achieve total eradication of disease and disease control in the pelvis . evidence for resection of metastases to the liver and lung have been extensively reported in the literature . the role of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal metastases is showing promise in achieving locoregional control of peritoneal dissemination . this paper summarizes the recent developments in approaches to rectal cancer surgery at all these time points of the disease natural history .

rectal cancer is often camouflaged to be part of colorectal cancer with australian statistics approximating 14 225 cases of bowel cancer in 2008 with a higher predominance among men and those above 50 years of age . meanwhile , the american counterpart recorded rectal and colon cancer to be the third leading malignancy plaguing both sexes in 2012 whereby 9% of deaths in each gender group was attributed to the disease . the risk of death before age 75 has declined over the years from a high of 1 in 50 in 1987 to 1 in 91 in 2007 and this is attributed to better early detection of precancerous lesions and management of the disease [ 1 , 2 ] . treatment of rectal cancer is primarily surgical and is highly dependent on the preoperative staging . early cancers , especially t1 tumors , have been controversially proposed for local excision either via transanal excision or transanal endoscopic microsurgery ( tems ) [ 46 ] . while some report good outcomes measurable to radical surgery , others beg to differ prompting the need for salvage therapy [ 47 ] . later stages of cancer usually follow the traditional approach of abdominoperineal resection ( apr ) or anterior resection and its success at reducing local recurrence is improved by total mesorectal excision ( tme ) [ 8 , 9 ] . however , these procedures are associated with their own set of morbidity and mortality [ 10 , 11 ] . other therapeutic modalities such as chemotherapy and radiotherapy have been included in conjunction with surgery particularly in advanced cancers . they can be used preoperatively or postoperatively either to increase the chances of a sphincter - preserving procedure to allow better quality of life or with a curative intent [ 1214 ] . an overall cancer - specific 5-year survival of 77% has been reported . in this paper , we seek to discuss the modern approach to rectal cancer surgery at all disease time points from preinvasive and early rectal cancer , resectable rectal cancer , and locally advanced and metastatic rectal cancer with an emphasis on presenting some of the controversies and the accepted standards of treatment . the advent of endoscopic mucosal resection ( emr ) for rectal tumours could not have been timelier as the race to provide a treatment that is highly efficacious and of low morbidity continues . emr has been in practice for a few decades now and is commonly used to address gastrointestinal pathologies such as a barrett 's oesophagus , and early gastric cancers ( egc ) with a reported local recurrence rate of 2% in egc [ 1618 ] . the procedure focuses on removal of diseased mucosal tissue or at most , the superficial submucosa instead of a full - thickness excision [ 16 , 19 ] . it can be done by means of a strip biopsy , local injection of hypertonic saline - epinephrine in the inject , lift and cut technique , cup and suction otherwise known as the emr - cap technique or emr with band ligation . if necessary , more than one of the aforementioned methods can be applied [ 16 , 20 , 21 ] . emr is an alternative to the locally - approached transanal excision ( tae ) and transanal endoscopic microsurgery ( tems ) . generally , local excision of rectal cancer is indicated for early disease that encompasses its precursor ( dysplasia or adenoma ) , t1 tumours that display moderate - to - well differentiation and no lymphatic or vascular invasion [ 6 , 16 , 17 , 22 ] . at present , it is known that emr can be performed on flat , sessile , or lateral spreading rectal tumours [ 2224 ] . the success of emr is reflected through its high overall cure rate with some reporting 91% for treatment - nave patients and others at 96% [ 23 , 25 ] . one of the common consequences of emr is bleeding which occurs from 1%45% of cases . the bleed can be minor and is easily manageable with haemostatic clips or severe requiring blood transfusions or even surgery [ 19 , 24 , 26 ] . 0.7% to 4% of cases are complicated with perforation which can appear later as abdominal distension and pain necessitating further operation [ 25 , 26 ] . while emr is an excellent therapeutic option for rectal tumours , it is operator dependent . identifying the lesion especially if it is a lateral spreading tumour can be challenging to the untrained eye as it displays subtle changes but a meta - analysis showed that the chances of a complete en bloc resection is directly proportional to experiences if the operator . the concern is that complete resection and a proper histological evaluation is rendered difficult with larger lesions , thus running a risk of local recurrence [ 23 , 25 , 28 ] . in spite of this , some studies showed that the success of the resection is not statistically different between the two techniques [ 26 , 29 ] . complicating failure of the mucosa to lift during the inject , lift and cut technique may occur secondary to submucosal fibrosis . the success rate of a repeated resection is reduced from 91.0% in a treatment - nave patient to 74.5% in a previously attempted lesion . it is reported that the time for conversion of a precursor lesion to cancer is variable , with dwelling times in the adenoma state ranging from 4 years to as long as 48 years . in fact , flat adenomas need not necessarily be treated as it is almost always noninvasive . this questions the need for emr in such lesions and more so , the implication on the patient who is no doubt heading for months of unwarranted anxiety . nevertheless , emr is a safe and effective treatment modality that is minimally invasive and only requires conscious sedation [ 25 , 26 , 28 ] . it is also efficient with moss et al . reporting a mean procedural duration of 25 minutes while another study accounted a range of 25137 minutes [ 25 , 29 ] . when possible , patients can be treated as an outpatient with discharges within the same day [ 31 , 32 ] . a comparison between tae and endoscopic resection showed that the latter was associated with a significantly shorter hospital stay of 2.7 1.1 days with a mean difference of 6.2 days . similarly , tems recorded a range of 044 days of hospitalization while emr had a range of 027 days . emr per se is not as costly as the other procedures available , thus allowing a cheaper and less invasive option for the patient . this translates into reduced expenditure for the spender [ 20 , 29 , 33 ] . on the other hand , tems and tae lesions larger than 8 cm in diameter can be removed with good effect via tems [ 34 , 35 ] . where emr may fail to completely remove the tumour , both tems and tae first attempt of tems for large rectal adenoma is reported to have a lower early local recurrence rate at 10.2% compared to emr at 31.0% ( p < 0.001 ) . meanwhile , low risk - t1 tumour recurrence rate following tems is 010% while santos et al . tae is estimated to have a 15% local recurrence rate of the malignancy at five years . it would seem that the recurrence rate following tae is high compared to emr but contradicting this is a 32-patient study by lee et al . which showed no recurrences of cancer following either endoscopic resection or tae at a median followup of 15 months ( range 699 ) . studies evaluating the disease - specific and overall survival rates ( tems versus radical surgery and tae versus radical surgery ) showed that the values were not statistically significant to one another indicating the efficacy of these procedures [ 5 , 38 ] . emr appears to be favourable as tems and tae have the added adverse effects including wound breakdown , incontinence , urgency , strictures , neuralgia , and anovaginal fistula [ 34 , 35 , 37 , 39 ] . barendse et al . studied both emr and tems and discovered that the former was associated with half the percentage ( 12% ) of postoperative complications compared to the latter ( 24% ) . however , given the variable recurrence rates , there is some apprehension towards emr . in view of this instead , it is a judgment call by the surgeon and more importantly the patient himself based on the local expertise and the pros and cons of each option available . as we push the fort of combination treatment , it is likely that in the future , local excisional strategies will become a more commonly adopted strategy in complete or subcomplete responding tumors after chemoradiotherapy . however , the limitation of mesorectal sampling may mean that more intensive followup is required to detect recurrence at the local excisional site and/or mesorectal lymph nodes . its proximity to the anal sphincter also makes this a major consideration into the surgical approach towards resection . in the localized setting , a multimodality approach has in recent years been developed and investigated in trials to improve local recurrence , disease - free and overall survival using a variety of sequences of chemotherapy and radiotherapy . prior to surgery , adequate local staging is paramount in the surgical planning and accurate prediction of the extent of bowel wall involvement may be obtained through endorectal ultrasonography and magnetic resonance imaging may serve the similar purpose but also identify the involvement of lymph node metastases . the aims of rectal cancer surgery are to remove the tumor with an adequate distal margin of a minimum of 2 cm in the case of a low rectal tumor with sphincter preservation or 5 cm in the case of a rectosigmoid / upper rectal tumor with restoration of intestinal continuity through an anastomosis . if a 2 cm distal margin can not be secured , an abdominoperineal excision with enbloc resection of the entire anorectum and an end colostomy is required . surgery should be performed using a no - touch technique with high ligation of the inferior mesenteric artery to achieve adequate lymphatic sampling through harvesting of the sigmoid mesentery and mesorectum . tumors of the middle and lower rectum require a total mesorectal excision ( tme ) . tme reduces the risk of local recurrence and although a prospective randomized trial has not been conducted to verify its efficacy , longitudinal data derived from the netherlands where a tme trial was conducted following rigorous training of colorectal surgeons demonstrated that there was an observed reduction in rate of local recurrence from 16% to 9% with tme surgery being an independent predictor of overall survival . further , data from the mrc cr07 and ncic - ctg co16 randomized trials demonstrated that the plane of surgery achieved in patients undergoing rectal cancer surgery impacted on local recurrences with a 3-year local recurrence rate of 4% for patients whose surgery was completed with achievement of the mesorectal plane , 7% for intramesorectal plane and 13% for muscularis propria plane . pertinent also in low rectal cancers requiring abdominoperineal excision , to avoid a coning effect in the deep pelvis as the tumor is approached from both the abdomen and perineum , extended abdominoperineal excision incorporating resection of the levator muscles to reduce inadvertent bowel perforation and breaching of the circumferential resection margin . today , in the current era of laparoscopic surgery where shorter postoperative hospital stays , reduction in pain scores , shorter time of return of bowel function , lower treatment cost , and improved cosmesis may be achieved , these standardized surgical resections have been demonstrated to be feasible in the laparoscopic approach . initially , the laparoscopic approach was first examined in colon cancer with at least 4 large randomized trials ; cost study group trial from the usa , color trial from europe , mrc clasicc trial from the uk , and a trial in rectosigmoid cancers from the prince of wales hospital in hong kong demonstrating equivalent oncologic efficacy with similar overall survival , disease - free survival and local and distant recurrences [ 4649 ] . these studies although predominantly examined in the setting of colon cancer were quick to translate into standard practice for rectal cancer despite limited large scale prospective randomized trials . however , there remain concerns over the ability to achieve adequate mesorectal excision and clear surgical margins in laparoscopic rectal cancer surgery . in the uk mrc clasicc trial , there was a 34% conversion rate from laparoscopic to open surgery in the rectal cohort , increase performance of tme surgery to ensure adequacy of the distal resection margin in the laparoscopic group because of the inability to palpate the tumor for localization . nonetheless , there was no difference in positive circumferential resection margin , local recurrence , disease - free , and overall survival in both the laparoscopic versus open anterior resection and abdominoperineal resection groups . the prince of wales hospital group from hong kong reported a small prospective randomized trial of laparoscopic assisted versus open abdominoperineal resection for low rectal cancer randomizing 99 patients ( 51 lap - assisted and 48 open ) demonstrating earlier return of bowel function , decrease time to mobilization , lesser analgesia requirement , longer operative time , and higher direct cost in the lap - assisted group without difference in morbidity and mortality . in an update of this trial , after 10 years of followup , the authors reported higher rates of bowel obstruction requiring hospitalization and intervention in the open group but similar oncologic outcomes were shown with 10-year survival of 83.5% and 78% ( p = 0.595 ) and 10-year disease - free survival 82.9% and 80.4% ( p = 0.698 ) in the lap - assisted and open group , respectively . in the corean trial that randomized 170 patients in each arm to laparoscopic and open surgery for mid or low rectal cancer after preoperative chemoradiotherapy , the laparoscopic group had lower amount of blood loss , longer operative time , quicker recovery of bowel function , and a lesser amount of analgesic requirement . surgical quality indicators including the circumferential resection margin , macroscopic quality of the tme specimen , number of harvested lymph nodes , and perioperative morbidity were similar between groups . in a spanish randomized trial of 204 patients of whom 78.6% in the open group and 76.2% in the laparoscopic group underwent sphincter - preserving surgery ; blood loss was greater in the open surgery group , operative time was longer in the laparoscopic group , and return to diet and hospital stay was longer in the open surgery group . complication rates were similar between groups but a larger number of lymph nodes were isolated in the laparoscopic group . together , these three small randomized trials suggest that the laparoscopic approach achieves improved short - term outcomes without compromising the surgical quality of rectal cancer operations in skilled hands but a longer operative time is required . longer followup of these trials and results of ongoing larger trials will confirm the long - term oncologic outcomes of laparoscopic rectal cancer surgery . thorough preoperative clinical staging is paramount in the sequencing of multimodality therapy for rectal cancer . for smaller tumors t1/t2 , surgery alone with wide surgical resection of low anterior resection or abdominoperineal resection for distal lesions not amendable to low anterior resection may be performed . this allows sampling of mesorectal lymph node for accurate pathological staging . in patients who are medically unfit or who adamantly refuse to undergo standardized resectional surgery , local excision with or without chemoradiotherapy this strategy fails to sample the mesorectal lymph nodes that are essential in disease staging . in a large single institution cohort study comparing their retrospective experience of 350 with stage i rectal cancer of whom 283 patients ( 80.9% ) underwent standardized resection and 67 patients ( 19.1% ) undergoing local excision , 5-year local recurrence was 14.1% in the local excision group compared to 3.3% in the standardized resection group . this significantly higher local recurrence rate may then be salvaged through multimodality approach combining preoperative chemoradiotherapy and surgery . however , at times , these local recurrences may not be resected with standardized resectional surgery and may require radical surgeries such as a pelvic exenteration . for clinically staged t3/t4 rectal tumors without clinically identified nodal disease ( stage ii ) who undergo tme surgery with either a low anterior resection or abdominoperineal resection with harvesting of at least 12 lymph nodes examined and staged as pn0 chemoradiation is not required . chemoradiation may be considered in the setting of pt3n0 tumors with adverse pathologic features , non - tme surgery or in those with fewer than 12 lymph nodes harvested . for t3/t4 tumors with lymph node metastases ( stage iii ) identified clinically , treatment involves both chemoradiation with fluorouracil ( 5-fu ) and total mesorectal excision ( tme ) based surgery . postoperative chemoradiation was shown to achieve superior results over postoperative radiation alone with a 34% reduction in recurrence rate with reductions observed for local recurrences and distant metastasis . when postoperative chemotherapy was compared to radiotherapy in the nsabp r-01 randomized trial , postoperative chemotherapy appeared to improve survival and radiotherapy reduced the incidence of locoregional recurrence without survival improvements . given the benefits of chemoradiation in achieving local control as an adjunct to surgery , the german rectal cancer study group then conducted a randomized trial of 421 patients to determine the perisurgical sequencing of chemoradiation for rectal cancer . these investigators compared preoperative to postoperative chemoradiation and demonstrated that the 5-year cumulative incidence of local recurrence was 6% in the preoperative arm compared to 13% in the postoperative arm ( p = 0.006 ) with fewer grad 3/4 acute and long - term toxic effects of chemoradiation observed in the preoperative arm compared to the postoperative arm . in a smaller korean trial , the improved effects of local control was not demonstrated in the preoperative compared to the postoperative chemoradiation arm , however , it was shown that an increased rate of sphincter preservative surgery could be achieved . the exact type of preoperative therapy was also recently debated with a short course radiation ( 25 gy in 5 fractions ) followed by surgery a week after or a long course chemoradiation ( 50.4 gy in 28 fractions combined with systemic chemotherapy ) followed by surgery four to six weeks after . the brief use of radiotherapy in the short course setting has been argued upon its role in providing adequate tumor response to allow sphincter preservative surgery . two randomized trials ; polish ( n = 312 ) and australian ( n = 326 ) compared these two regimens and both trials showed a lower rate of early acute toxicity and reduce cost of treatment without any difference in long - term oncologic outcomes [ 59 , 60 ] . after preoperative chemoradiation , guidelines recommend adjuvant chemotherapy for patients with node positive disease . however , the eortc 22921 trial of 785 clinically staged t3/t4 rectal cancer patients randomized to receive adjuvant fluorouracil based chemotherapy after preoperative ( chemo ) radiotherapy and surgery showed no survival benefit of chemotherapy on disease - free survival . however , specific subgroup analysis was performed to determine the appropriate role of adjuvant chemotherapy and showed that pathologically staged t0 - 2 ( ypt0 - 2 ) patients appeared to benefit in terms of both disease - free and overall survival from adjuvant chemotherapy compared to ypt3/t4 patients . importantly , adjuvant chemotherapy did not appear to demonstrate any difference in outcomes of patients with ypn0 or ypn+ disease . this demonstrates that further benefits of adjuvant chemotherapy are only observed in responding patients ( ypt0 - 2 ) . further trials are required in this area to determine the appropriate role of adjuvant chemotherapy and the selection of high - risk populations who may then benefit from other modern adjuvant agents . tumors extending beyond the rectal wall with invasion into surrounding viscera are considered locally advanced rectal cancer . often in patients who develop local recurrence , recurrent disease often similarly involve adjacent structures where the previously excised rectal tumor was located . although its incidence has decreased following total mesorectal excision and the incorporation of preoperative chemoradiation , when it occurs , it remains a debilitating condition that is difficult to treat . palliative radiotherapy may provide brief symptom relief for an average of 3 months with median survival in these patients being between 12 and 24 months [ 6264 ] . surgery may provide a long - term palliation to the debilitating symptoms of pelvic recurrences . in the curative setting , delivery of long course preoperative chemoradiotherapy may induce tumor down - staging to facilitate surgical resection . in a randomized trial comparing neoadjuvant radiotherapy to chemoradiotherapy in 207 patients with locally unresectable t4 primary rectal or local recurrent rectal cancer , chemoradiotherapy compared to radiotherapy facilitated higher potential for an r0 resection ( 84% versus 68% ; p = 0.009 ) , improved local control in patients who underwent a r0 or r1 resection ( 82% versus 67% at 5 years ; p = 0.03 ) , improved time to treatment failure ( 63% versus 44% ; p = 0.003 ) , cancer - specific survival ( 72% versus 55% ; p = 0.02 ) and overall survival ( 66% versus 53% ; p = 0.09 ) . the surgery involved necessitated pelvic exenteration where adjacent organs are resected with an aim to achieve clear margins . in patients with primary t4 rectal cancer and recurrent rectal cancer , 28% and 20% of patients in the chemoradiotherapy arm and 27% and 46% in the radiotherapy arm , respectively , required exenteration . intraoperative radiotherapy ( external - beam ) ( iort ) is another approach to improve local control . this treatment modality has been investigated in patients with locally advanced unresectable rectal cancer after chemoradiotherapy down - staging and surgery . valentini et al . reported 100 patients with t4m0 tumors undergoing r0 resection after down - staging by chemoradiotherapy and showed that 5-year local control was 90% in patients with r0 surgery and 100% in patients with r0 surgery and iort . further , iort did not appear to compensate for suboptimal surgery with 5-year overall survival of 68% observed in patients with r0 surgery compared to 22% in r1 or r2 surgery . such radical surgery however is not widely performed and only available in specialized institutions . in a pattern of care study of the united states population through data identified from the surveillance , epidemiology and end results ( seer ) registry , only 33% of patients with locally advanced adherent colorectal cancer underwent multivisceral resection for which was shown to be associated with improved overall survival . in patients with synchronous rectal cancer with liver metastases , there remains an enigma over the appropriate sequencing of chemotherapy , radiotherapy , and surgery . in a study from the erasmus university , van der pool et al . reported a consecutive series of 57 patients of whom 29 patients underwent resection of the primary tumor first , 8 patients underwent simultaneous rectal and liver resection and 20 underwent a liver - first approach achieving a median survival of 47 months and 5-year survival of 38% . this was achieved in a multidisciplinary setting where an individualized approach towards treatment was taken . in general , if resection of both primary tumor and liver metastases may be completed in one surgery , this approach may be favored . if the liver metastases are not completely resectable during the rectal surgery or are too advanced for hepatectomy irrespective , neoadjuvant chemotherapy is preferred followed by a liver - first approach followed by restaging and preoperative radiotherapy and rectal surgery . in patients with metachronous liver metastases , results of large clinical series have shown that median survival range from 43 months to 64 months with 5-year survival ranging between 37% to 51% [ 68 , 69 ] . of note , in a large international multi - institutional registry study , rectal primary tumor were associated with extrahepatic recurrences after hepatectomy for liver metastases , hence emphasizing the importance of local control in the pelvis . however , there is no difference in colon or rectal based primary tumor site impacting outcomes after hepatectomy for colorectal liver metastases . epidemiological and observatory data from the followup of patients with curatively treated colorectal cancer have shown that rectal cancer patients have a higher preponderance to developing recurrence in the lungs [ 7173 ] . in a large population based study of 30 years in burgundy ( france ) , mitry et al . reported that lung metastases often accompanied liver metastases with synchronous lung metastases being more common in left colonic and rectal cancers [ 7173 ] . surgery for lung metastases is indicated if the lung metastases are the only site of disease and a complete resection may be achieved . where extrapulmonary metastases are present , a highly selective approach should be taken often after adequate tumor response to systemic chemotherapy to select patients whose disease is amendable to resection of both lung and extra - pulmonary metastases . the highest level of evidence for resection of lung metastases comes from a large systematic review of 20 published studies for pulmonary metastasectomy for colorectal lung metastases . report a median 5-year overall survival of 40% in this selected group of patients who underwent surgery . however , given that the liver will often be involved when there is lung metastases , it is important that the selection of patients for pulmonary metastasectomy should include a sufficient disease - free interval from previous liver resection , use of prethoracotomy cea levels and the absence of mediastinal lymph node involvement as a separate selection criteria in this group of patients . presently , a randomized trial ( pulmicc ) funded by cancer research uk is seeking to investigate if pulmonary metastasectomy contributes to improved survival of patients with colorectal lung metastases by randomizing patients with a history of resected colorectal cancer who are found to have pulmonary metastases to be randomly allocated to active monitoring or active monitoring with pulmonary metastasectomy with overall survival , relapse - free survival , lung function , and patient - reported quality of life as endpoints of this clinical trial . shedding of tumor during the difficult abdominoperineal resection , low anterior resection operation , or invasion of a large t3/t4 tumor in the upper rectum above the peritoneal reflection may result in the shedding of free peritoneal tumor cells within the abdominopelvic peritoneal cavity . the growth and implantation of these cells may result in the development of peritoneal metastases ( carcinomatosis ) . results of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy ( hipec ) have shown that this combined modality technique allows complete excision of peritoneal tumors with locoregional control achieved through the chemoperfusate . in colorectal cancer , a randomized trial comparing cytoreductive surgery and hipec demonstrated a median survival of 22.3 months compared to 12.6 months in patients receive systemic chemotherapy with or without palliative surgery . however , in this trial of 105 patients , only 12 patients had rectal cancer . again , in a large registry study of the french experience of hipec in colorectal cancer , of 523 patients included , only 36 patients ( 7% ) had primary colorectal tumor of rectal origin . in another international registry of 506 patients with colorectal peritoneal metastases undergoing cytoreductive surgery and hipec , there were 40 patients ( 8% ) with the primary tumor of rectal origin and the median survival of these patients was 19.2 months compared to 24 months in patients with tumors of sigmoid origin , 17 months for patients with tumors of the right colon and 20 months for patients with tumors of the left colon . these results inform us that peritoneal metastases from rectal cancer is less common but prevent us from drawing any meaningful conclusion on whether there may be disparate survival outcomes for colon and rectal cancer patients with peritoneal metastases . based on the currently available evidence , selected rectal cancer patients with limited peritoneal disease burden may be considered for cytoreductive surgery and hipec . rectal cancer surgery has made significant advancement at all - time points of the natural history of this disease . there are now minimally invasive local excision options that are currently being tested for efficacy as we await further clinical trials to verify its efficacy for pre - invasive and early lesions . laparoscopic rectal cancer surgery incorporating total mesorectal excision is now emerging as the standard surgical approach with ongoing clinical trials that will confirm its short and long - term oncologic efficacy . there is now evidence based results from clinical trials for both preoperative short and long - course chemoradiation prior to surgery for resectable tumors for which has been shown to improve local control of disease albeit its aim achieving sphincter preservative surgery where possible . ultimately , the goal of this being to achieve adequate sampling of mesorectal lymph nodes to provide adequate information for tumor staging and prediction of local and distant recurrences for which will guide treatment decisions . there is now evidence for resecting metastases from rectal cancer from local recurrences , liver , lung , and peritoneal metastases based on a body of retrospective clinical data with long - term followup .

much progress has been made in understanding the pathophysiology and treatment of atypical hemolytic uremic syndrome ( ahus ) . plasma therapy is the mainstay of treatment for ahus . the availability of the first effective anti - complement therapeutic agent , eculizumab , has dramatically changed the outlook of this disease . however , its use in clinical practice raises important questions , such as who should receive the drug , when to start such therapy , and is it safe to stop treatment once the disease is controlled . we describe here for the 1st time in india , use of eculizumab in a 12-year - old boy with ahus . we also describe in this report challenges faced in procuring the drug , and an ideal , evidence - based method of treating ahus in children .

atypical hemolytic uremic syndrome ( ahus ) is a rare , and life threatening , disease . in most cases , the first line of treatment for ahus was plasmapheresis until september 2011 , when the food and drug administration approved eculizumab for the treatment of pediatric and adult patients with ahus . eculizumab has been available in the united states since 2007 for the treatment of paroxysmal nocturnal hemoglobinuria . this additional indication makes eculizumab the first drug to be approved for the treatment of ahus . we describe here for the first time in india , use of eculizumab in a 12-year - old boy with ahus , and the challenges faced . a 12-year - old caucasian boy was admitted in september 2014 , with complaints of pain abdomen for 4 days , petechial spots over face for 3 days , cola - colored urine and decreased urine output for 1 day . on evaluation , the child had hypertension ( blood pressure : 140/90 ) , anemia ( hemoglobin = 9 g / dl ) , severe thrombocytopenia ( platelet count 20,000/mm ) , acute kidney injury ( blood urea 183 mg / dl ; serum creatinine 4 mg / dl ) , lactate dehydrogenase 10,000 iu / ml , and schistocytes on peripheral smear suggestive of hemolysis . malarial antigen , dengue antigen , widal , leptospiral serology , and all cultures were negative . there was no history of diarrhea , dysentery , or fever prior to this illness . a diagnosis of ahus was made and plasma exchange ( pe ) initiated within 24 h of admission . following three daily pes , his hematuria , platelet count , serum creatinine , and urine output improved [ figure 1 ] . initially , the pe was done daily for 6 days , followed by alternate days for 2 weeks , then thrice a week for 2 weeks , according to the european pediatric study group guidelines for hus . the child was discharged on day 7 of admission , and the rest of the pes were done on an out - patient basis . a complement factor assay was sent to a collaborative laboratory in france which was found to be normal [ table 1 ] . since it was an unexplained ahus , the need for long - term pes or eculizumab and a complete genetic sequencing workup was discussed with the family . the treatment could only be stopped once the complete genetic work - up was negative . since the child was an american citizen , the american embassy , insurance agency and alexion pharmaceuticals , germany were contacted . the drug is not available in india and a special acquiring permission from the drug controller of india followed by custom duty exemption was taken . the child was already immunized for meningococci according to centers for disease control and prevention schedule . trend of hemolytic parameters with plasma exchanges and eculizumab results of the complement exploration performed in the family members subsequently , the child was started on eculizumab ( soliris ) 1200 mg q 2 weekly , along with penicillin prophylaxis for meningococci . after three doses , the child developed leukopenia ( total leukocyte count 3500/mm and absolute neutrophil count 800/mm ) . the child was put on cotrimoxazole and acyclovir prophylaxis , and the dose interval was increased to q 3 weekly . the child also developed hallucinations after three doses of eculizumab , which could be attributed to intensive care unit psychosis / drug , which improved on adding risperidone and increasing the drug interval . the child was continued on q 3 weekly eculizumab , with normal renal functions , and a close monitoring for ch50 and hemolysis . a trough level of eculizumab at 3-week dose interval was 135.8 mcg / ml ( normal trough used in paroxysmal nocturnal hemoglobinuria 100 mcg / ml ) . the gene sequencing results were available in february 2015 and it was normal [ table 1 ] . it was discussed with the family that the possibility of finding a major genetic defect has been ruled out , though there is still a possibility of relapse . hence , eculizumab was stopped in april 2015 , in view of the absence of any symptoms and normal complement factors and gene sequencing . currently the child is 9 months off eculizumab and is doing well , with a normal renal function , no proteinuria , and a normal urine examination with no relapses . despite poor quality evidence , the mainstay of treatment for ahus was plasma therapy till 2009 . the outcome for children with ahus is poor , and because of the rarity of these disorders , clinical experience is scanty . the guideline for the investigation and initial therapy in ahus published by the european pediatric study group for hus in 2009 , based on anecdotal case reports , retrospective series and expert consensus , advocated early , frequent , and high volume pes . the recently published audit of this guideline indicated considerable morbidity associated with plasma therapy in children . recently , eculizumab has been proposed as the first - line treatment for ahus , to avoid pe and the complications of central venous double lumen catheters . when possible , eculizumab treatment should be initiated within 2448 h of onset or admission . if eculizumab is not ( or not immediately ) available , pe ( or plasma infusions if pe is not possible ) should be started as recommended in the 2009 guideline . we switched our patient to maintenance eculizumab after the drug was procured from germany after getting all the necessary legal permissions . current evidence - based management of atypical hemolytic uremic syndrome ( adapted from loirat et al . ) eculizumab is a monoclonal antibody that binds to the complement protein c5 , inhibiting its cleavage to c5a and c5b . this prevents circulation of the pro - inflammatory c5a peptide and generation of the cytotoxic terminal complement complex c5b-9 ( referred to as the membrane attack complex [ mac ] ) . the most common adverse effects reported by the 37 adults and adolescents receiving eculizumab in the two prospective ahus studies were hypertension ( in 35% of patients ) , headache ( 30% ) , anemia ( 24% ) , leukopenia ( 16% ) , diarrhea ( 32% ) , vomiting ( 22% ) , and nausea ( 19% ) . our patient has significant leukopenia requiring increasing the dose interval and adding cotrimoxazole and acyclovir prophylaxis . the reduction in mac formation produced by eculizumab eliminates the body 's defense mechanisms against neisseria meningitidis under normal conditions . patients should be immunized with meningococcal vaccine at least 2 weeks prior to administering eculizumab unless the risks of delaying therapy outweigh the risk of a meningococcal infection . the risk of relapse in patients with ahus is influenced by the genetic background , and the interval between flares is extremely difficult to predict . the issue of the optimal duration of eculizumab treatment has not yet been properly addressed . the european medicines agency has approved life - long eculizumab treatment for patients with ahus . however , the high cost of the drug and the uncertainties surrounding the natural history of ahus in patients for whom eculizumab prevented the progression to end - stage renal disease , raise the question of whether life - long treatment is warranted for all patients with ahus . given that the natural history of ahus differs depending on the underlying genetic abnormalities , treatments could be tailored on the basis of an individual 's complement genetics . life - long treatment may be appropriate in patients with ahus who have mutations associated with poor outcomes ( e.g. cfh or c3/cfb gain - of - function mutations ) . we also carefully planned to stop eculizumab in our patient , since he had no major genetic mutations , with a normal complement panel . there is a need to completely evaluate all patients with ahus for complement abnormalities and antibodies to factor h. these patients should get early pe / eculizumab . hus being a common cause of acute kidney injury in children in india should get timely and appropriate management with lifesaving eculizumab or pes . to improve the availability of the drug in indian market , and subsequently decrease the cost of drug , collaboration among medical experts and health authorities must occur in order to implement a feasible plan of action .

[ purpose ] the purposes of this study were to investigate the influences of position on % mvic of spinal stability muscles to establish for the most effective breathing pattern for activation of spinal stability muscles in order to provide an additional treatment method for use in spinal stability exercise programs . [ subjects and methods ] thirty - three healthy subjects performed quiet breathing and four different forced respiratory maneuvers ( frm ) ; [ pursed lip breathing ( plb ) , diaphragmatic breathing ( db ) , combination breathing ( cb ) and respiration muscle endurance training ( rmet ) ] in both standing and sitting positions . % mvic of them ( the multifidus ( mf ) , erector spinae ( es ) , internal oblique / transversus abdominis ( io / tra ) , external oblique ( eo ) , rectus abdominis ( ra ) measured . [ results ] io / tra , mf and eo showed greater activation in standing than in sitting , while ra and es showed greater activation in sitting than in standing . rmet induced significantly greater activation of spinal stability muscles then other breathing patterns . % mvic changes of muscle activities induced by frm were independent of position with a few exceptions . [ conclusion ] the increased respiratory demands of frm induced greater activation of spinal stability muscles than qb . rmet was found to be the most effective breathing pattern for increasing the activation of the spinal stability muscles .

the diaphragm , the primary respiratory muscle , has been reported to act as one of spinal stability muscles and other spinal stability muscles are also activated to increase respiratory capacity1,2,3,4,5,6,7 . the muscles used in both spinal stability and respiratory tasks include the diaphragm , transversus abdominis ( tra ) , intercostals muscles , internal oblique muscle ( io ) and pelvic floor muscles ( pfm)1,2,3 , which are also known as core muscles . the spinal stability provided by these muscles is derived from their co - contraction which increases intra - abdominal pressure4 . these muscles function as respiratory muscles by increasing their activities when respiratory demand increases1,2,3 , 5,6,7 . we speculated that increasing the strengths of the respiratory muscles would have positive effects on spinal stability . the question was whether forced breathing pattern increased the spinal stability muscle activities enough to strengthen them . bridging exercises was the commonly used methods to increase spinal stability muscle strengths8 , 9 and prone position bridging exercise was the most effective among in other positions10 . the previous study showed % mvic of these muscles with forced breathing patterns were as high as with bridging exercises . in addition , the synergy ratios of these muscles with forced breathing patterns were similar with those with bridging exercises8 , 9 , 11,12,13 . however , these previous studies were performed in the standing position and it is not clear whether the increased muscle activations were due to increased postural demand or increased respiratory demand or both . in addition , it is necessary to determine whether there are any interaction effects between position and breathing patterns to suggest forced breathing pattern as one way to strengthen spinal stability muscles . thirty three subjects who had no history of low back pain within the last six months , musculoskeletal impairments of the lower limbs , or neurological or respiratory pathology were enrolled in this study . subjects with a cold , or excessive abdominal fat or a current or previous swimming habit were excluded . the data of 33 subjects ( 16 males , 17 females , 20.33 2.10 years old , height 1.66 0.08 m , weight 59.83 9.60 kg , bmi 21.57 2.31 kg / m ) were used for data analysis . prior to their participation , all the participants read and signed an informed consent form , in accordance with the ethical principles of the declaration of helsinki . the protocol for this study was approved by the ethics committee of catholic university of daegu . each participant performed five different breathing patterns in two positions , sitting and standing . the five different breathing patterns were quiet breathing ( qb ) and forced respiratory maneuvers ( frm ) : combination breathing ( cb ) , diaphragmatic breathing ( db ) , pulsed lip breathing ( plb ) and respiratory muscle endurance training ( rmet ) . % mvic of internal oblique abdominis / transversus abdominis ( io / tra)14 , external oblique abdominis ( eo)15 , rectus abdominis ( ra)15 , erector spinae ( es)16 and multifidus ( mf)8 were measured for comparison of muscle activities . measurements and data collection were performed following the procedures described in a previous study13 . two - way repeated anova was used to determine the main effects of breathing pattern and position as well as interaction effects between them . two - way repeated anova was performed in order to evaluate the effects of position and breathing pattern on five spinal stability muscle activities . the within - subjects factors were position ( two levels : sitting and standing ) and breathing patterns ( five levels : qb , cb , db , plb and rmet ) . the main effects of position on % mvic of io / tra , eo , ra , es and mf were significant [ f(1,32)=24.794 , p<0.001 ; f(1,32)=48.049 , p<0.001 ; f(1,32)=16.069 , p<0.001 ; f(1,32)=17.398 , p<0.001 ; f(1,32)=6.971 , p=0.013 ; respectively ] . in addition , the main effects of breathing patterns on % mvic of io / tra , eo , ra , es and mf were significant [ f(4,128)=25.150 , p<0.001 ; f(4,128)=29.943 , p<0.001 ; f(4,128)=28.974 , p<0.001 ; f(4,128)=61.043 , p<0.001 ; f(4,128)=22.487 , p<0.001 ; respectively ] . the interaction effects were tested and were significant on only io / tra , eo and mf , [ f ( 4,128 ) = 3.054 , p=0.034 ; f ( 4,128 ) = 3.281 , p=0.040 ; f ( 4,128 ) = 4.037 , p=0.013 ; respectively ] . the paired t - tests was performed to examine the significant main effects of position after controlling for the family - wise error rate across these tests using holm s sequential bonferroni approach . differences in mean % mvic of io / tra , eo , ra , es and mf between the two positions were significant : t(164)= 9.61 , p<0.001 ; t(164)= 10.06 , p<0.001 ; t(164)=5.53 , p<0.001 ; t(164)= 8.82 , p<0.001 ; t(164)= 5.27 , p<0.001 ; respectively . of particular interest , mean % mvic of io / tra , eo and mf were higher in standing than in sitting , whereas % mvic of ra and es were lower in standing than sitting . one - way anova was performed to examine the significant main effects of breathing patterns after controlling for the family - wise error rate cross these tests using holm s sequential bonferroni approach ( table 1table 1.results of the tests for the significant main effect of breathing patterns . unit ( % mvic)io / traeoraesmfqb5.816.12 * 2.811.25 * 2.361.13 * 3.401.70 * 2.891.37*cb8.318.404.002.01 * 2.761.293.841.86 * 3.461.61db9.388.834.292.262.791.243.851.87 * 3.271.56plb6.776.27 * 3.231.52 * 2.511.19 * 3.611.89 * 3.001.31**rmet12.9110.336.123.733.441.565.522.714.011.86*p < 0.001 different from rmet , * * p=0.003 different from rmet , * * * p=0.002 different from qb ) . for io / tra , ra and mf , rmet induced significantly greater activation of muscles than qb ( p < 0.001 ) and plb ( p<0.001 , p < 0.001 , p=0.002 , respectively ) . eo and es in rmet showed significantly higher % mvic than in cb , db , plb and qb ( p<0.001 ) . in addition , p < 0.001 different from rmet , * * p=0.003 different from rmet , * * * p=0.002 different from qb finally , the paired t - test was performed to examine differences among breathing patterns in each position , controlling for the family - wise error rate using holms sequential bonferroni approach . the mean % mvic of io / tra in plb and rmet showed a significantly greater increase in standing than sitting compared to qb ( p=0.002 , p=0.004 , respectively ) . the mean % mvic of mf in cb showed a significantly greater increase in standing than in sitting compared to db and plb ( p=0.003 , p=0.002 , respectively ) , however the mean % mvic of mf in qb showed a significantly greater increase in standing than in sitting compared to cb ( p<0.001 ) . the purposes of this study were to investigate the influences of position on % mvic of spinal stability muscles and to find the most effective breathing pattern for the activation of spinal stability muscles . the results of this study show that the positions and breathing patterns had important influences on spinal stability muscle activities and their influence was different depending on the muscles . the deep muscles and eo showed greater activation in standing than in sitting , while the superficial muscles showed greater activation in sitting than in standing . it is well - known that the deep muscle group provide segmental stability while the superficial muscle group provide torque production and general trunk stability17 . to maintain a standing posture , increased segmental stability might be needed rather than increased torque , which is provided by the superficial muscle group . some studies have reported that activation of the deep muscle group is more effective at increasing spinal stability than activation of the superficial muscle group17 , 18 . in other studies , subjects demonstrated optimal lower back stabilization during exercises with appropriate deep muscle activation19 , 20 . in addition , sitting posture showed increased trunk sway with decreased compensatory postural control21 ; therefore , more torque might be needed to minimize trunk sway in sitting , possibly explaining the increase in superficial muscles activities in sitting compared to standing . we suggest that the reason for this is that the lower extremities are unavailable to compensate for respiratory perturbation in sitting . in this study , the spinal stability muscles , except mf , were activated the most by rmet , db , cb , plb and qb in declining order , whereas mf was activated the most by rmet , in the order of cb , db , plb and qb . among the different breathing patterns , rmet induced significantly greater activation of the spinal stability muscles than the other breathing patterns . for io / tra , % mvic in db increased as much as in rmet , and % mvic in db was significantly higher than that in plb or qb . for mf , % mvic in cb was not significantly different from that in rmet , even though % mvic in cb was significantly higher than that in plb or qb . rmet was the breathing pattern that induced the greatest activation of the spinal stability muscles . however , for subjects who have difficulty performing rmet , db or cb could be used as an alternative method for activation and strengthening of the spinal stability muscles . although , position and breathing patterns showed meaningful impacts on spinal stability muscle activation , interaction effects between position and breathing patterns were found for a few muscles in a few breathing patterns . in general , % mvic changes in the spinal stability muscles induced by frm were independent of position with a few exceptions : io / tra in plb and rmet showed increased activation that was greater than the possible position effect compared to the change in qb , while mf in qb was activated more than the possible position effect compared to the change in cb . however , absolute % mvic of both of these muscles with rmet was the highest . in conclusion , increased respiratory demands of frm induced greater activation than qb , and rmet was found to be the most effective breathing pattern for increasing the activities of the spinal stability muscles regardless of position . in rmet , the increases in muscle activities induced by increase in respiratory demand was not different between sitting and standing . therefore , it can be expected that rmet in a sitting position activates the spinal stability muscles in a manner comparable to that of standing . subjects with difficulty in maintaining a standing posture could start a spinal stability muscle strengthening program using rmet in a sitting position and progress to rmet in a standing position as spinal stability improves . a previous study showed lumbar stability improved with increased tra contractility after 4 weeks of deep breathing exercises22 . however , further research will be required in order to determine whether spinal stability muscles are strengthened by rmet in sitting , and whether a spinal stability strengthening program with rmet can improve spinal stability .

lung atelectasis resulting after cardiopulmonary bypass ( cpb ) can result in increased intrapulmonary shunting and consequent hypoxemia . advanced pressure control modes of ventilation might have at least a theoretical advantage over conventional modes by assuring a minimum target tidal volume delivery at reasonable pressures , thus having potential advantages while ventilating patients with pulmonary atelectasis postcardiac surgery . however , the utility of these modes in the post - cpb setting have not been widely investigated , and their role in cardiac intensive care , therefore , remains quite limited .

pulmonary complications are frequently encountered after cardiac surgery with cardiopulmonary bypass ( cpb ) , and atelectasis is believed to be a major etiology . atelectasis results in a decrease in lung compliance and adversely affects oxygenation . significant lung collapse following cardiac surgery results in intrapulmonary shunting and hypoxemia . conventional mechanical ventilation might have additional deleterious influence on these atelectatic lungs , particularly with employment of high tidal volumes and pressures resulting in pulmonary hyperinflation . however , if a patient is hypoxemic , he will require positive pressure ventilation to sustain oxygenation . addition of continuous positive airway pressure in such patients , might improve functional residual capacity , and place the patient on a favorable part of the pressure - volume curve . if , however , this fails to improve oxygenation , then it becomes necessary to initiate positive pressure ventilation . it would be ideal , therefore , to ventilate patients at the top of the volume - pressure curve , at high lung volumes , but without accompanying phasic changes . in the absence of facilities for oscillatory ventilation , increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time ( ti ) in a pressure control mode . prolonged ti improves oxygenation . conversely , when ti exceeds the expiratory time , carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis . we know that generally patients tolerate respiratory acidosis very well , and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly , which could be a factor in patients undergoing cardiac surgery . pressure control ventilation ( pcv ) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation ( imv ) . that has changed in most cardiac surgical intensive care units , with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance , especially when combined with longer inspiratory times ( inverse ratio pcv , or pcirv ) . tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance . there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days , even in patients who do not have acute respiratory distress syndrome . especially , susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury . intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings . whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications , despite a paucity of studies confirming a substantial benefit from this mode . this mode is extremely uncomfortable for patients , who generally need to be heavily sedated , and often paralyzed . intensivists , however , prefer patients to be awake and interacting with the ventilator , leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv . airway pressure release ventilation ( aprv ) was described in 1987 by stock et al . as a mode for acute lung injury while limiting airway pressures . bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle [ figure 1 ] . the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided , thus enabling faster separation from mechanical ventilation . a study by rathgeber et al . compared duration of weaning between bilevel positive airway pressure ( synonymous with bilevel ) , volume controlled ( vc ) imv , and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal , yet significant decrease in weaning time . there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece , or to alternative modalities such as automatic tube compensation , proportional assist ventilation ( pav ) , adaptive support ventilation ( asv ) , or smartcare . however , putensen et al . have documented the benefits accrued from aprv , which include improvements in respiratory system compliance , pao2 , cardiac index , and delivery of oxygen , in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis . the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure . this results in redistribution of splanchnic blood from abdominal viscera to inferior vena cava , resulting in enhanced venous return . unlike other modes in which the physician presets a specific tidal volume or pressure , pav lets the patient determine the inspired volume and the flow rate . this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated . however , unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient , pav can only be used in active patients . while to the best of our knowledge , there are no published studies exploring the effects of pav in patients undergoing cardiac surgery , in at least one randomized controlled study , asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians , leading to better utilization of resources . slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation . as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury , there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation . like vc ventilation , delivery of a reasonable tidal volume is guaranteed , and at the same time , like pressure controlled ventilation , this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure . surprisingly , however , there are very few clinical trials related to their use in cardiac surgery , despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications . while it is entirely possible to infer that unfamiliarity with many of these modes , as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena , larger studies are needed to clearly identify strategies that will result in improved survival , decreased duration of mechanical ventilation , earlier icu discharge , earlier discharge from hospitals , and economic benefits . clinically , relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs , undergoing complex cardiac repairs , and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease . it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits . it would be ideal , therefore , to ventilate patients at the top of the volume - pressure curve , at high lung volumes , but without accompanying phasic changes . in the absence of facilities for oscillatory ventilation , increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time ( ti ) in a pressure control mode . prolonged ti improves oxygenation . conversely , when ti exceeds the expiratory time , carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis . we know that generally patients tolerate respiratory acidosis very well , and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly , which could be a factor in patients undergoing cardiac surgery . pressure control ventilation ( pcv ) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation ( imv ) . that has changed in most cardiac surgical intensive care units , with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance , especially when combined with longer inspiratory times ( inverse ratio pcv , or pcirv ) . tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance . there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days , even in patients who do not have acute respiratory distress syndrome . especially , susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury . intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings . whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications , despite a paucity of studies confirming a substantial benefit from this mode . this mode is extremely uncomfortable for patients , who generally need to be heavily sedated , and often paralyzed . intensivists , however , prefer patients to be awake and interacting with the ventilator , leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv . airway pressure release ventilation ( aprv ) was described in 1987 by stock et al . as a mode for acute lung injury while limiting airway pressures . bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle [ figure 1 ] . the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided , thus enabling faster separation from mechanical ventilation . a study by rathgeber et al . compared duration of weaning between bilevel positive airway pressure ( synonymous with bilevel ) , volume controlled ( vc ) imv , and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal , yet significant decrease in weaning time . there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece , or to alternative modalities such as automatic tube compensation , proportional assist ventilation ( pav ) , adaptive support ventilation ( asv ) , or smartcare . however , putensen et al . have documented the benefits accrued from aprv , which include improvements in respiratory system compliance , pao2 , cardiac index , and delivery of oxygen , in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis . the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure . this results in redistribution of splanchnic blood from abdominal viscera to inferior vena cava , resulting in enhanced venous return . pav allows automated modulation of airway pressure according to force generated by the patient . unlike other modes in which the physician presets a specific tidal volume or pressure this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated . however , unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient , pav can only be used in active patients . while to the best of our knowledge , there are no published studies exploring the effects of pav in patients undergoing cardiac surgery , in at least one randomized controlled study , asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians , leading to better utilization of resources . slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation . as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury , there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation . like vc ventilation , delivery of a reasonable tidal volume is guaranteed , and at the same time , like pressure controlled ventilation , this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure . surprisingly , however , there are very few clinical trials related to their use in cardiac surgery , despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications . while it is entirely possible to infer that unfamiliarity with many of these modes , as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena , larger studies are needed to clearly identify strategies that will result in improved survival , decreased duration of mechanical ventilation , earlier icu discharge , earlier discharge from hospitals , and economic benefits . clinically , relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs , undergoing complex cardiac repairs , and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease . it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits . it would be ideal , therefore , to ventilate patients at the top of the volume - pressure curve , at high lung volumes , but without accompanying phasic changes . in the absence of facilities for oscillatory ventilation , increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time ( ti ) in a pressure control mode . prolonged ti improves oxygenation . conversely , when ti exceeds the expiratory time , carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis . we know that generally patients tolerate respiratory acidosis very well , and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly , which could be a factor in patients undergoing cardiac surgery . pressure control ventilation ( pcv ) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation ( imv ) . that has changed in most cardiac surgical intensive care units , with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance , especially when combined with longer inspiratory times ( inverse ratio pcv , or pcirv ) . tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance . there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days , even in patients who do not have acute respiratory distress syndrome . especially , susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury . intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings . whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications , despite a paucity of studies confirming a substantial benefit from this mode . this mode is extremely uncomfortable for patients , who generally need to be heavily sedated , and often paralyzed . intensivists , however , prefer patients to be awake and interacting with the ventilator , leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv . airway pressure release ventilation ( aprv ) was described in 1987 by stock et al . as a mode for acute lung injury while limiting airway pressures . bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle [ figure 1 ] . the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided , thus enabling faster separation from mechanical ventilation . a study by rathgeber et al . compared duration of weaning between bilevel positive airway pressure ( synonymous with bilevel ) , volume controlled ( vc ) imv , and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal , yet significant decrease in weaning time . there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece , or to alternative modalities such as automatic tube compensation , proportional assist ventilation ( pav ) , adaptive support ventilation ( asv ) , or smartcare . however , putensen et al . have documented the benefits accrued from aprv , which include improvements in respiratory system compliance , pao2 , cardiac index , and delivery of oxygen , in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis . the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure . this results in redistribution of splanchnic blood from abdominal viscera to inferior vena cava , resulting in enhanced venous return . unlike other modes in which the physician presets a specific tidal volume or pressure , pav lets the patient determine the inspired volume and the flow rate . this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated . however , unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient , pav can only be used in active patients . while to the best of our knowledge , there are no published studies exploring the effects of pav in patients undergoing cardiac surgery , in at least one randomized controlled study , asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians , leading to better utilization of resources . slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation . as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury , there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation . like vc ventilation , delivery of a reasonable tidal volume is guaranteed , and at the same time , like pressure controlled ventilation , this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure . surprisingly , however , there are very few clinical trials related to their use in cardiac surgery , despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications . while it is entirely possible to infer that unfamiliarity with many of these modes , as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena , larger studies are needed to clearly identify strategies that will result in improved survival , decreased duration of mechanical ventilation , earlier icu discharge , earlier discharge from hospitals , and economic benefits . clinically , relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs , undergoing complex cardiac repairs , and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease . it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits .

a randomized crossover study explored whether viewing different scenes prior to a stressor altered autonomic function during the recovery from the stressor . the two scenes were ( a ) nature ( composed of trees , grass , fields ) or ( b ) built ( composed of man - made , urban scenes lacking natural characteristics ) environments . autonomic function was assessed using noninvasive techniques of heart rate variability ; in particular , time domain analyses evaluated parasympathetic activity , using root - mean - square of successive differences ( rmssd ) . during stress , secondary cardiovascular markers ( heart rate , systolic and diastolic blood pressure ) showed significant increases from baseline which did not differ between the two viewing conditions . parasympathetic activity , however , was significantly higher in recovery following the stressor in the viewing scenes of nature condition compared to viewing scenes depicting built environments ( rmssd ; 50.0 31.3 vs 34.8 14.8 ms ) . thus , viewing nature scenes prior to a stressor alters autonomic activity in the recovery period . the secondary aim was to examine autonomic function during viewing of the two scenes . standard deviation of r - r intervals ( sdrr ) , as change from baseline , during the first 5 min of viewing nature scenes was greater than during built scenes . overall , this suggests that nature can elicit improvements in the recovery process following a stressor .

since the 19th century , the natural environment has been considered important for ensuring a greater level of physical and mental health . gatherer past , present day humans have an innate affiliation with nature and living things . consequentially , nature is conducive to involuntary attention and does not require our directed attention , allowing recovery from mental fatigue and facilitating attention restoration . in the past decade , epidemiological studies in the netherlands have identified a positive correlation between improved health outcomes and amount of surrounding green space . subsequently , the diverse health benefits that maybe engendered by nature have become a focal point for research . two recent systematic reviews have concluded that exposure to nature is associated with improved mental well - being in comparison to indoor environments and synthetic or built environments . further support of these conclusions has been found in single studies where improvements in self - esteem , positive and negative mood , anxiety levels , and feelings of calmness and comfort have been observed . the studies reviewed by thompson - coon et al . did not report physiological variables , and bowler et al . both systematic reviews conclude that investigation regarding physiological changes during experiencing nature is lacking . however , there are individual studies investigating exposure to nature that identify changes in physiological health markers including decreased heart rate ( hr ) and decreased systolic ( sbp ) and diastolic blood pressure ( dbp ) . further , changes in endocrine markers such as reduced adrenaline , noradrenaline , and cortisol , as well as enhanced autonomic control ( indirectly measured using heart rate variability , hrv ) have also been reported . these findings have also been observed in a controlled and simulated environment indoors where potential confounding factors such as weather , climate , sounds , and smells are eliminated . there is , however , a lack of rigorous research providing empirical evidence of the physiological mechanisms that exist with exposure to nature . thus , the impact of environment on cardiovascular health needs to be explored further using controlled environments and outcome measures which reflect such physiological changes , e.g. , autonomic control . hrv provides a measure of autonomic nervous system ( ans ) functioning and can be used to explore physiological changes associated with nature exposure . the ans is important in controlling many bodily functions , and alterations are prevalent during relaxation and arousal . hrv is an easy to obtain , established noninvasive scientific and clinical measure and has prognostic value regarding cardiovascular health . it requires the measurement of interbeat differences in hr using either electrocardiogram ( ecg ) or mobile r - r interval monitors . the analysis of hrv reflects ans function by assessing the parasympathetic and sympathetic contributions to sino - atrial node regulation of hr . a higher hrv suggests an increased adaptability of the ans and is associated with better health . previous work that has observed hrv indicates that there is a tendency for higher hrv when nature is viewed in situ or simulated using projected images . the ans plays a central role in governing the response to stress and how the body recovers following a stressor . indeed , lane and thayer utilized functional magnetic resonance imaging ( fmri ) to examine the hypothesized heart brain connection and found concurrent associations between vagal influenced hrv and changes in blood flow through areas of the brain known to be involved in emotional responses , attention , and working memory . additionally , the prefrontal cortex has been observed to play a role in the top - down regulation of hrv , as demonstrated using direct current stimulation of the dorsolateral area of the prefrontal cortex during viewing of negative images compared to neutral images ( images of nature were not used in this study ) . the relaxation and restorative effect of nature might help combat the rising incidence of psychological stress by providing a potential resilience tool . previous analysis of the restorative effects of nature suggest that participants recover faster from induced stress ( in terms of hr ) when , during the recovery , they view nature through a window or view projected scenes of natural environments . hr recovery in the latter study was purported to occur due to , in part , an enhanced recovery of parasympathetic activity . it is , however , unknown whether the benefits of nature on cardiovascular reactivity continue to occur after exposure has ceased . it is further unknown whether a longer exposure to nature elicits greater responses during the exposure . during nature exposure , the first 5 min elicits the greatest improvements in mood and self - esteem . physiological alterations in hrv also occur during the first 5 min of viewing slides simulating nature . to date , there is no evidence to suggest that longer exposure times elicit better physiological responses and in particular greater changes in hrv during nature exposure . this time course is important to establish ; nature could be potentially used prior to a stressor and thus alter physiological outcomes in recovery from a stressor without the need of exposure to nature during the recovery period ; i.e. , nature could improve cardiovascular reactivity following a stressor by speeding the recovery process . the primary aim of this study was to investigate the effect that prior viewing of nature scenes had on ans function during recovery from a stressor . the hypothesis was that viewing nature scenes ( composed of trees , grass , fields ) prior to a stressor will lead to higher hrv in recovery when compared to viewing scenes of built environments ( composed of man - made , urban scenes lacking natural characteristics ) . a secondary aim was to compare ans function during the first and second 5 min of a 10 min exposure to nature images to see if hrv changes were sustained . ethical approval from the university ethics committee was granted , and participants ( n = 25 , 7 males , 18 females ) were recruited from university support staff who were independent from the research group conducting the study . informed consent was obtained from all participants who ranged in age from 19 to 65 years ( mean sd , 36.08 10.42 ) , with stature 169.05 7.33 cm ( mean sd ) and mass 71.66 13.11 kg ( mean sd ) . due to a technical problem , data were not recorded for two participants ( 1 female , 1 male ) during the stressor segment . the participants remaining ( n = 23 ) were aged 36.91 11.09 years ( mean sd ) , with stature 168.35 7.52 cm ( mean sd ) and mass 70.38 12.42 kg ( mean sd ) . the study was a within - subject randomized crossover design with all participants visiting the laboratory on two occasions one week apart . visits were at the same time of day to eliminate any effect of circadian rhythm on the dependent variables . participants were free from symptoms of disease and were not using medication that would affect the cardiovascular or ans . room temperature was set at 21 c ; artificial lighting was used , and blinds were drawn to avoid direct sunlight entering the room . each visit consisted of viewing one of two different groups of slides before being exposed to a mental stressor . participants were randomly assigned an order to either view the built scenes first or the nature scenes first . on arrival , participants completed a battery of psychological questionnaires ( described below ) . following this , participants were told to rest for 15 min in the semisupine position , allowing hr and blood pressure ( bp ) measures to stabilize . following the period of rest , participants viewed a set of slides ( either nature or built environment scenes ) for a 10 min period . immediately poststressor , a further 5 min of physiological data were recorded to capture recovery . after recovery , participants completed the same set of psychological questionnaires . at the next visit , a week later , participants repeated this protocol and viewed the other set of slides . the slideshows depicting scenes of nature and built environments involved still photographs representing each of the environments . the photographs were chosen from a central pool of photographs used within the research group . the photographs ( figure 1 ) depicting natural environments included natural elements , e.g. , trees , grass , or plant life , and were devoid of any man - made structures , e.g. , buildings , vehicles , roads . in contrast , the photographs depicting built environments contained man - made structures , e.g. , houses , flats , office buildings , brick walls . a critical element of the scenes of built environments was the lack of natural features . twenty photographs were selected and collated in a microsoft powerpoint slideshow , with each slide being shown for 30 s. each slideshow lasted a total of 10 min , and photographs were shown in the same order for every participant . before viewing the slideshow , participants were given the instructions to try and imagine they were in the environment depicted on the screen . the term condition will be used from this point to describe the viewing of either the nature or built area slide sets . the two slide sets will be referred to individually as the nature condition and built condition . examples of images used in the slideshows to depict scenes of nature environments ( a and b ) and scenes of built environments ( c and d ) . the mental stressor comprised a forward digit span test with an accompanying socio - evaluative threat and was designed specifically to elicit cardiovascular stress responses . a series of six numbers were displayed on a screen in front of the participant . after each series of six numbers , the participant had 10 s to write the numbers down in the correct order . the test is cognitively demanding and was designed to cause a stress response by adding a socio - evaluative component which has previously been reported to cause a stress response . to ensure a socio - evaluative threat , participants were informed that they would be carefully monitored during the test by the experimenter and a buzzer would sound when an incorrect answer was given . the buzzer was used twice in each session in order to achieve consistency irrespective of whether the participant had provided an incorrect answer . participants were informed at the end of the study that this was the case . they were asked if they were aware that they had in fact got the answer correct . all participants reported that they could not be sure that they had and had assumed they had answered incorrectly . an ecg modified lead ii configuration was used to record interbeat data throughout the protocol . bp oscillations were measured continuously throughout the protocol from a finger bp cuff attached to the middle finger of the nondominant hand ( portapres , fms , finapres medical systems bv , netherlands ) . continuous recording of blood pressure in this manner allows mean blood pressure to be calculated and is advantageous over using momentary measures which are susceptible to white coat syndrome or cuff response . sbp and dbp were calculated using labchart 7 software ( adinstruments , uk ) to transform the waveform data . sbp is recorded as the highest pressure and dbp as the lowest pressure during a single cardiac cycle . respiratory frequency and depth were measured using a respiratory strap ( pneumotrace , adinstruments , uk ) fitted around the participant s chest . all data were measured at 1000 hz and collected by a powerlab 8sp ( model ml785 , adinstruments , uk ) using labchart 7 software . data were then analyzed and averaged out into 5 min segments as it is most appropriate for hrv comparison that sections are equal in duration . these segments are categorized into : baseline ( base ) , first 5 min of viewing slides ( slides 1 ) , second 5 min of viewing slides ( slides 2 ) , mental stressor ( stress ) , and in recovery ( recv ) . in the time - domain standard deviation of r - r intervals ( sdrr ) was chosen to reflect overall hrv contributed to by both sympathetic and parasympathetic systems . to reflect parasympathetic system activity alone , the root - mean - square of successive differences ( rmssd ) was used . responses were coded on a 03 scale giving a range for total self - esteem score of 0 , representing the lowest level of self - esteem , and 30 , the highest level of self - esteem . although designed as a trait measure of self - esteem , rosenberg s scale has previously demonstrated changes as a state measure of self - esteem due to exposure to natural environments . mood - related adjectives ( e.g. , enthusiastic , inspired , hostile , afraid ) were rated on a scale of 1 to 5 for how well each adjective described participants current mood ( 1 = very slightly or not at all , 5 = extremely ) . self - esteem and mood were measured at the start and end of the protocol immediately after the recovery period . to test the primary hypothesis , a repeated measures anova was used to identify whether recovery relative to baseline differed between conditions for rmssd , sdrr , hr , sbp and dbp . for the secondary question , changes from baseline values were first calculated to explore if the expected short - term effect during the first 5 min of viewing is repeated in the second 5 min . data were not normally distributed ( assessed by kolomogorov - smirnov test for normality ) ; therefore , the friedman s test was performed with posthoc wilcoxon tests to explore significant differences . repeated measures anova was also used to examine whether the stress response ( hr and bp only ) compared to baseline differed between conditions . to assess if breathing frequency and depth was kept constant throughout the protocol , repeated measures anova was used . to identify whether changes in psychological measures changed from baseline to poststress and whether this differed between viewing condition , paired t tests were used to identify if performance in the mental stress task differed between condition and visit order . significance was set at an alpha of 0.05 , where appropriate posthoc paired t tests were used to investigate significant effects between conditions with a bonferroni corrected alpha level . all data are normally distributed except for rmssd ( assessed by kolomogorov - smirnov test for normality ) . planned paired t tests on baseline data ( table 1 ) showed there were no differences between the viewing conditions at baseline for rmssd , sdrr , hr , sbp , dbp , or breathing frequency or depth ( p > 0.05 ) . therefore , any subsequent differences are likely attributed to interventions and actions within the protocol . in all analyses , rmssd , root mean square of successive differences ; sdrr , standard deviation of r - r intervals ; self - esteem quantified using rosenberg s self - esteem , low scores = low self - esteem , range 030 ; negative and positive mood taken from the positive and negative affect scale ( panas ) , low scores = low negative or positive mood , range 040 . for the primary research question , repeated measures anova revealed an interaction effect for rmssd between the nature condition and the built condition over time ( f1 , 22 = 8.72 , p = 0.007 , p = 0.28 ) . this demonstrates increased levels above baseline during recovery for the nature condition compared to recovery in the built condition ( figure 2 ) where levels dropped below baseline . repeated measures anova on sdrr determined a main effect of view ( f1 , 22 = 11 , p = 0.003 , p = 0.33 ) and time ( f1 , 22 = 7.7 , p = 0.011 , p = 0.26 ) , with levels increasing during recovery irrespective of view and higher sdrr in the nature condition irrespective of time . mean ( sd ) heart rate and heart rate variability recovery from stress compared to baseline : , main effect for time ( p < 0.05 ) ; , interaction effect ( p < 0.05 ) ; , main effect for view ( p < 0.05 ) . for the secondary research question , exposure time was split in half to see if ans function during the first 5 min differed to the last 5 min according to condition ( figure 3 ) . test for multiple comparisons identified significant differences for sdrr during views ( ( 3 ) = 8.06 , p = 0.045 ) . change in sdrr from baseline was found to be significantly greater in the nature condition ( median = 1.20 ) compared to the built condition ( median = 2.44 ) during the first 5 min ( z = 2.56 , p = 0.011 ) . heart rate and heart rate variability means sd as change from baseline for the first 5 min and last 5 min of viewing : , significant difference between conditions . repeated measures anova revealed a main effect for time on hr ( f1 , 22 = 27.3 , p = 0.001 , p = 0.10 ) describing an increase from baseline to stress period ( 67.8 1.8 vs 73.4 1.8 bpm ) . there was also a main effect of time on sbp ( f1,22 = 34.1 , p = 0.001 , p = 0.61 ) and dbp ( f1,22 = 9.7 , p = 0.005 , p = 0.31 ) both revealing an increase from baseline to stress period ( 117.6 2.6 vs 128.8 3.1 mmhg and 60.1 1.4 vs 63.1 1.5 mmhg , respectively ) . change in these parameters supports the effectiveness of the mental stressor . repeated measures anova determined that time had an effect on breathing rate ( f4 , 84 = 6.2 , p = 0.001 , p = 0.23 ) . pairwise comparisons revealed that only the stress period was different from baseline ( 18.3 0.7 vs 14.3 0.7 , p < 0.0001 ) . repeated measures anovas revealed no main effect for condition on hr ( f1 , 22 = 1.9 , p = 0.187 , p = 0.08 ) , sbp ( f1 , 22 = 1.5 , p = 0.231 , p = 0.07 ) , dbp ( f1 , 22 = 0.0003 , p = 0.985 , p = 0.00001 ) , or breathing rate ( f1 , 21 = 0.9 , p = 0.356 , p = 0.04 ) suggesting that viewing condition did not influence the stress responses . a paired t test showed no difference was observed in the scores for the mental task between the conditions . however , a paired t test revealed that the second visit showed a significant improvement in the population mean score from 91.1 to 97.7 , ( t22 = 4.12 , p = 0.001 ) out of a possible 102 . a repeated measures anova determined an interaction effect on self - esteem ( f1 , 21 = 5.4 , p = 0.03 , p = 0.21 ) describing an enhanced self - esteem from baseline to poststress for the nature condition ( table 2 ) . conversely , the built condition showed deterioration in self - esteem from baseline to poststress values . repeated measures anova revealed a main effect of time on negative mood scores ( f1 , 21 = 7.3 , p = 0.013 , p = 0.26 ) describing a decrease in negative mood from baseline to poststress ( table 2 ) . self - esteem , quantified using rosenberg s self - esteem , low scores = low self - esteem , range 030 ; both positive and negative mood quantified using positive and negative affect scale ( panas ) , low scores = low positive or negative mood , range 1050 . significant interaction effect ( p < 0.05 ) . significant main effect for time , pre to post ( p < 0.05 ) . the majority of research exploring the impact of nature on ans mechanisms has included an exercise component which limits the conclusions drawn about the contribution of the nature component . as exercise has such positive effects on health parameters , it is often hard to isolate the effects solely from the nature component . therefore , this study looks at the nature contribution to ans function without complicating the effects by combining it with exercise . the main finding of this study was that hrv as a marker of ans function increased during stress recovery , if nature scenes were viewed prior to a stressor , compared to built scenes . this is the first study to suggest that simply viewing scenes of nature prior to a stressor enhances recovery of ans function poststressor . the interaction of nature and recovery from stress has been studied previously . however , previous research has assessed the restorative effects of viewing or interacting with nature during the actual recovery period following a stressor . for example , viewing video footage of nature scenes for 10 min directly after being exposed to a film of stressful images increased heart period ( i.e. , decreased hr ) , suggested to be due to enhanced parasympathetic system activity . viewing nature through a window during a 5 min rest period following cognitive tasks was also more effective at reducing hr . both authors postulate that these observations are a consequence of cognitive recovery or attention restoration occurring while nature scenes are viewed . the results of the present study suggest greater hrv during viewing nature scenes as contributed to by both sympathetic and parasympathetic systems . it is only during the recovery period that parasympathetic activity alone is greater in the nature views condition . therefore , viewing nature scenes may encourage future healthy stress responses and recovery patterns and could act as a vital tool in preventive health . research shows that a 5 min dose of nature offers the greatest increases in self - esteem and mood . physiologically , 5 min of viewing images of nature is known to increase hrv . however , it is unknown whether an additional 5 min would enhance the initial changes that occur in the first 5 min dose . within a laboratory setting , it appears that the second 5 min of exposure is less effective in inducing hrv changes . the strength of the current study is the use of 5 min segments for hrv analysis which is recommended in short - term analysis , i.e. , less than 24 h. furthermore , the division of exposure to nature images into 5 min segments enabled the observation of increased hrv as anticipated in an initial 5 min dose , thus supporting previous work . comparisons in the current study suggest an additional 5 min of exposure to nature does not enhance the greater hrv seen during the initial 5 min dose . in agreement with previous research , this finding suggests enhanced self - esteem associated with viewing nature shows robustness against exposure to a mild stress . in the current study , there was no change in mood associated with condition although this has been shown in previous studies . the changes in mood observed in these studies were measured using the profile of mood states ( poms ) . poms contains 5 subscales associated with negative mood and 1 subscale for positive mood . this restricts poms to predominantly reflect changes of negative mood rather than positive mood . in the current study , negative affect , measured using panas , showed negative mood improved irrespective of condition , therefore not replicating previous observations using poms the use of positive affect scores from panas was to ascertain if changes in positive mood occur that may not be so clearly identified using poms . positive mood did not differ between conditions nor did it change over time . in contrast , a meta - analysis of five studies did observe improvements in positive affect suggesting the manipulation in the current study was not strong enough to elicit positive changes . the use of panas in laboratory research of this nature might not be appropriate . previous literature lacks discussion as to the potential mechanisms behind observed changes in physiological function while viewing nature . one potential mechanism to explain alterations in physiological measures could be attributable to the restorative properties of the nature scenes . the concept of attention restoration occurring after exposure to nature has previously been demonstrated by way of improved performance in attention related cognitive tasks . viewing scenes of nature for 10 min , following a period of mentally fatiguing tasks , improved performance in a backward digit - span memory task . in the current study , there were no such observations of altered cognitive ability , i.e. , performance in the mental task , with different viewing conditions . this may be attributable to the lack of a mentally fatiguing task prior to the intervention , but the nature scenes acted as an effective buffer to ans function during recovery . to date , there are only a handful of studies that have measured or inferred changes in the ans associated with nature . we suggest that the different components of the environmental stimulus , e.g. , visual , cognitive , emotional , and restorative properties , induce changes in the regulation of different areas throughout the brain thus altering ans function . the findings of this study , alongside previous studies , suggest a top - down mechanism originating in higher centers of the brain . evidence for this was in part obtained from a study which utilized fmri while viewing urban scenes . the urban scenes caused increased activity in the amygdala compared to viewing nature scenes . this action is likely to cause alterations in ans control such as those seen in the current study , through changes in parasympathetic and sympathetic outputs . inhibition of the parasympathetic nervous system arises from the frontal cortex , and the pathways pass through the amygdala and then to the nucleus tractus solitarii and nucleus ambiguus . the prefrontal cortex is prominent in threat - avoidance situations , causing inhibition on hr via the vagus nerve . therefore , during periods of threat , parasympathetic activity is decreased ( increasing hr ) . the results of the current study suggest the absence of threat during nature viewing , without decreases in parasympathetic activity , while during built views overall variability decreased in the first 5 min . this interaction may be primarily due to alterations in both the frontal cortex and the amygdala . visual properties of an image may also play a role , as the composition of a picture can alter activity in the visual cortex . images of nature are less aversive and uncomfortable when examining their spectral properties compared to built images . indeed , recent research suggests that the primitive characteristic of color , in particular the greenness , of a nature image is associated with improved mood . through color perception and reduced impact on the visual system , images of nature may evoke lower activity in the amygdala and visual cortex culminating in increased parasympathetic activity as seen in the current study . it is unknown how long the physiological changes that nature evokes are maintained , but it will be vital to explore this further , especially if nature is to be considered as a therapy . in the current study , unlike previous studies , nature exposure was experienced 10 min before the stressor , not during the stressor or immediately following the stressor . the evidence from this study suggests that there does indeed appear to be a buffering effect of nature . a stronger stimulus , i.e. , within the environment itself , may prolong the buffering effect and also induce greater changes in cardiovascular measures . this may also be the case when nature is combined with exercise ( green exercise ) . exercising while viewing nature reduces bp in the 5 min following the exercise period in comparison to viewing built images . these effects again may be even greater following real exposure to nature . indeed , recent research suggests that adrenaline , noradrenaline , and bp still remain reduced in the evening following a daytime walk in a forest field . interestingly , forest walking increases natural killer cell activity for a period of 30 days in males and 7 days in females . the impact of individual beliefs on the regulation of emotions when viewing the different environments , and how this effects physiological modulations , is unknown and could pose a mediating factor to the effectiveness of viewing nature on improving stress recovery . in order to quantify individual relationships with nature , the nature relatedness scale could be used to indicate experience , beliefs , and contact with nature . complementary information could be gained by noting home postcode , and thus , the surrounding area could be assessed in terms of land usage to explore the potential impact it may have on participants perception of nature . the present study collected postcode data but does not have a sufficient population size to draw conclusions about all potential subsets . the majority of studies to date , including the present study , use extreme examples to depict natural and urban environments in order to examine the influence of nature . investigating a greater variety of environments ( including more urban green spaces ) would add population level validity to the results and account for individual landscape preferences . the restorative properties of nature ( images or within the location itself ) may evoke different psychological , cognitive , and physiological responses . further studies would benefit from the inclusion of a questionnaire to assess how restorative the scenes or places are perceived to be , as suggested by hartig , mang , and evans , in combination with physiological and psychological responses to these different images . the current study suggests that nature itself may evoke physiological responses , which may be in part driven by psychological reactions and restorative properties of nature . furthermore , the increase in parasympathetic activity in the recovery from a stressor may help to counteract a buildup of psychological stress and thus reduce the impact of stress on physical and mental health . this would likely occur by nature images encouraging a healthier stress recovery pattern . if nature increases autonomic recovery to stress and thus is an effective coping mechanism , this provides an argument for the need for more nearby nature . a green view through a workplace window , small pockets of greenspace in the home and workplace , and accessible local parks could be effective tools in altering ans control of the heart . the buffering effect of nature could have particular relevance for the workplace where it may be beneficial to utilize nature during the lunch break , prior to a stressful afternoon , to help enhance recovery of autonomic function .

objective : trocar - site incisional hernias and their complications are reported in 1% to 6% of patients . such hernias are attributed to the difficulty of applying standard suturing techniques to wound closure . we report our experience with a simple device , the deschamps ligature needle.methods:the deschamps needle has a handle and a tip ( sharp or blunt ) , with an opening to pass suture . the blunt tip is very effective for closing trocar sites . disposable needles are obviously sharp , but can bend on the needle holder and break in a deep , small incision . the deschamps needle is a rigid , noncutting instrument that can be forced through fascia and peritoneum ( around the surgeon 's fingertip ) avoiding loss of pneumoperitoneum . a full - thickness closure is accomplished . we perform closure under direct vision through the scope . tactile sense is provided by the surgeon 's finger . the last trocar site is closed in the same manner without the scope.results:we have used the deschamps needle since 1992 in all ( 1400 ) laparoscopic procedures . we close 10-mm and 5-mm trocar sites and have not observed wound dehiscence or hernias at these sites.conclusion:the deschamps needle is effective in preventing incisional hernias and wound dehiscence . it is cost - effective . disposable , single - use devices vary in price from $ 30 to $ 75 each . the deschamps needle is sold in italy at approximately $ 35 each . considering that it may have been in the trays of most operating rooms for years ( as in our case ) , and the number of procedures performed , we conclude that the real cost of this instrument is almost negligible

trocar - site incisional hernias and their complications are reported in 1% to 6% of patients . such hernias are attributed to the difficulty of applying standard suturing techniques to wound closure . the deschamps needle has a handle and a tip ( sharp or blunt ) , with an opening to pass suture . disposable needles are obviously sharp , but can bend on the needle holder and break in a deep , small incision . the deschamps needle is a rigid , noncutting instrument that can be forced through fascia and peritoneum ( around the surgeon 's fingertip ) avoiding loss of pneumoperitoneum . we have used the deschamps needle since 1992 in all ( 1400 ) laparoscopic procedures . we close 10-mm and 5-mm trocar sites and have not observed wound dehiscence or hernias at these sites . disposable , single - use devices vary in price from $ 30 to $ 75 each . the deschamps needle is sold in italy at approximately $ 35 each . considering that it may have been in the trays of most operating rooms for years ( as in our case ) , and the number of procedures performed , we conclude that the real cost of this instrument is almost negligible with the wide diffusion of laparoscopic surgery for many abdominal procedures , trocar site incisional hernias have become more frequent ( 1% to 6% ) , along with their related complications ( bowel or omentum incarceration and richter 's hernia ) . most complications occur with 10-mm trocars , but some have occurred at 5-mm trocar sites . the occurrence of these complications has been attributed to the difficulty in applying standard suturing techniques for wound closure . every surgeon who has performed traditional closure of fascia and peritoneum has found that closure of all layers in small , deep wounds is sometimes impossible , frequently unsafe , and never quick and easy . this is especially true in obese patients . immediately after the appearance of these reports of trocar site complications in gynecologic , urologic , and general surgery literature , many authors began to publish papers regarding original techniques and new devices to obviate the problem some have suggested that a foley catheter , spinal needle , hypodermic needle , or urologic instrument could solve the difficulties in closing trocar sites ; others developed new devices . the debut of numerous different approaches to closing trocar sites could have been controversial and disorientating to some , but elashry and associates in 1996 , published a perspective , randomized trial that compared many of these techniques . their final statement concluded that the preferred method of trocar site closure was one that utilized a new , disposable instrument . several companies have developed safer and smaller trocar tips while others have focused on developing new , disposable ( and frequently expensive ) wound closure instruments . at the beginning of our laparoscopic experience in 1990 we faced the problem of how to close trocar sites as did many of our colleagues . we struggled , we were frustrated , we doubted , and sometimes we renounced the techniques then available . although we experienced only two uncomplicated umbilical hernias at trocar sites in our initial period ( both following wound infections ) , we were very concerned about this problem . then one day the chief nurse of our operating room came with an old instrument from the forgotten storeroom . this instrument was a deschamps needle ( figure 1 ) , which was extensively used in the past for en masse ligature of pedicles . as shown in figure 2 , it has a handle and a tip , sharp or blunt , with a hole to pass a suture . it is probably present , and maybe forgotten , in most of the operating rooms in europe . the blunt type is very effective , in our experience , for the closure of trocar wounds . disposable needles although obviously sharp , can bend on the needle holder , and can sometimes break in a deep , small incision . the deschamps needle is a rigid , noncutting instrument that can be forced through the fascia and peritoneum in and out , turning ( bending ) around the finger tip of the surgeon . loss of pneumoperitoneum is thus avoided ( figures 3a , 3b , 3c , 3d ) and a full thickness closure of the trocar sites is accomplished . several techniques are available that are suitable for use in 5 mm incisions and in the deep fascial wounds of obese patients . the closure is performed under direct vision through the scope , and the tactile feedback provided by the surgeon 's finger allows quick , safe passage of the needle . the last trocar site , after removing the scope , is closed in the same manner , facilitated by maintenance of the pneumoperitoneum . no omentum or bowel is included in the suture , because passage of the instrument is felt by the surgeon 's finger . deschamps needle passes through the fascia and peritoneum in and out around the finger tip of the surgeon . since 1992 , we have used the deschamps needle in all of our laparoscopic procedures , even when only 5-mm trocars were used . to date , we have had no cases of wound dehiscence or herniation in approximately 1400 procedures . concerning financial matters , disposable , single - use devices have a price that varies from $ 30 to $ 75 per unit , and reusable instruments , frequently sold in a set of various sizes , can cost up to $ 2,500 . the deschamps needle is sold in italy at an average cost of $ 35 each . when one considers that the deschamps needle has been on the trays of most operating rooms for many years ( as in our case ) and when one considers the total number of procedures already performed , the real impact of this instrument on surgical costs is inconsequential . we conclude that the deschamps needle is a safe , cost - effective , readily available device to accurately close fascial defects that arise from the use of trocars and cannulas in laparoendoscopic surgery .

the aim of the present study was to assess the effects of an anticholinesterase agent , pyridostigmine bromide ( pyrido ) , on experimental chronic chagas heart disease in mice . to this end , male c57bl/6j mice noninfected ( control : con ) or chronically infected ( 5 months ) with trypanosoma cruzi ( chagasic : chg ) were treated or not ( nt ) with pyrido for one month . at the end of this period , electrocardiogram ( ecg ) ; cardiac autonomic function ; heart histopathology ; serum cytokines ; and the presence of blood and tissue parasites by means of immunohistochemistry and pcr were assessed . in nt - chg mice , significant changes in the electrocardiographic , autonomic , and cardiac histopathological profiles were observed confirming a chronic inflammatory response . treatment with pyrido in chagasic mice caused a significant reduction of myocardial inflammatory infiltration , fibrosis , and hypertrophy , which was accompanied by a decrease in serum levels of ifn with no change in il-10 levels , suggesting a shift of immune response toward an anti - inflammatory profile . lower nondifferent numbers of parasite dna copies were observed in both treated and nontreated chagasic mice . in conclusion , our findings confirm the marked neuroimmunomodulatory role played by the parasympathetic autonomic nervous system in the evolution of the inflammatory - immune response to t. cruzi during experimental chronic chagas heart disease in mice .

chagas disease , also known as american trypanosomiasis , an endemic parasitic disease caused by a flagellate protozoan ( trypanosoma cruzi ) , is highly prevalent throughout latin america . its major clinical manifestation is the chronic chagasic cardiomyopathy ( ccc ) , which affects 1/3 of the chronically infected patients and may present severe symptoms and signs , such as congestive heart failure , thromboembolic phenomena , cardiac arrhythmias , and sudden death [ 14 ] . pathological processes in the heart include mononuclear inflammatory infiltration , focal myocarditis , epicarditis , and neuroganglionitis , associated with variable focal fibrosis and a paucity of parasites , which is poorly correlated with myocardial inflammatory infiltration [ 2 , 4 ] . the pathogenesis of ccc includes the balance between parasite invasiveness and the host immune response , particularly the th1/th2 balance , which affects the resistance / susceptibility to t. cruzi infection [ 57 ] . even though an imbalanced , excessive production of th1 proinflammatory cytokines is critical to control of the parasite levels in blood and tissue , this type of response can also be capable of destroying functional cardiomyocytes and intracardiac autonomic neurons [ 2 , 4 , 9 ] this autonomic denervation , involving mainly parasympathetic postganglionic neurons causes a marked cardiac autonomic dysfunction [ 2 , 4 , 911 ] , which may be involved in initiating life - threatening cardiac arrhythmias and sudden death [ 2 , 4 ] . curiously , this vagal parasympathetic autonomic dysfunction is strongly associated with inflammatory infiltration and immune activation not only in chagas heart disease but also in other types of cardiopathy [ 1215 ] . although these data support a direct role of the immune system in mediating both cardiac and autonomic nervous disturbances , recent data indicate that changes in the autonomic nervous system also affect the pattern of immune response and inflammation in several cardiac and noncardiac disease states [ 1517 ] , including chagas disease . both the sympathetic and parasympathetic branches of the autonomic nervous system can exert substantial modulatory effects on the immune response , mainly by inhibiting the th1 response profile [ 17 , 1921 ] . this new evidence also indicates that cardiac sympathetic and particularly parasympathetic autonomic denervation / dysfunction may also contribute to an increased inflammatory response and possibly to enhanced parasite elimination . data from our laboratory collected in the context of acute chagas disease in mice has recently confirmed that cardiac autonomic denervation / dysfunction might contribute to increased inflammation . taking into account the concepts described above , new therapies based on manipulations of vagal neuroimmunomodulation of the heart may be beneficial for treating heart diseases in general and chronic chagasic cardiomyopathy in particular [ 17 , 23 ] . for example , therapeutic approaches for increasing cardiac vagal function , thereby potentiating or stimulating the vagal anti - inflammatory reflex , might have a positive impact on chronic chagasic cardiomyopathy . in fact , for other cardiopathies , these strategies , in both an experimental and clinical context , including chronic electric vagal stimulation or pharmacological potentiation , have successfully improved cardiac outcomes . pyridostigmine bromide , an anticholinesterasic agent that has been used for many years to treat myasthenia gravis , exhibits protective cardiovascular effects during short - term administration that lead to a reduction of cardiovascular risk markers and an improvement of autonomic dysfunction [ 26 , 26 , 27 , 2729 ] . by potentiating vagal parasympathetic function , this compound is thought to provoke sinus bradycardia , to reduce av nodal conduction and the refractory period of action potentials and to increase the cardiac excitation threshold , among other direct cardiac effects . it is also believed that pyridostigmine bromide , via potentiation of the cholinergic anti - inflammatory pathway , causes a reduction in myocardial inflammation and fibrosis associated with an improvement in cardiac hypertrophy and remodeling . however , there have been no reports regarding the effects of pyridostigmine bromide on the chronic phase of chagas heart disease . therefore , the main aim of the present study was to evaluate the effects of cholinergic potentiation with the anticholinesterase agent pyridostigmine bromide on electrocardiographical , cardiac autonomic , histological ( inflammatory infiltration and fibrosis ) , immunological , and parasitological parameters in c57bl/6j mice infected with the romildo strain of trypanosoma cruzi during the chronic phase of chagas heart disease . all experiments were performed on wild - type c57bl/6j mice obtained from the animal facility of the department of physiology of triangulo mineiro federal university , uberaba , mg . all animals were males , weighed 2030 g , and were maintained in the animal facility of the department of physiology at the triangulo mineiro federal university on a rodent diet ( nuvilab cr1 , nuvital nutrientes ltda , curitiba , pr , brazil ) and were given water ad libitum until the beginning of the experimental protocols . all the experiments were carried out according to the principles of laboratory animal care formulated by the national society for medical research , england , and the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication number 85 - 23 , revised in 1996 ) . all procedures were also submitted to and approved by the commission for ethics in the use of animals in research of the triangulo mineiro federal university . to induce experimental chagas disease , mice were intraperitoneally inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi . an additional set of animals matched for gender and weight received intraperitoneal injections of the vehicle , and these were designated as the control noninfected group . after inoculation , all of the animals were observed at least twice daily to monitor their general state and to assess mortality during the acute phase . on the 12th day after inoculation , levels of parasitemia were measured by the microhematocrit method for the peripheral tail blood of all inoculated animals , according to brenner 's technique , to confirm infection . all subsequent surgical procedures and experimental protocols were performed at the 5th and 6th months of infection , during the chronic phase of infection , which is characterized by light tissue invasion and low mortality . the experimental groups were divided according to the presence of chagas disease and the administration of pyridostigmine bromide during the month from the 5th to the 6th month of observation ( chronic phase ) , as described below . the following groups were investigated : group i con - nt : wild - type c57bl/6j mice were injected with vehicle as a control ( con ) , not treated ( nt ) with pyridostigmine bromide and evaluated after six months at the end of the observation period ; group ii con - pyrido : wild - type c57bl/6j were injected with vehicle as a control ( con ) and treated with 30 mg / kg pyridostigmine bromide ( pyrido ) , an anticholinesterase agent , dissolved in tap water , for 30 days from the 5th to the 6th month of observation ; group iii chg - nt : wild - type c57bl/6j mice were inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi ( chg ) , not treated ( nt ) with pyridostigmine bromide and evaluated after six months at the end of the observation period ; group iv chg - pyrido : wild - type c57bl/6j mice were inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi ( chg ) and treated with 30 mg / kg pyridostigmine bromide ( pyrido ) , an anticholinesterase agent , dissolved in tap water , for 30 days from the 5th to the 6th month of observation . the solution drinking volume was monitored daily , and the pyridostigmine bromide concentration ( approximately 0.084 mg / ml ) was adjusted daily according to the drinking volume to maintain a daily mean ingested dose of 30 mg / kg . immediately prior to inoculation ( ecg1 ) , at the 5th ( ecg2 , immediately prior to the pyridostigmine bromide treatment ) and at the 6th months of observation ( ecg3 , at the end of the experimental protocol , prior to the surgical procedures ) , all of the animals were submitted to a conventional ecg study under tribromoethanol ( 250 mg / kg , i.p . ) anesthesia . needle electrodes were placed under the skin to record the conventional bipolar limb leads ( i , ii , and iii ) , the unipolar limb leads ( avr , avl , and avf ) , and the unipolar precordial ( chest ) leads ( va : the needle was placed immediately to the right of the sternum in the 4th intercostal space ; vb : the needle was placed just to the left of the sternum in the 4th intercostal space ; and vc ; the needle was positioned in the 5th intercostal space at the midaxillary line ) . to avoid errors in the positioning of the leads , the ecg was recorded using an ecg amplifier ( model 8811a , hewlett - packard med . ma , usa ) coupled to a 12-bit analogue - to - digital interface ( di-720-usb , dataq instruments , inc . , akron , oh , usa ) at the sampling rate of 3 khz on an ibm personal computer with the analysis performed by windaq - pro+ software ( dataq instruments , inc . , the intervals and wavelengths ( ms ) were calculated automatically using customized software following wave identification and cursor placement . the ecg tracings were consistently analyzed by the same individual , who was blinded to the study protocol . the following ecg parameters were examined : ( 1 ) rr interval ( rri ) , ( 2 ) p wave duration ( pd ) , ( 3 ) pr interval ( pr ) , ( 4 ) qrs duration ( qrsd ) , ( 5 ) qt interval ( qt ) , and ( 6 ) corrected qt interval ( cqt , defined as the qt interval corrected for heart rate using bazett 's equation , where the corrected qtc was equal to qt ( in s)/rr ( in s ) ) . in contrast to humans , the t wave in small rodents is not well characterized and appears as a shoulder of the qrs complex . accordingly , to measure the qt interval , we used the apex of the t wave , which can be determined with high accuracy . the ecg parameters were determined from each lead and were averaged . additionally , the presence of cardiac arrhythmias and atrioventricular or intraventricular blockades , among other alterations , was analyzed by visual inspection of ecg tracings by a blinded ecg specialist . after the third ecg recording , at the 6th month of observation , the animals were reanesthetized with tribromoethanol ( 250 mg / kg , i.p . ) , and a pair of stainless steel electrodes were implanted inside the subcutaneous tissue to collect chronic recordings of conventional bipolar limb ecg lead ii . the animals were also cannulated with polyethylene tubing placed in the jugular vein for drug administration . after the surgical procedures , the animals were left to recover in individual cages for at least 4872 h. after 4872 h of surgical recovery and in the absence of anesthesia , the electrodes were connected to an ecg amplifier ( model 8811a , hewlett packard , waltham , ma , usa ) , and the baseline ecg was sampled continuously ( 3 khz ) for a period of 30 minutes with a personal computer ( ibm / pc ) equipped with a 12-bit analogue - to - digital interface ( di-720-usb , dataq instruments , inc . , all animals were always recorded between 8:00 am and 5:00 pm in a quiet condition and in a freely moving state . the time series of rr intervals derived from these chronic ecg recordings were used to study the cardiac autonomic modulation in heart rate variability . from the baseline chronic ecg recordings ( 30 minutes ) , the rr interval time series were derived automatically by the detection of the r wave peaks using customized linear analysis software , which was kindly provided by dr . the time series of the rr intervals were divided into contiguous segments of 300 beats overlapping by half ( welch protocol ) . after calculating the mean and variance for each segment , a model - based autoregressive spectral analysis was performed , as described elsewhere [ 31 , 32 ] . briefly , a model of the oscillatory components present in the stationary segments of the beat - to - beat time series of the rr intervals was calculated based on the levinson - durbin recursion , and the order for the model was chosen according to akaike 's criterion . this procedure allows for the automatic quantification of the center frequency and power of each relevant oscillatory component in the time series . the oscillatory components were labeled as very low frequency ( vlf ) , low frequency ( lf ) , or high frequency ( hf ) when the central frequencies were within the bands of 0.010.10 hz , 0.101.00 hz , or 1.005.00 hz , respectively . the power of the lf and hf components of the heart rate variability was also expressed in normalized units , which were obtained by calculating the percentage of the lf and hf variability with respect to the total power after subtracting the power of the vlf component ( frequencies < 0.10 hz ) . the normalization procedure tends to minimize the effect of changes in the total power on the absolute values of the lf and hf variabilities [ 31 , 32 ] . after 30 minutes of baseline chronic ecg recording , half of the animals were given an intravenous injection of atropine sulfate ( 1 mg / kg , i.v . ) followed by an injection of propranolol ( 1 mg / kg , i.v . ) 15 minutes later , whereas the other half received injections in the reverse sequence ( propranolol followed by atropine sulfate ) . this procedure allowed for the quantification of the cardiac parasympathetic and sympathetic autonomic effects , in the former and latter group , respectively , measured as the differences between heart rate ( hr ) after atropine and baseline hr ( parasympathetic effect ) or as the differences between hr after propranolol and baseline hr ( sympathetic effect ) , as well as the measurement of intrinsic pacemaker heart rate ( ihr ) , quantified as the heart rate after the double blockade with atropine sulfate followed by propranolol or propranolol followed by atropine sulfate . at the end of the experimental protocol , all animals were euthanized with an excess dose of sodium thiopental ( 100 mg / kg , resp . , i.p . ) , and the chest cavity was then opened to remove the heart for histopathological analysis . to evaluate the extent of inflammatory infiltration , tissue damage , fibrosis , and parasite nests , excised hearts from all animals were cleaned in 0.9% saline solution and fixed in phosphate - buffered 10% formalin solution for 48 h. after embedding the samples in paraffin , five 57 m thick longitudinal ( four - chamber ) sections of the hearts were stained with hematoxylin - eosin and analyzed using an upright light microscope ( axiolab , carl zeiss inc . the inflammatory infiltration and parasite nests were characterized using a semiquantitative approach and the scoring system described by chapadeiro et al . . a global myocardial inflammation score was defined for each animal as the sum of the scores from different regions of the heart . to quantify the fibrotic area in the myocardium , contiguous longitudinal sections of the hearts were stained with picrosirius red , which binds the collagen present in the tissue matrix , and the slides , germany ) . to enhance the visualization of parasite nests or antigens in cardiac tissue , an immunohistochemistry technique based on the detection of the diaminobenzidine ( dab- ) the chromogen was generated by a secondary antibody labeled with peroxidase , which binds to a primary antibody against t. cruzi antigens . after antibody labeling , peroxidase reaction , and costaining with hematoxylin , slides were analyzed with an upright light microscope ( axiolab , carl zeiss inc . serum samples for each animal were collected by means of cardiac puncture to perform cytokine profiling ( ifn- , tnf- , il-2 , il-4 , il-5 , and il-10 ) via elisa or the cytometric bead array ( cba ) technique serum concentrations of ifn- and il-10 were measured by elisa using pairs of monoclonal antibodies in accordance with the manufacturer 's specifications ( bd pharmingen ) . briefly , high - affinity 96-well plates ( nunc , roskilde , denmark ) were sensitized with cytokine - specific monoclonal antibodies followed by blocking with pbs containing 2% bsa ( sigma ) . the sera and recombinant cytokines were then added , and the plates were incubated for 4 h at room temperature . the plates were washed and incubated with 1 g / ml biotinylated anticytokine monoclonal antibody at 37c for 2 h followed by washing and incubation with alkaline phosphatase - conjugated streptavidin at 37c for 2 h. the reaction was developed using disodium - p - nitrophenyl phosphate ( sigma ) in diethanolamine buffer . absorbance was measured at 405 nm in a microplate reader ( bio - rad , 2550 reader eia , ca , usa ) . the cytokine concentration was calculated using a linear regression analysis of the absorbance values obtained for the recombinant cytokines , and it was expressed as pg / ml . the sensitivity of the tests ranged from 2 to 20 pg / ml . measurement of the cytokines tnf- and il-2 ( th1 cytokines ) and il-4 and il-5 ( th2 cytokines ) in serum samples was performed using the cytometric bead array ( cba ) kit ( bd biosciences , usa ) according to the manufacturer 's instructions . briefly , 50 l of bead populations with discrete fluorescence intensities and coated with cytokine - specific capture antibodies was added to 50 l of mice sera and 50 l of phycoerythrin - conjugated anti - mouse th1/th2 cytokine antibodies . simultaneously , standards for each cytokine ( 05000 pg / ml ) were mixed with cytokine capture beads and the phycoerythrin - conjugated reagent . the vortexed mixtures were incubated for 3 h. beads were washed and analyzed using flow cytometry ( facscalibur , bd biosciences , usa ) . the quantity ( pg / ml ) of each cytokine was calculated using cellquest software ( bd biosciences , usa ) . the lower limit of detection ranged from 1 to 2.1 pg / ml for different cytokines . for parasite dna isolation from the blood , total blood samples of mice were collected in tubes containing 6 m guanidine hcl with 0.2 m edta ( ph 8) ( v / v ) . dna was extracted using the phenol - chloroform - isoamyl alcohol method according to macedo et al . . for parasite dna isolation from the heart , half of the hearts excised at the end of the experimental protocol were homogenized , and dna was extracted by the alkaline lysis method . pcr specific for t. cruzi was performed according to wincker et al . using the following primers to amplify a fragment of 330 bp : 121 ( 5-aaa taa tgt acg gg(g / t ) gag atg cat ga-3 ) and 122 ( 5-ggt tcg att ggg gtt ggt gta ata ta-3 ) from a nonvariant region of the kinetoplast dna minicircles of t. cruzi . the cycling conditions were as follows : 95c for 5 minutes followed by 35 cycles at 95c for 1 minute and 65c for 1 minute . the products of the reaction were revealed by electrophoresis in a 6.0% polyacrylamide gel and stained with silver nitrate , which was photographed with a digital camera . all numerical data are expressed as the means ( s.e.m . ) , whereas the semiquantitative data from the histological examinations are expressed as the medians and the 25th and 75th percentiles . according to the normality and variance homogeneity of the distribution , a parametric statistic , such as two - way anova followed by tukey 's multiple comparison test , or a nonparametric test , such as the mann - whitney test , parasite detection in peripheral tail blood samples by the microhematocrit technique performed on the 12th day after infection was positive for all t. cruzi - inoculated animals and negative for all control noninfected mice . electrocardiographical parameters measured in the first ecg recording ( ecg1 ) at the beginning of the experimental protocol before t. cruzi inoculation and treatment did not show any significant difference among all experimental groups , as expected . however , after five months of infection and before the pyridostigmine bromide treatment , chagasic animals ( from chg - nt and chg - pyrido groups ) presented significant elongations of pd , qrsd , qt , and cqt ( table 1 ) , indicating a global functional disturbance of heart . interestingly , as shown in table 2 , after one month of the pyridostigmine bromide treatment and six months of infection , chg - pyrido mice presented a significant reduction in pd ( an atrial parameter ) and cqt ( a ventricular parameter ) , suggesting an improvement in the electrical function of the heart . in contrast , in chg - nt mice , most of the ecg parameters were significantly different compared to con - nt mice . it is worth noting that , in the con - pyrido group , a significant increase in pr was observed , as expected , because pyridostigmine bromide might be increasing vagal neural transmission in the atrioventricular node , with a consequent reduction in the conduction velocity of the action potential . these results suggest that pyridostigmine bromide treatment can be effective in improving electrical disorders induced by chronic chagasic cardiomyopathy . the results of heart rate variability analysis in time- ( variance ) and frequency - domain ( spectral ) parameters are shown in table 3 . a significant reduction in the absolute values of total variability ( variance ) and the spectral components vlf , lf , and hf was observed in chg - nt mice compared to con - nt and con - pyrido mice . the lf and hf components expressed in normalized units and the lf / hf ratio were not different from con - nt mice . in contrast , chagasic mice treated with pyridostigmine bromide ( the chg - pyrido group ) presented values of variance and vlf and hf spectral components that were significantly higher compared to chg - nt mice , suggesting an improvement of some autonomic parameters that changed during chronic chagasic cardiomyopathy ( table 3 ) . the autonomic dysfunction observed in the heart rate variability analysis of chg - nt mice and its improvement in chg - pyrido mice was confirmed by pharmacological autonomic blockade with atropine sulfate or propranolol . the use of these autonomic blockers showed that vagal parasympathetic effects significantly decreased without a change in sympathetic effects in chg - nt mice compared with con - nt mice ( figure 1 ) . chagasic mice treated with pyridostigmine bromide showed values of vagal parasympathetic effects that were significantly higher compared to chg - nt mice and similar to those found in con - nt mice ( figure 1 ) . additionally , noninfected control mice treated with pyridostigmine bromide showed vagal parasympathetic effects that markedly increased without any changes in sympathetic effects compared with con - nt mice ( figure 1 ) . no changes in the intrinsic pacemaker heart rate ( ihr ) were found in any of the experimental groups after the six - month observation period . the relative cardiac weight of chg - nt mice was significantly higher than that observed in the other groups ( figure 2 ) , indicating that cardiac hypertrophy was induced by chagas disease in mice and that pyridostigmine bromide treatment was able to reverse or impair the development of cardiac hypertrophy in chagasic mice . a semiquantitative examination of cardiac inflammatory infiltration indicated discrete to mild diffuse myocarditis ( average inflammatory score was equal to 0.5 , p25% = 0.5 , and p75% = 0.5 , p < 0.0001 versus con - nt ) , which was observed in the atria ( figures 3(g ) and 3(h ) ) , the ventricles ( figures 3(e ) and 3(f ) ) , and the neural ganglia ( figure 3(g ) ) in the chg - nt mice in comparison to normal con - nt mice ( figures 3(a ) , 3(b ) , 3(c ) , and 3(d ) ) , which presented no inflammatory infiltration ( average inflammatory score was equal to 0.0 , p25% = 0.0 and p75% = 0.0 ) . in chg - pyrido mice , reduced inflammatory infiltration , which was categorized as very discrete to discrete ( average inflammatory score was equal to 0.125 , p25% = 0.0 , and p75% = 0.25 , p < 0.001 versus chg - nt ) , was observed in the atria , the ventricles ( figures 3(i ) , 3(j ) , 3(k ) , and 3(l ) ) , and the neural ganglia . as expected in this mouse model of chronic chagas disease , no amastigote nests in the myocardium were found in either chg - nt or chg - pyrido mice . in different sections of the heart stained by picrosirius red , the morphometry of fibrosis revealed a marked and diffuse increase in the fibrotic area in both absolute ( m ) and relative ( % ) values in the chg - nt group compared to chg - nt mice ( figure 4 ) . interestingly , chg - pyrido mice presented significantly reduced fibrotic areas in all cardiac chambers ( figure 4 ) compared to chg - nt mice . despite these reduced values , the fibrotic areas were still larger than those found in con - nt mice ( figure 4 ) . the serum levels of the cytokines il-2 , il-4 , il-5 , and tnf- , measured with the cba technique , did not reveal any change in any of the experimental groups . in fact , the serum levels of il-2 and il-4 were undetectable , with values near 0 pg / ml . however , using the elisa technique , serum levels of ifn- were significantly higher in chg - nt mice compared to chg - pyrido mice ( p < 0.05 ) . serum levels of il-10 , measured with elisa technique , were significantly higher in both chg - nt and chg - pyrido mice compared to con - nt mice ( p < 0.05 ) . no differences were found in the serum il-10 levels between chg - nt and chg - pyrido mice ( figure 5 ) . the immunohistochemical technique using peroxidase to label t. cruzi parasite nests or antigens revealed a complete absence of parasites in control noninfected mice , whereas t. cruzi labeling was positive for 62.50% of chg - nt and 81.81% of chg - pyrido mice ( p = 0.603 , not significant using the fisher exact test ) . the parasite antigens were diffusely distributed throughout the heart tissue ( figure 6 ) . using a conventional pcr method to detect parasite dna in blood and heart during the chronic phase of chagas disease in mice , a complete absence of parasite dna in noninfected control mice was observed , whereas t. cruzi dna was found in 16.67% and 42.9% ( p = 0.559 ) in the blood of chg - nt and chg - pyrido mice , respectively , and in 72.73% and 86.67% ( p = 0.614 ) of heart samples from chg - nt and chg - pyrido mice , respectively . to our knowledge , the present study is the first to analyze the effects of the deliberate potentiation of cholinergic signaling using pyridostigmine bromide , an anticholinesterase agent , during the chronic phase of experimental chagas disease in mice . furthermore , this study evaluated the effects of pyridostigmine bromide treatment on electrocardiographic , autonomic , histopathological , immunoinflammatory , and parasitological parameters of chagas disease . the present study is , to our knowledge , the first to demonstrate autonomic dysfunction induced by chronic chagas disease in an experimental mouse model . this dysfunction was characterized by a marked reduction in heart rate variability , with reduced variance ( time - domain parameter ) and the vlf , lf , and hf components ( frequency - domain parameters ) of heart rate variability , as well as a concurrent reduction in the cardiac vagal effect , without an apparent change in sympathetic effects , as measured by pharmacological blockade in chronic control chagasic animals . only morphological reports describing the ganglia and nerve lesions in chagasic mice this finding follows our previous report of a similar autonomic dysfunction in mice with acute chagas disease . this autonomic dysfunction was accompanied by electrocardiographic changes associated with mild diffuse inflammatory infiltration of mononuclear cells as well as fibrosis and hypertrophy in the atrial and ventricular myocardium and the elevation of ifn- and il-10 in the blood serum of wild - type and untreated chagasic c57bl/6j mice , which confirms the presence of chronic myocarditis six months following infection with t. cruzi . this increase in serum levels of ifn- and il-10 in chronic chagasic mice matches similar previous reports in human beings [ 3739 ] . additionally , parasite antigens and dna were detected by immunohistochemical labeling in the heart and by pcr in blood and heart , respectively , reinforcing the role played by parasites in inducing and/or maintaining chronic infection . because our findings are consistent with previous observations in hamsters , rabbits , dogs , and humans during the chronic phase of chagas disease [ 10 , 40 ] , the mouse model used here appears to represent a suitable tool for future experimental studies in chagas heart disease . during the chronic phase of chagas heart disease , this report revealed a significant increase in cardiac vagal parasympathetic autonomic modulation associated with a significant reduction of inflammatory infiltration in the myocardium of infected mice treated with pyridostigmine bromide during the last month of a six - month observation period . because pyridostigmine bromide is an anticholinesterasic agent , which increases the bioavailability of acetylcholine in the synaptic varicosities , the increase in cardiac vagal parasympathetic autonomic function , demonstrated by the higher heart rate variability and cardiac vagal effect , was expected and was verified after treatment . the anti - inflammatory effect of pyridostigmine bromide was associated with a marked reduction of myocardial fibrosis and hypertrophy . serum levels of ifn- also decreased , whereas tna- trended to decrease , both of which are th1 proinflammatory cytokines , without any change in the augmented serum levels of il-10 . the combined analysis of these results suggests that pyridostigmine bromide may act on the heart , at least partially increasing the vagal parasympathetic activity , via the accumulation of acetylcholine in the myocardium . this accumulation was confirmed by improvements in heart rate variability and vagal parasympathetic effect , which , acting on immune cells , may exert immunomodulatory effects , thereby shifting the immune response toward the predominance of an anti - inflammatory response . these new findings in the chronic chagas disease context reinforce the idea of an intimate interplay between the immune and autonomic nervous systems and the potential use of parasympathetic stimulation to treat chagasic myocarditis [ 18 , 23 ] . additionally , the improvement of cardiac inflammation , fibrosis , and hypertrophy in pyridostigmine bromide - treated chronic chagasic animals suggests that the decrease of vagal autonomic function due to ganglionic neuronal lesions and denervation , which occur precociously during the acute phase , may play an important immunomodulatory role in the increased th1 immune response and inflammatory infiltration , as verified during the chronic phase of the disease . even though our results support the immunomodulatory role played by the cholinergic vagal parasympathetic nervous system in the heart , we can not rule out a possible role of pyridostigmine bromide in increasing local acetylcholine levels via its release from nonneural local structures , such as endothelial cells , cholinergic lymphocytes , and cardiomyocytes . in fact , these nonneural sources of acetylcholine , potentiated by pyridostigmine bromide treatment , may explain the substantial improvement in ecg abnormalities , inflammation , fibrosis , and hypertrophy observed in both ventricles of the heart , which are poorly innervated by vagal parasympathetic fibers . data from the immunohistochemical labeling of t. cruzi antigens and t. cruzi dna detection by pcr showed that parasites are present in the heart tissue and blood of the chagasic mice six months after infection , confirming previous reports [ 38 , 43 ] . the number of animals positive for parasite antigens or dna did not differ between pyridostigmine bromide - treated and nontreated chagasic animals . the persistence of the parasite in the chronic phase , even at a very low level , seems to play an important role in the pathogenesis of chronic chagas disease because the low parasite load may continuously stimulate an immune response [ 38 , 43 ] . although antigens or dna from t. cruzi were detected in the present study , no parasite pseudocysts or amastigote forms were observed in the heart tissue in either treated or nontreated chagasic mice . this lack of entire parasites at the site of cardiac lesions suggests the possibility of a remote niche for the parasites , such as smooth muscle cells , adipocytes , or skeletal muscle cells . in conclusion , our results support the notion that autonomic dysfunction is a primary cause of the pro-/anti - inflammatory imbalance observed in inflammatory diseases , favoring the shift of immune response toward the predominance of proinflammatory response , thereby contributing to the pathogenesis of these diseases in general and chagas heart disease in particular . additionally , our findings showed the potential beneficial effects of anticholinesterasic agents , particularly pyridostigmine bromide , in increasing cardiac autonomic modulation and reducing inflammatory response to the heart .

diffuse normolipemic plane xanthoma ( dnpx ) was first described by altman and winkelmann in 1962 . it is a rare and non - inherited form of xanthomatosis . clinically , the dermatosis is characterized by the presence of symmetric yellowish - orange plaques that favor the neck , upper trunk , flexural folds and periorbital region . it has been recognized to be associated with hematological diseases , especially with multiple myeloma and monoclonal gammopathy . we present a patient with diffuse plane xanthoma , normal lipid level , and monoclonal gammopathy .

an 85-year - old man presented with an 8-month history of slowly increasing diffuse yellow skin lesions on the torso and upper arms . he had been unaware of the discoloration until his internist noted it and referred him to dermatology . the patient had been followed by a hematologist for paraproteinemia that had not been treated . the examination showed well - demarcated yellow patches and plaques covering large portions of his upper trunk with some islands of sparing ( figure 1 , 2 ) . the dermoscopic evaluation showed a reticular pattern of yellow amorphous homogeneous structures with overlying branched and linear vessels ( figure 3 ) . a skin biopsy showed scattered foamy histiocytes within the reticular dermis , which were consistent with a plane xanthoma ( figures 4 , 5 , 6 ) . a complete blood count , comprehensive metabolic panel , fasting lipid panel were within normal range . diffuse normolipemic plane xanthoma ( dnpx ) was first described by altman and winkelmann in 1962 . dnpx is characterized by xanthelasma palpebrarum ; diffuse plane xanthoma of the head , neck , trunk , and extremities ; and normal plasma lipid levels . xanthelasma typically appears first , followed by involvement of the lateral parts of the neck and upper trunk . clinically , the dermatosis is characterized by the presence of symmetric yellowish - orange plaques that favor the neck , upper trunk , flexural folds and periorbital region . histologically , foam cells ( macrophages that have engulfed lipid droplets ) and variable numbers of touton giant cells , lymphocytes , and foamy histiocytes can be seen . while not all cutaneous xanthomas are associated with systemic diseases , dnpx has been associated with systemic diseases , particularly multiple myeloma and monoclonal gammopathy . however , other malignant hematological or lymphoproliferative disorders associated with dnpx include acute monoblastic leukemia , chronic myelomonocytic leukemia , chronic myloid leukemia , chronic lymphatic leukemia , non - hodgkin s lymphoma , adult t - cell lymphoma / leukemia , sezary syndrome , waldenstrom s macroglobulinemia , cryoglobulineimia and castleman s disease [ 2,3,512 ] . the pathogenesis of dnpx has not been fully elucidated , however , in gammopathy - associated dnpx , monoclonal igg is thought to bind to circulating ldl , rendering the antibody ldl complex more susceptible to phagocytosis by macrophages . dnpx can precede such disorders by several years ; therefore close follow - up with periodic laboratory tests for myeloproliferative disorders should be performed . the erbium : yag laser has been used successfully to treat facial xanthomas in one patient . in this patient , currently there are no associated systemic symptoms . , this case shows that dermatological lesions can be the first manifestation of important hematological diseases and so physicians should be familiarized with this rare entity .

objective : the objective of this study was to assess the effectiveness of different types of fixation in the enhancement of posttraumatic inferior alveolar nerve ( ian ) recovery in displaced mandibular angle fracture and to establish.patients and methods : thirty patients of displaced mandibular angle fracture were treated with preangulated plate and three - dimensional ( 3d ) matrix plate in two groups and were observed during follow - up at 04,06 and 12 weeks along with other parameters.results:fifteen patients were treated with preangulated plate and 15 patients with 3d matrix miniplate . there was early nerve recovery in group a than group b , with residual paresthesia 20% in group a and 26.6% in group b at the end of 12-week follow-up.conclusion:the displaced mandibular angle fracture with posttraumatic ian paresthesia treated with preangulated plate has shown evidence of early nerve recovery than those fractures were treated with matrix miniplate . the fracture fragments displaced more than 9 mm have shown poor nerve recovery in both groups .

angle region fracture is the most common fracture in developing countries , accounting 30% of all fractures . mandibular angle fracture line involves junction of ramus and body of the mandible to the third molar region and traverses through the inferior alveolar canal to reach the inferior border . . the traditional biomechanical model comprising tension at the superior border and compression at the lower border has been challenged , and it has been found that these tension compression zones reverse as the load position moves posteriorly . fracture involving inferior alveolar canal - associated nerve paresthesia may be transient or permanent type . thurmuller et al . reviewed the literature on nerve injuries in facial trauma and reported the overall incidence of inferior alveolar nerve ( ian ) injury in mandibular fractures with associated paresthesia to be 5.758.5% after injury without treatment and fractures in the mandibular body and angle region to be 4658.5% . the reported postoperative incidence of ian injury in fractures of the nerve - bearing area of the mandible ( angle , body ) was 76.191.3% . various factors such as fracture displacement , type of fixation , site , and type of fracture have been reported to influence the incidence of ian injury in mandibular fractures . the actual amount of fracture displacement due to trauma is not accurately estimated , and radiographs only demonstrated the posttrauma tissue recoil bony fragments position . this is not clear why some minimally displaced fractures resulted in permanent numbness . in blunt trauma involving the thorax , it is well known that major bony and visceral displacements occur in high - velocity impact , with recoil back to their original position soon after , nevertheless , resulting in injury . there is not much published literature supporting that greater fracture displacement can cause more nerve injury or vice versa . in this study , thirty patients of displaced mandibular angle fracture which are traversed by mandibular canal were included in the study . the university ethical board clearance was taken . patients who were reported at the oral and maxillofacial ( omf ) unit of trauma center and the outpatient department of omf surgery department of king george 's medical university were included in the study after their informed consent . in addition to demographic information ( i.e. gender age , cause ) , the following data of all the patients were also collected : location of fracture - left or right , presence of tooth in the fracture line , displacement of the inferior alveolar canal , and any major postsurgical complication ; routine antibiotics therapy and use of elastic for slight malocclusion were not considered in complication . clearance to conduct the study was obtained from the university ethical committee . written informed consent to participate in the study was obtained from each patient before enrolling in the study . patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram ( opg)displacement of the fractured fragments was measured on opg and was classified as 03 mm , 35 mm , and > 5 mm displacement between the fracture fragments . patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram ( opg ) displacement of the fractured fragments was measured on opg and was classified as 03 mm , 35 mm , and > 5 mm displacement between the fracture fragments . patients not willing to participate in the studypathological fracture , fractures with major tissue lossprevious surgery ( orthognathic surgery , implant surgery , surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery resulting numbness)panfacial trauma . patients not willing to participate in the study pathological fracture , fractures with major tissue loss previous surgery ( orthognathic surgery , implant surgery , surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery resulting numbness ) these thirty patients were divided into two groups , 15 patients in each group . in group b , the patients were treated using 2-mm three - dimensional ( 3d ) plate . group a patients were treated with six - hole preangulated plate as was applied along external oblique ridge transbucally . in group b , patients underwent osteosynthesis using 3d titanium strut plate transbucally . postoperatively , pain , swelling , and mobility between fracture fragments , nerve paresthesia , and recovery were assessed between two groups at various periods of follow - up . patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram ( opg)displacement of the fractured fragments was measured on opg and was classified as 03 mm , 35 mm , and > 5 mm displacement between the fracture fragments . patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram ( opg ) displacement of the fractured fragments was measured on opg and was classified as 03 mm , 35 mm , and > 5 mm displacement between the fracture fragments . patients not willing to participate in the studypathological fracture , fractures with major tissue lossprevious surgery ( orthognathic surgery , implant surgery , surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery resulting numbness)panfacial trauma . patients not willing to participate in the study pathological fracture , fractures with major tissue loss previous surgery ( orthognathic surgery , implant surgery , surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery resulting numbness ) these thirty patients were divided into two groups , 15 patients in each group . in group b , the patients were treated using 2-mm three - dimensional ( 3d ) plate . group a patients were treated with six - hole preangulated plate as was applied along external oblique ridge transbucally . in group b , postoperatively , pain , swelling , and mobility between fracture fragments , nerve paresthesia , and recovery were assessed between two groups at various periods of follow - up . a total of thirty patients of displaced angle fracture treated with different types of fixation were studied . there was significantly more prevalence of fracture in male patients with a mean age of 25 years . road traffic accident was the major cause ( 74% ) of fracture of the mandible and interpersonal violence and fall from height was less , incidence - wise . the mean duration of time lapse between injury and definitive treatment in both groups was 68 days . the failure to recognize or detect nerve paresthesia initially is due to poor cooperation of patients due to facial edema , lacerations and associated injury , or unconsciousness . nerve sensory function was evaluated by a light touch with cotton wool and two - point discrimination on the skin of chin and lip . postoperatively , there was significant in pain on a visual analog scale between both groups at different intervals . a decreasing trend was found with the time interval in both groups . at 6-week follow - up , the patients in both groups were pain - free . there was a gradual change in molar bite force from 7 day onward at different intervals in both groups ; however , in group a , this change was less than group b. preoperatively the mean displacement in group a was 7.86 6.75 mm and in group b was 8.43 6.65 mm preoperatively . the mean displacement in group a was 0.20 0.41 mm and in group b was 0.66 0.61 mm . the mean change was 7.66 4.48 mm and 7.76 6.54 mm , respectively . there was a significant change observed postoperatively in both groups ; t and p values were statistically significant , 2.43 and 0.02 , respectively . paresthesia was present in 46.6% cases of group a and 40% cases of group b preoperatively [ figure 1 ] . relation of paresthesia with degree of displacement of fractured fragments in both groups at 7 day postoperatively , 33.3% cases in group a and 40% cases in group b had paresthesia . at the end of 4 weeks , 20% cases in group a and 26% cases in group b had paresthesia while at the end of 12 weeks , 20% cases in group a and 26.6% cases in group b had paresthesia . there was no statistical significance ( p < 0.05 ) in anesthesia / paresthesia between both the groups at different time intervals . however , the presence of paresthesia was lower in group a than in group b. one patient in group a and two patients in group b had persistent paresthesia postoperatively at 12 weeks . paresthesia was present 28.5% cases when displacement was 13 mm , 62% cases when displacement was 39 mm , and 71% cases when displacement between fragments was > 9 mm . there was a complete recovery in both groups a and b when displacement was < 5 mm and persistent paresthesia 33% in group a and 66% in group b when displacement was > 9 mm after 12-week follow - up [ figure 2 ] . comparison of the persistence of paresthesia in both groups after fixation , showing greater percentage of recovery in group a the mandibular angle fracture usually resulted in pain , swelling , bleeding , and disturbed occlusion due to the displacement of fractured fragments . displaced fractures generally resulted in paresthesia , which causes problems such fluid drooling or food escape or accidental lip biting and occasionally shaving and applying makeup even kissing become difficult . angle fracture can be displaced by a number of ways as an open book , vertical overlap , and laterally . this can be explained on the basis that etiology of fracture shifting toward assault or interpersonal violence with a blow to side of the face . there is no accurate method to access the ian injury , but radiographically fractured fragments separation / gap and misalignment of inferior alveolar canal only suggest the ian injury . manipulation of fracture fragments during reduction and stabilization or extraction of the third molar and even screws placement may result in inferior nerve injury . persistent mobility at fracture site even in minimally displaced fracture can cause further nerve injury . in this study , group a patients had pretreatment sensory disturbance 46.6% paresthesia preoperatively , and postoperatively , 33% and 20% at 1 week , 4 weeks , and at the end of 12 weeks respectively . while in group b ( matrix plate ) , 40% patients had paresthesia preoperatively , and postoperatively , 26% and 26.6% at 4 weeks and the end of 12 weeks , respectively . postoperative difference in nerve injury recovery may be explained on the basis of fixation , that provides more firm fixation , which helps in early nerve recovery in group a than group b. similar observation was also reported by thurmuller et al . manipulation and reduction of fracture fragments can further result in starching or injury to the nerve . nagadia et al . found by computed tomography ( ct ) imaging that the mean distance from the outer buccal cortex to the inferior alveolar canal in chinese mandible was 6.97 mm ( min 4.8 mm ) ; the use of > 6 mm of monocortical screws can cause injury to nerve in this region . levine et al . reported that the distance of buccal cortical margin to the mandibular canal was 4.9 mm using ct imaging . demyelinating nerve lesion generally recovers during the first 4 months of injury ; however , after sustained axonal nerve injury , the nerve conduction velocity can also slow down permanently due to the schwann cell regeneration having shorter internodal interval than before injury and it may never reach baseline as happened probably in our few cases . due to wide separation of fractured fragments with persistent mobility at fractured may causes , repeated nerve injuries in the already stretched nerve . there is no direct evidence that suggested the relationship of fracture fragments displacement and time lapse in treatment resulted in paresthesia . early reduction and fragments fixation of fracture fragments may offer early nerve chances of nerve recovery margins . al - jandan et al . reported cone - beam ct observation that horizontal distances at the canine , first premolar , and second molar and reported that using 6 mm screw can cause 56% ian injury and 7 mm can cause 78% injury . hence , the routine use of 57 mm screws would have a high risk of injury to the root apex and the inferior alveolar canal if miniplate was placed along champy 's line of osteosynthesis . in this study when fractured fragment displacements were > 5 mm , then rate of recovery was poor as 33% in group a and 66% in group b , with persistent paresthesia at the end of 12-week follow - up . this may be explained on the basis that prolonged stretching of fragments may cause more nerve damage . while in < 5 mm displaced group , patients had complete nerve recovery ; robinson reported that most of the improvements of nerve injury occur in initial 4 weeks , but long - term 12-month follow - up will definitely be helpful . another study by queral et al . reported that fracture displaced > 5 mm had a 7-fold increased risk of ian after treatment compared with patients with < 5 mm fracture displaced . reported postoperatively 81% had poor sensation to thermal stimuli and 67% to von frey tactile stimulation . ardary and raveh et al . reported that the rate of postoperative sensory deficit has been lower varying from 0.9% to 34% . this variation may be explained on the basis as paresthesia diminished with the time and different follow - up of studies , and possibility studies had included fractures of noninferior canal region ( parasymphysis and symphysis ) . this study suggests that more stable fixation helps in early nerve recovery in displaced angle fracture . permanent ian neurosensory deficits may complain as pain , paresthesia dysesthesia , hypoesthesia , or anesthesia involving the chin , lower lip , and gums . early manual reduction , and temporary stabilization of fracture fragments will definitely prevent mobility of fragments and reduce bleeding , pain , and swelling causing injury to nerve .

purpose . obesity has reached epidemic proportions in the u.s . and has nearly doubled worldwide since 1980 . bariatric surgery is on the rise , but little focus has been placed on the psychosocial impacts of surgery . the purpose of this study was to explore experiences of patients who have undergone bariatric surgery at least two years before to gain an understanding of the successes and challenges they have faced since surgery . methods . this study used a phenomenological approach , to investigate the meaning and essence of bariatric patients with food after surgery . semi - structured interviews were conducted on a sample of nine participants who had undergone surgery at least two years prior . findings . two main themes regarding food intake emerged from the data : ( a ) food after the first year post - surgery and ( b ) bariatric surgery is not a magic pill . upon further analysis , food after the first year post - surgery had four subthemes emerge : diet adherence after the first year post - surgery , food intolerances , amount of food , and tendencies toward coping with food do not magically disappear . conclusion . findings revealed that post - operative diet and exercise adherence becomes increasingly difficult as weight loss slows . many participants find that only after the first year after surgery the work really begins .

obesity is now a global public health issue and has nearly doubled worldwide since 1980 . global obesity estimates from 2008 reported that 35% of adults over the age of 20 were overweight and 11% were considered obese . in addition , obesity rates continue to rise and remain epidemic in the united sates , as well . more than two - thirds of american adults ( 68.8% ) are considered overweight or obese and more than one - third ( 35.7% ) of adults are obese , constituting more than 78 million american men and women . individuals who are considered morbidly , or clinically severely , obese ( those with bmi of 35 in addition to documented health problems or those with a bmi over 40 ) are considered potential candidates for bariatric surgery . individuals elect to undergo bariatric surgery hoping to achieve long - term weight loss and to improve obesity - related comorbid conditions . in 2008 , nearly 350,000 bariatric procedures were performed globally , with an estimated 220,000 in the united states alone . however , questions still arise on long - term success and its effect on weight loss maintenance and prevention of the return of lifestyle diseases [ 4 , 7 ] . previous research indicated that , about a year and a half to two years after bariatric surgery , weight loss from surgery stops and a significant number of individuals begin to regain the weight they had lost . this is when support is needed most ; nevertheless , many longitudinal follow - up bariatric surgery studies are short - termed and incomplete . bariatric surgery is a life - changing procedure that requires a lifelong commitment from the individual limited research exists examining personal and psychosocial experiences , including satisfaction with weight loss and challenges patients experience following the first year after bariatric surgery . using a qualitative approach to explore the meaning of the bariatric experience and sharing it with others provides an opportunity for future patients , families of patients , and healthcare providers to gain an understanding of what it is like to undergo a bariatric procedure and to live with the persistent change that accompanies it , after the first two years post - surgery and beyond . this study sought to answer the question : what is the experience of bariatric patients who are at least two years post - surgery ? this study used a qualitative research design , guided by a phenomenological approach , to investigate and describe the meaning and essence of experience of patients at least two years after bariatric surgery [ 9 , 10 ] . phenomenological studies are primarily open - ended , searching for themes of meaning in participants ' lives . the researcher seeks to understand the deep meaning of a person 's experience and how one describes this experience . exploring the lived experience of patients at least two years after bariatric surgery provided a deeper understanding and awareness of the impact of weight - loss surgery . through the use of interview questions as a guide , the method of in - depth , phenomenological interviews applied to a sample of participants who have all experienced similar social conditions gives power to the stories of relatively few participants , such as life experience . the researchers used purposeful convenience sampling , which enabled relatively easy access to participants . by contacting participants who were acquainted with the researchers and met inclusion criteria for this study , the researcher was able to conduct semistructured interviews with nine bariatric surgery patients at least two years post - surgery . criteria for inclusion was set so that the participants who were at least two years after operation and over the age of 18 were recruited through email / telephone regardless of gender , age , surgery type , ethnicity , or socioeconomic status . the study sample included nine women who ranged from 27 to 57 years of age ( m = 42 ) . seven participants had the vertical sleeve gastrectomy ( vsg ) surgery and 2 had the roux en y gastric bypass ( rnyb ) . self - identified ethnicities of participants were caucasian ( n = 5 ) , hispanic ( n = 2 ) , and african - american ( n = 2 ) . participants ranged from two to seven years after operation ( m = 3 ) ( table 2 ) . weight before surgery following the preoperative diet ranged from 199 pounds to 298 pounds ( m = 247.4 ) . initial weight loss after surgery ranged from 58 pounds to 116 pounds ( m = 88 ) . weight gain after reaching plateau ranged from none to 48 pounds ( m = 8.7 ) ( table 3 ) . data analysis included the use of field notes , audiotapes , a reflexive journal , and peer debriefing . each audiotaped interview was transcribed verbatim and field notes were taken during each interview . after transcription , each interview was printed for clarity . the first transcript reading helped develop the coding categories , and then the second reading was conducted to start formal coding in a systematic way using colored post - it notes to group related data . the second transcript aided in identifying new categories of information and the two lists the transcript of the third interview was compared to the previous code list to see if new categories emerged and this process continued comparing each subsequent transcript for coding categories . themes were eventually developed into a written description of the participants ' experience with bariatric surgery to answer the research question . the researchers personal experience as bariatric patients included preconceived beliefs that bariatric surgery should be for obese individuals who had failed attempts at losing weight through traditional methods with diet and exercise and who need an additional weight - loss tool to aid in achieving a healthy body weight . being part of the weight - loss surgery community comes with a very pro - surgery mentality that the researchers acknowledged . to focus on the participants ' experience after bariatric surgery hays and wood define bracketing as setting aside any assumptions made in everyday life and expressed the need for the researcher to reserve all prejudgments of their experience and rely on intuition and imagination to obtain the picture of the experience . establishing trustworthiness of the findings was utilized by engaging in peer debriefing of instrument protocol and through prolonged discussions of the research project with peers . researcher reflexivity was engaged by keeping a journal and field notes , and simultaneous data collection and analysis , which involved collecting and analyzing data simultaneously . review of the data , including individual interviews and field notes , was conducted to analyze and identify themes of the participants ' experience with food after bariatric surgery . two main themes emerged from the data : ( a ) food after the first year post - op and ( b ) bariatric surgery is not a magic pill . the overarching theme of viewing weight - loss as work after the first year emerged from the data as participants described the mounting difficulty of adhering to the recommended post - op diet once the first year had passed . in addition , tendencies towards using food as comfort and emotional eating were still a struggle that many participants experienced.i know i have to keep staying on top of it , it 's just that i 'm sick of protein , i 'm sick of water , i 'm sick of working out , i 'm just sick of it . i just want eat normal sometimes , but i know i just need to suck it up and deal with it . finding that balance after the first year i know i have to keep staying on top of it , it 's just that i 'm sick of protein , i 'm sick of water , i 'm sick of working out , i 'm just sick of it . i just want eat normal sometimes , but i know i just need to suck it up and deal with it . finding that balance after the first year [ p7 ] the recommended diet after bariatric surgery varies among surgeons and across different bariatric surgeries , but there are some commonalities that all participants experienced . generally agreed upon nutritional guidelines are that supplemental minerals and vitamins should be taken daily , small bites of food should be chewed thoroughly before swallowing , liquids should be either ingested well before meals or at least 30 minutes afterwards , and at least 60 grams of proteins should be preferentially eaten before fats and carbohydrates [ 1215].consuming the right food and the vitamins and doing the right thing with food exist on a continuum it 's not a matter of being all right or all wrong or all good or all bad every day you 're striving to make the best choices you can in that moment . [ p3]in the second year , i noticed i had to be very careful about what i ate and i had to really focus on my vitamins every day and make sure to get my protein and water in every day the first year , you did not really have to think much and you 'd still lose weight . now i have to focus and pay attention to what i put in my mouth [ p9]i am more aware of what i 'm eating now because my intake is so small , you really have to pay attention to what you 're putting in your body . it 's quality now , i 'm going for the filet minion [ sic ] before the ground hamburger . [ p7 ] consuming the right food and the vitamins and doing the right thing with food exist on a continuum it 's not a matter of being all right or all wrong or all good or all bad every day you 're striving to make the best choices you can in that moment . [ p3 ] in the second year , i noticed i had to be very careful about what i ate and i had to really focus on my vitamins every day and make sure to get my protein and water in every day the first year , you did not really have to think much and you 'd still lose weight . now i have to focus and pay attention to what i put in my mouth [ p9 ] i am more aware of what i 'm eating now because my intake is so small , you really have to pay attention to what you 're putting in your body . it 's quality now , i 'm going for the filet minion [ sic ] before the ground hamburger . [ p7 ] potential long - term dietary complications include dumping syndrome ( e.g. , sweating , weakness , hypoglycemia , vomiting , diarrhea , and nausea ) related to eating foods high in concentrated sugar or fat [ 16 , 17 ] . all participants expressed problems with dumping syndrome and/or intolerances to various foods , namely , refined carbohydrates and sugar . most participants talked about food intolerances , especially to sugar.i think the one good thing is that i can not eat a huge amount of sweets without being nauseated i can eat a little that 's more cracker - ish or cookies but , like , big bakery pastries or pies , or a big hunk of cake or something , well , that 's not going to work very well [ p1]besides sweets , i have problems with all white carbs like rice , bread , and pasta . [ p9 ] i think the one good thing is that i can not eat a huge amount of sweets without being nauseated i can eat a little that 's more cracker - ish or cookies but , like , big bakery pastries or pies , or a big hunk of cake or something , well , that 's not going to work very well [ p1 ] besides sweets , i have problems with all white carbs like rice , bread , and pasta . [ p9 ] all participants discussed the amount of food they were able to consume in some way , noting reduced intake even years after surgery compared to presurgery amounts.as far as eating i can eat many more calories at one time than i used to be able to eat when recently post - op . [ p3]you do have more hunger , though you still can not eat like you used to and i have a bit more of a desire to eat than i did the first year i'm still restricted in how much i can eat and eat about 3 ounces of dense protein and 1 ounce of vegetables and 1 ounce of starch for a meal . [ p6 ] as far as eating i can eat many more calories at one time than i used to be able to eat when recently post - op . [ p3 ] you do have more hunger , though you still can not eat like you used to and i have a bit more of a desire to eat than i did the first year i'm still restricted in how much i can eat and eat about 3 ounces of dense protein and 1 ounce of vegetables and 1 ounce of starch for a meal . [ p6 ] many participants were identified as still grappling with food as a means to cope and it can be a challenge to find new ways to channel emotions without turning to food [ 18 , 19 ] . participants also identified that it was easier to manage the food cravings after surgery as well as the head hunger which involves not having physical hunger but the desire to eat anyway.i'm realizing now that there is a real emotional and physical effect that different foods have on your body . for example , the effect that sugar might have on your emotions or energy level , the way alcohol affects your body almost immediately post - op [ p3]i still have those gnawing cravings from time to time and i just find it easier to say you do not need it right now , you can wait . it 's not like before where i had to have the food otherwise i was gon na die . [ p6]if i 'm going to have something bad like sugar or something , i make sure i have protein first so i 'm full , then it 's not such a big deal . i try not to tell myself no on anything because the minute i say no , i 'm feeling depressed today and i wish i could just go eat a couple of pieces of cheesecake and that would only make me happy temporarily [ p4 ] i 'm realizing now that there is a real emotional and physical effect that different foods have on your body . for example , the effect that sugar might have on your emotions or energy level , the way alcohol affects your body almost immediately post - op [ p3 ] i still have those gnawing cravings from time to time and i just find it easier to say you do not need it right now , you can wait . it 's not like before where i had to have the food otherwise i was gon na die . [ p6 ] if i 'm going to have something bad like sugar or something , i make sure i have protein first so i 'm full , then it 's not such a big deal . i try not to tell myself no on anything because the minute i say no , then i start have mental issues with it . i 'm feeling depressed today and i wish i could just go eat a couple of pieces of cheesecake and that would only make me happy temporarily it kind of conditioned me to not do that anymore like , hey , this is bad for you [ p4 ] this theme was common across all participants and it deserves to be noted that many patients who passed the first two years of surgery realize ( e.g. , by experiencing weight regain ) that bariatric surgery is intended to be used only as a tool to aid in weight loss or to reduce physical hunger . all participants offered words of advice and/or caution to those considering bariatric surgery.i think that the surgery is not a fix all it is a tool to help you achieve weight loss , but it is only a tool . it helps you with the amount you can eat and can definitely help you lose weight but if you still do not pick the right [ food ] choices to eat and exercise , it 's still not going to do you any good in the long term . i mean , everybody has this misconception that you have surgery and then get magically skinny and that 's not how it works . you still have to put in the work and your everything into this every day . [ p4]i think people have it in their heads that you go into surgery and have it done and come out instantly thin . they do not realize that you can not eat and drink at the same time or you can only eat 3 ounces of food at once nobody talks about all that and all the working out and that you 've been gutted like a fish , they just think you 're instantly skinny within a month [ p7 ] i think that the surgery is not a fix all it is a tool to help you achieve weight loss , but it is only a tool . it helps you with the amount you can eat and can definitely help you lose weight but if you still do not pick the right [ food ] choices to eat and exercise , it 's still not going to do you any good in the long term . [ p2 ] weight loss is not something that 's going to happen on its own . i mean , everybody has this misconception that you have surgery and then get magically skinny and that 's not how it works . you still have to put in the work and your everything into this every day . [ p4 ] i think people have it in their heads that you go into surgery and have it done and come out instantly thin . they do not realize that you can not eat and drink at the same time or you can only eat 3 ounces of food at once nobody talks about all that and all the working out and that you 've been gutted like a fish , they just think you 're instantly skinny within a month experience of participants was profiled in an attempt to share their qualities through thick description . the experience of all participants ' lives after bariatric surgery provided a glimpse of what role food now plays after the first two years after operation . food was still considered a huge part of life , though limited in quantity , even years after bariatric surgery . participants discussed not consuming their recommended daily intake of protein and not taking their vitamins . low iron , b12 , and vitamin d were consequences of poor supplementation . participants compared food cravings to addictions to alcohol or drugs and there was still grief felt in regard to being unable to enjoy the same meals socially and with family as before surgery . participants adjusted to changes in diet in different ways , and some were more successful than others , though many still fight the urges to use food to cope in certain situations . many found eating a simple food such as a cracker ( carbs ) or any refined sugar may make them become unpleasantly ill . these patients face medical and psychosocial challenges that require continuing care for years after surgery , and the hard work begins post - surgery . it is also pointed out that not much has been written on the topic of follow - up care , but rather the focus has been on who is a good candidate for surgery rather than what patients go through after the procedure . a qualitative study using phenomenological approach was used for this study in order to gain a better understanding of the experience of participants who were at least two years after bariatric surgery . the use of semistructured interviews allowed the participants to express their feelings and experience of life after bariatric surgery which many follow - up surveys lack and may expand the knowledge base from the participants own lived encounters with bariatric surgery . the themes around food generated from the data collected in this study provided a glimpse of life once the magic of the first year has passed and the reality of the surgery had set in . additional emerging themes from this study to be analyzed include the role of social support and perceptions of self and society . while obesity threatens to explode into a reverberating pandemic , there is a need to understand and support those individuals who have made the decision to have bariatric surgery . studies such as this can offer health care professionals needed insight and information to be supportive and provide the care necessary to this vulnerable population . one participant who has struggled more than two years post - surgery with weight regain , and who stands apart from the others in current postoperative bmi ( table 3 ) , summed up the lifelong impact of diet for the bariatric patient.it's lifelong and a lifestyle change i mean you can not have your stomach cut out and then think you can eat crap food like you did before . i noticed that it is very easy to eat junk food like chips and soda these foods go down easily also if you drink your calories i just think that if you want long - term success with bariatric surgery then it 's a complete lifestyle change and it 's not a temporary thing , i know that now . you can not lose all the weight and then think you can go back to unhealthy eating like you did before surgery . i mean you can not have your stomach cut out and then think you can eat crap food like you did before . i noticed that it is very easy to eat junk food like chips and soda these foods go down easily also if you drink your calories i just think that if you want long - term success with bariatric surgery then it 's a complete lifestyle change and it 's not a temporary thing , i know that now . you can not lose all the weight and then think you can go back to unhealthy eating like you did before surgery .

carcinoma showing thymus - like differentiation ( castle ) is a rare carcinoma of the thyroid or adjacent soft tissue of the neck with a histologic resemblance to thymic epithelial tumors . although the fine - needle aspiration ( fna ) plays a central role in the initial evaluation of thyroid nodules , few reports about the cytologic findings of castle have been found according to a review of literatures . we report cytologic findings of a case of castle . a 34-year - old woman presented with a 2-month history of sore throat . the fna showed that the smear was composed of three dimensional clusters and sheets . the tumor cells were round to ovoid with high nuclear : cytoplasmic ratios . the nuclei were vesicular with small nucleoli . there were some tumor cells showing keratinization . some lymphocytes were found on the background and within clusters . the presence of poorly - differentiated tumor cells with a focal keratinization and a lymphocytic background on the fna is suggestive of castle .

ultrasound showed a 1.7-cm , well demarcated , heterogeneous , low echoic solid mass in the lower pole of the right thyroid ( fig . 1 ) . a computed tomography scan of the neck revealed a 3.3-cm , exophytic , low - attenuated mass in the lower pole of the right thyroid . the fna biopsy of the mass was performed , which was accompanied by the standard papanicolaou stain . the smear was composed of not only cohesive three dimensional clusters and sheets but also singly scattered cells ( fig . the tumor cells showed mild anisonucleosis and high nuclear : cytoplasmic ( n / c ) ratios . nuclei were round to ovoid with irregular nuclear contours and small , distinct nucleoli ( fig . some tumor cells showed dense orangeophilic cytoplasm , which is suggestive of individual cell keratinization ( fig . 2c ) . some lymphocytes were found on the background and within clusters , which was accompanied by the presence of a few stripped nuclei ( fig . the aspirate was interpreted as a high - grade malignant thyroid neoplasm without further definitive classification . the mass had a lobular , tan - colored cut surface and a firm consistency ( fig . the tumor was well - circumscribed and it was composed of variably sized and irregularly shaped lobules of cohesive polygonal tumor cells which were separated by bands of dense fibrous stroma ( fig . the tumor cells had high n / c ratios , eosinophilic cytoplasm and ill - defined cell borders . the tumor cells showed positivity for cd5 , carcinoembryonic antigen , high molecular weight keratin ( hmwk ) , cytokeratin 5 ( ck5 ) , and p63 . one regional lymph node was found and involved by tumor . over a 27-month follow - up period , the cytological diagnoses were mentioned in previous 14 reports consisting of 40 cases . malignant tumor or poorly - differentiated carcinoma were the most frequent cytological diagnosis in cases of castle , except for six cases ( table 1).2 - 15 one case was diagnosed as castle for which no cytological findings have been described.11 in previously reported cases of castle , the most common cytological findings include tight clusters and sheets of round tumor cells with high n / c ratios , vesicular nuclei , prominent nucleoli , amphophilic cytoplasm , and lymphocytic background , which correspond well with the present case.2 - 4,6 in the present case and those of hirokawa et al.,2 keratinized cells were present . youens et al.6 reported indistinct cell borders , intranuclear grooves and sparse mitotic figures , which were also seen in the present case . youens et al.6 reported not only intranuclear cytoplasmic inclusions and papillary - like structures but also granular chromatin and rosette - like structures . the differential diagnoses of castle include papillary carcinoma , hrthle cell neoplasm , undifferentiated carcinoma , medullary carcinoma and metastatic lymphoepithelioma - like carcinoma . castle may show intranuclear pseudoinclusions or papillary - like structures , but it does not show fine pale chromatin and monolayer sheets of cells with dense cytoplasm . although hrthle cell neoplasm contains cellular aggregates with abundant cytoplasm and prominent nucleoli , lymphocytic background of castle is lacking . it is very difficult to distinguish castle from metastatic lymphoepithelioma - like carcinoma , which also shows sheets of poorly differentiated round cells intermixed with small lymphocytes . subtle differences , such as frequent mitotic activity and a polymorphous inflammatory background , favor metastatic lymphoepithelioma - like carcinoma.4 castle may resemble undifferentiated thyroid carcinoma with squamous differentiation , but it does not show marked cytologic atypia , frequent mitotic activity and necrotic background characteristic of undifferentiated carcinoma . although castle may show rosette - like structures and granular chromatin , along with an abundance of single tumor cells as in medullary carcinoma , it is lacking of typical plasmacytoid cells , pink cytoplasmic granules and amyloid deposition.6 immunocytochemical study for cd5 is helpful to differentiate castle from other malignant thyroid neoplasm . cd5 is a surface glycoprotein expressed on mature t cells and a subset of b cells.16 thymic carcinoma cells are also known to be positive for cd5.17 cd5 is almost always expressed in castle . castle is positive for hmwk , ck5 , and p63 because it is also a squamous cell carcinoma . however , thyroid squamous cell carcinoma and poorly - differentiated carcinoma were negative for cd5.17 ito et al.11 reported that the sensitivity and specificity for cd5 immunohistochemistry were 82% and 100% , respectively , in making a diagnosis of castle . in conclusion , the presence of poorly - differentiated tumor cells with a focal keratinization and a lymphocytic background on the fna biopsy is suggestive of castle .

adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration . the integration profiles of vectors based on mlv , aslv , siv , and hiv have all been shown to be non - random , and novel vectors with a safer integration pattern have been sought . recently we developed a producer cell line called catpac that packages standard momlv vectors with felv gag , pol and env gene products . we now report the integration profile of this vector , asking if the felv integrase and capsid proteins could modify the momlv integration profile , potentially resulting in a less genotoxic pattern . we transduced rhesus macaque cd34 + hematopoietic progenitor cells with catpac or standard momlv vectors , and determined their integration profile by lam - pcr . we obtained 184 and 175 unique integration sites ( is ) respectively for catpac and standard momlv vectors , and these were compared to 10 000 in silico - generated random is . the integration profile for catpac vector was similar to momlv and equally non - random , with a propensity for integration near transcription start sites and in highly dense gene regions . we found an is for catpac vector localized 715 nucleotides upstream of lmo-2 , the gene involved in the all developed by x - scid patients treated via gene therapy using momlv vectors . in conclusion , we found that replacement of momlv env , gag , and pol gene products with felv did not alter the basic integration profile . thus there appears to be no safety advantage for this packaging system . however , considering the stability and efficacy of catpac vectors , further development is warranted , utilizing potentially safer vector backbones , for instance those with a sin configuration .

insertional mutagenesis after gene transfer in animal models1,2 as well as in x - scid clinical trials35 has led numerous laboratories to focus on the safety of the virus - based vectors used to transfer and express genes of interest . a better understanding of the mechanisms that define the characteristics of the integration of virus - based vectors should allow the development of new vectors with increased biosafety . taking advantage of the subgroup c feline leukemia virus ( felv - c ) as a platform for efficient transduction of target human cell types , including cd34 + stem and progenitor cells hek293 cells were engineered to stably package momlv vectors with felv 61e gag - pol gene products , in addition to felv - c sarna env , resulting in a completely felv - derived packaging system6 . in the current study we assessed the integration profile of catpac vector in comparison to the same momlv vector packaged with momlv gag - pol and amphotropic env in order to elucidate the influence of the felv component packaging proteins on viral integration . both primary vector sequences as well as integrase proteins have been implicated in genomic integration site selection for retroviruses , and thus we asked whether this hybrid vector might have a potentially less genotoxic integration pattern than standard momlv , due to the presence of the alternative felv integrase machinery . felv - c and momlv are both members of the mammalian c - type retrovirus family , but there is no prior information on the integration profile for either felv - c or hybrid momlv / felv packaging and vector systems . two rhesus macaques ( rq4984 & rq4972 ) were treated with a combination of g - csf and scf and mobilized circulating hematopoietic progenitor and stem cells were collected by apheresis . pbmncs contained in apheresis product were purified by density gradient centrifugation over lymphocyte separation media ( lsm , mp biomedicals ) and cd34 + cells were positively immunoselected using the 12.8 igm anti - cd34 biotinylated antibody and macs streptavidin microbeads ( miltenyi biotec ) . the purified cd34 + cells were stimulated for 48 hrs in dmem supplemented with 10% fcs , scf , flt3-l , and tpo and then transduced twice on fibronectin - treated plates ( retronectin , takara ) with the catpac or control momlv vector supernatants , using our standard in vitro transduction conditions8 . the vectors contained the gfp marker gene , allowing assessment of transduction efficiency by flow cytometry . in both animals , the transduction efficiencies of cd34 + cells for catpac and control momlv packaging systems were equivalent : for rq4984 8.1 % and 8.8 % of gfp positive cells for catpac and momlv vectors respectively , and for rq4972 39.7 % and 20.3% respectively . genomic dna of transduced cd34 + cells was isolated using qiagen dneasy blood & tissue kit ( # 69506 ) and lam - pcr was carried out as previously described9 with 100 ng of total genomic dna . the first exponential pcr was carried out with primer lci ( 5-gacccgggagatctgaat-3 ) and ltr - r1 primer ( 5-cagctgttccatctgttc-3 ) , whereas the second exponential pcr was carried out with lciii ( 5-agtggcacagcagttagg-3 ) and ltr - r2 primer ( 5-gctagcttgccaaaccta-3 ) . sequences obtained were aligned by blat or blast to the rhesus macaque genome assembly ( mmul 1.0 , jan 2006 ) . we obtained 200 and 187 valid integration sites ( is ) for catpac and momlv vectors respectively ( supplemental information ) . sites mapping to 2 or more genomic positions as well as sites mapping within repetitive genomic sequences were omitted to finally obtain 184 and 175 unique is respectively for catpac and momlv vectors . we also compared both profiles to in silico - randomly generated is sets as previously described9 . briefly , 10 000 sets of 184 or 175 random is were designed in silico as follows : an aatt ( tasi ) site in the genome was selected at random using a random number generator . the in silico is was placed either upstream or downstream ( p=0.5 ) of the aatt site , at a distance matching the size of one of the sequences obtained experimentally . the in silico is was validated only when a blast alignment of the genomic sequence between the aatt and the is returned a unique sequence in the genome . this operation was repeated 184 times and 175 times respectively for catpac and momlv vectors to obtain a single matching random dataset . these control datasets were subjected to the same analyses as the experimental datasets , and the results were used to generate empiric p - values . we generated two groups of control genomic coordinates , one with 10 000 sets of 184 coordinates each to mimic the catpac vector is , and one with 10 000 sets of 175 coordinates each to mimic the momlv is . for gene annotation , ensembl release 54 ( may 2009 ) comprising 38 146 predicted gene transcripts was used . the association between the catpac and momlv vectors integration sites was tested using a chi - square test . the results obtained for momlv are comparable to previously reported integration patterns for standard momlv derived vectors10,11 . we confirmed the preference of momlv for integrating near the tss , a feature also shared by catpac vector . like other retroviral vectors already studied such as momlv10,11 , aslv12 , siv10,13 , and hiv12,14 , catpac vector exhibited a profile of integration significantly different from random integrations as can be seen in all the tables and figures presented in this report . as shown in figure 1 , out of the 114 catpac vector and 112 momlv is located in a window of 60 kb centered on transcription start sites ( tss ) , 44% ( catpac vector ) and 35% ( momlv ) occurred in a 10 kb window centered on tss , implicating a preference for integration near tss . with regard to inter or intragenic insertions , table 1 shows that approximately half of the vector integrations were found inside of a gene ( 49.5 and 54.3% for catpac vector and momlv vectors respectively ) . one quarter of all integrations were found within a 30 kb window upstream of a gene ( table 1 ) . in these upstream regions as shown in table 2 integration of both vectors in or around ( 1 kb and 5 kb ) cpg islands was significantly different from the random datasets . a quarter of the insertion sites were found 5 kb around cpg islands for catpac ( 27.7 % ) and momlv ( 25.7 % ) vectors . however , this higher percentage probably represents the propensity of catpac and momlv vectors to integrate close to the tss , rather than a preference for cpg islands . looking at integrations with regard to gene density , table 2 shows that less than half of the integrations for both vectors were found in lower gene - density regions ( 0 to 10 genes / mb ) of the genome , and thus 53.8 % and 57.7 % of integration respectively for catpac and momlv vectors in gene dense regions ( 11 genes / mb and up ) . although this integration profile is significantly different from in silico - generated integrations , there were no obvious differences in the integration pattern between catpac and momlv vectors with respect to gene density . theoretically , virus - based vectors can lead to tumor formation by insertional mutagenesis either by activating an oncogene or by disrupting a tumor suppressor gene15 . most , if not all , cases of malignant disease development following gene therapy for hematological disease have been associated with the activation of a proto - oncogene 25 . we therefore looked at integration of catpac and momlv vectors in the proximity of oncogenes ( table 3 ) . we assessed both the proximity of proto - oncogenes to is , and conversely the proximity of is to proto - oncogenes . we defined proto - oncogenes based on the most recent version of the sanger oncogene file ( dated 2008 - 12 - 16 , at http://www.sanger.ac.uk/genetics/cgp/census/ ) . three hundred eighty four human entries were mapped to 368 unique ensembl rhesus macaque gene identifiers . as shown in table 3 , with this size dataset , the momlv integrations were not notably different from randoms with regard to proximity to oncogenes . however , the catpac vector integrations were more likely than the random datasets to be in proximity to oncogenes . the 12 catpac vector is in or within a 30 kb window of an oncogene were located : upstream of mllt11 , mutyh , cytsb , and lmo2 ; downstream of arnt ; in the intron of lasp1 , q4w6x8_macmu , akap9 , erg , cbfb , and pcm1 ; and in the exon of sufu . the 7 momlv is were located : upstream of mllt11 and tal1 ; downstream of kras and stil ; in the intron of cdk6 , pms1 , and ciita . the only oncogene in proximity to both catpac vector and momlv integrations was mllt11 , myeloid / lymphoid or mixed - lineage leukemia translocated to chromosome 11 , also termed af1q . expression of this gene has been shown to be linked to poor prognosis in aml ( reviewed in16 ) and has a function in apoptosis and drug resistance . we did not find any integrations in or near the mds1-evi1 gene complex , previously reported to be over - represented in engrafting human , murine , and rhesus macaque hematopoietic stem and progenitor cells transduced with momlv vectors17 . interestingly , in the 12 oncogenes targeted by the catpac vector we found an integration 715 nucleotides upstream of lmo2 , the gene activated in x - scid trials and causing leukemia through a t cell clonal expansion35 . lmo2 was also reported as an mlv insertion site in the ada - scid and cgd gene therapy trials , but so far , no clonal expansion has been observed1820 . in order to analyze if there was a significant clustering of vector insertions we used the definition of common integration sites ( cis ) reported by suzuki and coworkers21 . as shown in figure 2 , there is a difference between experimental data obtained for catpac vector and random integrations . we found 7 second order cis for catpac vector , a higher value than the frequency of second order cis in the matching random datasets . none of the cis identified were localized within 200 kb of a known proto - oncogene . these investigations contribute to knowledge regarding the determinants of retrovirus integration into the genome . at least two vector - related elements are involved : the integrase / capsid proteins and/or the primary sequence of the vector backbone itself . the relative contributions of each element in determining integration preferences remain under intense investigation , but it appears that the integrase core region is critical . swapping the integrase between even closely related viruses , such as hiv and fiv , results in a change in the integration pattern determined primarily by the integrase , along with a contribution from gag - encoded capsid proteins22,23 . the path of entry into the cell , determined by the env gene product , appears to have little impact on integration preferences24 . in a previous study , pseudotyping of a standard momlv retroviral vector with the felv - c env protein only resulted in more efficient transduction of primitive cd34 + cells compared to a standard galv env pseudotype , but no change in integration profile , based on a limited number of integration sites retrieved from myeloid cells following in vivo engraftment25 . in the present study , we went on to study the impact of using felv gag and pol to package an momlv backbone and compared it to an momlv backbone packaged with standard momlv components , to determine if the integrase and capsid proteins of felv would change the integration pattern of momlv vector backbones . utilization of felv gag , pol , and env components did not alter the integration profile of momlv vectors , in contrast to the prior results comparing the impact of swapping integrase between hiv and fiv viruses . both standard momlv and catpac vectors integrated within genes and in areas of high gene density , near tss . other studies have indicated that vector backbone sequences are also important determinants of integration patterns . the pre - integration complex contains viral integrase and capsid proteins , reverse - transcribed vector dna , and host cell factors , all implicated in both site - selection and the actual integration process2628 . there are no large - scale integration profiles published for felv , however cis identification in tumors induced by felv infection indicate activation of an overlapping but not identical set of genes by felv as compared to momlv29 . swapping the u3 region of the molv ltr with the same region of felv results in a partial shift in cis in tumors30 . however , the integration profile of cis in tumors may relate more to the enhancer activity of a specific u3 region in a target cell type as opposed to actual integration characteristics , since analysis of tumor cis relies to a large degree on in vivo selection of clones with activating insertions , from an initial highly polyclonal pool of cells . in conclusion , retroviral vectors packaged using the catpac system integrate into the genome in a non - random fashion , with a general profile very similar to vectors packaged using standard momlv gag and pol gene products , unfortunately characterized by a preference for integration within genes and near tss . although the study of a larger number of integration sites might uncover subtle differences between felv and momlv integrase and capsid determinants of integration , it is clear from the data we obtained in this pilot study that their general profiles are similar and characterized by the same risks . therefore , while catpac vector may have advantages in terms of ease of high titer vector production and potential improved efficiency of gene transfer to some target cell populations , the integration profile we have characterized does not suggest any increased safety of these vectors compared to standard retrovirus packaged with momlv components .

the genome of lager brewer 's yeast is a hybrid , with saccharomyces eubayanus and saccharomyces cerevisiae as sub - genomes . due to their specific use in the beer industry , relatively little information is available . the genome of brewing yeast was sequenced and annotated in this study . we obtained a genome size of 22.7 mbp that consisted of 133 scaffolds , with 65 scaffolds larger than 10 kbp . with respect to the annotation , 9939 genes were obtained , and when they were submitted to a local alignment , we found that 53.93% of these genes corresponded to s. cerevisiae , while another 42.86% originated from s. eubayanus . our results confirm that our strain is a hybrid of at least two different genomes .

there is evidence that the production and consumption of beer began in egypt in the early dynastic period ( 55003100 bce ) . high because yeast floats to the top of the tank at a temperature between 15 c and 25 c during production of ale beer with the yeast saccharomyces cerevisiae . this yeast is capable of fermenting at a temperature lower than 10 c and flocculates at the bottom of the tank . the saccharomyces sensu stricto complex includes six parented species : s. cerevisiae , saccharomyces bayanus , saccharomyces cariocanus , saccharomyces kudriavzevii , s. mikatae and s. paradoxus ; however , it has been observed that lager - brewing yeast is a hybrid species of two combined genomes of s. eubayanus and s. cerevisiae , , , , , , , . this provides an important source of chromosomal rearrangements , leading to the gene number and the size of the complete genome , , , . it has been proposed and recently demonstrated that lager yeast is the product of two independent hybridization events that can be divided into two groups : saaz and frohberg , or group i and group ii , respectively , , , , . with the use of next generation sequencing ( ngs ) technologies , such as the illumina platform , 40,175 prokaryote and eukaryotes genomes have been reported , including 210 different strains of the saccharomyces complex ( http://www.ncbi.nlm.nih.gov/genome/browse/ - revised july 22 , 2015 ) . to obtain a higher level of understanding of the sequenced organism the brewing yeast saccharomyces sp . strain 790 and a reference sequence of 76 scaffolds from s. eubayanus de c.v . the s. cerevisiae s288c reference genome sequence was retrieved from the yeast genome database ( www.yeastgenome.org ) . the brewing yeast genome was sequenced using the flx 454 titanium ( roche ) and miseq ( illumina ) massive sequencing platforms according to the manufacturer 's protocols . we obtained 0.8 million reads from flx 454 titanium ( 454 life sciences , branfort , ct ) with an average size of 400 bp ; 6 million pair - end reads from illumina ( illumina , san diego , ca ) with an average size of 150 bp ; 5 million mate - pair reads from illumina with an insert size of 350 bp and a size of 101 2 bp ; and 11.7 million mate - pair reads from illumina with an insert of 8 kb and a size of 51 2 bp . approximately 454 illumina pair - end reads were assembled with a newbler denovo assembler ( roche ) . this whole genome shotgun project has been deposited at ddbj / ena / genbank under the accession lsmh00000000 . the sequencing quality data were analyzed with fastqc 0.10.1 software with a value q30 . likewise , alignments were made against reference sequences ( s. cerevisiae s288c and s. eubayanus ) with the mummer 3.23 software package . the bioinformatics analysis for the annotation was performed with maker v2.31.8 software ( university of utah ) . the brewing yeast saccharomyces sp . strain 790 and a reference sequence of 76 scaffolds from s. eubayanus de c.v . the s. cerevisiae s288c reference genome sequence was retrieved from the yeast genome database ( www.yeastgenome.org ) . the brewing yeast genome was sequenced using the flx 454 titanium ( roche ) and miseq ( illumina ) massive sequencing platforms according to the manufacturer 's protocols . we obtained 0.8 million reads from flx 454 titanium ( 454 life sciences , branfort , ct ) with an average size of 400 bp ; 6 million pair - end reads from illumina ( illumina , san diego , ca ) with an average size of 150 bp ; 5 million mate - pair reads from illumina with an insert size of 350 bp and a size of 101 2 bp ; and 11.7 million mate - pair reads from illumina with an insert of 8 kb and a size of 51 2 bp . approximately 454 illumina pair - end reads were assembled with a newbler denovo assembler ( roche ) . this whole genome shotgun project has been deposited at ddbj / ena / genbank under the accession lsmh00000000 . the sequencing quality data were analyzed with fastqc 0.10.1 software with a value q30 . likewise , alignments were made against reference sequences ( s. cerevisiae s288c and s. eubayanus ) with the mummer 3.23 software package . the bioinformatics analysis for the annotation was performed with maker v2.31.8 software ( university of utah ) . the read assembly yielded 133 scaffolds with a ~ 70 depth and a n50 of 568,800 bp , suggesting a complete genome size of ~ 22.7 mbp ( table 1 ) , similar to previous reports of other lager beer yeasts , , , . approximately 65/133 scaffolds had a size > 10 kbp , which represents 99.667% of the assembled genome ( table 2 ) . table 3 shows a comparison of the assembly level of the sequenced genomes of the saccharomyces species ( as of august 2015 ) . the alignments against the reference genome , s. cerevisiae s288c , assigned scaffolds to each of its 16 chromosomes , and some scaffolds covered different portions of more than one chromosome ; for example , scaffold01 ( sf01 ) aligns with two chromosomes : a small portion with chromosome 1 and with chromosome 12 . the estimated size matches the previous and known information ; this is due to the presence of 16 chromosomes of the s. cerevisiae sub - genome and 16 of s. eubayanus . likewise , its size is close to the sum of the aforementioned genomes ( ~ 12 mbp each ) . ( 2014 ) , who reported the sequence and assembly of the lager brewing yeast genomes saccharomyces carlsbergensis ( 78 scaffolds , 29 chromosomes with a 19.5 mbp length ) , and saccharomyces pastorianus weihenstephan 34/70 ( 985 scaffolds , ~ 29 chromosomes , and 22.9 mbp ) . the annotation yielded 9939 cds and a gff file with their locations in the scaffolds of the assembly ( fig . the protein and transcript sequences , were subjected to a local alignment with the blast tool against a local database using the sequence of s. cerevisiae s288c as a reference . the transcripts were considered to be genes because previous reports showed that only approximately 5% of the yeast genome contains introns , . the scaffolds were classified using the results obtained from blastn according to the mean identity percentage in all of the genes contained in the same scaffold , as follows ( table 4 ) : % i d > 99.0% and e value < 10 = scaffold belongs to s. cerevisiae . % i d < 90.0% and e value < 10 = scaffold does not belong to s. cerevisiae . 99.0% > % i d > 90.0% and e value < 10 = hybrid scaffold . our identity criterion was validated by subjecting the gene sequences from s. cerevisiae s288c to a local alignment against s. eubayanus , and we found that the % i d between these strains was < 90% ( supplementary table s1 ) and the average size of the cds was 1550 bp . approximately 96.8% of the genome was annotated ; 53.93% corresponded to s. cerevisiae , 42.86% were non - cerevisiae and 3.20% remained un - annotated fig . its nuclear genome consists of approximately 32 chromosomes , 16 of which correspond to the s. cerevisiae genome and 16 to the s. eubayanus genome , without considering ploidy . nine scaffolds presented continuous translocations ( scaffolds 1 , 4 , 6 , 23 for the s. cerevisiae sub - genome and 26 , 11 , 17 , 22 and 32 for the s. eubayanus sub - genome ) , which indicate homologous recombination events . data on the chromosome number and size , as well as the number of scaffolds obtained , are consistent with previous reports on lager yeast , , . the following are the supplementary data related to this article.supplementary table s1comparative analysis of saccharomyces cerevisiae s288c and saccharomyces eubayanus.supplementary table s1supplementary table s2saccharomyces sp . 790 and saccharomyces cerevisiae comparative results.supplementary table s2 comparative analysis of saccharomyces cerevisiae s288c and saccharomyces eubayanus .

fragments of tracheal epithelium alone or in continuity with connective tissues , can be maintained in culture medium and used for short term or long term studies of toxicity of a variety of chemicals . large numbers of uniform cultures are prepared with the aid of a slicing device or by application of simple method for dissecting sheets of epithelium free from underlying cartilage . the cultures may be placed in an exposure chamber - incubator mounted on a microscope stage and monitored continually for ciliostasis and exfoliation of cells . morphology is further studied by fixation of selected specimens and preparation for light microscopy and electron microscopy . synthetic functions are evaluated by autoradiographic measurement of incorporation of radioactive precursors into macromolecules and other dynamic features are indirectly assessed by histochemical and histoenzymatic methods . short - term studies using these several techniques have shown that ciliostasis does not correlate with cell injury in all instances , and a long - term study has demonstrated dose dependence of a cytotoxic agent when duration of culture viability is measured . the method lends itself to a broad range of investigations in which dose , period of exposure , and role of cofactors must be independently and quantitatively assessed.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12 .

radiotherapy is one of the major clinical approaches for treatment of bone cancer pain . activation of camp - pka signaling pathway plays important roles in bone cancer pain . here , we examined the effects of radiotherapy on bone cancer pain and accompanying abnormal activation of camp - pka signaling . female sprague - dawley rats were used and received tumor cell implantation ( tci ) in rat tibia ( tci cancer pain model ) . some of the rats that previously received tci treatment were treated with x - ray radiation ( radiotherapy ) . thermal hyperalgesia and mechanical allodynia were measured and used for evaluating level of pain caused by tci treatment . pka mrna expression in dorsal root ganglion ( drg ) was detected by rt - pcr . concentrations of camp , il-1 , and tnf- as well as pka activity in drg and the spinal cord were measured by elisa . the results showed that radiotherapy significantly suppressed tci - induced thermal hyperalgesia and mechanical allodynia . the level of pka mrna in drg , camp concentration and pka activity in drg and in the spinal cord , and concentrations of il-1 and tnf- in the spinal cord were significantly reduced by radiotherapy . in addition , radiotherapy also reduced tci - induced bone loss . these findings suggest that radiotherapy may suppress bone cancer pain through inhibition of activation of camp - pka signaling pathway in drg and the spinal cord .

pain is one of the most prevalent symptoms in patients with primary bone sarcomas and with the distant metastases of nonbone primary tumors [ 1 , 2 ] . studies have indicated that bone cancer pain has complex and unique mechanisms that may involve both inflammatory and neuropathic pain . clinically , most patients with bone cancer have already passed the optimal time for radical surgery and multidisciplinary therapies . however , with radiotherapy , majority of these patients experience pain relief , control of tumor growth , and prolonged survival . radiotherapy is not only an effective method in the clinical treatment of bone cancer , but also an important approach for treatment of the severe pain associated with bone cancer . however , mechanisms underlying radiotherapy for bone cancer pain have not been well investigated and remain elusive . cyclic adenosine monophosphate - protein kinase a ( camp - pka ) signaling pathway plays important roles in a number of cellular processes , including immune function , growth , differentiation , and metabolism , and is essential to the plasticity in neural synapses in cns . activation of camp - pka pathway has been reported to enhance presynaptic neurotransmitter synthesis and vesicular transportation probably through phosphorylation of key transcriptional factors ( i.e. , camp response element - binding protein ) and synaptic vesicle proteins [ 911 ] . in addition , recent studies found that camp - pka signaling pathway is involved in both inflammatory pain [ 1214 ] and neuropathic pain [ 1517 ] . the peripheral hyperalgesic actions of inflammatory mediators are mediated by the camp - pka signaling pathway . we have recently demonstrated that the camp - pka pathway is crucial for the maintenance of dorsal root ganglia ( drg ) neuronal hyperexcitability and behaviorally expressed hyperalgesia , in an in vivo neuropathic pain animal model of chronic compression of the drg ( ccd model ) , as well as in an in vitro model of acute drg dissociation [ 15 , 16 , 18 ] . recently , we have further found that activation of the camp - pka signaling pathway plays an important role in both induction and maintenance of bone cancer pain in rats . however , it remains unknown whether and then how camp - pka signaling would contribute toward radiotherapy treatment for bone cancer pain . this study provides evidence supporting an idea that radiotherapy may suppress bone cancer pain through inhibition of abnormal activation of camp - pka signaling pathway in drg and the spinal cord . female adult sprague - dawley rats ( 160180 g at the start of the experiment ) were housed in a controlled lighting environment with free access to food and water . all experiments were approved by the institutional animal care and use committees in oriental hospital and conducted in accordance with the declaration of the national institutes of health guide for the care and use of laboratory animals ( publication number 85 - 23 , revised 1985 ) . surgery was performed under anesthesia with intraperitoneal injection of sodium pentobarbital ( 50 mg / kg , i.p . ) . the protocols of the bone cancer pain model were similar to that described previously [ 1921 ] . in brief , following induction of general anesthesia with intraperitoneal injection of sodium pentobarbital , rats were placed abdominal side up . after disinfecting with 75% v / v ethanol , a one - centimeter rostrocaudal incision was made in the skin directly above the top half of the tibia . tumor cells ( 1 10 cells/l , 5 l ) , extracted from the ascetic fluid of female rats that received walker 256 mammary gland carcinoma cells , were injected ( tumor cell implantation , tci ) into the intramedullary space of the right tibia to induce bone cancer . rats in the sham group were injected with the same number of boiled tumor cells . rats were immobilized in an acryl jig , and a dose of 6 gy was delivered to the right tibia area using a collimator system with 6 mv x - rays . sham radiotherapy applied in tci rats was using the same protocol , except that they were not given the real x - ray radiation . on the 17th day after tci treatment , radiographic images were taken ( exposure setting : 12 ms , 31 kvp ) using a philips digital radiographer system ( digital diagnost vm ; philips medical systems dmc gmbh , hamburg , germany ) . bone destruction was evaluated on a scale of 05 [ 20 , 23 ] : 0 = normal bone structure without any sign of deterioration ; 1 = small radiolucent lesions in the proximal epiphysis ( <3 ) ; 2 = increased number of radiolucent lesions ( > 3 ) indicating loss of medullary bone ; 3 = loss of medullary bone , plus erosion of the cortical bone ; 4 = full - thickness unicortical bone loss ; and 5 = full - thickness bicortical bone loss and displaced fracture . thermal hyperalgesia was indicated by a significantly shortened latency of foot withdrawal in response to heat stimulation . to determine thermal hyperalgesia , a radiant heat source was focused and delivered on a portion of the hind paw ; the thermal stimuli shut off automatically when hind paw moved ( or after 20 s to prevent tissue damage ) . mechanical allodynia was indicated by a significant decrease in the threshold of paw withdrawal to mechanical indentation of the plantar surface of each hind paw , with a sharp , cylindrical probe . the probe was applied to 6 designated loci distributed over the plantar surface of the foot . the minimal force that induced paw withdrawal was read off the display [ 19 , 24 ] . the experimenters who performed these behavioral tests were blinded to the treatment condition of the animals . the total rna isolated with trizol reagent ( invitrogen , usa ) was reverse transcribed using m - mlv reverse transcriptase ( takara ) . primer sets were synthesized by integrated dna technologies ( sangon biotech ) ; their sequences are shown in table 1 . the amplification conditions were set as follows : 94c for 2 min and 30 cycles of 94c for 30 s and 58c for 40 s. pcr products were analyzed on agarose gel electrophoresis and were verified by dna sequencing . the gels were imaged with tanon 2500 imaging systems and analyzed by analysis software imagej 1.48u . the drg and the spinal cord at segments of l4-l5 ipsilateral to tci were collected on postoperative days 10 and 14 for further neurochemical analysis . commercial enzyme - linked immunosorbent assay kits were used to determine the concentrations of camp , il-1 , and tnf- and activity of pka , according to the manufacturers ' instructions . elisa kit for camp was purchased from cayman chemical ( ann arbor , michigan , usa ) ; elisa kits for il-1 , tnf- , and activated pka were purchased from r&d systems ( minneapolis , minnesota , usa ) . all statistical analyses were carried out using statistical product and service solutions , ver . 15.0 ( spss inc . , alterations in camp mrna and concentration , pka activity , and the levels of il-1 and tnf- were tested using one - way analysis of variance ( anova ) followed by bonferroni post hoc tests . two - way repeated - measures anova ( days groups ) was used to test the behavioral responses to thermal and mechanical stimuli , followed by bonferroni post hoc tests . . the thresholds of thermal and mechanical withdrawal ipsilateral , but not contralateral , to the tci treatment decreased approximately by 50% during postoperative days 917 compared to sham control . to determine whether radiotherapy treatment affects bone cancer pain , a single dose of x - radiation ( 6 gy ) was applied on the 9th day after operation . such radiotherapy produced long - lasting inhibition of tci - induced thermal hyperalgesia and mechanical allodynia . the inhibition lasted for a week with 1 d delay following the x - radiation application . the peak values of inhibition of the thermal hyperalgesia and mechanical allodynia were approximately 2550% . the thermal and mechanical withdrawal of the hind paw contralateral to tci treatment was not altered following the radiotherapy . no radiographic change ( score = 0 , no bone destruction ) was found in the group of sham without radiotherapy . bone destruction was seen clearly in groups of tci with and without radiotherapy , respectively . score of the tci group with sham therapy was 3.75 0.43 ( range : 35 ) . the score in the group of tci with radiotherapy dropped to 2.38 0.52 ( range : 13 ) , which was significantly less than the score in tci group with sham therapy . our previous studies have shown that expression of pka - rii and pka - c mrnas was increased after tci treatment in a time - dependent manner . to test whether radiotherapy has an effect on this tci - induced pka mrna expression , we measured the levels of pka - rii and pka - c mrnas on day 1 and day 5 after radiotherapy treatment ( postoperative days 10 and 14 , resp . ) using rt - pcr . we found that radiotherapy treatment greatly inhibited tci - induced increase of expression of pka - rii and pka - c mrna . the expression of pka - rii and pka - c mrna in the tci + radiotherapy group was significantly reduced compared with the tci + sham group ( figure 3 ) . we have previously shown that camp concentration and pkg activity in drg and the spinal cord were significantly increased in a time - dependent manner after tci treatment . to test the hypothesis that camp - pka signaling pathway might be altered by radiotherapy , we measured camp level and pka activity in drg and the spinal cord following radiotherapy treatment . the results showed that radiotherapy treatment significantly reduced tci - induced increase of camp concentration as well as pka activity in drg and the spinal cord ( figure 4 ) . we have recently shown that il-1 and tnf- levels in the spinal cord were significantly increased in a time - dependent manner after tci treatment . to test whether radiotherapy could affect tci - induced increase of il-1 and tnf- , we measured levels of il-1 and tnf- in the spinal cord after radiotherapy on postoperative days 10 and 14 ( 1 and 5 days after radiotherapy ) . our results showed that radiotherapy treatment significantly reduced tci - induced increase of il-1 and tnf- in the spinal cord ( figure 5 ) . this study demonstrates that radiotherapy is an effective treatment approach for treating bone cancer pain . the camp - pka signaling pathway may be a mechanism that underlies the analgesic effect of radiotherapy in bone cancer pain . radiotherapy suppresses tci - induced painful behaviors , thermal hyperalgesia , and mechanical allodynia and alleviates tci - induced massive bone destruction . radiotherapy reduces tci - induced increased expression of pka mrnas in drg as well as the increased level of camp concentration and pka activity in both drg and the spinal cord . in addition , radiotherapy results in a significant decrease of il-1 and tnf- activity in the spinal cord . these findings suggest that radiotherapy treatment may suppress tci - induced hyperalgesia and allodynia by inhibiting the camp - pka signaling pathway in drg and the spinal cord . approximately 50% of these patients experience moderate to severe pain . mechanisms of cancer pain are thought to be complex and may involve a combination of inflammation , nerve injury , and other unique factors . considerable evidence of cancer pain treatment shows that radiotherapy effectively relieves pain in up to 95% of patients and can maintain the level of analgesia in more than 70% of the patients for up to three months [ 2729 ] . previous studies have shown that activation of the camp - pka signaling pathway contributes to noxious stimulus - induced peripheral and central sensitization [ 3032 ] . we have found that in vivo chronic compression of drg or in vitro acute drg dissociation resulted in activation of the camp - pka signaling pathway . continued activation of the camp - pka signaling pathway is required to maintain hyperexcitability of the drg neurons and behaviorally expressed hyperalgesia in these two different injury - related stress conditions [ 15 , 17 ] . recently , we have confirmed that activation of the camp - pka pathway plays an essential role in the induction and maintenance of bone cancer pain [ 19 , 25 ] . here , we provide direct evidence that the elevated activity of camp - pka signaling pathway is significantly decreased by radiotherapy in a bone cancer pain model . these results support an idea that radiotherapy may suppress bone cancer pain through inhibition of abnormal activation of camp - pka signaling pathway , suggesting a new mechanism for the radiotherapy of bone cancer pain . the proinflammatory cytokines are activators of the camp - pka pathway in primary afferent neurons [ 25 , 3335 ] . during tumor growth and development of bone cancer pain , certain proinflammatory cytokines such as tnf- and il-1 are activated and released from the astrocytes and microglial cells and contribute to bone cancer pain [ 25 , 36 , 37 ] . radiotherapy can reduce these proinflammatory cytokines and inhibit activation of the camp - pka signaling pathway and thus result in relief of bone cancer pain . these findings suggest that the cytokines are also targets that may be responsible for radiotherapy - induced analgesia . in addition , our results showed that radiotherapy resulted in less bone loss in tci rats . this is consistent with the previous finding that radiotherapy with high dose may kill part of the tumor cells or reduce their activation and thus delay the destruction of the bone structure [ 38 , 39 ] . these findings indicate that radiotherapy may reduce both pain and loss of bone structure following tci treatment . our results demonstrate that radiotherapy can effectively suppress bone cancer pain probably through inhibition of activation of camp - pka signaling pathway in the primary sensory neurons and the spinal cord . this study may suggest a new mechanism underlying radiotherapy - induced analgesia of bone cancer pain and support the clinical use of radiotherapy in treatment of certain cancer pain conditions .

creatine kinase ( ck ; ec 2.7.3.2 ) is related to several skin diseases such as psoriasis and dermatomyositis . ck is important in skin energy homeostasis because it catalyzes the reversible transfer of a phosphoryl group from mgatp to creatine . in this study , we predicted ck binding proteins via the use of bioinformatic tools such as protein - protein interaction ( ppi ) mappings and suggest the putative hub proteins for ck interactions . we obtained 123 proteins for brain type ck and 85 proteins for muscle type ck in the interaction networks . among them , several hub proteins such as nfkb1 , fhl2 , myoc , and asb9 were predicted . determination of the binding factors of ck can further promote our understanding of the roles of ck in physiological conditions .

creatine kinase ( ck ) ( atp : creatine kinase n - phosphotransferase , ec 2.7.3.2 ) is thought to be crucial for intracellular transport and the storage of high energy phosphate because it catalyzes the reversible transfer of a phosphoryl group from mgatp to creatine , which leads to the creation of phosphocreatine and mgadp . ck plays an important role in the cellular energy metabolism of vertebrates , and it is widely distributed in tissues that require a lot of energy . several types of ck are expressed in various tissues : the muscle and brain types of ck are the most common , and three different isoenzymes that include ck - mm ( the muscle type homodimer ) , ck - bb ( the brain type homodimer ) , and ck - mb ( the muscle plus brain type heterodimer ) originate from these two common types . various types of cks ( the muscle , brain , and mitochondrial types ) are thought to be important not only in the diagnosis of myocardial infarction , cardiac hypertrophy , and muscular dystrophy but also for studies of some other serious diseases , including alzheimer 's disease , parkinson 's disease , and psoriasis [ 38 ] . ck - bb is associated with several pathologies , including neurodegenerative and age - related diseases . recently , chang et al . reported an important role for ck - bb in osteoclast - mediated bone resorption , which was found using a proteomics approach . they found that ck - bb is greatly increased during osteoclastogenesis and suggested that it represents a potential target for antiresorptive drug development . ck - bb interacts with the potassium - chloride cotransporter 3 , which is involved in the pathophysiology of hereditary motor and sensory neuropathy with agenesis of the corpus callosum . previous studies [ 11 , 12 ] have reported that ck - bb is involved in alzheimer 's disease ( ad ) as an oxidatively modified protein . this suggests that oxidatively damaged ck - bb may be associated with aging and age - related neurodegenerative disorders such as ad . ck - mm is a good model to use for studying folding pathways because of several characteristics : ( i ) it is a dimer that consists of two identical subunits , each with an n - terminal domain with about 100 residues and a c - terminal domain with about 250 residues connected by a long linker ; ( ii ) extensively denatured ck can be renatured spontaneously with restoration of its enzymatic activity in the absence of any external assistance ; ( iii ) its folding pathway is complicated and involves several intermediates [ 15 , 16 ] ; ( iv ) conformational changes of the secondary and tertiary structures can be easily measured by monitoring activity changes [ 14 , 15 ] ; ( v ) protein - protein interactions , including molecular chaperones , are observed during refolding [ 17 , 18 ] . in this study , we obtained computational predictions of the binding proteins by using two types of ck ( ck - bb and ck - mm ) as hub proteins in bioinformatic algorithms . as a result , we obtained 208 protein lists in the interaction networks via application of both muscle and brain types of ck . determination of the binding factors and functions of ck can further promote our understanding of the physiological roles of ck . we present the functionally classified protein - protein interactions on the basis of the cell cycle , cell transport , oxidoreductase , and apoptosis . the protein interaction resources included six databases : dip , bind , intact , mint , hprd , and biogrid . they are ( 1 ) protein structural interactome map ( psimap ) , a method that uses the structural domain of the scop ( structural classification of proteins ) database and ( 2 ) protein experimental interactome map ( peimap ) , a common method that uses public resources of experimental protein interaction information such as hprd , bind , dip , mint , intact ; and biogrid . the basic procedure of psimap is to infer interactions between proteins by using their homologs . interactions among domains or proteins for known pdb ( protein data bank ) structures are the basis for the prediction . if an unknown protein has a homolog to a domain , then psimap assumes that the query has the probability to interact with its homolog 's partners . we carried out a redundancy check to remove identical protein sequences from the source interaction databases . we identified potential candidates through protein - protein interaction predictions made using various protein interaction resources . by analyzing the hub protein of the networks with metrics such as degree and centrality , we detected 123 potential candidates for ckb interacting ( direct or indirect ) factors and 85 candidates for ckm . in figure 1 , interacting factors such as nfkb1 ( np_003989 , nuclear factor of kappa light polypeptide gene enhancer in b - cells 1 ) , myoc ( np_000252 ; myocilin , trabecular meshwork inducible glucocorticoid response ) , myom2 ( np_003961 ; myomesin ( m - protein ) 2 , 165 kda ) , fhl2 ( np_001034581 , four - and - a - half lim domains 2 ) , hif1an ( np_060372 , hypoxia - inducible factor 1 , alpha subunit inhibitor ) , asb9 ( np_076992 , ankyrin repeat and socs box - containing 9 ) , and ckm ( np_001815 , creatine kinase , muscle ) were elucidated . interestingly , nfkb1 was detected as a hub protein interacting with ck - bb in our results . in figure 2 , we obtained results similar to those from figure 1 , where nfkb1 , myoc , myom2 , fhl2 , hif1an , asb9 , and ckm were detected as interacting factors that were directly or indirectly associated with ckb . nfkb1 , ckm , and asb9 interacted with ckb directly . in the same way , we detected the ckm - associated proteins as shown in figure 3 with 80% sequence identity . as a result , we found that ckb , fhl2 , myoc , asb9 , hif1an , nfkb1 , ttn ( np_596870 , titin ) , myh9 ( np_002464 , myosin , heavy chain 9 , non - muscle ) , and itga7 ( np_002197 , integrin , alpha 7 ) mainly interacted with ckm at 80% sequence identity . at the level of 100% identity , we found that myom2 , ckb , fhl2 , and myoc directly interacted with ckm as shown in figure 4 . in addition to these factors , complete lists of factors that interacted with ckb and ckm in a direct or indirect manner are shown in tables 1 and 2 . after overlapping the results from figures 1to 4 , we found that nfkb1 , fhl2 , and myoc were still detected as hub proteins in figure 5 . nfkb1 ( also known as p50 or nf - kappab ) is a well - known transcription regulator that is responsible for the expression and regulation of many genes for immune response , cell adhesion , differentiation , proliferation , angiogenesis , and apoptosis [ 2731 ] . it translocates into the nucleus and stimulates the expression of many genes involved in various biological functions . nfkb1 is also associated with a number of inflammatory diseases such as lymphoma , alzheimer disease , psoriatic arthritis , breast cancer [ 35 , 36 ] , and rheumatoid arthritis . activation of nfkb1 requires binding of nf - kappab essential modulator ( nemo ) to ubiquitinated substrates . with respect to an association with ck , it has been reported that nfkb1 is mostly associated with myocardial ischemia / reperfusion . during reperfusion , the absence of poly(adp - ribose ) polymerase-1 ( parp-1 ) leads to a reduction of myocardial apoptosis , which is associated with reduced nfkb1 activation [ 39 , 40 ] , and proteasome inhibition ablates activation of nfkb1 in myocardial reperfusion and reduces reperfusion injury . myocardial injury was assessed by measuring the serum levels of ck , and ck was reduced in serum along with reduction of nfkb1 activation . fhl2 is a member of the human four - and - a - half - lim - only protein family , which consists of the members fhl1 , fhl2 , fhl3 , fhl4 , and act . these proteins function in various cellular processes , including regulation of cell survival , transcription , and signal transduction . fhl2 contains an lim domain , one of the protein - protein interaction motifs , which allows specific proteins to combine with certain partners . the specificity of a protein - protein interaction can be obtained by an interaction code predicted by conserved amino acid sequences . the interaction of fhl2 with transcription factors and other proteins involved in cancer development was examined . since transcription factors control all fundamental developmental and homeostatic processes , transcriptional cofactors such as fhl2 are likely to contribute to human carcinogenesis and are of clinical importance in various forms of cancer , including leukemia . with respect to an association with ck , chung et al . reported that fhl2 ( developmentally enhanced phosphotransfer enzyme - anchoring protein ) amalgamated the myofibrillar ck metabolic signaling circuit , providing an energetic continuum between mitochondria and the nascent contractile machinery in a murine embryonic stem cell cardiac differentiation model . they reported that ck - m clustered around developing myofibrils , sarcolemma , and the perinuclear compartment , whereas ck - b was tightly associated with myofibrillar alpha - actinin , forming wire - like structures extending from the nuclear compartment to the sarcolemma . fhl2 was also increased in myocardial ischemia - reperfusion injury , where il-6 and il-8 mrna are upregulated in human cardiac myocytes . the ankyrin repeat domains of asb9 can associate with the substrate binding site of ck in a socs box - independent manner . asb9 is a member of the ankyrin repeat and is a suppressor of the cytokine signaling ( socs ) box protein family . it can interact with the socs box domain of the elongin b - c adapter complex and can further complex with the cullin and ring box proteins to form e3 ubiquitin ligase complexes . these complexes may be involved in specific substrate - recognition for ubiquitination and degradation and mediate the substrate - recognition of the e3 ubiquitin ligases . however , myoc has a cytoskeletal function , and this implies that it may interact with ck somehow . myoc is expressed in many ocular tissues including the trabecular meshwork , which is a specialized eye tissue that is essential in regulating intraocular pressure . myoc mutations have been identified as the cause of hereditary juvenile - onset open - angle glaucoma . researchers could apply computational prediction by ppi mapping to help determine target proteins . since the next step in the functional study of interesting proteins / genes is a time- and cost - consuming process , the number of target proteins is limited ; hence , for the right choice , computational prediction on the basis of database information could be critical at this step . functional studies can be further conducted using a mouse model and a large number of clinical samples . final confirmation and ck mechanisms could then be more clearly evaluated for developing drugs to effectively treat ck - related diseases . the functions of most of the candidate proteins predicted in this study have not been well reported in skin diseases or in the pathogenesis of other diseases . we provide new information regarding these candidate proteins ' interaction with ck , as well as the involvement of several hub proteins such as nfkb1 , fhl2 , asb9 , and myoc . although we do not suggest a direct role of any candidate protein in skin diseases , we provide candidate proteins to be targeted in further studies of ck - associated diagnostic markers and/or treatment of corresponding skin conditions . furthermore , we also provide some insights into understanding the responses of ck in skin .

acrometastasis to the hand is an unusual presentation which might mimic an infectious , inflammatory , or a metabolic pathology . we herein describe a case series of three patients of acrometastasis to the hand . we encountered three cases of acrometastasis to the hand attending the departmental clinics from 2007 to 2010 . the median age at presentation was noted to be 55 years . all were males . the primaries included squamous cell carcinoma of the skin , larynx , and esophagus . in two patients , acrometastasis was detected at presentation and in one it was detected 2 years postcompletion of radical therapy . two patients were offered palliative radiation to acrometastasis , and best supportive care was given to one . palliation achieved after radiation was noted to be modest to good . the brief report highlights the importance of the clinical awareness of metastatic dissemination to unusual sites in the face of increasing cancer survivorship . acrometastasis portends a poor prognosis with limited survival , and optimal integration of the best supportive care is mandatory . a short course of hypofractionated palliative radiation therapy results in modest to good palliation .

we herein describe a series of three cases of acrometastasis to the hand [ table 1 ] . we have also reviewed the pathogenesis , clinical presentation , and therapeutic management of acrometastasis to the hand . a 60-year - old gentleman presented to our clinic with the chief complaints of a nonhealing ulcer in the right groin for the last 8 years and a growth in the right thumb for the last 6 months . on examination , an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin was noted [ figure 1 ] . there was a presence of two firm , mobile , and tender lymph nodes ( 4 4 cm and 2 2 cm , respectively ) in the left horizontal inguinal chain . besides , a 3 3 cm ulceroproliferative growth involving the distal phalanx of the right thumb with subungual and periungual involvement was seen [ figure 2 ] . an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin a 3 3 cm ulceroproliferative growth involving the distigal phalanx of the right thumb with subungual and periungual involvement fine needle aspiration cytology ( fnac ) of the left inguinal lymph node was suggestive of metaststic squamous cell carcinoma . a skiagram of the hand revealed a lobulated , homogenous soft tissue mass around the distal phalanx of the right thumb with no definite evidence of bony erosion . a contrast - enhanced ct scan of the chest and whole abdomen was unremarkable excepting the findings of enlarged left inguinal lymphadenopathy . a short course of palliative radiation 30 gy/10 fractions for 2 weeks to the primary lesion and metastatic lymphadenopathy was delivered . this was followed by radiation of 54 gy/27 fractions for 5.5 weeks to the site of acrometastasis . a 55-year - old gentleman was diagnosed with carcinoma supraglottic larynx ( stage t4n1m0 ) . he was treated with combined modality therapy : radiation 66 gy/33 fractions for 6.5 weeks with concurrent cisplatin 100 mg / mq 3 week . subsequently , he presented with swelling of the tip of all fingers of the left hand with nail bed involvement [ figure 3 ] , and multiple subcutaneous nodules in the upper and lower limb . fnac of acral lesions was suggestive of metastatic carcinoma . a contrast - enhanced ct scan of the chest , abdomen , and pelvis revealed metastasis in bilateral lungs and liver [ figures 4 and 5 ] in view of the poor performance status ( ecog 3 ) and widespread dissemination of disease , he was offered best supportive care . metastatic swelling of all five distal phalanges of the left hand multiple bilateral lung metastasis multiple liver metastasis a 52-year - old gentleman presented to our clinic with the complaints of dysphagia to solids progressing to liquids , headache , and a nodule in the little finger of the left hand for the last 6 months [ figure 6 ] . on endoscopy , a friable tumor involving half of the circumference of the esophageal lumen was seen 25 - 30 cm from the incisors . endoscopic biopsy revealed moderately differentiated squamous cell carcinoma . a contrast - enhanced ct scan of the chest showed circumferential mural thickening of the midesophagus with mediastinal lymphadenopathy . fnac of the nodule involving the distal phalanx of the little finger of the left hand was suggestive of metastatic squamous cell carcinoma . he underwent palliative radiation to esophagus and acrometastasis , 30 gy/10 fractions for 2 week . a 60-year - old gentleman presented to our clinic with the chief complaints of a nonhealing ulcer in the right groin for the last 8 years and a growth in the right thumb for the last 6 months . on examination , an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin was noted [ figure 1 ] . there was a presence of two firm , mobile , and tender lymph nodes ( 4 4 cm and 2 2 cm , respectively ) in the left horizontal inguinal chain . besides , a 3 3 cm ulceroproliferative growth involving the distal phalanx of the right thumb with subungual and periungual involvement was seen [ figure 2 ] . an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin a 3 3 cm ulceroproliferative growth involving the distigal phalanx of the right thumb with subungual and periungual involvement fine needle aspiration cytology ( fnac ) of the left inguinal lymph node was suggestive of metaststic squamous cell carcinoma . a skiagram of the hand revealed a lobulated , homogenous soft tissue mass around the distal phalanx of the right thumb with no definite evidence of bony erosion . a contrast - enhanced ct scan of the chest and whole abdomen was unremarkable excepting the findings of enlarged left inguinal lymphadenopathy . a short course of palliative radiation 30 gy/10 fractions for 2 weeks to the primary lesion and metastatic lymphadenopathy was delivered . this was followed by radiation of 54 gy/27 fractions for 5.5 weeks to the site of acrometastasis . a 55-year - old gentleman was diagnosed with carcinoma supraglottic larynx ( stage t4n1m0 ) . he was treated with combined modality therapy : radiation 66 gy/33 fractions for 6.5 weeks with concurrent cisplatin 100 mg / mq 3 week . subsequently , he presented with swelling of the tip of all fingers of the left hand with nail bed involvement [ figure 3 ] , and multiple subcutaneous nodules in the upper and lower limb . fnac of acral lesions was suggestive of metastatic carcinoma . a contrast - enhanced ct scan of the chest , abdomen , and pelvis revealed metastasis in bilateral lungs and liver [ figures 4 and 5 ] there was however no evidence of locoregional disease on clinical examination and laryngoscopy . in view of the poor performance status ( ecog 3 ) and widespread dissemination of disease metastatic swelling of all five distal phalanges of the left hand multiple bilateral lung metastasis multiple liver metastasis a 52-year - old gentleman presented to our clinic with the complaints of dysphagia to solids progressing to liquids , headache , and a nodule in the little finger of the left hand for the last 6 months [ figure 6 ] . on endoscopy , a friable tumor involving half of the circumference of the esophageal lumen was seen 25 - 30 cm from the incisors . endoscopic biopsy revealed moderately differentiated squamous cell carcinoma . a contrast - enhanced ct scan of the chest showed circumferential mural thickening of the midesophagus with mediastinal lymphadenopathy . fnac of the nodule involving the distal phalanx of the little finger of the left hand was suggestive of metastatic squamous cell carcinoma . he underwent palliative radiation to esophagus and acrometastasis , 30 gy/10 fractions for 2 week . due to the increasing longevity of patients afflicted with cancer , there is a surge in the metastatic dissemination of disease , often to unusual sites . the underlying mechanism of the deposition of metastatic cells within the hand is unclear , but an increase in vascularity and trauma has been suggested in the past . it is because of the above - mentioned reasons , healey and colleagues have reported an increased incidence of acrometastasis to the dominant hand . in most patients , the tumor initially metastasizes to the bone and subsequently spreads to the adjacent soft tissue , though the reverse may also be occasionally observed . in a review of 257 cases of acrometastasis to the hand by flynn and colleagues , the median age at presentation was noted to be 58 years and men were twice likely to be affected compared to females . the most common primaries were in the lung ( 44% ) , kidney ( 12% ) , and breast ( 10% ) whereas the remaining cases had primaries in the colon , stomach , liver , prostate , and rectum . the common sites of involvement in the hand were noted to be distal phalanx ( 74 lesions ) , metacarpals ( 56 lesions ) , proximal phalanx ( 26 lesions ) , and middle phalanx ( 16 lesions ) . bronchogenic carcinoma usually led to monoostotic lytic lesions , whereas polyostotic sclerotic , lytic , or mixed lesions were preponderant in carcinoma of the breast . acrometastasis to the hand can be asymptomatic or can present with painful swelling and movement restriction . subungual metastasis may present with a painful erythematous enlargement of the distal digit , or a red , violaceous nodule leading to nail dystrophy . the common differentials include acute infection in the form of abscess , felon , or paronychia ; underlying osteomyelitis ; or metabolic conditions like gout and pseudogout . as acrometastasis generally accompanies widespread disease , the prognosis is poor with an anticipated survival of 6 months . amputation , wide excision , curettage , cementation , radiotherapy , and chemotherapy are the therapeutic options in this rare presentation . solitary acrometastasis to the thumb from cutaneous squamous cell carcinoma of the groin is indeed a rarity.[68 ] hematogenous dissemination to the skin and subcutaneous tissue of the thumb without underlying osseous involvement is all the more striking . tumor implantation due to contact is not unknown in squamous cell carcinoma and is another possibility . taking into account the advanced age , performance status ( ecog 2 ) , and lack of family support , a course of palliative radiation to the primary and acrometastasis was considered in the patient . cutaneous metastasis has been reported to occur in 12 % of patients with squamous cell carcinoma of the head and neck and accounts for less than 10% of all distant metastasis in such cases . a review of the surgical literature revealed only seven previously reported cases of cutaneous metastases from squamous cell carcinoma of the larynx . the common sites of cutaneous metastasis include neck , chest , scalp , face , lips , axilla , areola , back , arms , and digits . it is evident on the literature search that multiple metastases from laryngeal carcinoma involving all five distal phalanges of hand , bilateral lungs , and liver have not been reported till date . considering the poor performance status and widespread metastases in the second case , the best supportive care was offered to the patient . the third patient in the series had an esophageal primary with simultaneous acrometatsasis . following palliative radiation to esophagus and acrometatsasis , a brief course of systemic chemotherapy the patient had progressive disease and the best supportive care was considered in his case . acrometastasis to the hand is an unusual presentation which might mimic an infectious or inflammatory pathology . clinical awareness of unusual sites of metastatic dissemination is a must in the face of increasing cancer survivorship . acrometastasis portends a poor prognosis with limited survival , and optimal integration of the best supportive care is mandatory .

we report on the thermally induced changes of the nano - structural and optical properties of hydrogenated nanocrystalline silicon in the temperature range 200700 c . the as - deposited sample has a high crystalline volume fraction of 53% with an average crystallite size of ~3.9 nm , where 66% of the total hydrogen is bonded as si h monohydrides on the nano - crystallite surface . a growth in the native crystallite size and crystalline volume fraction occurs at annealing temperatures 400 c , where hydrogen is initially removed from the crystallite grain boundaries followed by its removal from the amorphous network . the nucleation of smaller nano - crystallites at higher temperatures accounts for the enhanced porous structure and the increase in the optical band gap and average gap .

hydrogenated nanocrystalline silicon ( nc - si : h ) has been the subject of intense scientific and technological interest over the past decade , mainly due to its reduced photo - induced degradation , efficient visible photoluminescence , tailored optical band gap , increased conductivity and greater doping efficiency . it has been highlighted that these unique features are a direct cause of the quantum size effects of the silicon nano - crystallites . these improvements make nc - si : h a potential candidate for application in photovoltaic and opto - electronic devices . the hot - wire chemical vapour deposition ( hwcvd ) technique , based on the catalytic decomposition of the precursor gasses by a heated transition metal filament , has been established as a viable deposition technique for nc - si : h thin films . the structural and opto - electronic properties of the thin films are dependent on the deposition parameters , of which the hydrogen dilution and substrate temperature are the most crucial . it has been established that the etching effect of atomic hydrogen , created by the catalytic decomposition of h2 , is responsible for the termination of weak si si bonds from the surface and sub - surface regions and that the nucleation of the nano - crystallites are improved by increasing the hydrogen dilution [ 7 - 10 ] . it has also been reported that the hydrogen dilution during deposition determines the concentration and the distribution of hydrogen in nc - si : h , which is closely related to the nano - structural features ; i.e. crystallite size and crystalline volume fraction [ 11 - 14 ] . in particular , the quantum size effects of the si nano - crystallites and the hydrogen concentration have a strong correlation with the optical band gap . an investigation into the role of hydrogen in nc - si : h is therefore crucial for the understanding of its relation to the nano - structure and the optical properties . in this contribution , we investigate the effects of the hydrogen concentration and bonding configuration in nc - si : h deposited by hwcvd on the nano - structural features and the optical properties . the nc - si : h thin film was deposited by the hwcvd process simultaneously on single - side polished 100 crystalline silicon and corning 7059 glass substrates , using a mixture of 4 sccm sih4 and 26 sccm h2 decomposed by seven parallel tungsten filaments , 15 cm apart and 36 cm away from the substrates . the filament temperature , substrate temperature and deposition pressure were fixed at 1600 c , 420 c and 60 bar , respectively . the as - deposited nc - si : h thin film was ~1140 nm - thick , as measured using a veeco profilometer . subsequent annealing was performed under high - purity , flowing n2gas in a tube furnace at annealing temperatures ( ta ) ranging from 200 to 700 c in 100 c increments . the n2flow rate , heating rate and dwell time for all temperatures amounted to 300 sccm , 10 c / min and 30 min , respectively . after each annealing temperature , the thin film was allowed to cool to room temperature in the tube furnace , while maintaining the n2flow rate . thereafter the required analytical techniques were performed . fourier transform infrared ( ftir ) absorption spectra were collected in transmission geometry from 400 to 4000 cmwith a spectral resolution of 1 cm , using a perkin - elmer spectrum 100 ftir spectrophotometer . the structural properties were investigated using a jobin - yvon hr800 micro - raman spectrometer in backscattering geometry at room temperature . the raman spectra were collected in the region 1001000 cmwith a spectral resolution of 0.4 cm , using an excitation wavelength of 514.5 nm . x - ray diffraction ( xrd ) spectra were collected in reflection geometry at 2-values ranging from 10 to 90 with a step size of 0.02 , using a phillips pw 1830 x - ray powder diffractometer operating at 45 kv and 40 ma . copper k1radiation with a wavelength of 1.5406 was used as the x - ray source . optical transmission spectra were measured from 200 to 900 nm with a spectral resolution of 1 nm , using a perkin - elmer lamda 750s uv / vis spectrophotometer . hydrogen bonding configurations and to calculate the hydrogen concentration in nc - si : h and related material . the ftir absorption spectrum of the sample in the as - deposited state is shown in fig . the strong absorption bands in the region 9201250 cm is associated with the asymmetric si o si stretching vibration , whereas the peak centred around 2250 cm is assigned to the h sio3 vibration . this is indicative of an oxidation effect caused by its porous - like microstructure , which is a typical feature for nc - si : h thin films . the enhanced absorption band centred around 640 cm is attributed to the rocking vibrations of all bonding configurations of si hx . the absorption bands in the region 19002150 cm is a result of the convolution of several absorption bands associated with the stretching vibrations of si hx in different configurations . the absorption peaks centred around 1985 cm and 2090 cm are assigned to the stretching vibrations of si h monohydrides in the amorphous network ( isolated ) and on the surface of the si nano - crystallites ( clustered ) , respectively , . the weak absorption band centred at ~2130 cm is assigned to the existence of ( = si = h2)n polyhydride complexes on si nano - crystallite grain boundaries . ftir absorption spectrum of the as - deposited sample and the deconvolution of the stretching vibrations ( insert ) to quantify the fraction of h bonded on the surface of nano - crystallites in nc - si : h , we define a structure factor , where i denotes that integrated intensity of each decomposed peak . the total bonded hydrogen concentration ( ch ) was estimated from the integrated absorption of the 640 cm rocking mode using previous reported procedures . in the as - deposited state , ch amounts to ~2 at.% , characteristic for nc - si : h deposited with high hydrogen dilution , where ~66% thereof is bonded on the surface of the nano - crystallites . we propose that this relatively high value for rs is indicative of a high crystalline volume fraction . figure 2shows the plots of the hydrogen concentration and the structure factor as a function of annealing temperature . the hydrogen concentration and structure factor are relatively constant at temperatures below 400 c , demonstrating that the nano - structure is stable in this temperature regime . after annealing at 400 c most of the ( = si = h2)npolyhydride bonds on the grain boundaries of the si nano - crystallites have been terminated and consequently results in an increase in the structure factor . annealing at higher temperatures induce a significant decrease inch , coupled with an increase inrs . we propose that the instability induced atta400 c is related to the growth of the native nano - crystallites and to the nucleation of nano - crystallites in the amorphous network , thereby resulting in an increase in the crystalline volume fraction . it should be noted that no si hxabsorption peaks were identified after annealing at 700 c . ahydrogen concentration andbthe structure factor as a function of annealing temperature raman spectroscopy provides direct nano - structural information quantitatively related to the average nano - crystallite size and the crystalline volume fraction in nc - si : h . figure 3shows the raman spectra of the sample in the as - deposited state and after annealing at specific temperatures . all spectra display the following main features : ( i ) a sharp peak centred around 515 cm , associated with the transverse optic ( to ) mode of the nc - si phase ; ( ii ) the broad shoulder centred around 480 cm , due to the to - mode of the amorphous silicon ( a - si ) phase ; and ( iii ) a smaller shoulder around 505 cm , corresponding to the distribution of crystalline grain boundaries in the sample . raman spectra of the sample in the as - deposited state and after annealing at specific temperatures the crystalline volume fraction , can be estimated from the integrated areas of the afore - mentioned deconvoluted gaussian peaks . the crystallite size is empirically calculated from where is the shift of the 515 cm peak relative to the c - si peak at 520 cm and b = 2.0 cm . the quantitative raman results are summarized in table 1 . in the as - deposited state , the average crystallite size and the crystalline volume fraction amounts to about 3.9 nm and 53% , respectively , and remain relatively constant after annealing at 300 c . these observations reiterate that the nano - structure of the sample remains stable at temperatures below 400 c . a blue shift of the nc - si to - peak , accompanied with a reduction in the intensity of the a - si to - peak is observed at annealing temperatures 400 c , indicative of an increase in the crystallite size and the crystalline volume fraction , respectively , and supports the claims based on the ftir results . crystallite size , crystalline volume fraction and optical properties after specific annealing temperatures xrd was employed as a complimentary method to qualitatively probe the changes in the crystallinity as a function of annealing temperature ( see fig . 4 ) . three preferential orientations in the 111 , 200 and 311 directions are observed . the crystallite size in the as - deposited state , estimated from the full - width - half - maximum ( fwhm ) of the ( 111)-peak , amounts to ~19.5 nm . a narrowing in the fwhm of the ( 111)-peak , accompanied with an increase in its intensity is observed with an increase in annealing temperature . this confirms the increase of the crystallite size and crystalline volume fraction , as probed by raman spectroscopy . xrd spectra of the sample in the as - deposited state and after annealing at specific temperatures the thermally induced nano - structural changes of the nc - si : h thin film can be interpreted as follows , based on the variation of the si the crystalline volume fraction is relatively large and therefore the majority of h is bonded to the surface of the nano - crystallites . the nano - structural properties are stable at temperatures below 400 c , attributed to its large crystalline volume fraction . an initial increase in the native crystallite size is observed after annealing at 400 c , resulting in the removal of hydrogen from the grain boundaries . it is also feasible that smaller crystallites have coalesced into larger crystallites . at higher temperatures , hydrogen is removed preferentially from the amorphous phase , indicative of the nucleation of smaller nano - crystallites of size <3 nm in the amorphous network , undetected by raman spectroscopy and xrd . the optical properties were determined from uv visible transmission measurements performed on the thin film deposited on the corning 7059 glass substrate , using the method proposed by swanepoel . the thickness of the as - deposited sample was calculated to be ~1180 nm , which concurs to that measured by profilometry . the refractive index n( ) of a material is an important optical parameter , since it is directly proportional to density . figure 5 shows the spectral dependence of the calculated refractive index for the sample in the as - deposited state and after annealing at specific temperatures . a slight increase in the refractive index is observed after annealing at 400 c , followed by a decrease at higher temperatures . the initial increase can be ascribed to the increase in the native crystallite size and possibly due to the coalescence of smaller nano - crystallites . furthermore , the presence of ( = si = h2)n complexes in the as - deposited state is indicative of a disordered , porous material and the removal thereof after 400 c would therefore result in a more compact material . the subsequent decrease of the refractive index at higher temperatures is attributed to a more porous structure , possibly caused by the nucleation of smaller nano - crystallites in the amorphous network . similar trends in the refractive index at zero photon energy ( no ) are observed ( see table 1 ) . refractive index spectra of the sample in the as - deposited state and after annealing at specific temperatures detailed analysis of the refractive index spectra were performed using the model suggested by wemple et al . . at energies below than of the optical band gap , the refractive index is related to the square of the photon energy ( h ) by:(1 ) where em and ed is the average gap and dispersion energy , respectively . the plot of 1/n()-1 ] versus ( h ) allows for the determination of emed and no . the extrapolated results of no and em , calculated from the linear fit through the data , are listed in table 1 . the spectral dependence of the absorption coefficient ( ) for the sample in the as - deposited state and after annealing at specific temperatures is depicted in fig . the optical band gap , referred to as e04 , is defined as the photon energy where ( ) = 10 cm , and the values are reported in table 1 . a red shift in e04 is observed for ta 600 c followed by an unexpected blue shift after annealing at 700 c . it is established that the optical band gap of hydrogenated amorphous silicon ( a - si : h ) deposited by hwcvd and pecvd increases with an increase in the hydrogen concentration . it should be noted that the optical band gap for the sample in the as - deposited state is larger than that of a - si : h with similar ch values . this discrepancy is due to the presence of nano - crystallites in the amorphous network , which lowers the absorption in nc - si : h and shifts the optical band gap towards higher energies . the quantum size effect size also predicts that an increase in crystallite size is associated with a decrease in the optical band gap . the initial decrease in e04 after 400 c is due to the combined effect of the decreased ch and the growth in the crystallite size . after annealing at 600 c the initial hydrogen concentration has decreased by ~90% with about the same incremental increase in the crystallite size as at 400 c , and therefore a more notable decrease in e04 is expected . however , a minute 0.03 ev decrease in e04 is observed and is attributed to the nucleation of smaller nano - crystallites in the amorphous network , which explains the competing increasing effect on e04 . after annealing at 700 c , where no hydrogen was detected by ftir spectroscopy , this effect is more pronounced in that an increase in the optical band gap is observed . absorption coefficient spectra of the sample in the as - deposited state and after annealing at specific temperatures the optical band gap and the average gap ( em ) have similar behaviours with respect to annealing temperature , thereby implying that the growth of the native nano - crystallites and the nucleation of smaller crystallites in the amorphous network have similar effects on the band edges and on the conduction and valence bands . therefore , the average gap can be used to describe the thermal induced changes in the optical properties of nc - si : h . hydrogen bonding configurations and to calculate the hydrogen concentration in nc - si : h and related material . the ftir absorption spectrum of the sample in the as - deposited state is shown in fig . the strong absorption bands in the region 9201250 cm is associated with the asymmetric si o si stretching vibration , whereas the peak centred around 2250 cm is assigned to the h sio3 vibration . this is indicative of an oxidation effect caused by its porous - like microstructure , which is a typical feature for nc - si : h thin films . the enhanced absorption band centred around 640 cm is attributed to the rocking vibrations of all bonding configurations of si hx . the absorption bands in the region 19002150 cm is a result of the convolution of several absorption bands associated with the stretching vibrations of si hx in different configurations . the absorption peaks centred around 1985 cm and 2090 cm are assigned to the stretching vibrations of si h monohydrides in the amorphous network ( isolated ) and on the surface of the si nano - crystallites ( clustered ) , respectively , . the weak absorption band centred at ~2130 cm is assigned to the existence of ( = si = h2)n polyhydride complexes on si nano - crystallite grain boundaries . ftir absorption spectrum of the as - deposited sample and the deconvolution of the stretching vibrations ( insert ) to quantify the fraction of h bonded on the surface of nano - crystallites in nc - si : h , we define a structure factor , where i denotes that integrated intensity of each decomposed peak . the total bonded hydrogen concentration ( ch ) was estimated from the integrated absorption of the 640 cm rocking mode using previous reported procedures . in the as - deposited state , ch amounts to ~2 at.% , characteristic for nc - si : h deposited with high hydrogen dilution , where ~66% thereof is bonded on the surface of the nano - crystallites . we propose that this relatively high value for rs is indicative of a high crystalline volume fraction . figure 2shows the plots of the hydrogen concentration and the structure factor as a function of annealing temperature . the hydrogen concentration and structure factor are relatively constant at temperatures below 400 c , demonstrating that the nano - structure is stable in this temperature regime . after annealing at 400 c most of the ( = si = h2)npolyhydride bonds on the grain boundaries of the si nano - crystallites have been terminated and consequently results in an increase in the structure factor . annealing at higher temperatures induce a significant decrease inch , coupled with an increase inrs . we propose that the instability induced atta400 c is related to the growth of the native nano - crystallites and to the nucleation of nano - crystallites in the amorphous network , thereby resulting in an increase in the crystalline volume fraction . it should be noted that no si hxabsorption peaks were identified after annealing at 700 c . ahydrogen concentration andbthe structure factor as a function of annealing temperature raman spectroscopy provides direct nano - structural information quantitatively related to the average nano - crystallite size and the crystalline volume fraction in nc - si : h . figure 3shows the raman spectra of the sample in the as - deposited state and after annealing at specific temperatures . all spectra display the following main features : ( i ) a sharp peak centred around 515 cm , associated with the transverse optic ( to ) mode of the nc - si phase ; ( ii ) the broad shoulder centred around 480 cm , due to the to - mode of the amorphous silicon ( a - si ) phase ; and ( iii ) a smaller shoulder around 505 cm , corresponding to the distribution of crystalline grain boundaries in the sample . raman spectra of the sample in the as - deposited state and after annealing at specific temperatures the crystalline volume fraction , can be estimated from the integrated areas of the afore - mentioned deconvoluted gaussian peaks . the crystallite size is empirically calculated from where is the shift of the 515 cm peak relative to the c - si peak at 520 cm and b = 2.0 cm . the quantitative raman results are summarized in table 1 . in the as - deposited state , the average crystallite size and the crystalline volume fraction amounts to about 3.9 nm and 53% , respectively , and remain relatively constant after annealing at 300 c . these observations reiterate that the nano - structure of the sample remains stable at temperatures below 400 c . a blue shift of the nc - si to - peak , accompanied with a reduction in the intensity of the a - si to - peak is observed at annealing temperatures 400 c , indicative of an increase in the crystallite size and the crystalline volume fraction , respectively , and supports the claims based on the ftir results . crystallite size , crystalline volume fraction and optical properties after specific annealing temperatures xrd was employed as a complimentary method to qualitatively probe the changes in the crystallinity as a function of annealing temperature ( see fig . 4 ) . three preferential orientations in the 111 , 200 and 311 directions are observed . the crystallite size in the as - deposited state , estimated from the full - width - half - maximum ( fwhm ) of the ( 111)-peak , amounts to ~19.5 nm . a narrowing in the fwhm of the ( 111)-peak , accompanied with an increase in its intensity is observed with an increase in annealing temperature . this confirms the increase of the crystallite size and crystalline volume fraction , as probed by raman spectroscopy . xrd spectra of the sample in the as - deposited state and after annealing at specific temperatures the thermally induced nano - structural changes of the nc - si : h thin film can be interpreted as follows , based on the variation of the si the crystalline volume fraction is relatively large and therefore the majority of h is bonded to the surface of the nano - crystallites . the nano - structural properties are stable at temperatures below 400 c , attributed to its large crystalline volume fraction . an initial increase in the native crystallite size is observed after annealing at 400 c , resulting in the removal of hydrogen from the grain boundaries . it is also feasible that smaller crystallites have coalesced into larger crystallites . at higher temperatures , hydrogen is removed preferentially from the amorphous phase , indicative of the nucleation of smaller nano - crystallites of size <3 nm in the amorphous network , undetected by raman spectroscopy and xrd . the optical properties were determined from uv visible transmission measurements performed on the thin film deposited on the corning 7059 glass substrate , using the method proposed by swanepoel . the thickness of the as - deposited sample was calculated to be ~1180 nm , which concurs to that measured by profilometry . the refractive index n( ) of a material is an important optical parameter , since it is directly proportional to density . figure 5 shows the spectral dependence of the calculated refractive index for the sample in the as - deposited state and after annealing at specific temperatures . a slight increase in the refractive index is observed after annealing at 400 c , followed by a decrease at higher temperatures . the initial increase can be ascribed to the increase in the native crystallite size and possibly due to the coalescence of smaller nano - crystallites . furthermore , the presence of ( = si = h2)n complexes in the as - deposited state is indicative of a disordered , porous material and the removal thereof after 400 c would therefore result in a more compact material . the subsequent decrease of the refractive index at higher temperatures is attributed to a more porous structure , possibly caused by the nucleation of smaller nano - crystallites in the amorphous network . similar trends in the refractive index at zero photon energy ( no ) are observed ( see table 1 ) . refractive index spectra of the sample in the as - deposited state and after annealing at specific temperatures detailed analysis of the refractive index spectra were performed using the model suggested by wemple et al . . at energies below than of the optical band gap , the refractive index is related to the square of the photon energy ( h ) by:(1 ) where em and ed is the average gap and dispersion energy , respectively . the plot of 1/n()-1 ] versus ( h ) allows for the determination of emed and no . the extrapolated results of no and em , calculated from the linear fit through the data , are listed in table 1 . the spectral dependence of the absorption coefficient ( ) for the sample in the as - deposited state and after annealing at specific temperatures is depicted in fig . the optical band gap , referred to as e04 , is defined as the photon energy where ( ) = 10 cm , and the values are reported in table 1 . a red shift in e04 is observed for ta 600 c followed by an unexpected blue shift after annealing at 700 c . it is established that the optical band gap of hydrogenated amorphous silicon ( a - si : h ) deposited by hwcvd and pecvd increases with an increase in the hydrogen concentration . it should be noted that the optical band gap for the sample in the as - deposited state is larger than that of a - si : h with similar ch values . this discrepancy is due to the presence of nano - crystallites in the amorphous network , which lowers the absorption in nc - si : h and shifts the optical band gap towards higher energies . the quantum size effect size also predicts that an increase in crystallite size is associated with a decrease in the optical band gap . the initial decrease in e04 after 400 c is due to the combined effect of the decreased ch and the growth in the crystallite size . after annealing at 600 c the initial hydrogen concentration has decreased by ~90% with about the same incremental increase in the crystallite size as at 400 c , and therefore a more notable decrease in e04 is expected . however , a minute 0.03 ev decrease in e04 is observed and is attributed to the nucleation of smaller nano - crystallites in the amorphous network , which explains the competing increasing effect on e04 . after annealing at 700 c , where no hydrogen was detected by ftir spectroscopy , this effect is more pronounced in that an increase in the optical band gap is observed . absorption coefficient spectra of the sample in the as - deposited state and after annealing at specific temperatures the optical band gap and the average gap ( em ) have similar behaviours with respect to annealing temperature , thereby implying that the growth of the native nano - crystallites and the nucleation of smaller crystallites in the amorphous network have similar effects on the band edges and on the conduction and valence bands . therefore , the average gap can be used to describe the thermal induced changes in the optical properties of nc - si : h . the effect of isochronal annealing on the nano - structural and optical properties of nc - si : h , with the emphasis on its relation to the hydrogen distribution and concentration , was investigated . initial changes in the nano - structure are observed after annealing at 400 c , as evident by termination of ( = si = h2)npolyhydrides from the grain boundaries caused by the growth of the native nano - crystallites . at higher temperatures , a further increase in the native nano - crystallite size and the crystalline volume fraction is observed , accompanied with the nucleation of smaller nano - crystallites and the subsequent removal of hydrogen from the amorphous network . at temperatures 600 c the nucleation of the smaller nano - crystallites results in a porous material with an increased optical band gap and average gap , explained by the quantum size effect . the authors acknowledge the financial assistance of the department of science and technology , the national research foundation and the council for scientific and industrial research ( project no : hgera2s ) of south africa .

aims : one of the most common fungal infections infecting humans is candidiasis . belonging to the group of opportunistic infections , it often affects individuals with various debilitating diseases . fluconazole and clotrimazole are two of the commonly used anti - fungal agents for the treatment of oral candidiasis . hence , we planned this study to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis.materials and methods : a total of 180 participants were enrolled in the present study . all the patients of candidiasis were divided broadly into two study groups . group i included patients who were treated with fluconazole mouthrinse whereas group ii included patients who were treated with clotrimazole mouth paint . grading of patient discomfort was done as noted from readings given by the patients . specimen was collection by a swab from the lesional area of the oral cavity from the patients and were incubated in sabouraud 's dextrose agar medium and assessed . all the patients were treated with medication as give to their respective groups . patients were recalled as assessed . all the readings were recorded and analyzed.results:for group i patients , the fungal eradication was 89.5% , whereas for group ii patients , the fungal eradication was 86.7% . no significant results were obtained while comparing the mycological eradiation in patients of the two study groups.conclusion:approximately similar effectiveness in terms of treatment was noted with fluconazole and clotrimazole in treating patients with candidiasis .

candida genus , in particular candida albicans , is the one of the common cause of yeast - associated oral infections . there occurrence has been widely described in the literature . over the last four to five decades the main reason that might attribute for such an increase in its incidence might be the increase in the prevalence of human immunodeficiency virus ( hiv ) and other immuno - compromising states and diseases the distribution of all the members of this genus is ubiquitous and consists of inhabitation of soil as saprophytes , aquatic environment , and even colonization of various animal reservoirs . at 37c , the growth of most of candida spices is prohibited , and therefore , human colonization is not associated with it under normal conditions . within humans , several species persist as commensal microorganism and can act as potential opportunistic pathogen in compromised body status . with respect to the treatment of these oral fungal infections , the most common line of treatment is the removal of the etiologic factor along with suitable anti - fungal therapy . there is a paucity of data regarding the comparative evaluation of the abovementioned anti - fungal agents in the treatment of oral candidiasis . hence , we planned this study to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis . the present study was conducted in the department of oral medicine and radiology of the dental institute and included assessment of 180 participants with chief problem of oral candidiasis from 2010 to 2015 . ethical approval was obtained from the institutional ethical committee and written consent was obtained after explaining in detail the entire research protocol . complete physical examination of the patients was done before starting the treatment therapy of the patients . recording of the time duration of the candidiasis was done along with other personal details of the patients . exclusion criteria for the present study included : pregnant patients , patients who underwent any kind of anti - fungal therapy in the past 1 month , patients who were on barbiturates or anticoagulants in the past 1 month , patients with any known drug allergy , patients with alcohol history , patients with history of any psychiatric disorder . patients who underwent any kind of anti - fungal therapy in the past 1 month , patients who were on barbiturates or anticoagulants in the past 1 month , patients with any known drug allergy , patients with alcohol history , patients with history of any psychiatric disorder . group i included patients who were under treatment with fluconazole therapy whereas group ii included patients who were on clotrimazole therapy . based on the clinical history , visual examination of the lesion area and presence of clinical signs and symptoms , diagnosis of the candidiasis both the signs and symptoms of candidiasis were graded by patients into the following categories based on the severity of the lesion and individual perception : mild , moderate , andsevere . symptoms severity was categorized on the basis patient 's response to discomfort whereas sign 's severity was categorized on the basis of extent of lesion . mild referred to cases which involved localized involvement to one or two oral sites , moderate referred to localized involvement of more than two oral sites , whereas severe cases involved generalized oral candidiasis . swab was taken from the lesion area of the patients and was transferred to the microbiological laboratory in the transport medium for culturing . they were incubated in sabouraud 's dextrose agar medium for assessment of culture growth characteristics . i patients , preparation of fluconazole suspension was done and were given to all the patients in the form of prepared moth rinse . patients were instructed to use the suspension mouth rinse three times a day . in group ii patient , suspension of clotrimazole was given in the form of moth paint and patients were instructed to use it thrice daily . recalling of the patients was done after 2 weeks of continuation of the treatment therapy and was examined thoroughly for the presence of clinical signs and symptoms ; microbial growth was assessed by culturing swab specimens as done earlier before the starting of the treatment . chi - square test and student 's t - test was used for the assessment of level of significance . all samples were examined twice to avoid intraobserver variability and kappa value came out to be 0.8 . in both the study groups , prolonged antibiotic therapy was the most common compromised state associated with the presence of fungal infections [ graph 1 ] . mean age of the patients in groups i and ii was 49.5 years and 51.2 years , respectively [ table 1 and graph 2 ] . in group i , 65% of the patient population was of males whereas remaining 35% were females . among group ii , 62% of patient population was of males while the reaming was of females . among group i patients , before starting the treatment , 81% of the patient population had moderate severity of clinical symptoms , whereas in group ii , 75% of the patient population had moderate severity of symptoms [ table 2 and graph 3 ] . among group i participants , after commencing the treatment , in 98% of the patients , clinical signs and symptoms were absent whereas in group ii participants , 88% of the patients showed absence of clinician signs . significant results were obtained while comparing the signs and symptoms of the patients in the two study groups after the treatment . in group i patients , before and after the treatment , the colony count was 1325.25 and 9.51 , respectively , whereas in the group ii patients , the mean colony count was 996.52 and 21.82 , respectively [ table 3 ] . significant results were obtained while comparing the mean colony count before and after the treatment in both study groups . distribution of all the medically compromised patients in the study groups comparative assessment of the two study groups in terms of various parameters comparative assessment of the two study groups in terms of various parameters comparison of various clinical signs and symptoms in between the patients of the two study group comparison of various clinical signs and symptoms in between the patients of the two study group comparison of colony counts before and after the treatment therapy over the past few decades , there has been an increase in the incidence of occurrence of fungal infections . furthermore , there has been as simultaneous increase in the incidence and occurrence of the predisposing factors . some of the important predisposing factors included xerostomia , antibiotic therapy for prolonged period of time , local chronic trauma or irritation , endocrinal disorders , medically compromised states , etc . treatment of the fungal infections included removal of the etiologic systemic or local factor along with antifungal therapy . fluconazole and clotrimazole are the two commonly used anti - fungal agents for the treatment of fungal lesions . hence , we aimed to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis . in the present study , we observed significant results while comparing the mean difference of colony culture growth before and after the treatment in both the study groups . similar results were obtained by sholapurkar et al . who observed similar findings in their study . o - prasertsawat et al . comparatively evaluated the effectiveness of fluconazole and clotrimazole in the treatment of vulvovaginal candidiasis . they assessed 103 female patients in a single blinded randomized trial and divided them broadly into two study groups . first group consisted of 53 patients and included participants in whom treatment was done by fluconazole , while the other group consisted of 50 patients and included those in which treatment was done by clotrimazole . they did not observe any significant difference in relation to the clinical characteristic in between the two study groups . they observed approximately 79% and 80% mycological cure rates in the two study groups , respectively . they concluded that for the treatment of cases of vulvovaginal candidiasis , fluconazole can be given as an alternative line of treatment . comparatively evaluated the effectiveness of single dose of fluconazole and intravaginal clotrimazole 200 mg per day for 6 days in the patients undergoing treatment for the acute episode of vulvovaginal candidiasis ( vvc ) . they prospectively analyzed 142 patients with were diagnosed with vvc and divided them randomly into two study groups . first group consisted of 70 patients and included those patients who received intravaginal tablet whereas the other group included 72 patients and consisted of participants who were given single dose oral fluconazole . they observed that , at the time of follow - up , during the second visit of the patients , approximately 85% and 81% of the patients of the group receiving fluconazole were cured clinically and mycologically , respectively . similarly in the other study group , approximately 83% and 70% of the study group patients were cured clinically and mycologically , respectively . from the results , they concluded that for the treatment of cases of vvc , oral fluconazole single dose appeared to be a valid mode of treatment . goins et al . compared the effectiveness of nystatin and fluconazole in treating the cases of oral candidiasis . one of the common conditions affecting the young infant group of individuals is oral thrush . one of the frequent problems associated with the administration of nystatin is the frequent cases of recurrence and difficulty encountered in its administration . they randomly analyzed 34 infants and were randomly subjected to either nystatin oral suspension four times a day for 10 days or fluconazole suspension 3 mg / kg in a single daily dose for 7 days . they observed that among the patients treated with nystatin , the clinical curing rate was 32% , while in the other group , a success rate of 100% was observed . significant results were obtained while comparing the two anti - fungal agents . from the results assessed the efficacy of fluconazole mouthrinse and clotrimazole mouthpaint in the treatment of cases of oral candidiasis . they observed significant difference while comparing the fungal growth on culture media before and after the treatment . authors advocate the use of fluconazole and clotrimazole for treating oral candidiasis patients , thereby helping in improving the quality of life of the patients . sample size was smallunderlying etiologic factors for the occurrence of candidiasis were not explored . sample size was small underlying etiologic factors for the occurrence of candidiasis were not explored . the treatment therapy might differ depending upon the etiologic cause of candidiasis which was not taken as a parameter for the present study . authors advocate the use of fluconazole and clotrimazole for treating oral candidiasis patients , thereby helping in improving the quality of life of the patients . sample size was smallunderlying etiologic factors for the occurrence of candidiasis were not explored . sample size was small underlying etiologic factors for the occurrence of candidiasis were not explored . the treatment therapy might differ depending upon the etiologic cause of candidiasis which was not taken as a parameter for the present study . from the above results , the authors obtained significant results while using both the fluconazole and clotrimazole in treating patients with oral candidiasis . however , future studies are required in the same field for better exploration of results .

objective . to determine the prevalence of left ventricular hypertrophy ( lvh ) and its associated risk factors in lupus nephritis ( ln ) patients . methods . 287 ln patients ( age : 38.54 13.31 , 262 female ) were recruited . echocardiography and serum high - sensitivity c - reactive protein ( hs - crp ) were measured . their relationship was evaluated by univariate correlation analysis and multivariate regression analysis . results . the prevalence of lvh in this cohort was 21.25% ( n = 61 ) . serum hs - crp level was significantly elevated in patients with lvh compared to those without ( 8.03 ( 3.2230.95 ) versus 3.93 ( 1.489.48 ) mg / l , p < .01 ) , and correlated with left ventricular mass index ( lvmi ) ( r = 0.314 , p = .001 ) . multivariate regression analysis further confirmed that hs - crp was an independent risk factor ( = 0.338 , p = .002 ) for lvh in patients with ln . conclusions . our findings demonstrated that serum hs - crp level is independently correlated with lvmi and suggested that measurement of hs - crp may provide important clinical information to investigate lvh in ln patients .

alterations in left ventricular structure and function have been reported among the cardiac manifestations of systemic lupus erythematosus ( sle ) , especially in those who have renal complications . these alterations include echocardiographic evidence of increases in lv wall thicknesses and mass , a decrease in lv ejection fraction , and impaired diastolic filling [ 13 ] . however , it is currently uncertain whether these abnormalities are disease - related effects or a result of other predisposing conditions , such as inflammation , hypertension , anemia , and disorder of mineral metabolism . in recent years , there has been a growing interest in the hypothesis that atherosclerosis may be an inflammatory disease . it has been noted that c - reactive protein ( crp ) , a marker of the reactant plasma protein component of the inflammatory response , is a major predictor of cardiovascular disease ( cvd ) in apparently healthy subjects [ 46 ] . previous reports have found the association between crp and left ventricular hypertrophy ( lvh ) in several pathologic states such as hypertension , insulin resistance , and chronic kidney disease ( ckd ) [ 79 ] . in this study , we investigated the potential interrelationships among hs - crps , a more sensitive marker of systemic inflammation and lv mass index ( lvmi ) in patients with lupus nephritis ( ln ) by using the clinical cutoff levels of crp . a total of 287 incipient ln patients were consecutively enrolled from january 2005 to december 2008 . all participants met the diagnostic criteria of the american college of rheumatology [ 10 , 11 ] . exclusion criteria included ischemic heart disease , acute coronary syndrome , congestive heart failure ( chf ) ( new york heart association ( nyha ) class ii or greater ) , old cerebral infarction , history of transient ischemic attack , secondary hypertension , receipt of any immunosuppressant and/or an anti - inflammatory drug ( aspirin or nonsteroidal anti - inflammatory drug ( nsaid ) ) , chronic infection , cancer , and pregnancy . the study protocol was approved by the local ethics committee , and all participants gave their written informed consent to participate in this study . after fasting overnight , bp was measured with an appropriate arm cuff and a mercury column sphygmomanometer on the left arm after a resting period of at least 10 min in the supine position . the following parameters were also determined : serum creatinine , serum lipids including cholesterol , triglyceride , and lipoprotein(a ) , measurement of serum complement c3 and c4 , high - sensitivity c - reactive protein , and antibody testing . high - sensitivity crp ( hs - crp ) was measured by autoimmune scattering rate nephelometry ( bnp nephelometer , dade behring ) . double - stranded dna ( ds - dna ) was detected by farr assay ( euroimmun ag , germany ) , and antiphospholipid antibodies ( acl ) were measured by enzyme - linked immunosorbent assay ( elisa ) ( euroimmun ag , germany ) . echocardiography was performed by an experienced research technician using standard techniques who was unaware of the clinical characteristics of the patients . studies were performed using phased - array echocardiography with m - mode , 2-dimensional , pulsed , and color - flow doppler capabilities . lv mass ( lvm ) was calculated using the following formula : lvm = 0.8 ( 1.04 ( lvst+lvpwt+lvdd)lvdd)+ 0.6 , where lvst is lv septal wall thickness , and lvpwt is lv posterior wall thickness , lvdd is lv diastolic diameter . lvmi was indexed for body surface area ( bsa ) , and lvh was defined by an lvmi of over 110 g / m in women and 125 g / m in men . data were described as means sds for those with normal distribution and as medians and interquartile ranges for asymmetrical distribution . comparisons between patients divided by crp cutoff level and with or without lvh were performed by unpaired t - tests in normally distributed data and by nonparametric mann - whitney test in asymmetrically distributed data , or by x test in categorical data . the cut - off level of hs - crp was defined according to the aha / cdc recommendations , in which crp levels 3 mg / l were defined as average- and high - risk groups for cvd . all variables that had significant relations were evaluated for inclusion in a model predicting lv mass using multivariable regression analysis ; unstandardized regression coefficients all of the statistics were performed by spss version 13.0 , and a 2-tailed p < .05 was considered to indicate statistical significance . the 287 subjects were predominantly female ( 91.29% ) , with a mean age of 38.5 13.3 at their entry . totally 223/239 patients ( 93.30% ) showed positive ana , 177/200 patients ( 88.50% ) had positive ds - dna , and 29/153 patients ( 18.95% ) showed acl antibodies . renal biopsy was obtained from 135 ( 47.04% ) patients , which showed minimal mesangial ln ( class i ) in 4 ( 3.0% ) , mesangial proliferative ln ( class ii ) in 7 ( 5.2% ) , focal ln ( class iii ) in 16 ( 11.9% ) , diffuse ln ( class iv ) in 77 ( 57.0% ) , membranous ln ( class v ) in 28 ( 20.7% ) , and advanced sclerotic ln ( class vi ) in 3 ( 2.2% ) patients according to international society of nephrology / renal pathology society ( isn / rps ) 2003 classification . we compared the baseline characteristics of patients with and without lvh , as showed in table 1 . patients with lvh were much older , had significantly elevated hs - crp level and higher uric acid level , lower hemoglobin level , and egfr . however , bmi , blood pressure , and serum lipids were not significantly different between the two groups . meanwhile , autoantibody parameter positive incidence including ds - dna , ana , and acl did not differ between patients with and without lvh . to further explore the extent to which inflammation augment lvh , the patients were subdivided into low- and average - to - high risk groups according to hs - crp cutoff level . among those who had higher hs - crp levels ( 3 mg / l ) , lvmi was significantly increased ( 132.68 57.84 versus 113.67 29.17 , p = .018 ) ( figure 1 ) . in addition , these patients had elder age ( 39.37 14.09 versus 34.97 11.55 , p = .02 ) , lower hemoglobin level ( 93.04 24.91 versus 106.57 23.65 , p < .001 ) , lower cholesterol level ( 5.60 1.89 versus 6.41 2.76 , p = .018 ) , higher esr ( 56.00 ( 31.5083.00 ) versus 32.00 ( 19.7558.50 ) , p < .001 ) and higher serum fibrinogen level ( 4.17 1.59 versus 3.54 1.12 , p = .004 ) ( table 2 ) . in univariate analysis involving the entire sample ( table 3 ) , significant correlates of lvmi included age , body mass index , blood pressure , hemoglobin level , hs - crp , uric acid level , and egfr . after introducing all these significant variables into multivariate regression analysis , hs - crp ( = 0.228 , p = .009 ) , along with uric acid ( = 0.382 , p < .001 ) , was further confirmed to have positive associations with lvmi . our cross - sectional study revealed a linear relationship between low - grade chronic inflammation estimated by high - sensitivity crp levels and lvmi , independent of several other important covariates , such as adipose tissue distribution bmi , bp levels , serum lipids , renal function , age , and gender . the observation of this independent association between hs - crp level and lvmi is consistent with previous findings [ 15 , 16 ] , and the present study extended to ln patients . as far as we know , these findings are new and potentially important for refining cvd risk stratification in this population . at the initial stage of the atherosclerotic process , systemic inflammation would appear most importantly associated with subclinical cardiovascular disease development , such as lvh occurrence . a raised baseline crp value has been associated with inflammation , endothelial dysfunction , obesity , the metabolic syndrome [ 18 , 19 ] , diabetes mellitus , insulin resistance , and severity of hypertension , and thus , various metabolic disorders may occur by increasing crp level and simultaneously promote an increase in lv mass . on the other hand , local crp synthesis and secretion by smooth muscle cells , including those of the human coronary artery , it is possible to speculate that crp may play a direct role in promoting lvh through these mechanisms , including ( 1 ) increasing phosphatidylinositol3- kinase activity ; ( 2 ) upregulating inducible nitric oxide synthase , certain cell signal transduction pathways including the mitogen - activated protein kinase pathway , and nuclear factor -b ; ( 3 ) upregulating angiotensin ii type 1 receptor in vascular smooth muscle cells , and directly quenching the production of nitric oxide by endothelial cells [ 24 , 25 ] , resulting in increased production of endothelin-1 ; ( 4 ) elevation of von willebrand factor , which is known to be associated with endothelial dysfunction . thus , cardiac hypertrophy may be , at least in part , attributable to an increase in crp itself , via activated transcriptional regulatory mechanisms , proinflammatory and proatherogenic effects , and stimulation of endothelial dysfunction first , it is not possible to conclude from this observational research whether crp stimulates higher lvmi or whether crp is increased before the development of lvh . the cross - sectional design prevents the demonstration of the mechanisms by which lvh is related to inflammation . second , it may be better to introduce sledai score into multivariate regression analysis to further estimate the effect of disease itself on lvh , and inflammation status as well . third , it is very regrettable that some of our patients ' autoantibodies data were missing and incomplete . this cohort will be followed and expanded to further observe the prevalence and correlative factors of lvh , especially after intervention therapy . in conclusion , in ln subjects initially free of cvd , hs - crp showed a significant association with lvmi , which suggested that assessment of hs - crp level may help to refine cvd risk stratification in this population .

the platelet - rich plasma ( prp ) has been advocated as a way to introduce increased concentrations of growth factors and other bioactive molecules to injured tissues in an attempt to optimize the local healing environment . a 94-yr - old woman with various comorbidities presented with a two - week history of severe cutaneous ulcer on the left dorsum of foot . it was caused by recurrent mechanical trauma and did not respond to several wound debridement and simple dressings . however , after she was completed on seven times of autologous prp treatments , we observed complete healing of the skin lesion within 3 months . herein , we report a case of recalcitrant cutaneous ulcer with various comorbidities and discuss about the promising possibility of autologous prp as an effective alternative therapeutic modality .

in recent years the problem of " cutaneous ulcers " ( venous , arterial , diabetic , and pressure sores ) has become increasingly important , in particular because of the progressive increase in the elderly population and , therefore , of chronic disorders ( 1 ) . chronic cutaneous ulcers of the comorbid elderly patient tend to resist on standard therapy and therefore the management is difficult , time - consuming and expensive ( 2 ) . optimally , a short period of advanced therapy to stimulate healing can result in the transition of an intractable wound to a treatable one , which can then be treated with less expensive , standard care ( 3 ) . platelet - rich plasma ( prp ) is an autologous preparation of platelets in concentrated plasma and contains various growth factors . due to the presence of high concentrations of these growth factors , prp has been used in a wide variety of surgical procedures and clinical treatments ( 4 ) . we herein report a comorbid patient with refractory cutaneous ulcer which did not respond to formal therapeutic methods . the patient was treated with autologous prp and could achieve complete response of the skin lesion within 3 months . on november 25 , 2011 , a 94 yr - old woman was referred for the management of erythematous to violaceous patches with bullae on dorsum of left foot . she repeatedly hit her left foot in which intravenous line was situated with the opposite side for days . she had a various underlying disease including alzheimer 's dementia , angina pectoris , type 2 diabetes , hypertension , chronic kidney disease ( stage iv ) and was in a bed ridden state . because of the poor oral intake and noncompliance to the levin - tube , the percutaneous endoscopic gastrostomy tube had been inserted on her abdomen and only minimal amount of nutrition was maintained . despite of the daily simple dressing and intermittent debridement about 2 weeks , the status of the wound was deteriorated and advanced to painful ulcerative patches with eschar and granulation tissues ( fig . autologous prp were prepared for the alternative management of her worsening wound . 12 ml peripheral autologous blood was obtained and collected into tubes containing acid - citrate - dextrose solution formula ( acd - a ) anticoagulant . subsequently , the yellow plasma ( containing buffy coat with platelets ) was separated from other components . the upper 3 to 4 ml plasma called platelet poor plasma was removed by micropipette . the patient underwent a total of 7 prp treatments over a span of 8 weeks ( performed twice a week until 5th treatment and then weekly until 7th treatment ) . as described above , these treatments involved topically application of activated prp as well as packing of the wound with aseptic film for the sustained effect of growth factors . the film was removed on next day and the top layer was covered with hydrofoam dressing material until subsequent prp treatment . over the course of the prp treatment , the results of the changes in wound surface area and depth are shown in fig . the wound was essentially filled in with granulation tissue after seven prp treatments with only subtle epithelialization of the new tissue remaining . there were neither signs of infection and allergic reaction nor discomfort of the patient during the treatment . after 2 months from the last ( 7th ) treatment , the complete epithelialization was seen and no further simple dressing was required for the wound . the complete response of the recalcitrant cutaneous ulcer using prp therapies implies that this stimulation therapy could be an appropriate alternative modality when the degree of healing with conventional treatment appears to be unsatisfactory . the ulcer bed more rapidly demonstrated margin in - growth , granulation tissue development , vascularization and epithelialization ( 5 , 8) . first of all , the growth factors and cytokines released from prp are able to stimulate the proliferation , migration and differentiation of dermal fibroblasts and endothelial cells . prp also increased type i collagen and mmps gene expression , suggesting that prp also have the potential to promote the tissue remodeling of aged skin ( 4 ) . the second component is the mesh of fibrin that constitutes the " structure " of the platelet gel : this mesh forms a biological scaffold that helps and guides the migration of the mesenchymal cells , derived from populations of resident stem cells or circulating precursors , from the base and the margins of the wound . the third component is the antibacterial properties of the factors released by the platelets and , probably , by the leucocytes contained in the prp ( 1 ) . on the basis of these molecular understandings , prp therapy has been successfully used in the treatment of various dermatological defect including pressure ulcer ( 1 , 5 ) , diabetic ulcer ( 1 , 2 , 6 ) vascular ulcers , post - traumatic ulcers ( 1 ) and lipodermatosclerosis ( 7 ) . however , a few studies have reported in the treatment of severe cutaneous ulcers associated with old age , hypomobility and many underlying diseases ( 2 , 9 ) . although further studies are required for better understandings of the mechanism , efficacy , and safety of autologous prp therapy , it could be considered as an effective alternative option for the recalcitrant cutaneous ulcer with various comorbidities .

backgroundlow contact force and force time integral ( fti ) during catheter ablation are associated with ineffective lesion formation , whereas excessively high contact force and fti may increase the risk of complications . we sought to evaluate the optimal fti for pulmonary vein ( pv ) isolation based on atrial wall thickness under the ablation line.methods and resultscontact force parameters and fti during anatomical ipsilateral pv isolation for atrial fibrillation and atrial wall thickness were assessed retrospectively in 59 consecutive patients for their first pv isolation procedure . the pv antrum was divided into 8 segments , and the wall thickness of each segment under the ablation line was determined using multidetector computed tomography . the fti for each ablation point was divided by the wall thickness of the pv antrum segment where each point was located to obtain fti / wall thickness . in total , 5335 radiofrequency applications were delivered , and 85 gaps in pv isolation ablation lines and 15 dormant conductions induced by adenosine were detected . the gaps or dormant conductions were significantly associated with low contact force , radiofrequency duration , fti , and fti / wall thickness . among them , fti / wall thickness had the best prediction value for gaps or dormant conductions by receiver operating characteristic curve analysis . fti / wall thickness of < 76.4 gramseconds per millimeter ( gs / mm ) predicted gaps or dormant conductions with sensitivity ( 88.0% ) and specificity ( 83.6% ) , and fti / wall thickness of < 101.1 gs / mm was highly predictive ( sensitivity 97.0% ; specificity 69.6%).conclusions fti / wall thickness is a strong predictor of gap and dormant conduction formation in pv isolation . an fti / wall thickness 100 gs / mm could be a suitable target for effective ablation .

recent progress and technical advances in catheter ablation have dramatically improved the success rate and safety of pulmonary vein isolation ( pvi ) for atrial fibrillation ( af ) . realtime monitoring of tiptotissue contact force ( cf ) is a useful technique for confirming that the ablation electrode is applying appropriate pressure . the development of cf has allowed control of the quality of lesions during radiofrequency ( rf ) ablation.1 , 2 a low cf during catheter ablation is associated with ineffective lesion formation , whereas excessively high cf may result in an increased risk of steam pop , thrombus formation , or cardiac perforation,3 particularly an atrioesophageal fistula in the posterior wall.4 a recent study reported that cf and the force time integral ( fti ) during rf ablation are predictors of transmural lesion , with the best cutoff fti value of > 392 gramseconds ( gs).5 the efficas i study reported that ablation with a minimum fti of < 400 gs showed an increased likelihood of reconnection and that gap occurrence showed a strong trend with lower average cf and average fti6 ; however , the left atrial ( la ) wall under the catheter ablation line is not of uniform thickness , and it can be particularly thick in the left lateral ridge ( llr ) . although the llr does not only compose the myocardial fiber,7 it has been reported that llr thickness is associated with the recurrence of af after pvi.8 for these reasons , we speculated that the optimal cf parameters for each lesion may differ according to the anatomical site and atrial wall thickness . the aim of this study was to evaluate the optimal cf or fti for anatomical ipsilateral pvi based on la wall thickness under the catheter ablation line . the study participants were 59 consecutive patients ( 118 ipsilateral veins ) with symptomatic drugrefractory af who were referred to our hospital between september 2014 and august 2015 for rf catheter ablation for their first procedure . we included patients who underwent anatomical ipsilateral pvi and who were assessed for dormant conduction ( dc ) by an intravenous bolus of adenosine . the study was approved by the ethics committee at the national hospital organization , kanazawa medical center . prior to the procedure , la thrombus was excluded using laboratory data and cardiac computed tomography ( ct ) angiogram or transesophageal echocardiogram . antiarrhythmic agents were discontinued before the procedure , allowing a washout period of 5 halflives , although atrioventricular blocking agents were permitted in symptomatic patients . we excluded cases that required rf application in carina other than the pulmonary vein ( pv ) antrum to achieve pv isolation . a multielectrode catheter was transvenously inserted and positioned in the coronary sinus . a 10f intracardiac echocardiography catheter ( 64element , 5.510.0 mhz , soundstar ; biosense webster ) threedimensional ultrasound images of the left atrium and pvs were acquired and processed using the carto 3 system ( biosense webster ) . the reconstructed 3dimensional ct data sets were merged with the 3dimensional ultrasound derived geometries . after a double transseptal puncture was performed under intracardiac ultrasound guidance , heparin was intravenously administered to maintain activated clotting time of > 300 seconds . through transseptal accesses , nonsteerable sheaths jude medical , inc ) were placed into the left atrium . the irrigated cf ablation catheter ( navistar thermocool smarttouch ; biosense webster ) was advanced into the left atrium through a steerable sheath , and extensive encircling pvi was performed guided by the 3dimensional ultrasound geometries and 3dimensional merged ct images . ablations were performed in a temperaturecontrolled mode with the temperature limited to 43c and the power limited to 25 w in the posterior segments and 30 w in the other segments . luminal esophageal temperature was monitored using a luminal esophageal temperature probe ( sensitherm ; st . jude medical ) , and we considered a luminal esophageal temperature of > 39c as requiring cessation of ablation . the irrigation flow rate was set to 2 ml / min during mapping and 1730 ml / min during ablation . ablations were performed with continuous dragging or point by point except for the regions of the posterior wall where the esophagus makes contact . the operator 's decision to move 1 site to the next ablation site was based mainly on electrogram abatement and luminal esophageal temperature increase . the circumferential isolation was first performed anatomically without the circular catheter ( lasso ) or the 1mm multielectrodemapping catheter ( pentaray ) , using an exclusively anatomical approach . the rf application time at each site varied , and pv antrum isolation was verified ( as the absence of any pv potential or la potential in the antral ablation area ) using the lasso or pentaray catheter and/or the ablation catheter electrograms . all examinations were performed using a 64detectorrow ct ( multidetector ct [ mdct ] ) scanner ( brilliance ct 64 ; philips electronics ) . for the contrastenhanced scan , 2021 mg of iodine per kg / s of contrast medium the scan was started with a delay of 11 seconds after the detection of contrast in the main pulmonary artery . volume data were reconstructed into axial images with a slice thickness of 0.9 mm and were transferred to a workstation for postprocessing ( ziostation2 ; ziosoft , inc ) . for each pv , we divided the pv antrum into 8 segments under the ablation line , and quantitative measurements for each region were performed , as reported previously.8 at the llr and the carina between the superior and inferior pvs , we used images orthogonal to each region . the transmural myocardial thicknesses of the llr at the superior and inferior pv were each calculated as the mean of 2 measurements taken from 2 directions . the carina when several branches were present on the sameside pv , we adopted the thickest value of the carina . the pv antrum was defined as the region consisting of a 10mm space between the pv ostium and the left atrium . in the segments other than the llr , we measured the wall thickness of the pv antrum estimated to be on the catheter ablation line , using sagittal section . after ipsilateral anatomical pv antrum isolation , all pvs were mapped to detect ablation gaps of pvtoleft atrium conduction . in the absence of gaps , the presence of dcs was assessed in each vein with an intravenous administration of 20 mg adenosine . the positions where rf application resulted in a change of sequence or abolition of the pv potential were defined as gap sites . a dc was defined as the reappearance of pv conduction , demonstrated by associated pv spikes of > 1 beat , as recorded by the circular catheter . the positions where rf application resulted in a change of sequence or the abolition of dc were defined as dc sites . when 1 gap or dc site included multiple ablation points with the smallest vigitag ( biosense webster ) , we counted all of these points as gaps or dcs . ablation points were assigned to each segment of the pv antrum where they were located . fti , maximum cf , average cf , minimum cf , rf duration , and rf power were obtained for each ablation point . when one ablation site overlapped to the next site , even with the smallest vigitag size , the higher fti site was selected as an ablation point . the fti for each ablation point was divided by the wall thickness of the pv atrium segment where the ablation point was located to calculate the ablation fti required for every 1 mm of wall thickness ( fti / wall thickness ) . the predictive values of different thresholds of cf and fti for gaps and dcs were assessed using sensitivity , specificity , and receiver operating characteristic ( roc ) curve analysis . all procedures were performed under conscious sedation or general anesthesia . a multielectrode catheter was transvenously inserted and positioned in the coronary sinus . a 10f intracardiac echocardiography catheter ( 64element , 5.510.0 mhz , soundstar ; biosense webster ) threedimensional ultrasound images of the left atrium and pvs were acquired and processed using the carto 3 system ( biosense webster ) . the reconstructed 3dimensional ct data sets were merged with the 3dimensional ultrasound derived geometries . after a double transseptal puncture was performed under intracardiac ultrasound guidance , heparin was intravenously administered to maintain activated clotting time of > 300 seconds . through transseptal accesses , nonsteerable sheaths jude medical , inc ) were placed into the left atrium . the irrigated cf ablation catheter ( navistar thermocool smarttouch ; biosense webster ) was advanced into the left atrium through a steerable sheath , and extensive encircling pvi was performed guided by the 3dimensional ultrasound geometries and 3dimensional merged ct images . ablations were performed in a temperaturecontrolled mode with the temperature limited to 43c and the power limited to 25 w in the posterior segments and 30 w in the other segments . luminal esophageal temperature was monitored using a luminal esophageal temperature probe ( sensitherm ; st . jude medical ) , and we considered a luminal esophageal temperature of > 39c as requiring cessation of ablation . the irrigation flow rate was set to 2 ml / min during mapping and 1730 ml / min during ablation . ablations were performed with continuous dragging or point by point except for the regions of the posterior wall where the esophagus makes contact . the operator 's decision to move 1 site to the next ablation site was based mainly on electrogram abatement and luminal esophageal temperature increase . the circumferential isolation was first performed anatomically without the circular catheter ( lasso ) or the 1mm multielectrodemapping catheter ( pentaray ) , using an exclusively anatomical approach . the rf application time at each site varied , and pv antrum isolation was verified ( as the absence of any pv potential or la potential in the antral ablation area ) using the lasso or pentaray catheter and/or the ablation catheter electrograms . all examinations were performed using a 64detectorrow ct ( multidetector ct [ mdct ] ) scanner ( brilliance ct 64 ; philips electronics ) . for the contrastenhanced scan , 2021 mg of iodine per kg / s of contrast medium the scan was started with a delay of 11 seconds after the detection of contrast in the main pulmonary artery . volume data were reconstructed into axial images with a slice thickness of 0.9 mm and were transferred to a workstation for postprocessing ( ziostation2 ; ziosoft , inc ) . for each pv , we divided the pv antrum into 8 segments under the ablation line , and quantitative measurements for each region were performed , as reported previously.8 at the llr and the carina between the superior and inferior pvs , we used images orthogonal to each region . the transmural myocardial thicknesses of the llr at the superior and inferior pv were each calculated as the mean of 2 measurements taken from 2 directions . the carina when several branches were present on the sameside pv , we adopted the thickest value of the carina . the pv antrum was defined as the region consisting of a 10mm space between the pv ostium and the left atrium . in the segments other than the llr , we measured the wall thickness of the pv antrum estimated to be on the catheter ablation line , using sagittal section . after ipsilateral anatomical pv antrum isolation , all pvs were mapped to detect ablation gaps of pvtoleft atrium conduction . in the absence of gaps , the presence of dcs was assessed in each vein with an intravenous administration of 20 mg adenosine . the positions where rf application resulted in a change of sequence or abolition of the pv potential were defined as gap sites . a dc was defined as the reappearance of pv conduction , demonstrated by associated pv spikes of > 1 beat , as recorded by the circular catheter . the positions where rf application resulted in a change of sequence or the abolition of dc were defined as dc sites . when 1 gap or dc site included multiple ablation points with the smallest vigitag ( biosense webster ) , we counted all of these points as gaps or dcs . ablation points were assigned to each segment of the pv antrum where they were located . fti , maximum cf , average cf , minimum cf , rf duration , and rf power were obtained for each ablation point . when one ablation site overlapped to the next site , even with the smallest vigitag size , the higher fti site was selected as an ablation point . the fti for each ablation point was divided by the wall thickness of the pv atrium segment where the ablation point was located to calculate the ablation fti required for every 1 mm of wall thickness ( fti / wall thickness ) . the predictive values of different thresholds of cf and fti for gaps and dcs were assessed using sensitivity , specificity , and receiver operating characteristic ( roc ) curve analysis . of the 59 patients , 20 ( 33.9% ) were shown during the ct scan to have af . complete pvi was achieved in 23 patients ( 39% ) after ablation of single continuous circular lesions around ipsilateral pvs . all targeted pvs ( 118 ipsilateral veins ) were successfully isolated by the end of the procedures . characteristics of the study population af indicates atrial fibrillation ; bnp , btype natriuretic peptide ; egfr , estimated glomerular filtration rate ; mdct , multidetector computed tomography ; noac , nonvitamin k antagonist oral anticoagulants ; rf , radiofrequency . as shown in figure 1 , the llr was the thickest part of the ablation line , with mean thicknesses of 3.90.9 mm for the left lateral superior ridge , 3.90.8 mm for the left lateral inferior ridge , and 4.30.9 mm for the left lateral carina ridge . the thinnest atrial wall was the left posterior wall ( 1.80.3 mm in the left posterior superior and 1.90.3 mm in the left posterior middle and the left posterior inferior ) . the wall thickness of each pv segment was not significantly affected by the presence of af rhythm during ct scanning ( figure s1 ) . myocardial thickness of each segment under the ablation line ( lower panels ) and distribution of acute gaps and dormant conductions ( dcs ) after pulmonary vein isolation ( upper panels ) . libt indicates left inferior bottom ; lipv , left inferior pulmonary vein ; llcr , left lateral carina ridge ; llir , left lateral inferior ridge ; llsr , left lateral superior ridge ; lpi , left posterior inferior ; lpm , left posterior middle ; lps , left posterior superior ; lspv , left superior pulmonary vein ; lsrf , left superior roof ; rac , right anterior carina ; rai , right anterior inferior ; ras , right anterior superior ; ribt , right inferior bottom ; ripv , right inferior pulmonary vein ; rpi , right posterior inferior ; rpm , right posterior middle ; rps , right posterior superior ; rspv , right superior pulmonary vein ; rsrf , right superior roof . the highest average cf ( 23.810.2 g ) was applied to the right anterior inferior , and the lowest average cf was applied to the left lateral carina ridge ( 15.86.2 g ) . rf duration time was the shortest and fti was the lowest of all segments at the left posterior inferior ( 15.46.4 seconds and 234.7100.0 gs , respectively ) as a result of avoiding an increase in luminal esophageal temperature . fti / wall thickness was higher in the anterior wall of the right pvs and lower in the llr . the distribution of gaps and dcs was concentrated in the llr and right anterior wall ( figure 1 ) . cf parameters for each segment under the ablation line cf indicates contact force ; fti , force time integral ; gs , gramseconds ; max , maximum ; min , minimum ; pv , pulmonary vein ; rf , radiofrequency . all cf parameters including cf , rf duration , fti , and fti / wall thickness were significantly lower in the lesions with gaps or dcs compared with the lesions without them . these correlations were similarly observed within each patient . the delivered rf power was not significantly different between the 2 groups ( p=0.08 ) ( table 3 ) . ablation parameters at each point with a gap or dc compared with those without cf indicates contact force ; dc , dormant conduction ; fti , force time integral ; gs , gramseconds ; rf , radiofrequency . roc curves were made for fti , average cf , maximum cf , rf duration , and fti / wall thickness to determine the thresholds that best predict gaps or dcs ( figure 2 ) . fti / wall thickness showed the best prediction value with an area under the curve of 0.9242 ( 95% ci 0.90600.9425 ) . the areas under the curve for fti , average cf , maximum cf , and rf duration were 0.8101 , 0.7046 , 0.6246 , and 0.7161 , respectively . the best threshold for predicting gaps or dcs , defined by the position on the roc curve at the minimum distance from the left corner of the roc space , was an fti / wall thickness of 76.4 gs / mm ( sensitivity 88.0% ; specificity 83.6% ) . an fti / wall thickness of < 101.1 gs / mm was highly predictive of gaps or dcs ( sensitivity 97.0% ; specificity 69.6% ) . an fti / wall thickness of < 127.3 gs / mm predicted gaps or dcs with 100% sensitivity ( specificity 55.01% ) . conversely , an fti of < 475.5 gs showed high sensitivity but low specificity ( sensitivity 97.0% ; specificity 26.6% ) . subgroup analysis revealed that fti / wall thickness also showed the best prediction value with an area under the curve in the patients whose ct scan was obtained during af rhythm ( figure s2 ) . receiver operating characteristic curve analysis for acute gap and dormant conduction ( dc ) predictability . fti / wall thickness showed the best prediction value with an area under the curve ( auc ) of 0.9242 ( 95% ci 0.90600.9425 , p<0.001 vs aucs of fti and the other contact force [ cf ] parameters ) . fti , average cf , maximum cf , and rf duration had aucs of 0.8101 , 0.7046 , 0.6246 , and 0.7161 , respectively . the best threshold for fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm ( sensitivity 88.0% ; specificity 83.6% ) . an fti / wall thickness of < 101.1 gs / mm was highly predictive of acute gap or dc ( sensitivity 97.0% ; specificity 69.6% ) . avg indicates average ; fti , force time integral ; gs , gramseconds ; max , maximum . as shown in figure 1 , the llr was the thickest part of the ablation line , with mean thicknesses of 3.90.9 mm for the left lateral superior ridge , 3.90.8 mm for the left lateral inferior ridge , and 4.30.9 mm for the left lateral carina ridge . the thinnest atrial wall was the left posterior wall ( 1.80.3 mm in the left posterior superior and 1.90.3 mm in the left posterior middle and the left posterior inferior ) . the wall thickness of each pv segment was not significantly affected by the presence of af rhythm during ct scanning ( figure s1 ) . myocardial thickness of each segment under the ablation line ( lower panels ) and distribution of acute gaps and dormant conductions ( dcs ) after pulmonary vein isolation ( upper panels ) . libt indicates left inferior bottom ; lipv , left inferior pulmonary vein ; llcr , left lateral carina ridge ; llir , left lateral inferior ridge ; llsr , left lateral superior ridge ; lpi , left posterior inferior ; lpm , left posterior middle ; lps , left posterior superior ; lspv , left superior pulmonary vein ; lsrf , left superior roof ; rac , right anterior carina ; rai , right anterior inferior ; ras , right anterior superior ; ribt , right inferior bottom ; ripv , right inferior pulmonary vein ; rpi , right posterior inferior ; rpm , right posterior middle ; rps , right posterior superior ; rspv , right superior pulmonary vein ; rsrf , right superior roof . the highest average cf ( 23.810.2 g ) was applied to the right anterior inferior , and the lowest average cf was applied to the left lateral carina ridge ( 15.86.2 g ) . rf duration time was the shortest and fti was the lowest of all segments at the left posterior inferior ( 15.46.4 seconds and 234.7100.0 gs , respectively ) as a result of avoiding an increase in luminal esophageal temperature . fti / wall thickness was higher in the anterior wall of the right pvs and lower in the llr . the distribution of gaps and dcs was concentrated in the llr and right anterior wall ( figure 1 ) . cf parameters for each segment under the ablation line cf indicates contact force ; fti , force time integral ; gs , gramseconds ; max , maximum ; min , minimum ; pv , pulmonary vein ; rf , radiofrequency . all cf parameters including cf , rf duration , fti , and fti / wall thickness were significantly lower in the lesions with gaps or dcs compared with the lesions without them . these correlations were similarly observed within each patient . the delivered rf power was not significantly different between the 2 groups ( p=0.08 ) ( table 3 ) . ablation parameters at each point with a gap or dc compared with those without cf indicates contact force ; dc , dormant conduction ; fti , force time integral ; gs , gramseconds ; rf , radiofrequency . roc curves were made for fti , average cf , maximum cf , rf duration , and fti / wall thickness to determine the thresholds that best predict gaps or dcs ( figure 2 ) . fti / wall thickness showed the best prediction value with an area under the curve of 0.9242 ( 95% ci 0.90600.9425 ) . the areas under the curve for fti , average cf , maximum cf , and rf duration were 0.8101 , 0.7046 , 0.6246 , and 0.7161 , respectively . the best threshold for predicting gaps or dcs , defined by the position on the roc curve at the minimum distance from the left corner of the roc space , was an fti / wall thickness of 76.4 gs / mm ( sensitivity 88.0% ; specificity 83.6% ) . an fti / wall thickness of < 101.1 gs / mm was highly predictive of gaps or dcs ( sensitivity 97.0% ; specificity 69.6% ) . an fti / wall thickness of < 127.3 gs / mm predicted gaps or dcs with 100% sensitivity ( specificity 55.01% ) . conversely , an fti of < 475.5 gs showed high sensitivity but low specificity ( sensitivity 97.0% ; specificity 26.6% ) . subgroup analysis revealed that fti / wall thickness also showed the best prediction value with an area under the curve in the patients whose ct scan was obtained during af rhythm ( figure s2 ) . receiver operating characteristic curve analysis for acute gap and dormant conduction ( dc ) predictability . fti / wall thickness showed the best prediction value with an area under the curve ( auc ) of 0.9242 ( 95% ci 0.90600.9425 , p<0.001 vs aucs of fti and the other contact force [ cf ] parameters ) . fti , average cf , maximum cf , and rf duration had aucs of 0.8101 , 0.7046 , 0.6246 , and 0.7161 , respectively . the best threshold for fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm ( sensitivity 88.0% ; specificity 83.6% ) . an fti / wall thickness of < 101.1 gs / mm was highly predictive of acute gap or dc ( sensitivity 97.0% ; specificity 69.6% ) . avg indicates average ; fti , force time integral ; gs , gramseconds ; max , maximum . we evaluated the utility of an optimal value for fti in relation to the wall thickness under the pvi ablation line to predict gaps or dcs . our major findings were as follows : ( 1 ) mdct analysis revealed that the wall thickness was different at each part of the ablation line and was thickest at the llr ; ( 2 ) the gaps or dcs were significantly associated with low cf , rf duration , fti , and fti / wall thickness ; and ( 3 ) roc curve analysis identified fti / wall thickness as the best predictor for gaps and dcs . in this study , the wall thicknesses of the llr , the right anterior carina , and the posterior walls were 4 , 3.4 , and < 2 mm , respectively . these results are generally consistent with previous reports regarding japanese patients.8 in european studies using heart specimens by dissection and histological sections or by magnetic resonance angiography , the mean width of the llr was greater.7 , 9 although the modalities used to measure wall thickness in those studies differed from ours , the different width may be related to race , age , or sex . a lower average cf has been reported as a strong predictor of gap formation.6 , 10 , 11 it has been reported that cf at the llr tends to be low12 and that the majority of conduction gaps after single continuous circular lesions around ipsilateral pvs were located at the llr and the anterior wall of the right pv.13 in the present study , the cf for the llr was low , and most of the gaps and dcs were located at the llr or the right anterior carina , which was consistent with previous reports.12 , 13 we speculate that the lower cf and insufficient fti against the thick atrial walls at the llr and anterior right pv wall led to the formation of gaps and dcs . although rf power is a known predictor of lesion size,3 , 14 , 15 , 16 it had no significant impact on gap or dc formation in this study . this result might be derived from the fixed power output setting , which is uniform across the ablation line ( 25 w in posterior wall segments and 30 w in the other segments ) . further study is required to test whether the addition of a power output parameter improves prediction performance of fti / wall thickness . it has been reported that a minimum fti of 400 gs for each lesion was necessary to avoid reconnection6 , 10 or to create transmural lesions in pvi.5 conversely , the relationship between fti and electrogram attenuation plateaued at 500 gs , and fti and impedance drop also plateaued at 500 gs.17 beyond this plateau point , continuation of ablation is unlikely to produce further gains but may increase the potential risk of complications such as perforation , steam pops , or damage to extracardiac structures.17 nevertheless , these studies did not take wall thickness into account , and it differs at each ablation point . in the present study , most of the gaps or dcs were concentrated at the llr and the right anterior carina regardless of high fti . conversely , few gaps or dcs were observed at the posterior walls , where fti were relatively low . these observations imply that fti sufficient for creating transmural legions is higher for the thicker wall . conversely , excess fti for thin walls may bring a potential risk of damage to extracardiac structures such as the esophagus at the posterior wall of the lpv . in this study , an average rf application number was 90 points per patient . considering that even a few remaining gaps or dcs from these ablation points can lead to af recurrence , more sensitive fti / wall thickness values should be targeted to avoid formation of gaps and dcs . although fti / wall thickness of > 76.4 gs / mm has a best predictive value on the basis of roc analysis , with the best balance between sensitivity and specificity , fti / wall thickness of 101.1 gs / mm with higher sensitivity could be a more suitable target to avoid formation of gaps and dcs . first , this study was a singlecenter retrospective study with a relatively small number of patients . our participants were relatively old japanese patients with small physiques , and it is not known whether the present results would be applicable to other races . second , our measurements of wall thickness were made on the basis of the assumed ablation lines on mdct data and might not match the actual ablation line . in addition , the af rhythm observed in 33.9% of the patients during ct scanning may have affected the quality of ct images and the measurements of wall thickness . third , the number of gaps and dcs observed in the present study is greater than the number observed in the previous report13 ; however , complete pvi was achieved in 39% of the patients after ablation of single continuous circular lesions around ipsilateral pvs , which is consistent with the previous report . we suppose that the larger number of gaps and dcs was related to our definition . in this study , when 1 gap or dc site included multiple ablation points with the smallest vigitag size , we counted all of these points as gaps or dcs . finally , we evaluated the effect of fti only on acute success and the elimination of dcs . although it has been reported that the presence of dcs is associated with a higher risk of recurrence after pvi,18 it is still unclear whether the higher fti / wall thickness actually leads to the better outcome of af ablation . first , this study was a singlecenter retrospective study with a relatively small number of patients . our participants were relatively old japanese patients with small physiques , and it is not known whether the present results would be applicable to other races . second , our measurements of wall thickness were made on the basis of the assumed ablation lines on mdct data and might not match the actual ablation line . in addition , the af rhythm observed in 33.9% of the patients during ct scanning may have affected the quality of ct images and the measurements of wall thickness . third , the number of gaps and dcs observed in the present study is greater than the number observed in the previous report13 ; however , complete pvi was achieved in 39% of the patients after ablation of single continuous circular lesions around ipsilateral pvs , which is consistent with the previous report . we suppose that the larger number of gaps and dcs was related to our definition . in this study , when 1 gap or dc site included multiple ablation points with the smallest vigitag size , we counted all of these points as gaps or dcs . finally , we evaluated the effect of fti only on acute success and the elimination of dcs . although it has been reported that the presence of dcs is associated with a higher risk of recurrence after pvi,18 it is still unclear whether the higher fti / wall thickness actually leads to the better outcome of af ablation . this study demonstrated that the optimal fti for achieving effective ablation for ipsilateral anatomical pvi varies at each point along the ablation line . atrial wall thickness is not uniform under the ablation line , and fti / wall thickness is a strong predictor of gap and dc formation . fti / wall thickness 100 gs / mm could be a suitable target value to achieve effective ablation . figure s1 . the effect of atrial fibrillation ( af ) during computed tomography scanning on the measured myocardial wall thickness of each segment under the ablation line . the wall thicknesses of the pulmonary vein segments were not significantly different between the sinus group and the af group . figure s2 . the impact of af during computed tomography scanning on predictability of acute gap and dc . fti / wall thickness showed the best prediction value , with an auc of 0.9325 . the best threshold of the fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm ( sensitivity 93.2% ; specificity 83.0% ) . the lower panel shows roc curve analysis in af rhythm . in af rhythm , fti / the best threshold of the fti / wall thickness for predicting acute gaps or dcs was 89.7 gs / mm ( sensitivity 92.6% ; specificity 77.6% ) . af indicates atrial fibrillation ; auc , area under the curve ; dc , dormant conduction ; fti , force time integral ; gs , gramseconds ; roc , receiver operating characteristic .

epidemiological and experimental studies report associations between overweight mothers and increased obesity risk in offspring . it is unclear whether neonatal leptin regulation mediates this association between overweight mothers and offspring obesity . we investigated the effect of neonatal treatment with a leptin antagonist ( la ) on growth and metabolism in offspring of mothers fed either a control or a high fat diet . wistar rats were fed either a control ( con ) or a high fat diet ( mhf ) during pregnancy and lactation . male con and mhf neonates received either saline ( s ) or a rat - specific pegylated la on days 3 , 5 , and 7 . offspring were weaned onto either a control or a high fat ( hf ) diet . at day 100 , body composition , blood glucose , -hydroxybutyrate and plasma leptin and insulin were determined . in con and mhf offspring , la increased neonatal bodyweights compared to saline - treated offspring and was more pronounced in mhf offspring . in the post - weaning period , neonatal la treatment decreased hf diet - induced weight gain but only in con offspring . la treatment induced changes in body length , fat mass , body temperature , and bone composition . neonatal la treatment can therefore exert effects on growth and metabolism in adulthood but is dependent upon interactions between maternal and post - weaning nutrition .

obesity and metabolic - related disorders are considered major health issues worldwide . over the last decade , the incidence of obesity and overweight has almost doubled in developed countries and the trend is mirrored in developing nations that are transitioning to first - world economies . obesity results from an interaction of many factors including genetic , physiologic , behavioural , and environmental influences . however , the rapid increases in the rates of obesity suggest that environmental and behavioural influences , rather than genetic causes , are fuelling the present epidemic . increasing evidence from both clinical and animal studies has highlighted the link between altered maternal nutrition and the risk of offspring developing obesity and the metabolic syndrome [ 25 ] . initial epidemiological studies suggested that fetal growth restriction is correlated with later disease , implying that fetal nutritional deprivation may be a strong stimulus for developmental programming . however , although maternal nutrient deprivation has been well characterized in this context , in many societies , maternal and postnatal nutrition can be excessive . as a result , excessive weight gain and/or obesity are common nutritional problems complicating pregnancy in developed countries . as such , there is now accumulating evidence from human and animal studies suggesting that excess maternal caloric intake has adverse effects on the health and well - being of offspring , independent of postnatal diet and exerted transgenerational effects . maternal obesity has many adverse outcomes , including labour and delivery complications , fetal and neonatal death , maternal hypertension , and preeclampsia and gestational diabetes [ 912 ] . in addition to acute risks to the obese mother , negative outcomes extend to offspring , including obesity and cardiovascular disease in adulthood [ 1316 ] . on one hand , while a shared postnatal environment and genetic susceptibility are likely contributors , maternal bmi is reported in some cohorts as having greater influence on offspring than paternal bmi , highlighting the independent influence of the intrauterine environment on offspring adiposity . in a rodent model , we recently reported that offspring born to high - fat - fed mothers showed an obese phenotype in adulthood characterised by hyperinsulinemia and hyperleptinemia , independent of postnatal diet [ 7 , 19 ] . although the mechanistic drivers underlying this obesogenic phenotype are still unclear , experimental data in rodents suggest that leptin plays a critical role in underpinning early life influences on postnatal phenotypic development [ 2022 ] . early studies demonstrated that leptin , an adipokine produced primarily by adipocytes , plays a key role in regulation of energy homeostasis and food intake via its action on specific hypothalamic nuclei . leptin has been since demonstrated to exert other important functions , including its regulation of bone growth , skeletal metabolism , and linear growth via direct effects on osteoblast and osteocalcin release and growth hormone secretion , respectively [ 24 , 25 ] . in rodents , a characteristic of the neonatal period is a leptin surge , which normally peaks in the second week of neonatal life . it has been shown that early life nutritional insults affect this surge resulting in altered hypothalamic development [ 27 , 28 ] . maternal undernutrition in rats has been shown to result in a blunted and altered timing of the leptin surge in neonatal pups , and leptin administration during the neonatal period in ob / ob mice normalised hypothalamic development and partially normalised orexigenic behaviour . importantly , postweaning leptin administration had no effect , emphasising that , in the rat , the critical stage of hypothalamic leptin regulation is during the first 2 weeks of neonatal life . our group has previously shown that neonatal leptin treatment can reverse the deleterious effects of maternal undernutrition on postnatal outcomes in male and female offspring [ 29 , 30 ] . in contrast , kirk et al . reported that maternal high - fat diet led to an amplified leptin surge in neonatal pups during the first 2 weeks of life , resulting in altered hypothalamic regulation of food intake . there is growing evidence that leptin plays a significant role in the development of an obesogenic phenotype after early - life exposure to an imprudent diet . despite this , no studies have investigated whether maternal high - fat - diet - induced changes in neonatal leptin action regulate this association . we , therefore , hypothesized that leptin blockade , using a specific leptin antagonist , during the critical neonatal period of leptin sensitivity would ameliorate maternal high - fat - induced obesogenic effects on offspring . we investigated the effect of leptin antagonist administration during the time of the critical neonatal leptin surge on weight gain , food intake , and body composition in male offspring born to mothers fed either a control or a high - fat diet . the animal model of maternal high - fat nutrition has been described in detail previously [ 7 , 19 ] . briefly , female wistar rats were time - mated using a rat estrus cycle monitor ( ec40 , fine science tools , foster city , ca , usa ) to assess the stage of estrus of the animal before introducing the male . upon confirmation of mating , rats were randomly assigned to one of two maternal diets : the control chow diet throughout pregnancy and lactation ( con , n = 11 litters , diet 2018 , harlan - teklad , oxon , uk ) or a high - fat diet ( mhf , n = 13 litters , 45% kcals from fat , d12451 , research diets , new brunswick , nj , usa ) to be fed ad libitum throughout pregnancy and lactation . females were housed individually , with free access to water , and bodyweight and food intakes were measured every two days until the end of lactation . at birth , pups were weighed , and on postnatal day 2 , litter size was adjusted to 8 pups per litter to ensure adequate and standardized nutrition until weaning . at postnatal day 3 , mhf and con litters were randomly assigned to receive either saline ( s ) or pegylated rat leptin antagonist ( la , mutant l39a / d40a / f41a , protein laboratories rehovot , israel ) . the la or saline was administrated by subcutaneous injection at postnatal days 3 , 5 , and 7 at a dose of 12.5 g / g . dosage and timing of la administration was derived from calculated half - life ( approximately 20 hours ) and prior cited publications . male pups were weighed daily during the treatment period and then every 2 days thereafter until weaning ( p22 ) . at weaning , saline- and la - treated con and mhf male offspring were housed two per cage , and randomly placed on either the control rat chow ( c ) or high - fat ( hf ) diet until the completion of the trial ( day 110 ) . body weights and caloric intakes were recorded in offspring every 3 days until the end of the study . body composition ( fat mass , bone mineral content ( bmc ) , and bone mineral density ( bmd ) ) was measured by dual - energy x - ray absorptiometry ( dexa ) at p100 under light isoflurane ( 2% ) anaesthesia and using a dedicated small animal software package ( lunar hologic , waltham , ma ) . rats were culled at p110 by decapitation following anaesthesia with sodium pentobarbitone ( 60 mg / kg ) . a tail blood sample was taken for fasting glucose and -hydroxybutyrate ( bhb ) measurements ( roche accucheck ) and a rectal temperature measurement recorded . trunk blood was collected into heparinised vacutainers , centrifuged and plasma stored at 20c for later analysis . all animal work was approved by the animal ethics committee of the university of auckland . plasma leptin and insulin concentrations were analysed using commercially available rat - specific elisas ( no . cary , nc , usa ) and r software ( v.2.9.0 , r foundation for statistical computing , vienna , austria ) for windows . all models were statistically validated for assumptions of normality of residuals and absence of heteroscedasticity . nonnormal data were log transformed to normalize where necessary . maternal pregnancy data and neonatal data at birth were analysed using one - way anova . maternal caloric intakes ; weight and body composition during lactation was analysed by two - way anova with maternal diet and treatment group as factors and litter as covariate . although the growth analysis was performed on absolute body weight data , growth figures are shown as relative changes for sake of clarity given the number of experimental groups involved . preweaning data for pups was analysed by two - way factorial anova with maternal diet and la administration as factors , and their interactions . data from adult offspring were analyzed by three - way factorial anova with maternal diet , postweaning diet , and la treatment as factors , and the interaction between these factors ( litter included as a covariate ) . consistent with our previous observations , maternal hf diet during pregnancy and lactation resulted in a transient increase in caloric intake from day 2 ( p < 0.001 ) to day 15 of gestation when intakes returned to levels similar to those observed in con dams ( data not shown ) . increased caloric intake in mhf dams was reflected in an increased maternal weight gain by gestational day 7 ( p = 0.04 , figure 2 ) , which persisted until birth . mhf dams remained heavier than controls from the early neonatal period until mid - lactation when body weights returned to match those of controls . there was no overall significant effect of neonatal la administration on weight or caloric intake of dams during pregnancy and lactation . at weaning , despite a similar maternal body weight , total fat mass ( % ) and fat : lean ratios ( f / l ) were significantly increased in mhf dams compared to con ( % fat : con 14.7 1.7% versus mhf 22.9 2.3 , p = 0.01 ; f / l : con 0.18 0.08 versus mhf 0.31 0.16 , p = 0.01 ) . there was no significant effect of neonatal la treatment or an interaction between treatment and maternal diet , on body composition of dams . birthweights were slightly but significantly reduced in male offspring of mhf dams compared to con ( con 6.2 0.1 g ; mhf 5.9 0.1 g , p < 0.001 ) . at p3 and prior to start of la administration , pups born to mhf mothers remained lighter than con neonates ( con 7.1 0.1 g ; mhf 6.8 0.1 g , p < 0.05 ) . la administration leads to an increased neonatal weight gain in con and mhf offspring compared to their saline treated counterparts ( p < 0.005 , figure 3 ) . the increased weight gain in la - treated neonates was more pronounced in offspring of mhf dams reflected in a maternal diet la treatment interaction ( p < 0.005 ) . by weaning ( p22 ) , mhf offspring were slightly but significantly heavier than con offspring and la treatment further increased weaning weights in cont and mhf offspring ( p22 : con - s 59.9 0.9 g , con - la 62.0 1.1 g , mhf - s 61.8 1.7 g , mhf - la 66.3 1.1 g , p < 0.05 for effect of maternal diet and la treatment , no interactions ) . an mhf diet had no significant effect on adult body weight at postnatal day 110 but resulted in significantly increased total percent body fat and decreased lean body weight percentage compared to con animals as quantified by dexa scanning ( table 1 ) . a postweaning hf diet increased body weight and total body fat mass in all hf - fed groups . neonatal la treatment had a significant overall effect on reducing total percent body fat mass , increasing lean mass and a decreased fat : lean ratio ( table 1 ) . a significant maternal diet la treatment postnatal diet interaction ( p < 0.001 ) revealed that body weights were significantly reduced in offspring of con dams that were treated as neonates with la and fed a postweaning hf diet as compared to saline treated con offspring fed the hf diet ( figure 4(c ) ) . la treatment in con neonates reduced hf diet - induced obesity by approximately 10% and equated to an absolute bodyweight difference of 72.6 g ( figure 4(a ) ) . dexa analysis of body fat content showed that this reduction in body fat was paralleled by a reduction in fat mass in these animals compared to saline treated ( figure 4(d ) ) . this effect was not observed in mhf offspring where neonatal la failed to significantly impact on postweaning hf - induced changes in final bodyweight or fat mass ( figures 4(b ) and 4(d ) and table 1 ) . this may reflect a more marked increase in relative lean mass in la - treated con - hf offspring as compared to la treated mhf - hf offspring compared to relative saline - treated groups ( table 1 ) . there were no significant effects of mhf diet , neonatal la treatment , or postweaning hf diet on total caloric intake ( expressed as kcals consumed per gram body weight ) across any of the treatment groups ( figures 5(a ) and 5(b ) ) . there were significant overall effects of maternal diet and la treatment on nose - anus ( na ) length ( table 1 ) . post hoc analysis revealed la treatment increased na length in mhf offspring but not con offspring as reflected in a maternal diet la treatment interaction ( p < 0.05 ) . a postweaning hf diet increased na length only in mhf offspring ( table 1 ) . maternal diet had no overall effect on nose - tail ( nt ) length ( table 1 ) . there were overall significant effects of neonatal la treatment and postweaning hf diet on increasing nt length . a significant maternal diet la treatment interaction revealed that increases in nt length as a result of neonatal la treatment were greater in mhf offspring compared to con offspring for both chow and postweaning hf diets ( table 1 ) . there was no effect of mhf diet on bmd ( table 1 ) . neonatal la treatment reduced bmd in all treatment groups and a postweaning hf diet increased bmd in all offspring . an mhf diet had the overall effect of increasing bmc in all offspring ( table 1 ) . neonatal la treatment reduced bmc in all treated groups and a postweaning hf diet increased bmc in all offspring . a postweaning hf diet had no significant effect on rt ( p = 0.092 ) . an mhf diet significantly increased plasma leptin levels in all mhf offspring ( table 2 ) . there was a strong trend toward increased fasting plasma insulin levels in mhf offspring , but this difference did not reach statistical significance ( p = 0.068 ) . a postweaning hf diet increased plasma insulin levels in all hf - fed groups ( table 2 ) . fasting blood bhb levels were not altered by mhf diet or neonatal la treatment but were significantly increased in all hf - fed offspring compared to chow - fed offspring ( table 2 ) . these results have demonstrated for the first time that early - life manipulation of the leptin axis via neonatal leptin antagonism can exert marked effects on growth and body composition , which are dependent upon prior maternal nutrition status and postweaning diet . investigators using neonatal leptin treatment given to offspring of normally fed dams have shown increased adiposity and leptin and insulin resistance in offspring in later life [ 3335 ] . control offspring , given a leptin antagonist prior to being fed an obesogenic hf diet postweaning , show an amelioration of a diet - induced fat accumulation and reduced linear body growth . the marked contrast in adult phenotype in offspring of normally nourished mothers , based on exposure to either leptin or leptin antagonism during early - life development , further serves to highlight how critical the maintenance of leptin threshold levels is during this period of developmental plasticity . as we have shown previously , maternal high - fat nutrition resulted in increased adiposity , leptin , and insulin concentrations in offspring compared to offspring of control mothers , independent of postweaning diet . there is a well - characterized leptin surge in the first two weeks of life in the rodent although the source of the leptin is yet to be defined with the surge occurring independently of changes in neonatal body weight trajectory and milk leptin intake . this concurs with the increased sensitivity to body weight gain in mhf neonates treated with the la as compared to con offspring . in other studies , hypoleptinemic offspring of mothers undernourished during pregnancy have either a delayed or premature leptin surge . however , there is little known about the leptin surge in models of maternal obesity . recently reported that rat offspring of mothers fed an obesogenic diet had normal serum leptin levels at birth but displayed an amplified and prolonged neonatal leptin surge , which was accompanied by an elevation in leptin mrna expression in abdominal white adipose tissue . however , it is unknown whether the leptin surge in the mhf offspring of the present study is altered . although inborn leptin deficiency causes weight gain , it is unclear whether induced leptin deficiency in adult wild - type animals would be orexigenic . leptin antagonists have only recently become commercially available and provide an invaluable tool for investigating central and peripheral leptin deficiency and exploring the involvement of leptin in metabolic processes . previous reports using a nonpegylated leptin antagonist have been problematic . the extremely short half - life of the antagonist necessitated administration of supraphysiological doses to induce a clinical response and was not sufficient to induce a true metabolic state of leptin deficiency [ 32 , 37 ] . hormones with molecular masses similar to that of leptin are cleared primarily via the kidney with a half life of only 830 minutes . the effect of early postnatal leptin blockade in normal rat neonates has previously been reported in the study by attig et al . . in this work , the authors studied the long - term effect of neonatal therapy with a non - pegylated leptin antagonist ( day 2 to day 13 ) in female wistar rats . in contrast to the present study , they showed that leptin antagonism induced a decrease in neonatal weight gain , which has previously been commonly associated with neonatal leptin treatment [ 30 , 40 ] . later in life , the leptin disruption led to a higher sensitivity to diet - induced obesity , as shown by a higher body weight gain when challenged with a high - energy diet , associated with increased adiposity and leptinemia . these animals also displayed a phenotype of leptin resistance at 4 months , characterized by the inability of treated animals to respond to leptin by failing to reduce food intake and showing reduced birth weight . overall , the long - term effect in the attig study was paradoxically similar to that reported for rats treated with leptin during neonatal life [ 30 , 40 ] . importantly , the molecule used as the leptin antagonist was different from the one used in our study . indeed in this work , the authors used the leptin mutein , a molecule acting as an antagonist , with in vivo effects previously validated only using intracerebroventricular , but not subcutaneous , administration [ 4144 ] . furthermore , this antagonist , obtained by alanine mutagenesis of amino acids 39 to 41 - 42 , has an extremely short half - life and high doses are required to produce a clinical response that is similar to a true metabolic state of leptin resistance . the present study utilised a recently developed rat - specific pegylated la whereby the attachment of polyethylene glycol increased the overall molecule size to 70 kda . pegylation of the la results in an approximate 30-fold increase in in vivo half - life , thus true states of induced leptin deficiency are possible at physiologic doses . to date , only one prior study has examined the effects of the pegylated la moiety , albeit in normal postweaning animals where it was shown that treatment with the pegylated la to postweaning mice results in a rapid and dramatic increase in food intake and weight gain . the blood brain barrier ( bbb ) in the neonatal rat is relatively immature ; pegylated leptin antagonist has been shown to block circulating leptin from crossing the bbb , an action that would attenuate the anorexigenic effect of leptin . the windows of treatment are different , bbb permeability is at different developmental stages ( neonatal versus postweaning ) , and offspring responsiveness to leptin intervention is known to elicit sexually dimorphic responses [ 29 , 30 ] . in addition , the work in the mouse examined the immediate phenotypic response to la treatment , whereas the present study examines a postnatal phenotype derived from an early - life neonatal intervention . however , consistent with the reports from mice , the present result demonstrated that la treatment induced a significant increase in body weight over the neonatal treatment period . in the present study , neonatal leptin antagonism , despite having significant effects on pre - weaning weights in offspring of mhf mothers , had no effect on postnatal weight gain in con or mhf offspring fed the standard chow diet . there was , however , a marked effect of neonatal la treatment in reducing body weight gain in con offspring fed the hf diet after weaning . conversely , la treatment to mhf offspring subsequently fed the hf diet had no significant effect on body weight ; independent of changes in body weight and circulating plasma leptin concentrations . interestingly , neonatal la treatment did not alter postweaning caloric intake , thus the observed changes in body weight gain are independent of food intake and suggest a lack of effect of la administration on the arcuate nucleus and related feeding circuitry , as has been reported with neonatal leptin treatment in the ob / ob mouse . nose - anus lengths were increased in mhf offspring but not con offspring , which may suggest that altered effects on the growth - hormone- ( gh- ) insulin - like growth factor ( igf ) axis are mediated by neonatal la exposure . the observed change in tail length in con and mhf la - treated offspring was unexpected but may have resulted from altered thermoregulatory set - point processes as reflected in the significant differences in basal body temperature . in the rat , a significant portion of total body heat loss occurs through sympathetically mediated changes in tail blood flow . however , since rectal temperature was decreased in la - treated con and mhf offspring , it is difficult to explain the disparate changes in tail length to thermoregulatory processes and , as with nose - anus length , may reflect la - induced alterations in the gh - igf axis in mhf offspring as compared to controls or development of a thrifty metabolic phenotype as regards thermogenesis and energy expenditure . future independent studies looking at brown fat thermogenesis and uncoupling proteins may further explain this observation . bone morphology was significantly altered in adult offspring following neonatal la treatment with overall significant reductions in bmc and bmd . it is well established that leptin treatment can result in enhanced bone formation and promotion of pro - osteogenic factors in bone marrow [ 46 , 47 ] , and the current data suggests that the reverse holds true for leptin antagonism and further work investigating specific bone markers is now warranted . this is the first study designed to examine the efficacy of neonatal leptin antagonism following altered maternal nutrition and its interaction with differing levels of postweaning nutrition , on offspring phenotype development . responsiveness to neonatal leptin antagonism is dependent upon both maternal and postweaning nutrition , with minimal efficacy in chow - fed offspring of either con or mhf mothers . more studies are now required to further understand the mechanistic underpinnings of the present observations , including characterization of the effects of leptin antagonism on the timing and magnitude of the leptin surge in offspring of mothers with different dietary backgrounds . however , it is important to recognise that leptin - mediated development of feeding circuits occurs postnatally in the rodent and occurs in utero in primates , including humans , and thus timing of intervention strategies may be different . nonetheless , taken together , the data on both neonatal leptin treatment and leptin antagonism in the setting of both normal and nutritionally challenged pregnancies serves to highlight the important role of leptin regulation during critical early - life windows of development on lasting growth and metabolic function in offspring .

among 101 feet that presented with symptoms and signs similar to morton s neuroma , intermetatarsal rheumatoid nodules were found in five feet ( three patients ) . two patients had bilateral involvement . histology of the excised tissue showed the presence of a rheumatoid nodule and morton s neuroma in four feet and a rheumatoid nodule with unremarkable nerve bundles in one . a rheumatoid nodule can coexist with morton s neuroma , as seen in our patients , and the presentation is often similar to that of a morton s neuroma . our patients were rendered asymptomatic with surgical treatment and went on to have appropriate management of rheumatoid arthritis . rheumatoid nodule should be considered in the differential diagnosis of morton s neuroma in not only rheumatoid arthritis patients but also asymptomatic patients who have never been tested for rheumatoid antibodies .

morton s neuroma is a common paroxysmal neuralgia affecting the forefoot , typically in the third interdigital space . the clinical syndrome of morton s neuroma was described over a century ago , but its etiopathology remains poorly understood [ 1 , 8 , 9 ] . recent studies suggest that morton s neuroma is a mechanically induced degenerative neuropathy which has a strong predilection for the third common digital nerve . clinical examination is the most sensitive and specific , and is superior to imaging modalities like ultrasound or magnetic resonance imaging scan . various treatment options have been described in the literature for the treatment of morton s neuroma , including infiltration of a local anaesthetic and steroid combination into the interdigital space , sonography - guided alcohol injection , and surgery . the results of local steroid injections and ultrasound - guided alcohol injections are comparable to those of surgery , but limited literature is available on long - term results . some authors still recommend therapeutic injections prior to surgery . however , surgical resection has shown good long - term results , with improvement in 80 % of cases . rheumatoid synovitis and nodules producing symptoms mimicking morton s neuroma have been reported in the literature [ 1 , 3 , 9 ] , but are still rare . most of the cases reported in the literature were found in rheumatoid arthritis patients [ 1 , 7 , 9 ] . we report three patients ( five feet ) presenting with symptoms and signs of morton s neuroma due to underlying rheumatoid nodules . histology of the excised tissue showed the presence of a rheumatoid nodule and morton s neuroma in four feet and a rheumatoid nodule and unremarkable nerve bundles in one . our patients were rendered asymptomatic with surgical treatment and went on to have appropriate management of rheumatoid arthritis . informed consent was obtained from the patients prior to being included in the case series . case 1 . a 38-year - old lady presented to the foot and ankle clinic with pain and a burning sensation over the third intermetatarsal space of the right foot and the second intermetatarsal space of the left foot . clinical examination revealed fullness in the interdigital space with splaying of the corresponding toes ( fig . 1 ) and tenderness over the plantar aspect of the corresponding intermetatarsal space with a positive mulder s click . the patient underwent surgery through a single dorsal linear incision in the corresponding intermetarsal space . intraoperatively , a large soft tissue mass 2 cm in size involving the digital nerve and capsule of the second metatarsophalangeal ( mtp ) joint was noted on the left side , along with necrosis of the underlying fat pad . a soft tissue swelling originating from the dorsum of the capsule of the third mtp joint , filling the intermetatarsal space , was noted on the right side.fig . 1fullness in the second intermetatarsal space , with splaying of the surrounding toes fullness in the second intermetatarsal space , with splaying of the surrounding toes histology of the operative specimen revealed fibrinoid necrosis rimmed by palisaded histiocytes and fibroblasts , confirming the diagnosis of rheumatoid nodule ( fig . 2 ) . it also showed thickened nerve bundles and blood vessels which were surrounded by fibrous tissue consistent with morton s neuroma ( fig . 3 ) in the left foot , and a rheumatoid nodule and unremarkable digital nerve bundles in the right foot.fig . 2hematoxylin and eosin 100 . 3hematoxylin and eosin 25 . thickened blood vessels and nerve bundles that are rimmed by fibrous tissue consistent with morton s neuroma hematoxylin and eosin 100 . central fibrinoid necrosis rimmed by palisaded fibroblasts and histiocytes indicating a rheumatoid nodule hematoxylin and eosin 25 . thickened blood vessels and nerve bundles that are rimmed by fibrous tissue consistent with morton s neuroma she was subsequently tested for rheumatoid arthritis , and gave an equivocal result on ra assay and a strong positive result for anti - mutated citrullinated vimentin antibodies ( anti - mcv ) suggestive of seropositive rheumatoid arthritis . during follow - up , she developed symptoms of rheumatoid arthritis seven months after her first attendance at the foot and ankle clinic . case 2 . a 37-year - old lady with a past medical history of rheumatoid arthritis presented to the foot and ankle clinic with pain and swelling in the second intermetatarsal space bilaterally . clinical examination revealed splaying of the 2/3 toes bilaterally , with a positive mulder s click . a clinical diagnosis of morton s neuroma was made and the feet were operated on , one side at a time . intraoperatively , large fluid - filled bursae were noted in the intermetatarsal spaces bilaterally , with nerves lying underneath and tethered to bursae . case 3 . a 38-year - old lady presented to the foot and ankle outpatient clinic with an 18-month history of pain over the plantar aspect of the third and fourth metatarsal heads . a clinical diagnosis of synovitis of the third and fourth metatarsophalangeal joints was made , and an ultrasound scan was organized . her blood work was normal , with an erythrocyte sedimentation rate ( esr ) of 2 , c - reactive protein ( crp ) of 3 , and a normal white cell count . the ultrasound scan revealed a hypoechoic mass in the third intermetatarsal space suggestive of a morton s neuroma . histology revealed the presence of morton s neuroma and a rheumatoid nodule in the specimen . she was tested positive on ra assay and was referred to rheumatologists for further management . between january 2004 and october 2008 , 101 cases of morton s neuroma were operated on by two orthopedic surgeons with a special interest in foot and ankle surgery . among these , histology revealed rheumatoid nodules in five cases . morton s neuroma can be a local manifestation of a generalized disease such as rheumatoid arthritis [ 5 , 9 ] . the incidence of interdigital neuroma in rheumatoid arthritis patients was reported to be 1 in 520 , with a female preponderance . rheumatoid synovitis and nodules producing symptoms mimicking morton s neuroma have been reported in the literature , but most of the cases were found in previously diagnosed rheumatoid arthritis patients [ 1 , 9 ] . awerbuch et al . presented a series of 50 patients with morton s neuroma , and found that 24 % had rheumatoid arthritis at the time of diagnosis , 16 % developed rheumatoid arthritis over a follow - up period ranging from two months to 15 years , and an inflamed bursa in the intermetatarsal space was the first sign of rheumatoid arthritis in 8 % . in this series , out of 20 patients , a histological diagnosis of rheumatoid arthritis without any evidence of morton s neuroma was made for ten patients , of which seven were asymptomatic at presentation but three developed symptoms with four years of the initial presentation . suggested seeking histological evidence of rheumatoid disease in all tissues excised in the surgical treatment of morton s neuroma , as they believed that rheumatoid disease is the basic etiology in a significant number of patients . in the senior author s view , this is a rather interesting and unusual finding , and may be due to selection bias . in a series by vainio , performing prophylactic metatarsophalangeal synovectomy in rheumatoid arthritis patients resulted in a decrease in morton s neuroma operations . the distribution of the location of the neuroma in rheumatoid arthritis patients in this series was almost the same ( in interspaces ii iii and iii iv ) , in contrast to nonrheumatoid patients , in whom the iii iv interspace is more common . vainio also suggested that metatarsalgia associated with rheumatoid arthritis should preferably be called morton s metatarsalgia , as the nerve changes in this group of patients are not typical of neuroma . in our case series , out of three patients , one had a history of rheumatoid arthritis , and two patients were later diagnosed with seropositive rheumatoid arthritis after rheumatoid nodules were identified on a histological examination of the excised tissue . our case series demonstrates that a rheumatoid nodule presents with symptoms similar to morton s neuroma . however , the number of cases in our series is too small to suggest that patients with ii iii intermetatarsal neuralgia may be rheumatoid in origin , although the literature seems to suggest that view . we therefore suggest that a nodule of rheumatoid origin should be considered in the differential diagnosis of morton s neuroma , not only in patients with a known history of rheumatoid arthritis but also in patients who present with symptoms of morton s neuroma and have atypical intraoperative findings . the presence of a rheumatoid nodule in the foot can be the first manifestation of rheumatoid arthritis , and requires prompt referral to the rheumatology team .

pfeiffer syndrome is a rare autosomal dominant disorder characterized by coronal craniosynostosis , brachycephaly , mid - facial hypoplasia , and broad and deviated thumbs and great toes . pfeiffer syndrome occurs in approximately 1:100,000 live births . clinical manifestations and molecular genetic testing are important to confirm the diagnosis . mutations of the fibroblast growth factor receptor 1 ( fgfr1 ) gene or fgfr2 gene can cause pfeiffer syndrome . here , we describe a case of pfeiffer syndrome with a novel c833_834gc > tg mutation ( encoding cys278leu ) in the fgfr2 gene associated with a coccygeal anomaly , which is rare in pfeiffer syndrome .

pfeiffer syndrome is a rare autosomal dominant disorder , characterized by premature fusion of cranial sutures that prevents the skull from growing normally and affects the shape of the head and face , resulting in brachycephaly , hypoplastic maxilla , shallow orbits , proptosis , and exophthalmos , and accompanied with broad and deviated thumbs and big toes1 ) . mutations in the fibroblast growth factor receptor ( fgfr1 ) gene2 ) or fgfr2 gene3 ) cause pfeiffer syndrome . here we describe a case of pfeiffer syndrome with a novel c833_834gc > tg mutation ( encoding cys278leu ) in the fgfr2 gene , with an everted coccyx . his mother 's obstetric history was gravida 1 , para 0 , and was unremarkable . his parents were both korean , phenotypically normal , and did not have any history of consanguinity . he weighed 3,360 g ( 75 - 90 percentile ) , was 50 cm long ( 75 percentile ) , and had a head circumference of 34 cm ( 75 - 90 percentile ) . he had brachycephaly , maxillary hypoplasia , exophthalmos , proptosis , low - set ears , preauricular skin tags , atresia of the external auditory canal , high arched palate , radially deviated broad thumbs and medially deviated big toes , ankylosed elbows , and a mass on the coccygeal area ( fig . three dimensional skull ct revealed closure of the bilateral coronal sutures and left lambdoidal suture , midface hypoplasia , and shallow orbits ( fig . a lateral spine view noted the disappearance of normal curvature of spine ; spinal magnetic resonance imaging showed an outwardly everted coccyx ( fig . 2c ) , and plain radiographs of his hands and feet showed widening of both 1st phalangeal bones , metacarpal bones , and metatarsal bones . with informed consent , pcr was performed for exon 7 and exon 8 to analyze major mutation of fgfr2 gene , and revealed a novel amino acid substitution of leucine coded by tks , for cysteine coded by tgc , at codon 833_834 ( cys278leu ) of the fgfr2 gene ( fig . pfeiffer syndrome was first described by pfeiffer in 1964 , and 60 cases have been reported . it affects about 1 in 100,000 individuals1 ) , but is more rare in the asian population , with only a few cases reported in korea5 - 8 ) . pfeiffer syndrome involves the cranial bones and thumbs and great toes , which are broad and bend away from the other digits , occasionally accompanied by an ankylosed elbow or radiohumeral synostosis9 ) . type 1 consists of brachycephaly , a hypoplastic mid - face , and finger and toe abnormalities with normal to near - normal intelligence ( classic type ) . type 2 is characterized by a cloverleaf - shaped head , severe proptosis , and central nervous system involvement caused by more extensive fusion of the skull bones , with potential elbow ankylosis or synostosis . type 3 is similar to type 2 but without a cloverleaf - shaped head . both types 2 and 3 are more severe and have poor neurodevelopmental outcomes . the patient in this case report was pfeiffer syndrome type 1 , the classic type . cranial sutures normally close in a synchronized manner after birth , allowing the skull to achieve normal size and shape . when this normal development is disrupted , premature cranial fusion or delayed cranial closure occurs . mutations in fgfr1 , 2 , or 3 can affect craniofacial and skeletal development10 ) . more than 60 mutations in fgfr , a majority of which occur in fgfr2 , are associated with craniosynostosis syndrome such as antley - bixler syndrome , apert syndrome , bearse - stevenson syndrome , crouzon syndrome , muenke syndrome , and pfeiffer syndrom11 ) . mutations in the fgfr1 at chromosome 8p11.2-p12 were only detected in pfeiffer syndrome type 22 , 12 ) . mutations in the fgfr2 at chromosome 10q25-q26 were reported in all three subtypes13 ) , including 1036t->c3 ) and 1037g->a which were also detected in crouzon syndrome14 ) . we report a novel c833_834gc > tg mutation ( encoding cys278leu ) in pfeiffer syndrome . spinal anomalies such as the cervical spine fusion are rare in pfeiffer syndrome15 ) , but coccygeal anomaly occurred in one other case in korea7 ) . 90% of alp in young infants is produced from bone , and reflects osteoblastic activity , bone turnover , and rapid bone growth , and positively correlates with bone mineral accretion . alp is normal at birth and rises gradually in the first 2 - 3 weeks of life16 ) . osteoblast progenitors in bone marrow were significantly increased in fgfr2cc342y/+ heterozygote mice , which had a phenotypically shortened face , protruding eyes , and premature fusion of cranial sutures17 ) . patients with apert syndrome with the s252w mutation in the fgfr2 gene enhanced osteoblastic marker gene expression and alp activity18 ) . increased alp from birth might reflect prenatally increased osteoblastic marker gene expression resulting from mutations in fgfr2 gene , but further study is needed . the prognosis of pfeiffer syndrome depends on accompanying anomalies , and multiple surgeries are needed to release the prematurely closed sutures19 , 20 ) . in conclusion , this report describes a case of pfeiffer syndrome with a novel c833_834gc > tg mutation ( encoding cys278leu ) in the fgfr2 gene , with an everted coccyx resembling a tail and increased alp activity .

in this report , we present a case of in vitro maturation ( ivm ) with surgical retrieved testicular sperm in a normo - ovulatory female . human chorionic gonadotropin - primed ivm , testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed . fourteen cumulus - oocyte complexes were obtained in germinal vesicle or metaphase i stage , eight oocytes reached metaphase ii , seven presumptive zygotes were obtained , and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy . a single healthy newborn was obtained . our results suggest that ivm may be an alternative for in vitro fertilization in normo - ovulatory women even if surgical retrieval of sperm is needed . further research is required to depict contributing factors to the success of ivm in indications different from polycystic ovaries syndrome and the role of male gamete .

in vitro maturation ( ivm ) is a technique described in the 90s primarily as alternative technique to conventional stimulation for patients with polycystic ovaries syndrome ( pcos ) because the decreased risk of ovary hyperstimulation . however , ivm has additional benefits such as a reduced cost compared to regular in vitro fertilization ( ivf ) procedures and represents a friendly approach for female patients . recent reviews of the literature confirmed that ivm is inferior to conventional stimulation in terms of pregnancy and take - home baby rates . this is why ivm is considered as an experimental procedure according to the american society for reproductive medicine and its application is limited in the united states . despite ivm is routinely applied in some european countries and it is a flagship for the mild approaches movement . one potential application is the use of ivm as a less expensive alternative to stimulated ivf in case of severe male infertility . the experience of using ivm with non - ejaculated sperm is limited , case reports of pregnancies are published from sperm obtained by testicular biopsy , testicular fine - needle aspiration , percutaneous epididymal sperm extraction , and microtesticular sperm extraction . nevertheless , some of the reports used a low stimulation including low doses of follicle - stimulating hormone ( fsh ) , menotropins , or clomiphene citrate that constitute mild stimulation cycles and not ivm . in our private center in leon mexico , we started to offer ivm routinely in 2007 . in this report , we describe a case where human chorionic gonadotropin ( hcg)-primed ivm was performed for a male infertility indication using a testicular sperm sample from a patient with nonobstructive azoospermia . a 28-year - old male with nonobstructive azoospermia with normal karyotype , no y chromosome microdeletions , or common mutations in cftr gene and fsh 12 mui / ml , luteinizing hormone ( lh ) 9.0 mui / ml , and testosterone 300 ng / ml . female partner 34 years with primary infertility , normal menstrual cycles , basal fsh 7.4 mui / ml , lh 2.5 mui / l , e2 : 22.5/l , testosterone 0.2 ng / ml , 14 follicles at antral follicle count , and endometrial thickness of 9 mm . taking into account that no female factors were detected , and the prognosis was good , we decided offer ivm as treatment option to the couple . the couple had chosen ivm for their desire of a lower cost in ivf procedures . a transvaginal ultrasound scan on day 3 revealed ten antral follicles . on day 7 of the cycle , a follow - up of follicle growth when one follicle reached 1214 mm and endometrial thickness was 7 mm , the patient received a single intramuscular injection of 250 g of recombinant hcg ( ovidrel , merck serono , switzerland ) . thirty - six hour after hcg injection , oocyte retrieval was performed under patient intravenous ( iv ) sedation using a 17-gauge 20 single lumen needle ( kitazato oocyte pickup needle ) . fourteen cumulus - oocytes complexes were retrieved , cumulus cells were removed using hyaluronidase , no mature ( metaphase ii ) oocytes were found . immature oocytes ( germinal vesicle or metaphase i oocytes ) were cultured in ivm media ( sage ) supplemented with 0.75 iu / ml of recombinant fsh ( rfsh ) and 0.75 iu / ml of recombinant lh . testicular biopsy was performed under iv sedation 1 day after oocyte capture , fresh spermatozoa obtained by testicular biopsy . the testicular tissue was minced into small pieces using two scalpels in human tubal fluid ( htf)-hepes , the supernatant was centrifuged at resuspended in fresh htf until intracytoplasmic sperm injection ( icsi ) . injected oocytes were transferred to global medium ( lifeglobal , usa ) incubated at 37c in 6% co2 . embryos were culture in 20 l droplets of global medium at 37c in 6% co2 , three cleavage embryos in day 2 were transferred . a singleton live birth at 37 weeks was obtained with a weight of 2700 g and length of 50 cm , and apgar score was 9/9/9 . a 28-year - old male with nonobstructive azoospermia with normal karyotype , no y chromosome microdeletions , or common mutations in cftr gene and fsh 12 mui / ml , luteinizing hormone ( lh ) 9.0 mui / ml , and testosterone 300 ng / ml . female partner 34 years with primary infertility , normal menstrual cycles , basal fsh 7.4 mui / ml , lh 2.5 mui / l , e2 : 22.5/l , testosterone 0.2 ng / ml , 14 follicles at antral follicle count , and endometrial thickness of 9 mm . taking into account that no female factors were detected , and the prognosis was good , we decided offer ivm as treatment option to the couple . the couple had chosen ivm for their desire of a lower cost in ivf procedures . a transvaginal ultrasound scan on day 3 revealed ten antral follicles . on day 7 of the cycle , a follow - up of follicle growth when one follicle reached 1214 mm and endometrial thickness was 7 mm , the patient received a single intramuscular injection of 250 g of recombinant hcg ( ovidrel , merck serono , switzerland ) . thirty - six hour after hcg injection , oocyte retrieval was performed under patient intravenous ( iv ) sedation using a 17-gauge 20 single lumen needle ( kitazato oocyte pickup needle ) . fourteen cumulus - oocytes complexes were retrieved , cumulus cells were removed using hyaluronidase , no mature ( metaphase ii ) oocytes were found . immature oocytes ( germinal vesicle or metaphase i oocytes ) were cultured in ivm media ( sage ) supplemented with 0.75 iu / ml of recombinant fsh ( rfsh ) and 0.75 iu / ml of recombinant lh . testicular biopsy was performed under iv sedation 1 day after oocyte capture , fresh spermatozoa obtained by testicular biopsy . the testicular tissue was minced into small pieces using two scalpels in human tubal fluid ( htf)-hepes , the supernatant was centrifuged at resuspended in fresh htf until intracytoplasmic sperm injection ( icsi ) . injected oocytes were transferred to global medium ( lifeglobal , usa ) incubated at 37c in 6% co2 . embryos were culture in 20 l droplets of global medium at 37c in 6% co2 , three cleavage embryos in day 2 were transferred . a singleton live birth at 37 weeks was obtained with a weight of 2700 g and length of 50 cm , and apgar score was 9/9/9 . in this report , a live birth from an hcg - primed ivm using testicular sperm is reported , this as part of routine offer of ivm to selected patients in our clinic in mexico . because pregnancy rates are lower that for conventional ivf , it is difficult to introduce this procedure in private practice where patients cover completely with the cost of assisted reproduction procedures . however , ivm offers several advantages for the case of male infertility such as a friendly procedure for a female partner , the absence of risk related with ovary stimulation in otherwise healthy women , and the reduction in costs of treatment . the published experience of ivm using testicular sperm is quite heterogeneous , and in some reports , the stimulation is more intense than simple hcg priming , contradicting the main benefit of ivm . on the other hand , apparently ivm in normo - ovulatory women and pregnancy rates the biggest experience of icsi with testis biopsies from non - obstructive azoospermia in normo - ovulatory women using rfsh + hcg indicated that pregnancy rates are similar . however , in pcos patients using only hcg lower fertilization and pregnancy rates are reported . in the present case , pregnancy was obtained at the first ivm attempt ; but further research is required to understand factors impacting the results of ivm , such as hormonal priming , ovary function , and the etiology of azoospermia when ivm oocytes are fertilized using testicular spermatozoa . summarizing ivm may be an option for normo - ovulatory women in cases of male infertility that requires icsi with sperm obtained from testicular biopsies . repa is supported by conacyt ( 329705 ) and elphinstone scholarships and a fellowship from ibsa foundation . repa is supported by conacyt ( 329705 ) and elphinstone scholarships and a fellowship from ibsa foundation .

purpose . the pathogenesis of rhegmatogenous retinal detachment ( rrd ) remains incompletely understood , with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy . here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach . materials and methods . retinal detachment was created in the right eyes of six new zealand red pigmented rabbits . sham surgery was undertaken in five other rabbits that were used as controls . after seven days the eyes were enucleated and the vitreous was removed . the vitreous samples were evaluated with two - dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry . results . ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups . protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments , and those downregulated were found to be peroxiredoxin 2 , collagen - i1 fragment , and -1-antiproteinase f. conclusions . proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications .

rhegmatogenous retinal detachment ( rrd ) is characterized by the accumulation of subretinal fluid between the neurosensory retina and retinal pigment epithelium following the formation of a retinal break . the pathogenesis of rrd is complex and incompletely understood , involving age - related and/or inherited structural and molecular changes of the vitreous extracellular matrix and vitreoretinal interface , and the process of posterior vitreous detachment . the annual incidence of the condition has been estimated at 12.05 per 100,000 , and although primary surgical reattachment is successful in the great majority of cases , photoreceptor cell death , subretinal fibrosis , and proliferative vitreoretinopathy ( pvr ) continue to be significant causes of reduced visual outcomes [ 3 , 4 ] . pvr involves the proliferation and migration of various cell types including retinal pigment epithelial ( rpe ) cells , mller cells , inflammatory cells , and hyalocytes , which contribute to the formation of vitreal and periretinal membranes that can impede photoreceptor regeneration following surgical reattachment and cause tractional retinal detachment . the postulated epithelial - to - mesenchymal transition of rpe cells and mller cell activation and growth onto the retinal surfaces are believed to be pivotal events in pvr [ 4 , 5 ] . it appears that the exposure of such cells to the vitreous and associated growth factors as a result of rrd significantly contributes to the pathogenesis of pvr , though the basic cause as well as a clinically effective therapeutic approach for this condition remains elusive [ 5 , 6 ] . proteomics studies proteins on a large scale in pursuit of a global and integrated view of disease processes at the protein level , which may potentially lead to the identification of novel biomarkers and therapeutic targets useful in clinical practice [ 711 ] . rrd would likely be associated with alterations in the proteomic profiles of both the retina and vitreous . indeed , we initially undertook the first such retinal study from which a number of potentially important proteins were identified . the present study extends the proteomic investigation to the vitreous of this rabbit model of retinal detachment , building upon previous such analyses of human vitreous [ 1316 ] , in order to add further knowledge of the underlying pathophysiology [ 10 , 17 ] . inferior retinal detachment was created in the right eyes of six new zealand red pigmented rabbits . combined injections of xylazine ( 6.7 mg / kg ) and ketamine ( 33.3 mg / kg ) were administered intramuscularly to induce anesthesia and analgesia . the pupils were dilated with topical drops of atropine and tropicamide ( 1% solutions ) . a pipette tip , with an external diameter of approximately 100 m , sodium hyaluronate ( healon , 0.25% in a balanced salt solution ; pharmacia , piscataway , nj ) was infused via a glass pipette between the neurosensory retina and retinal pigment epithelium . healon was necessary to prevent spontaneous retinal reattachment , and 0.25% is the most dilute solution that maintains the detachment for extended periods . approximately 50% of the retina beneath the medullary rays , which included the central retina , was detached ( figure 1 ) . sham surgery was performed in the right eyes of five other rabbits that were used as controls , which involved surgical entry of the vitreous cavity without disruption of the retina . seven days postoperatively the animals were euthanized by the administration of sodium pentobarbital ( 120 mg / kg ; butler schein , dublin , oh ) and the eyes enucleated . after removal of the cornea and lens , the associated vitreous of the sham and detached retinas was extracted and immediately snap - frozen in liquid nitrogen within separate vials . there was no gross evidence of blood or other contamination of the vitreous samples at the time of tissue harvesting . all of the animal experiments undertaken in this study were in accordance with the standards of the national institutes of health animal care and use committee protocols , the arvo statement for the use of animals in ophthalmic and vision research , and the guidelines of the animal resource center , university of california , santa barbara . the rabbit vitreous samples were homogenized and dissolved in a lysis buffer containing 9 m urea , 2% ( v / v ) triton x-100 , 2% ( v / v ) immobilized ph gradient ( ipg ) buffer ( ph 310 nonlinear ) , and 2% ( w / v ) dithiothreitol ( dtt ) . the total protein content in each vitreous sample was determined with non - interfering protein assay ( calbiochem , san diego , ca ) . the extracted proteins were first fractionated by isoelectric focusing ( ief ) using ph 310 nonlinear 18 cm ipg strips ( ge healthcare , chalfont st . the ipg strips were rehydrated for 20 h at room temperature in 200 l lysis buffer each containing 20 g protein from individual vitreous samples and 150 l rehydration buffer ( 8 m urea , 2% ( w / v ) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( chaps ) , 0.3% ( w / v ) dtt , and 2% ( v / v ) ipg buffer ) , using the immobiline drystrip reswelling tray ( ge healthcare ) . the ief was undertaken on a multiphor ii electrophoresis system ( ge healthcare ) at 500 v for 5 h and 3500 v in two steps for 5 h and 9.5 h in a gradient mode at 17c with the use of a multitemp iii thermostatic circulator ( ge healthcare ) . before the second - dimension sodium dodecyl sulfate ( sds ) polyacrylamide gel electrophoresis ( page ) , the ipg strips were equilibrated firstly for 10 min with gentle agitation in 20 ml of equilibration solution ( 0.6% ( w / v ) tris - hcl , ph 6.8 , 6 m urea , 30% ( v / v ) glycerol , 1% ( w / v ) sds , and 0.05% ( w / v ) dtt ) and secondly using 4.5% ( w / v ) iodoacetamide and bromophenol blue . the ipg strips were then transferred to 12% polyacrylamide gels for electrophoresis , which was performed at a maximum voltage of 50 v for approximately 20 h to separate the proteins vertically on the basis of molecular mass . the two - dimensional ( 2d ) gels were silver stained using a protocol optimized for protein identification with mass spectrometry . in brief , the gels were fixed overnight in 50% ( v / v ) ethanol , 12% ( v / v ) acetic acid , and 0.0185% ( v / v ) formaldehyde . the gels were washed 3 times for 20 min in 35% ( v / v ) ethanol and pretreated for 1 min in 0.02% ( w / v ) na2s2o35h2o . they were then rinsed in water and stained for 20 min in 0.2% ( w / v ) agno3 and 0.028% ( v / v ) formaldehyde . following further rinsing with water , development was undertaken for approximately 3 min in 6% ( w / v ) na2co3 , 0.0185% ( v / v ) formaldehyde , and 0.0004% ( w / v ) na2s2o35h2o . the development was arrested in a fixative solution of 40% ( v / v ) ethanol and 12% ( v / v ) acetic acid . silver stained 2d gels were scanned on a gs-710 calibrated imaging densitometer ( bio - rad , hercules , ca ) using the quantity one program ( bio - rad ) , and the pdquest software ( bio - rad ) was used to define , quantify , and match the protein spots on each of the 2d gels . all well - defined protein spots that were at least twofold ( mann - whitney u test , p < 0.05 ) differentially expressed between the sham and retinal detachment vitreous groups were selected for identification with nanoliquid chromatography - electrospray ionization tandem mass spectrometry ( lc - ms / ms ) . the selected protein spots were carefully excised from the gels with a scalpel and subjected to in - gel digestion with trypsin gold ( mass spectrometry grade ; promega , madison , wi ) . the peptide samples that were obtained were analyzed by lc - ms / ms as previously described . in brief , peptides generated by trypsin digestion were separated on an inert nano - lc system ( lc packings , san francisco , ca ) connected to a q - tof premier mass spectrometer ( waters , milford , ma ) . the masslynx 4 sp4 ( waters ) was used to obtain spectra and the raw data was processed using proteinlynx global server 2.1 ( waters ) . the processed data were used to search the total part of the swiss - prot database using the online version of the mascot ms / ms ions search facility ( matrix science , ltd . ) . the search was undertaken with doubly and triply charged ions with up to two missed cleavages , a peptide tolerance of 50 ppm , one variable modification , carbamidomethyl - c , and a ms / ms tolerance of 0.05 da . contaminating peptides such as trypsin , keratin , bovine serum albumin , and all peptides originating from previous samples were disregarded . individual peptide ions scores above approximately 36 indicated identity or extensive homology giving a less than 5% probability that the observed match was a random event . all peptides for the protein hits are reported ( table 1 ) . in each case three micrograms of vitreous sample protein was separated on novex 1020% gradient tris - glycine polyacrylamide gels ( invitrogen corporation , carlsbad , ca ) and subsequently transferred to nitrocellulose hybond - c extra membranes ( ge healthcare ) . the membranes were blocked overnight with 5% skimmed milk in 80 mm na2hpo4 , 20 mm nah2po4 , 100 mm nacl , and 0.05% tween 20 buffer , ph 7.5 . membranes were incubated with anti - albumin ( genway biotech , ca , usa ; 1 : 5000 ) and anti - peroxiredoxin 2 ( abcam , cambridge , uk ; 1 : 200 ) . no suitable antibodies were commercially available for the rabbit f isoform of -1-antiproteinase or the rabbit collagen - i1 fragment that was identified with lc - ms / ms . following washing , the membranes were further incubated with appropriate horseradish peroxidase - conjugated secondary antibodies : p0163 sheep and p0260 mouse ( both 1 : 1000 ; dako , glostrup , denmark ) . proteins were visualized with the enhanced chemiluminescence system ( ge healthcare ) and imaging system ( fujifilm las-3000 , tokyo , japan ) . up to approximately 340 protein spots were clearly resolved on each of the 11 2d gels . ten protein spots were found to be significantly and at least twofold differentially expressed between the sham and detachment vitreous groups ( figure 2 ) . three protein spots were upregulated and seven spots were downregulated . from the three upregulated protein spots , two were identified as fragments of albumin ( spots 5104 and 6101 ) , whilst spot 6205 could not be identified . four of the seven downregulated protein spots were identified as fragment of collagen - i1 ( spot 0503 ) , -1-antiproteinase f ( spots 0703 and 1707 ) , and peroxiredoxin 2 ( spot 0705 ) . protein spots 0102 , 0815 , and 1302 could not be identified ( figure 2 ; table 1 ) . western blotting developed with anti - albumin showed a heavy band at approximately 60 kda , which is likely to represent the full length protein , whilst multiple bands below this suggest the presence of several fragments , some of which may correspond with those identified with the 2d - page analysis ( figure 3 , left ) . however , spot 0705 containing peroxiredoxin 2 migrates with a molecular mass around 60 kda with 2d - page ( figure 2 ) , and this size was verified by western blot analysis ( figure 3 , right ) . though a single and specific band was achieved with anti - peroxiredoxin 2 , western blotting could not be reliably used for quantification due to a weak signal near the detection limit and variable background reaction . analysis with 2d - page revealed fragments of albumin to be upregulated in the vitreous following retinal detachment . albumin is the most abundant protein in plasma , aqueous , and vitreous humor , where in the latter it constitutes around 6070% of total protein [ 1921 ] . serum proteins such as albumin are present in the aqueous and vitreous humor at a relatively lower level compared to the vascular circulation from where they may have in part originated [ 21 , 22 ] . western blot analysis showed an intense band at approximately 60 kda corresponding with the full length albumin protein , with multiple lower molecular mass bands that are likely its fragments . increase of albumin and its fragments may signify increased proteolysis and the passage of albumin into the vitreous . indeed , the breakdown of the blood - retinal barrier that occurs with retinal detachment has also been implicated in the increase of other such proteins in the vitreous [ 12 , 2328 ] . it is also possible that albumin in the vitreous may arise from de novo synthesis in the retina , similar to the reported increased gene and protein expression of albumin in the corneal epithelium during wound healing [ 29 , 30 ] . extraocular albumin is known to have diverse and important functions , which include maintenance of colloid osmotic pressure , transport of biomolecules , and inactivation of toxins through intermolecular binding [ 31 , 32 ] . albumin can also act as an antioxidant by scavenging reactive oxygen species and sequestration of metal ions and has anti - inflammatory and apoptotic regulatory abilities [ 3235 ] . vitreal albumin has been proposed to transport long chain fatty acids into the lens for biosynthesis of lenticular lipids [ 21 , 31 , 36 ] . indeed , albumin is likely to have many such important roles in the eye , which requires further investigation . in the present study we observed peroxiredoxin 2 to have a molecular mass above 60 kda using both 2d - page and western blot analyses . however , the predicted molecular mass of the peroxiredoxin family of proteins is approximately 22 kda31 kda . this variation may represent the well - studied property of these proteins to undergo oligomerization , which can be promoted by a number of factors including overoxidation of cysteine residues of peroxiredoxin . although the present experiments were conducted in standard reducing conditions that aim to break cysteine bonds , we obtained a band well above 20 kda . this is in keeping with another study , which also showed some peroxiredoxin 2 western blot bands appearing at molecular mass much higher than 20 kda that was suggested to result from oligomerization or posttranslational modification . our finding could also represent a novel alternative splicing variant of peroxiredoxin 2 , as reported for peroxiredoxin 5 . the peroxiredoxins are a group of ubiquitous antioxidant proteins that currently comprise six members in mammals . these proteins are primarily found at high levels intracellularly , mainly within the cytosol , but are also present in the mitochondria , peroxisomes , and nuclei , and they may be exported . furthermore , presence of peroxiredoxin 2 has been shown in plasma , not only as a result of hemolysis but also possibly by secretion from the t lymphocytes [ 41 , 42 ] . these multifunction enzymes act as antioxidants by using redox active cysteines for the reduction and degradation of hydrogen peroxide , peroxynitrite , and organic hydroperoxides [ 37 , 43 ] . oxidative stress is thought to result from an imbalance between reactive oxygen species production and antioxidant ability and is recognized to be an important factor in the pathogenesis of a number of age - related and neurodegenerative diseases , which include age - related cataract , age - related macular degeneration , glaucoma , diabetic retinopathy , retinal detachment , and pvr [ 4447 ] . indeed , the present study showed a decrease in the vitreal levels of peroxiredoxin 2 following retinal detachment . this may be in keeping with reported reductions in the levels of other members of the antioxidant defense system such as glutathione and ascorbic acid both in vitreal and in blood samples of patients suffering from pvr [ 44 , 45 ] . furthermore , apart from their role as antioxidants , the peroxiredoxins can affect a diverse range of biological processes that include cellular proliferation , differentiation , and apoptosis by influencing signal transduction pathways that employ hydrogen peroxide as a secondary messenger [ 43 , 48 ] . recent studies on tears from patients with glaucoma have also identified peroxiredoxin 1 as having a possible involvement in inflammation [ 49 , 50 ] . indeed , peroxiredoxin 2 and other members of this family of proteins are liable to have a significant role in the pathophysiology of retinal detachment . a fragment of collagen - i1 was identified in the vitreous of the rabbit ; however , type i collagen has not previously been identified as a natural component of the mammalian vitreous and is rather known to be a constituent of early pvr membranes [ 5153 ] and retinal blood vessels [ 54 , 55 ] . a mixture of type ii , ix , and v / xi hybrid collagen fibrils , which are separated out mainly by water and ions attracted to hyaluronan , characterizes the vitreous body . collagen , possibly with the aid of adhesive - like intermediate molecules , may provide the basis of vitreoretinal adhesion by connecting the vitreous with the retinal inner limiting membrane ( ilm ) . this attachment is extremely strong in the vitreous base since the fibrils pass through the ilm to merge into underlying collagen networks and crypts [ 2 , 56 , 57 ] . collagen is also a significant component of both epiretinal and subretinal pvr membranes [ 53 , 58 ] , and type i collagen is recognized to be a principal constituent during their early development [ 5153 ] . the presence of collagen in the subretinal space , a place normally devoid of this protein , suggests that certain cells , particularly the rpe and mller cells associated with membranes , are able to synthesize collagen under certain pathological conditions such as retinal detachment and pvr [ 5861 ] . however , the present analysis suggests collagen - i1 fragment to be found in sham vitreous , which furthermore showed a decreased concentration following retinal detachment that may indicate perturbed proteolytic activity . matrix metalloproteinases ( mmp ) and other proteolytic enzymes that are able to degrade and remodel vitreal collagen have been found to be increased with rrd and pvr [ 6265 ] , which could be in keeping with the decrease in -1-antiproteinase shown in the present study . further studies will be necessary to confirm the source and nature of collagen - i1 in the vitreous and the possible mechanisms of collagen fragmentation that may be an important feature of vitreous liquefaction and rrd [ 2 , 62 , 66 ] . 2d - page showed -1-antiproteinase ( also called -1-antitrypsin or -1-proteinase inhibitor ) at two closely positioned spots , which were largely in keeping with their predicted molecular mass but differing by their charge . currently , four isoforms of -1-antiproteinase have been identified in the rabbit , termed f , s1 , s2 , and e , which is a similar picture to the multiple variants identified in humans [ 67 , 68 ] . alpha-1-antiproteinase is an acute phase protein and archetypal member of the superfamily of serine protease inhibitors ( serpin ) , which are involved in a wide range of biological processes that includes inflammation , angiogenesis , blood coagulation , ecm remodeling , and tumor suppression . this protein has the ability to inhibit a large number of serine proteases though its principle target is neutrophil elastase . indeed , -1-antiproteinase originally received much attention because its deficiency increases the risk of a variety of clinical conditions , such as chronic obstructive pulmonary disease , which can result from unrestrained elastase activity . we found the f isoform of rabbit -1-antiproteinase to be downregulated in the vitreous following retinal detachment . so far identified that has been shown to have the oxidizable methionine residue site that is present in human -1-antiproteinase . the oxidation of methionine to methionine sulfoxide , which can occur during episodes of inflammation as a result of oxygen - free radicals secreted by leucocytes , has an inhibiting effect upon -1-antiproteinase function . this process is thought to enhance the ability of proteinases such as elastase to locally degrade tissue debris that occurs at sites of inflammation [ 67 , 68 ] . alpha-1-antiproteinase is primarily produced in the liver and circulated to the rest of the body tissues via the blood ; however , extrahepatic sites of its synthesis have been identified , which include blood monocytes , alveolar macrophages , bronchial and gastrointestinal epithelial cells , and the cornea [ 7073 ] . the protein has also been localized to the tear film , aqueous humor , and vitreous , where in the latter a phosphorylated form of -1-antiproteinase has been suggested as a potential biomarker of idiopathic macular hole and rhegmatogenous retinal detachment [ 7375 ] . it has been postulated that one of the main functions of corneal -1-antiproteinase is to protect against the damaging effects of neutrophil elastase produced during corneal inflammation , and it may be expected that a similar role in addition to others is applicable to vitreal -1-antiproteinase , though this requires further investigation . this proteomic investigation of the rabbit vitreous has identified a set of proteins that assist our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications . certain proteins , such as those of low abundance and at the extremes of molecular mass , together with membrane proteins , can be difficult to resolve and detect using the 2d - page technique . therefore , complementary proteomic methods such as gel - free mass spectrometry should be considered in future work in order to help address these limitations .

primary breast lymphoma ( pbl ) is a rare entity . it represents 0.4% of malignant breast lesions and 2% of extranodal lymphomas . a 23-year - old woman presented with a left breast lump followed by palpable left axillary lymphadenopathy . fine - needle aspiration cytology ( fnac ) and histopathology were diagnostic of non - hodgkin lymphoma - large cell type . immunohistochemistry was positive against leukocyte common antigen and cd20 . because pbls are uncommon malignant lesions and they usually do not have characteristic clinical and imaging findings , fnac may prove to be a simple , rapid , reliable , and cost - effective procedure for successful diagnosis of the pbl .

primary breast lymphoma ( pbl ) is a rare neoplasm that accounts for 0.4% of malignant breast lesions and 2% of extranodal lymphomas . diagnosis of pbl by fine - needle aspiration cytology ( fnac ) is reported infrequently . a 23-year - old non - lactating ( para 2 , gravida 0 ) lady came with a left breast lump and lymphedema of the left arm of 1-month duration . she recently had lumpectomy done in the same breast 1-month earlier at a peripheral hospital , which was diagnosed as breast abscess on histopathology . her ultrasound examination previous to lumpectomy was available that showed an irregular hypoechoic mass of size 2.6 2.1 1.6 cm in the left breast having both solid and cystic components , perilesional inflammatory changes , and architectural distortion . the right breast was normal and bilateral axillary lymphadenopathy was absent at this point of time . her examination at the present visit revealed an irregular hard tender fixed mass of 6 4 cm in the outer lower and upper quadrants of the left breast . the skin overlying the mass was erythematous , nodular , and showed a surgical scar mark just above the areola [ figure 1a ] . fnac of the left breast mass and left axillary lymph nodes was done in department of pathology . the breast mass fnac showed richly cellular monomorphic population of loosely cohesive single lymphoid cells of two to three times the size of mature lymphocytes with moderate anisonucleosis , 1 - 3 prominent nucleoli and fragile cytoplasm [ figure 1b ] . similar cytomorphologic features were also present in the aspirate from left axillary lymph nodes . a cytological diagnosis of non - hodgkin lymphoma- large cell type was given . a tru - cut needle biopsy of the breast mass also showed features of non - hodgkin lymphoma . her formalin - fixed , paraffin - embedded blocks of the prior lumpectomy specimen was then retrieved from the peripheral laboratory and reviewed . hematoxylin and eosin ( h and e)-stained sections showed replacement of the breast parenchyma by diffuse sheets of monomorphic lymphoid cells [ figure 1c ] . on higher magnification , the lymphoid cells showed moderate anisokaryosis , clumped nuclear chromatin , prominent nucleoli , and minimal amount of eosinophilic cytoplasm on a background of lymphoglandular bodies [ figure 1d ] . lymphoepithelial lesion was characterized by infiltration of lymphoma cells into the sparsely present duct epithelium of the breast [ figure 2a ] . immunohistochemical studies were performed on formalin fixed , paraffin - embedded blocks of the lumpectomy specimen . cytokeratin immunostaining showed strong cytoplasmic reactivity in the duct epithelium while the lymphoma cells stained negative [ figure 2b ] . in contrast , the lymphoma cells expressed diffuse strong membranous positivity with antibodies against leukocyte common antigen [ figure 2c ] and cd 20 [ figure 2d ] while negative for cd 15 , cd 30 , and cd 3 . there was no primary elsewhere as evidenced by the absence of hepatosplenomegaly and the absence of any lymph node enlargement other than left axillary lymph nodes . the final diagnosis was primary diffuse large b - cell lymphoma of the breast , stage ii . the patient was planned for chemotherapy , her baseline laboratory investigations and ultrasound examination of abdomen was normal . however , further management and follow up of the patient could not be done as she escaped from the hospital against the medical advice . ( b ) fnac showing dispersed large non - cleaved lymphoma cells aspirated from the breast lump ( pap , x400 ) . ( c ) diffuse infiltration of the lymphoma cells resulting in destruction and fibrosis of breast parenchyma ( h and e , x200 ) . ( d ) magnified view of lymphoma cells against a background of lymphoglandular bodies ( h and e , x400 ) ( a ) lymphoepithelial lesion demonstrating invasion of ductal epithelium of the breast by lymphoma cells ( h and e , x200 ) . ( b ) cytokeratin immunostaining strongly positive in ductal epithelium , negative in surrounding lymphoma cells ( ihc , x200 ) . ( c ) diffuse strong lca positive immunostaining in tumour cells ( ihc , x400 ) . ( d ) lymphoma cells showing diffuse strong positivity against cd 20 ( ihc , x400 ) the deceptive benign appearance on clinical and ultrasound examinations was diagnosed as non - hodgkin lymphoma . fnac provided the earliest clue to diagnosis of pbl which was later confirmed by histology and immunohistochemistry in this case . because breast lymphoma is a rare condition , however , clinician should be aware of the deceiving benign appearance of pbl in contrast to much more commonly seen breast carcinoma . on physical examination , pbl often appears as a rapidly growing single palpable nodule of mean size 3.5 cm that is mobile and non - tender . this probably results from the relatively rapid growth of these lesions in comparison to breast carcinomas , especially in the younger patients . . clinical signs of advanced breast malignancy are rare , such as inflammatory changes , nipple retraction , puckering of the skin or tumor fixation . breast lymphoma can occasionally present as a diffuse rapid breast enlargement in the younger age group , or as breast skin thickening due to lymphatic blockage by lymphoma resulting in retrograde edema . on diagnostic mammography , pbl often appears as a well - defined mass with smooth margins and homogeneous appearance . again , signs of breast malignancy such as microcalcification , spiculation , and distortion of surrounding tissue are usually absent . likewise pbl will most often demonstrate a well - defined hypoechoic lesion without enhancement suggesting a wrong notion of benignity . the specific criteria for diagnosis of pbl were first defined by wiseman and liao in 1972 . these are : ( a ) the clinical site of appearance is the breast ; ( b ) a history of prior lymphoma or evidence of widespread disease are absent at diagnosis ; ( c ) demonstration of lymphoma in proximity to breast tissue ; and ( d ) involvement of ipsilateral lymph nodes may be present if they develop simultaneously with the primary breast tumor . the entire criteria for diagnosis of pbl are satisfactorily present in our case . at the time of initial lumpectomy , however , when patient came to us she had significant enlargement of the ipsilateral axillary lymph nodes in association with the recurring breast lump ( stage ii disease ) . this offers evidence that excision biopsy alone is not curative in patients with pbl ; rather such a procedure may upstage the disease process in these patients . thus , if a young patient presents with a rapidly growing breast tumor , lymphoma should be considered before undertaking any surgical intervention . in such cases , a meticulously performed fnac is vital for an accurate preoperative diagnosis of the breast lymphoma . in summary , pbl is a rare entity that accounts for only a small fraction of the breast malignancies . as none of the clinical or imaging features of the disease is characteristic , a rapidly growing breast lump in a young female should warn the attending clinician of this rare possibility . fnac is very useful , simple , rapid , reliable , and cost - effective procedure for an accurate diagnosis of the lesion .

objectivein type 1 diabetes , small studies have found that resistance exercise ( weight lifting ) reduces hba1c . in the current study , we examined the acute impacts of resistance exercise on glycemia during exercise and in the subsequent 24 h compared with aerobic exercise and no exercise.research design and methodstwelve physically active individuals with type 1 diabetes ( hba1c 7.1 1.0% ) performed 45 min of resistance exercise ( three sets of seven exercises at eight repetitions maximum ) , 45 min of aerobic exercise ( running at 60% of vo2max ) , or no exercise on separate days . plasma glucose was measured during and for 60 min after exercise . interstitial glucose was measured by continuous glucose monitoring 24 h before , during , and 24 h after exercise.resultstreatment-by-time interactions ( p < 0.001 ) were found for changes in plasma glucose during and after exercise . plasma glucose decreased from 8.4 2.7 to 6.8 2.3 mmol / l ( p = 0.008 ) during resistance exercise and from 9.2 3.4 to 5.8 2.0 mmol / l ( p = 0.001 ) during aerobic exercise . no significant changes were seen during the no - exercise control session . during recovery , glucose levels did not change significantly after resistance exercise but increased by 2.2 0.6 mmol / l ( p = 0.023 ) after aerobic exercise . mean interstitial glucose from 4.5 to 6.0 h postexercise was significantly lower after resistance exercise versus aerobic exercise.conclusionsresistance exercise causes less initial decline in blood glucose during the activity but is associated with more prolonged reductions in postexercise glycemia than aerobic exercise . this might account for hba1c reductions found in studies of resistance exercise but not aerobic exercise in type 1 diabetes .

the study was approved by the research ethics boards of the university of ottawa and ottawa hospital . two of the participants were competitive athletes training 6 days per week , while those remaining were recreationally active . all participants had been regularly performing both aerobic and resistance exercise at least three times weekly for a minimum of 6 months . participants were using either multiple daily injections ( mdis ) of insulin or continuous subcutaneous insulin infusion with an insulin pump . the same cohort of participants also took part in a previously published study from the same research group ( 20 ) . testing took place in the human and environmental physiology research unit at the university of ottawa . participants provided written informed consent prior to being tested for vo2max , muscular strength ( eight repetition maximum ) , and hba1c as previously described ( 20 ) . the cgms system gold ( medtronic , northridge , ca ) was used in this study so that participants would be blinded to their glucose values and would not change their behavior based on real - time glucose monitoring . onetouch ultrasmart handheld glucose meters ( lifescan ; johnson & johnson , milpitas , ca ) and coded strips ( same code throughout the study ) were provided for capillary glucose tests . twenty - four hours after the end of the exercise / no - exercise control session , cgm units were retrieved and data were downloaded ( minimed solutions v.3.0c ; medtronic , northridge , ca ) . over each monitoring period , participants consumed the same self - selected breakfast , lunch , and dinner daily at the same times of day and recorded food and insulin intake on study log sheets . participants refrained from exercise for 24 h before insertion of the sensor ( 48 h before the experimental session ) and avoided caffeine and alcohol during the monitoring period . participants arrived at the laboratory at 4:00 p.m. on the day after the sensor insertion . the following sessions were performed , separated by at least 5 days : 1 ) resistance exercise , three sets of eight repetitions maximum of seven different exercises with 90-s rest between sets ( duration 45 min ) ; 2 ) aerobic exercise , 45 min of treadmill exercise ( 60% of vo2max ) ; and 3 ) no - exercise control , 45 min of seated rest . testing sessions for the female participants , who were using monophasic oral contraceptives , took place during the active pill - consumption phase . participants reduced their insulin doses on exercise days by making either a 10% decrease in intermediate or long - acting insulin ( mdi ) or a 50% decrease in basal rate starting 1 h before exercise and maintained until the end of exercise for pump users . if blood glucose was < 5 mmol / l upon arrival , those using insulin pumps decreased their basal rate a further 25% . participants consumed a standard snack ( glucerna chocolate graham snack bars , 150 calories , 25 g carbohydrate ; abbott laboratories , abbott park , il ) at 4:00 p.m. every day , including the exercise day , with the bar consumed upon arrival at the laboratory . capillary glucose was checked 60 and 30 min before exercise and immediately prior to exercise to ensure glucose levels 5.5 and 13.9 mmol / l . venous blood samples were collected through an intravenous catheter at baseline and 5 , 10 , 15 , 30 , and 45 min during all three testing sessions ( resistance exercise , aerobic exercise , and no - exercise control ) and at the 50- , 55- , 60- , 65- , 75- , 85- , 95- , and 105-min blood was immediately mixed by inversion , centrifuged ( 4,000 revolutions / min for 4 min ) , and stored at 80c . the hexokinase timed end point method was used to determine plasma glucose levels using the beckman coulter unicel dxc600 synchron clinical analyzer ( beckman coulter , fullerton , ca ) and synchron cx systems glucose reagent ( cat . glucose levels were compared among sessions using two - way repeated - measures ( time and condition ) anova . exercise and recovery periods were examined separately among the three sessions ( aerobic , resistance , and no - exercise control ) . the exercise period consisted of the 5- , 10- , 15- , 30- , and 45-min time points , while the recovery period consisted of the remaining time points . paired sample t tests were used to perform pairwise post hoc comparisons for each time point between conditions ( aerobic , resistance , or no - exercise control ) within exercise and recovery separately and to examine changes from baseline and changes from the end of exercise within each exercise condition . cgm data were examined as 15-min averages in the following windows : 24-h pre - exercise , overnight ( 12:00 a.m. to 6:00 a.m. ) pre - exercise , 16 h postexercise , overnight postexercise , and 24 h postexercise . a two - way ( time and condition ) repeated - measures anova was used to compare among conditions in the 16-h postexercise period . paired sample t tests were then used to perform pairwise post hoc comparisons for each 15-min segment . the minimum , maximum , and mean blood glucose ; amount of time spent in hypoglycemic and hyperglycemic states ; and areas under the curve ( aucs ) for time spent in hypo- and hyperglycemic states were determined for each window . pre - exercise values were compared with postexercise values within exercise conditions using related - samples wilcoxon signed rank tests . differences among conditions were examined using related - samples friedman two - way anova by ranks . agreement between cgm data and capillary glucose over the 3 days was determined by performing pearson correlations between sensor glucose and self - recorded capillary glucose values . daily total insulin and carbohydrate intake was calculated based on the information provided in participant logs . comparisons among conditions for each day were made using related - samples friedman two - way anova by ranks . where significant results were found , related - samples wilcoxon signed rank tests ensued for determination of where the differences lie . analyses were performed using spss 18.0 for windows ( spss , chicago , il ) . testing took place in the human and environmental physiology research unit at the university of ottawa . participants provided written informed consent prior to being tested for vo2max , muscular strength ( eight repetition maximum ) , and hba1c as previously described ( 20 ) . the cgms system gold ( medtronic , northridge , ca ) was used in this study so that participants would be blinded to their glucose values and would not change their behavior based on real - time glucose monitoring . onetouch ultrasmart handheld glucose meters ( lifescan ; johnson & johnson , milpitas , ca ) and coded strips ( same code throughout the study ) were provided for capillary glucose tests . twenty - four hours after the end of the exercise / no - exercise control session , cgm units were retrieved and data were downloaded ( minimed solutions v.3.0c ; medtronic , northridge , ca ) . over each monitoring period , participants consumed the same self - selected breakfast , lunch , and dinner daily at the same times of day and recorded food and insulin intake on study log sheets . participants refrained from exercise for 24 h before insertion of the sensor ( 48 h before the experimental session ) and avoided caffeine and alcohol during the monitoring period . participants arrived at the laboratory at 4:00 p.m. on the day after the sensor insertion . the following sessions were performed , separated by at least 5 days : 1 ) resistance exercise , three sets of eight repetitions maximum of seven different exercises with 90-s rest between sets ( duration 45 min ) ; 2 ) aerobic exercise , 45 min of treadmill exercise ( 60% of vo2max ) ; and 3 ) no - exercise control , 45 min of seated rest . testing sessions for the female participants , who were using monophasic oral contraceptives , took place during the active pill - consumption phase . participants reduced their insulin doses on exercise days by making either a 10% decrease in intermediate or long - acting insulin ( mdi ) or a 50% decrease in basal rate starting 1 h before exercise and maintained until the end of exercise for pump users . if blood glucose was < 5 mmol / l upon arrival , those using insulin pumps decreased their basal rate a further 25% . participants consumed a standard snack ( glucerna chocolate graham snack bars , 150 calories , 25 g carbohydrate ; abbott laboratories , abbott park , il ) at 4:00 p.m. every day , including the exercise day , with the bar consumed upon arrival at the laboratory . capillary glucose was checked 60 and 30 min before exercise and immediately prior to exercise to ensure glucose levels 5.5 and 13.9 mmol / l . venous blood samples were collected through an intravenous catheter at baseline and 5 , 10 , 15 , 30 , and 45 min during all three testing sessions ( resistance exercise , aerobic exercise , and no - exercise control ) and at the 50- , 55- , 60- , 65- , 75- , 85- , 95- , and 105-min time points during recovery . blood was immediately mixed by inversion , centrifuged ( 4,000 revolutions / min for 4 min ) , and stored at 80c . the hexokinase timed end point method was used to determine plasma glucose levels using the beckman coulter unicel dxc600 synchron clinical analyzer ( beckman coulter , fullerton , ca ) and synchron cx systems glucose reagent ( cat . glucose levels were compared among sessions using two - way repeated - measures ( time and condition ) anova . exercise and recovery periods were examined separately among the three sessions ( aerobic , resistance , and no - exercise control ) . the exercise period consisted of the 5- , 10- , 15- , 30- , and 45-min time points , while the recovery period consisted of the remaining time points . paired sample t tests were used to perform pairwise post hoc comparisons for each time point between conditions ( aerobic , resistance , or no - exercise control ) within exercise and recovery separately and to examine changes from baseline and changes from the end of exercise within each exercise condition . cgm data were examined as 15-min averages in the following windows : 24-h pre - exercise , overnight ( 12:00 a.m. to 6:00 a.m. ) pre - exercise , 16 h postexercise , overnight postexercise , and 24 h postexercise . a two - way ( time and condition ) repeated - measures anova was used to compare among conditions in the 16-h postexercise period . paired sample t tests were then used to perform pairwise post hoc comparisons for each 15-min segment . the minimum , maximum , and mean blood glucose ; amount of time spent in hypoglycemic and hyperglycemic states ; and areas under the curve ( aucs ) for time spent in hypo- and hyperglycemic states were determined for each window . pre - exercise values were compared with postexercise values within exercise conditions using related - samples wilcoxon signed rank tests . differences among conditions were examined using related - samples friedman two - way anova by ranks . agreement between cgm data and capillary glucose over the 3 days was determined by performing pearson correlations between sensor glucose and self - recorded capillary glucose values . daily total insulin and carbohydrate intake was calculated based on the information provided in participant logs . comparisons among conditions for each day were made using related - samples friedman two - way anova by ranks . where significant results were found , related - samples wilcoxon signed rank tests ensued for determination of where the differences lie . analyses were performed using spss 18.0 for windows ( spss , chicago , il ) . twelve ( 10 male and 2 female ) nonobese ( bmi 25.3 3.0 kg / m ) , physically active ( vo2max 51.2 10.8 ml kg min ) individuals aged 1762 years ( mean age 31.8 15.3 years ) took part in the study . mean diabetes duration was 12.5 10.0 years , and participants were in moderate to good control of their blood glucose levels ( hba1c 7.1 1.1% ) . five participants were receiving insulin by mdi , while seven were using continuous subcutaneous insulin infusion . 1 . information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1 . a significant interaction between time and exercise modality was observed ( p < 0.001 ) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions ( fig . 1 ) . there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration . a gradual decline in plasma glucose concentration occurred with resistance exercise ( from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session ) , resulting in levels that were significantly lower than baseline by the end of exercise ( p = 0.008 ) . no changes from baseline were detected throughout the first 45 min of the no - exercise session ( from 8.4 3.5 to 8.6 3.8 mmol / l [ p = 0.585 ] ) . in contrast , during the aerobic exercise , plasma glucose levels declined rapidly and more dramatically ( from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min [ p = 0.001 ] ) , resulting in significant changes from baseline within 10 min . glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity . mean se plasma glucose during the experimental sessions ( represented by box ) and 60 min of recovery ( n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise ) . , no - exercise control ; , resistance exercise , , aerobic exercise . differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons . during exercise , participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l . a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery ( p < 0.001 ) . plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise . the number of participants requiring glucose tablets during the testing session were two , nine , and three for the no - exercise control , aerobic , and resistance exercise sessions , respectively ( supplementary table 2 ) . differences were significant between no - exercise control and aerobic exercise ( p = 0.007 ) . there were no significant differences in carbohydrate intake among conditions on the day before or the day after the laboratory session or in the 6 h after exercise ( table 1 ) ; however , carbohydrate intake was higher on the exercise testing day in the aerobic exercise session compared with the resistance exercise session ( p = 0.013 ) , mostly because of differences in supplementation during exercise . two participants using insulin pumps chose to omit their usual insulin bolus with the glucerna bar before exercise , and one insisted on suspending basal insulin ( instead of a 50% reduction ) when learning upon arrival at the laboratory that it was the day for aerobic activity . daily insulin intake did not differ significantly among conditions on any day of sensor wear . insulin and carbohydrate intake during the 6 h after exercise * pearson correlations between capillary glucose levels measured on handheld meters and interstitial glucose levels measured by cgm were 0.95 , 0.90 , and 0.94 during nonlaboratory periods in the resistance exercise , aerobic , and no - exercise control sessions , respectively . during the 24 h before either exercise trial or no - exercise control , there were no significant differences among sessions in the total time spent in hypoglycemia , auc for hypoglycemia , number of hyperglycemic events , time spent in a hyperglycemic state , auc for hyperglycemia , or mean blood glucose . postexercise cgm data were only available for 11 and 10 of 12 participants in the no - exercise and aerobic exercise sessions , respectively , because of equipment malfunction in the remaining three sessions . data were available for all 12 participants in the resistance exercise session . in total , there were 124 paired handheld meter and cgm values for the no - exercise control condition , 113 for the aerobic condition , and 115 for the resistance exercise condition . a marginal effect of time ( p = 0.073 ) was found in the analysis of the cgm data from 1 to 6 h postexercise . higher mean interstitial glucose concentrations were found in the fourth and fifth hours after the aerobic exercise session compared with the resistance exercise session ( p = 0.018 at 5 h postexercise ) ( fig . , no - exercise control session ; , aerobic exercise session ; , resistance exercise session . the box represents the period of time where glucose was significantly higher after aerobic exercise compared with resistance exercise ( p < 0.05 ) . n = 11 ( no - exercise control ) , n = 10 ( aerobic ) , and n = 12 ( resistance ) . although there were twice as many nocturnal hypoglycemic excursions ( table 2 ) detected by cgm devices after resistance exercise ( nine in total ) versus aerobic exercise and no exercise ( four for each ) , differences among conditions were not statistically significant . there was , however , a trend of more episodes of nocturnal hyperglycemia after resistance exercise ( p = 0.059 ) compared with the pre - exercise night , but differences in mean glucose levels were not significant . summary of overnight cgm data for the night after resistance exercise , aerobic exercise , and no - exercise control 1 . information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1 . a significant interaction between time and exercise modality was observed ( p < 0.001 ) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions ( fig . 1 ) . there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration . a gradual decline in plasma glucose concentration occurred with resistance exercise ( from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session ) , resulting in levels that were significantly lower than baseline by the end of exercise ( p = 0.008 ) . no changes from baseline were detected throughout the first 45 min of the no - exercise session ( from 8.4 3.5 to 8.6 3.8 mmol / l [ p = 0.585 ] ) . in contrast , during the aerobic exercise , plasma glucose levels declined rapidly and more dramatically ( from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min [ p = 0.001 ] ) , resulting in significant changes from baseline within 10 min . glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity . mean se plasma glucose during the experimental sessions ( represented by box ) and 60 min of recovery ( n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise ) . , no - exercise control ; , resistance exercise , , aerobic exercise . differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons . during exercise , participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l . a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery ( p < 0.001 ) . plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise . 1 . information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1 . a significant interaction between time and exercise modality was observed ( p < 0.001 ) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions ( fig . 1 ) . there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration . a gradual decline in plasma glucose concentration occurred with resistance exercise ( from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session ) , resulting in levels that were significantly lower than baseline by the end of exercise ( p = 0.008 ) . no changes from baseline were detected throughout the first 45 min of the no - exercise session ( from 8.4 3.5 to 8.6 3.8 mmol / l [ p = 0.585 ] ) . in contrast , during the aerobic exercise , plasma glucose levels declined rapidly and more dramatically ( from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min [ p = 0.001 ] ) , resulting in significant changes from baseline within 10 min . glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity . mean se plasma glucose during the experimental sessions ( represented by box ) and 60 min of recovery ( n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise ) . , no - exercise control ; , resistance exercise , , aerobic exercise . differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons . during exercise , participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l . a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery ( p < 0.001 ) . plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise . the number of participants requiring glucose tablets during the testing session were two , nine , and three for the no - exercise control , aerobic , and resistance exercise sessions , respectively ( supplementary table 2 ) . differences were significant between no - exercise control and aerobic exercise ( p = 0.007 ) . there were no significant differences in carbohydrate intake among conditions on the day before or the day after the laboratory session or in the 6 h after exercise ( table 1 ) ; however , carbohydrate intake was higher on the exercise testing day in the aerobic exercise session compared with the resistance exercise session ( p = 0.013 ) , mostly because of differences in supplementation during exercise . two participants using insulin pumps chose to omit their usual insulin bolus with the glucerna bar before exercise , and one insisted on suspending basal insulin ( instead of a 50% reduction ) when learning upon arrival at the laboratory that it was the day for aerobic activity . daily insulin intake did not differ significantly among conditions on any day of sensor wear . pearson correlations between capillary glucose levels measured on handheld meters and interstitial glucose levels measured by cgm were 0.95 , 0.90 , and 0.94 during nonlaboratory periods in the resistance exercise , aerobic , and no - exercise control sessions , respectively . during the 24 h before either exercise trial or no - exercise control , there were no significant differences among sessions in the total time spent in hypoglycemia , auc for hypoglycemia , number of hyperglycemic events , time spent in a hyperglycemic state , auc for hyperglycemia , or mean blood glucose . postexercise cgm data were only available for 11 and 10 of 12 participants in the no - exercise and aerobic exercise sessions , respectively , because of equipment malfunction in the remaining three sessions . , there were 124 paired handheld meter and cgm values for the no - exercise control condition , 113 for the aerobic condition , and 115 for the resistance exercise condition . a marginal effect of time ( p = 0.073 ) was found in the analysis of the cgm data from 1 to 6 h postexercise . higher mean interstitial glucose concentrations were found in the fourth and fifth hours after the aerobic exercise session compared with the resistance exercise session ( p = 0.018 at 5 h postexercise ) ( fig . 2 ) . mean se glucose as measured by cgm from 1 to 12 h postexercise . , no - exercise control session ; , aerobic exercise session ; , resistance exercise session . the box represents the period of time where glucose was significantly higher after aerobic exercise compared with resistance exercise ( p < 0.05 ) . n = 11 ( no - exercise control ) , n = 10 ( aerobic ) , and n = 12 ( resistance ) . although there were twice as many nocturnal hypoglycemic excursions ( table 2 ) detected by cgm devices after resistance exercise ( nine in total ) versus aerobic exercise and no exercise ( four for each ) , differences among conditions were not statistically significant . there was , however , a trend of more episodes of nocturnal hyperglycemia after resistance exercise ( p = 0.059 ) compared with the pre - exercise night , but differences in mean glucose levels were not significant . summary of overnight cgm data for the night after resistance exercise , aerobic exercise , and no - exercise control resistance exercise resulted in much smaller declines in blood glucose during exercise than aerobic exercise or no exercise in individuals with type 1 diabetes . less carbohydrate supplementation was required during resistance exercise versus aerobic exercise , which would have attenuated some of the hypoglycemic effects of the aerobic activity . in contrast to resistance exercise and no exercise , aerobic exercise was associated with greater increases in glucose levels during early recovery , which resulted in a trend toward higher glucose concentrations in late recovery ( as measured by cgm 36 h postexercise ) . these trends were observed in the absence of any significant differences in insulin dosage or carbohydrate intake during this time . mean blood glucose levels after resistance exercise were similar to those when no exercise was performed : more stable during early recovery and within a healthier range ( 56 mmol / l ) during late recovery . as such , performance of resistance exercise may represent an alternative strategy to prevent the acute decline in blood glucose levels observed with aerobic exercise while maintaining more favorable postexercise glucose levels . there was , however , a tendency toward more frequent , albeit mild , nocturnal hypoglycemia after resistance exercise sessions , which deserves further scrutiny . the mechanisms for the more dramatic reduction in blood glucose levels during aerobic versus resistance exercise are unclear , but the reliance on anaerobic sources of fuel production during resistance exercise rather than aerobic sources ( i.e. , less reliance on blood glucose ) ( 21,22 ) may have played a role . previous studies involving anaerobic activity in individuals with type 1 diabetes ( intermittent 4-s sprints or a 10-s sprint pre- or postexercise ) found slower declines in blood glucose concentration during exercise and smaller decreases in postexercise glucose concentrations in comparison with low - intensity aerobic exercise alone . insulin and cortisol levels were comparable across conditions in these studies and were therefore unlikely to be responsible for the differential patterns of blood glucose response ( 1114 ) . growth hormone and catecholamines , meanwhile , were elevated after sprinting , potentially enhancing lipolysis and glycogenolysis , respectively , thereby potentially stabilizing blood glucose levels ( 1114 ) . it is undetermined whether these hormones are responsible for stabilizing blood glucose levels after resistance exercise in individuals with type 1 diabetes ; however , both growth hormone and catecholamines are known to increase significantly in individuals without diabetes during resistance exercise protocols similar to the one used in the current study ( 23,24 ) . attenuated declines in blood glucose concentration may also be related to increased lactate production during resistance exercise . in comparing the hormonal responses to various resistance exercise protocols , smilios et al . ( 23 ) found that two sets of 10 repetitions of chest press , lateral pull down , and squat ( a stimulus of smaller magnitude than the one used in the current study ) resulted in a fourfold increase in blood lactate levels , with elevated lactate persisting for at least 30 min postexercise in individuals without diabetes ( 23 ) . while we are unaware of published data on lactate production during resistance exercise in individuals with type 1 diabetes , there is no reason to believe that lactate production would be impaired in this population . indeed , other anaerobic activity ( high - intensity cycling ) produced elevated lactate levels persisting up to 30 min postexercise in individuals with type 1 diabetes ( 1114,25 ) . we did not measure lactate in the current study but can surmise that blood lactate levels would have increased more during resistance exercise where glycolysis predominates ( 22 ) than during aerobic exercise where lipolysis generates much of the energy required ( 26 ) , especially in physically fit individuals ( 21 ) . overall , there were no significant differences among the conditions with respect to any measures of hypoglycemia or mean nocturnal blood glucose levels ( table 2 ) , although resistance exercise was associated with a nonsignificant trend for more nocturnal hypoglycemia . while we are unaware of any study examining nocturnal blood glucose levels after resistance exercise in type 1 diabetic subjects , mcmahon et al . ( 16 ) found that adolescents with type 1 diabetes had a higher glucose infusion requirement to maintain euglycemia between midnight and 4:00 a.m. after performing evening aerobic exercise than if no exercise had been performed . this coincides with the time when the lowest nocturnal glucose levels were found after both exercise sessions in our study ( fig . 2 ) , although differences among conditions were not significant . as mcmahon et al . ( 16 ) surmised that delayed increases in postexercise glucose needs relate to replenishment of glycogen stores , a higher frequency of low blood glucose after resistance exercise ( which relies more on glycogen for fuel ) ( 22 ) might be expected . it is also possible that differences in food and insulin intake ( table 2 ) , while not statistically significant , could have had a minor effect on postexercise glucose profiles . in addition , while participants were asked to match their food and insulin intake both pre- and postexercise as closely as possible among the sessions , some differences may not have been reported . this does not , however , detract from the findings , as patient decisions regarding insulin dosage and carbohydrate intake play an essential role in diabetes management . as there is currently very little information available with respect to insulin adjustments for resistance exercise , participants in the current study were relying to a great extent on personal experience and judgment . higher physical activity levels in individuals with type 1 diabetes have been associated with lower frequency and severity of diabetes complications ( 1 ) ; however , fear of hypoglycemia is generally the strongest barrier to physical activity for this population ( 27 ) . resistance exercise is associated with improvements in muscular strength ( 4 ) , improved lipid profiles ( 4 ) , lower insulin needs ( 4,5 ) , and lower self - monitored blood glucose levels ( 4,5 ) in individuals with type 1 diabetes . it also carries many of the same benefits as aerobic exercise ( higher bone mineral density , increased insulin sensitivity , and improved cardiovascular function ) ( 28 ) and may therefore be a safe and effective option for this population . interestingly , we observed more exercise - associated glycemic fluctuation with aerobic exercise compared with resistance exercise . during the activity , aerobic exercise was associated with greater reductions in glycemia , while in early recovery there was more rebound hyperglycemia compared with resistance exercise . thus , one could conclude that resistance exercise may be more beneficial as far as glucose stability is concerned . moreover , as individuals with type 1 diabetes may also have an increased risk of myopathy ( 29 ) and complications associated with insulin resistance ( 29,30 ) , performing regular resistance exercise may help maintain or improve muscle mass and metabolism . meanwhile , it should also be noted that postexercise hypoglycemia might occur more frequently in individuals who have changed their exercise routine to incorporate resistance training or for patients unaccustomed to exercise ( 15 ) . in summary , our findings suggest that in trained individuals with type 1 diabetes who habitually practice both aerobic and resistance exercise , resistance exercise may result in more stable glucose levels both during and after exercise than aerobic exercise , which may explain the beneficial effects on hba1c found in previous intervention studies involving resistance exercise . the trend toward more frequent , albeit mild , nocturnal hypoglycemia after resistance exercise reported in our study , however , indicates the possible need to develop more effective clinical management protocols for different forms of exercise .

plants respond to many environmental changes by rapidly adjusting their hydraulic conductivity and transpiration rate , thereby optimizing water - use efficiency and preventing damage due to low water potential . a multiple - load - cell apparatus , time - series analysis of the measured data , and residual low - pass filtering methods were used to monitor continuously and analyse transpiration of potted tomato plants ( solanum lycopersicum cv . ailsa craig ) grown in a temperature - controlled greenhouse during well - irrigated and drought periods . a time derivative of the filtered residual time series yielded oscillatory behaviour of the whole plant 's transpiration ( wpt ) rate . a subsequent cross - correlation analysis between the wpt oscillatory pattern and wet - wick evaporation rates ( vertical cotton fabric , 0.14 m2 partly submerged in water in a container placed on an adjacent load cell ) revealed that autonomous oscillations in wpt rate develop under a continuous increase in water stress , whereas these oscillations correspond with the fluctuations in evaporation rate when water is fully available . the relative amplitude of these autonomous oscillations increased with water stress as transpiration rate decreased . these results support the recent finding that an increase in xylem tension triggers hydraulic signals that spread instantaneously via the plant vascular system and control leaf conductance . the regulatory role of synchronized oscillations in wpt rate in eliminating critical xylem tension points and preventing embolism is discussed .

stomata play a major role in water loss and carbon gain by regulating diffusive conductance in leaves . it is widely accepted that stomata respond to perturbations by many soil - plant - atmosphere signals but there is little agreement regarding the mechanism(s ) by which they sense and react to such perturbations ( buckley , 2005 ) . the phenomenon of autonomous , cyclic opening and closing movements of stomata within a period of less than 3 h was observed over 40 years ago ( barrs , 1971 ) . those measurements showed that when the periodic variations in transpiration are plotted against time , a sine wave with a period of 1090 min is obtained ( barrs and klepper , 1968 ; cox , 1968 ; barrs , 1971 ; teoh and palmer , 1971 ) . such movements are most readily detected in a stable environment , but there seems to be no a priori reason why such movements can not persist , albeit with some modifications , in non - stable environments ( barrs , 1971 ) . traditionally , two approaches have been taken to study the effect of environmental conditions on oscillatory transpiration . the first involves tracking the variations in transpiration rate of a small part of , or a whole leaf . the second focuses on the behaviour of individual stomata . adopting the first approach , ehrler et al . ( 1994 ) , naidoo and von willert ( 1994 ) , herppich and von willert ( 1995 ) , jarvis et al . ( 2003 ) studied oscillations in whole leaves from a large variety of species , most often using gas - exchange techniques . measurements of chlorophyll a fluorescence ( siebke and weis , 1995 ) , leaf temperature or leaf water potential in terms of water relations ( ehrler et al . , 1965 ; barrs and klepper , 1968 ; lang et al . , 1969 ; mcburney and costigan , 1984 ; naidoo and von willert , 1994 ; herppich and von willert , 1995 ; prytz et al . , 2003 ) have also been used to record stomatal oscillations . a common conclusion in all of these studies was that in - phase oscillations are induced by various agents , bringing about abrupt stomatal opening or closure . reports of the synchronous rhythmic pattern of stomatal activity in different leaves on the same plant were reviewed by hopmans ( 1969 ) and lang et al . these authors found oscillations in leaf - xylem water potential and proposed that these are a necessary condition for the stomata of all leaves to oscillate in phase . oscillations in root - xylem pressure were measured by wegner and zimmermann ( 1998 ) and prytz et al . ( 2003 ) , who noted that the synchronous behaviour observed during oscillatory transpiration indicates strong coupling among stomata ; however , they did not suggest a coupling agent . herppich and von willert ( 1995 ) measured pronounced oscillations in stomatal apertures under certain climatic conditions and noted that changes in transpirational water loss during these oscillations are closely accompanied by changes in leaf water potential ( l ) . on a stomatal scale , the simultaneous existence of different stomatal apertures , extents of stomatal opening across a leaf , and the possible implications of cross - co2 exchange were first discussed by laisk and co - workers ( laisk et al . terashima ( 1992 ) argued that under certain physiological conditions , the spatial distribution of different stomatal conductances is non - random ( patchy ) , i.e. certain areas of a leaf can have a very different conductance from others . patchy stomatal closure has been observed , particularly under water stress ( downton et al . , 1988 ; beyschlag et al . , 1992 ) and low humidity ( mott and parkhurst , 1991 ; mott et al . , 1993 , 1999 ) , and following abscisic acid application ( daley et al . , 1989 ; terashima , 1992 ; mott , 1995 ) . several models of the mechanism responsible for the oscillations in stomata aperture have been described ( lang et al . , 1969 ; , 1997 ; mcainsh et al . , 1997 ; shabala et al . , 1997 ; meleshchenko , 2000 ; bohn et al . , 2001 ) , presenting a common basic assumption : that the oscillation behaviour is part of a feedback loop in which evaporation rate influences the stomatal aperture which , in turn , affects evaporation rate . some have suggested that stomatal cycling is induced by internal fluctuations in water potential and flow resistance within the plant ( steudle , 2000 ) . despite the fact that oscillations have been intensively studied in single stomata , leaf patches and whole leaves , oscillations on a whole - plant scale have been rather neglected . the studies of rose and co - authors ( rose and rose , 1994 ; rose et al . , 1994 ) and steppe et al . ( 2006 ) are , to the best of our knowledge , the only ones in which oscillations in whole - plant transpiration ( wpt ) have been measured . a recent study , which revealed that the long - distance signal triggering the reduction in leaf conductance under water stress is hydraulic rather than root - produced abscisic acid ( christmann et al . , 2007 ) , led us to hypothesize that synchronized stomatal activity at the whole - plant level could be the result of a hydraulic signal spreading rapidly via the plant 's vascular system . consequently , this study aimed to isolate and characterize the autonomous , self - regulated oscillations in wpt rate that develop with increasing water stress . the experimental study was conducted in greenhouses at the faculty of agriculture , food and environment in rehovot , israel . the experimental set - up included 3.9 l growing pots placed on temperature - compensated load cells ( tadea - huntleigh , natanya , israel ) connected to a cr10 data logger ( campbell scientific inc . , logan , ut ) . the pots were filled with a commercial growing medium ( a mixture of peat and tuff scoria ) and each contained one plant . each pot was immersed in a plastic container ( 1321.531.5 cm , hwl ) through a hole in its top cover . the water level fell to 2 cm above the pot base after the irrigation events due to a drainage hole in the container side wall . the pot - container system ensured that water was available to the plant throughout the day following irrigation , without supplemental irrigation . a commercial fertilizer solution ( super grow 6 - 6 - 6 + 3 hortical , kadima , israel ) was added at 0.2% ( v / v ) daily with the irrigation water ( fertigation ) . this set - up ensured that ( i ) the plants would not be subjected to water stress throughout the following day , and ( ii ) the container weight on the following day would decrease monotonically solely due to plant transpiration . ailsa craig ) were grown in a controlled - environment greenhouse , with the temperature set at 18 c during the night and 35 c during the midday hours with periods of gradual variation between them . the effect of water stress on the patterns of wpt - rate oscillations was studied in a dehydration experiment using the tomato plants ( at the age of 1012 weeks ) . the plants were dehydrated by stopping irrigation , removing the containers in which the pots were immersed and allowing the plant to deplete the water from the growth medium in the pot for five successive days . average and momentary changes in atmospheric demand were assessed by measuring the weight loss from a vertical woven floor rag ( 0.14 m ) , the lower part of which was submerged in water in a container ( noted hereafter as wet wick ) that was placed on a load cell . to isolate intrinsic noise associated with the measuring and data - acquisition systems from the short - term fluctuations in plant transpiration , a constant load of 6 kg ( typical of the container+pot+plant weight ) was placed on the load cells in the greenhouse for several days , during which the weight variation was monitored . the data from the load cells with plants , wet wick and constant load were analysed by the following time - series analysis . weight readings were taken every 10 s. the load - cell reading stabilized after 2 s following stimulation by dropping a 70-g steel ball from a height of 700 mm ( manufacturer 's data ) . thus , a 10 s weight - sampling interval ensured that the maximum rate of weight decrease ( 0.5 g per 10 s ) was appropriately followed . the data analysis consisted of two steps : the first included averaging 10 s readings over 3 min periods . this stage was aimed at decreasing the number of data points in further data analysis ( second stage ) . the 3 min periods are clearly lower than the oscillation frequency ( 2040 min ) and higher than the nyquist frequency ( the highest frequency at which meaningful information can be obtained from a set of data ) . leaves were excised from each plant separately and their areas were measured using a leaf area scanner ( licor 3210 , lincoln , ne ) . the rate of water loss from the system , being the negative value of the wpt rate , was calculated by the first derivative of the measured weight time series(1)where wk and wk+1 are , respectively , the measured weights at time tk and the following time step tk+1 in general , differentiation acts as a high - pass filter , thereby significantly amplifying the high - frequency noise , and noises are amplified as the measurement ( sampling ) interval ( tk+1tk , equation 1 ) decreases . consequently , a signal treatment is essential when the noise associated with the transpiration - induced weight decrease is differentiated together with the high - frequency noises associated with the load - cell and data - acquisition systems , as both are embedded in the measured time series , owing to amplification of the noise introduced by the latter . in fact , measurement errors , which can never be avoided , complicate the differentiation , because these amplify the noise to such an extent that additional signal treatment is essential . the noise associated with the differentiation of noisy signals has stimulated a large number of investigations which have led to several solutions , in both the time and frequency domains ( savitzky and golay , 1964 ; cullum , 1971 ; anderssen and bloomfield , 1974a , b ; wahba , 1975 ; rice and rosenblatt , 1983 ; scott and scott , 1989 ) . noise can be reduced or eliminated by smoothing ( detrending ) the measured data ( time series ) so that the smoothed data becomes stationary prior to the subsequent stage of spectral analysis . many methods are used for smoothing noisy data in time - series analyses ( harvey and shephard , 1993 ) . these methods can be categorized as non - parametric smoothing [ e.g. moving average , savitzky - golay ( s - g ) , and fast fourier transform ( fft ) filtering ] and non - parametric regression ( fitting polynomials of various orders , exponential functions , symmetrical and asymmetrical - transition functions , etc to the measured data ] . smoothing should be performed with care since substantially different results can be obtained after differentiation , despite the high r values that are usually obtained for the different smoothing methods . the savitzky and golay ( 1964 ) method ( s - g ) was used in this study to smooth the measured time series . this smoothing method is based on least - squares quadratic polynomial fitting ( although higher orders can also be used ) across a moving window within the data ( press et al . the s - g method can be applied for various breadths of filtering windows , and it is considered a very good way of producing accurate smooth derivatives . the fit improves and includes fluctuations of higher frequencies and amplitudes with decreasing breadth of the filtering window . s - g with 30 data points was ultimately chosen for the window breadth as its time derivative provided a smooth ( but not too smooth ) pattern . it is assumed herein that the container weight - time series follows an additive model(2)where w is the value that the weight at time tk would have if it varied smoothly with time , and k is the deviation from that value . the system 's weight oscillations superimposed on the smoothed time series also constitute a time series , designated residual time series ( residuals are the differences between the measured data and the fitted curve ) . when the mean of the residual time series is zero , the residual time series k ( equation 2 ) is a superposition of two time series : one made up of residuals that originate from the noises related to data acquisition , and the other of residuals originating from either the ambient conditions and/or the intrinsic oscillations in wpt . the time series for the constant - weight , wet - wick , and whole - plant runs ( which were independently measured ) were used to examine its randomness ( white noise ) . the randomness of the noise originated from the data - acquisition system and the repeating patterns ( periodicity ) in the wet - wick and plant oscillations were examined by the autocorrelation function . this is a mathematical tool for finding repeating patterns , such as the presence of a periodic signal which has been buried under the noise function , by expressing the similarity between observations as a function of the time separation between them . if the noise is a white noise , the autocorrelation function should be near zero for any and all time - lag separations . if not a white noise , then one or more ( except at lag=0 ) of the autocorrelations will be significantly non - zero . the spectrum analysis of k was used to explore the existence of cyclical patterns by decomposing the complex time series with cyclical components into a few underlying sinusoidal ( sine and cosine ) functions of particular wavelengths . the characteristics of the phenomenon of interest become apparent when the important underlying cyclical components are identified . the spectrum analysis enabled us to uncover a few cycles of different lengths in the time series of interest , which at first looked more or less like random noise . the spectrum ( amplitudes versus frequencies ) of the residual time series was calculated by fast fourier transform ( fft ) , which decomposes a time - domain signal or time series into complex exponentials ( sines and cosines ) . the spectrum of the constant - load residual time series was used to determine the frequency threshold that would be used to filter out the high - frequency noise ( low - pass filter ) from the plant - weight residual time series . subsequently , the filtered spectrum was reconstructed back to a time series ( in the time domain ) by inverse fft . the calculations of the above were executed by tablecurve 2d ( systat software inc , ca ) . the time derivative of the reconstructed low - pass - filtered time series d(k)/dt ( ( k ) is the low - pass filter of k ) provided the oscillatory transpiration rate that superimposes the smoothed wpt rate . the evaporation rate ( smoothed and oscillatory ) from the wet wick integrates the effect of the ambient conditions on both the wick and the plant . a high correlation between the two time series is expected under optimal growth conditions , while deviations are likely to occur when the plants are exposed to stresses . the wet - wick evaporation rate - time series is regarded as the input to the plant system and the wpt rate - time series as the output . hence , a comparison between the patterns of smoothed and low - pass oscillations in wpt rate and wet - wick evaporation rate can reveal whether the synchronized oscillations in wpt rate are mainly driven by fluctuations in ambient conditions in the greenhouse , or if they tend to become autonomous under certain independent conditions ( e.g. water stress ) . the relationship between the two time series can be interpreted by quantitative and qualitative means . a qualitative estimation of the correlation between the transpiration- and evaporation - rate time series can be obtained by plotting the two time series on the same plot and a quantitative estimation is obtained by the cross - correlation function . a plot of the time series enables isolating periods throughout the day during which the two time series deviate , and relating these periods to the variation of the smoothed patterns of these variables or to other independent variables in the system . a cross - correlation function with a single and pronounced peak indicates that the transpiration and wet - wick evaporation oscillatory patterns are related , and that the oscillatory wpt rate is driven by the fluctuations in ambient conditions ( evaporative demand ) . a peak value close to 1 located at lag=0 indicates that the changes in wpt rate are fully and instantaneously driven by the fluctuations in evaporative demand , as reflected by the wet wick . as the wpt rate of a well - watered plant responds to environmental conditions , a good correlation coefficient is expected between well - watered plants and the wet wick , the latter closely simulating the evaporative demand . however , a perfect correlation coefficient was not anticipated as the boundary - layer resistances between the wet wick and the plant crown are dissimilar , the thermal properties and heat capacity of the two systems differ , and the plant can control its diffusive resistance by changing stomatal aperture . on the other hand , a rippled , low - value cross - correlation function , with no clear peak , can be interpreted as a lack of dependence between the two time series , namely , the oscillations in wpt rate are autonomous and self - regulated , independent of the fluctuations in ambient conditions . the experimental study was conducted in greenhouses at the faculty of agriculture , food and environment in rehovot , israel . the experimental set - up included 3.9 l growing pots placed on temperature - compensated load cells ( tadea - huntleigh , natanya , israel ) connected to a cr10 data logger ( campbell scientific inc . , logan , ut ) . the pots were filled with a commercial growing medium ( a mixture of peat and tuff scoria ) and each contained one plant . each pot was immersed in a plastic container ( 1321.531.5 cm , hwl ) through a hole in its top cover . the water level fell to 2 cm above the pot base after the irrigation events due to a drainage hole in the container side wall . the pot - container system ensured that water was available to the plant throughout the day following irrigation , without supplemental irrigation . a commercial fertilizer solution ( super grow 6 - 6 - 6 + 3 hortical , kadima , israel ) was added at 0.2% ( v / v ) daily with the irrigation water ( fertigation ) . this set - up ensured that ( i ) the plants would not be subjected to water stress throughout the following day , and ( ii ) the container weight on the following day would decrease monotonically solely due to plant transpiration . ailsa craig ) were grown in a controlled - environment greenhouse , with the temperature set at 18 c during the night and 35 c during the midday hours with periods of gradual variation between them . the effect of water stress on the patterns of wpt - rate oscillations was studied in a dehydration experiment using the tomato plants ( at the age of 1012 weeks ) . the plants were dehydrated by stopping irrigation , removing the containers in which the pots were immersed and allowing the plant to deplete the water from the growth medium in the pot for five successive days . average and momentary changes in atmospheric demand were assessed by measuring the weight loss from a vertical woven floor rag ( 0.14 m ) , the lower part of which was submerged in water in a container ( noted hereafter as wet wick ) that was placed on a load cell . to isolate intrinsic noise associated with the measuring and data - acquisition systems from the short - term fluctuations in plant transpiration , a constant load of 6 kg ( typical of the container+pot+plant weight ) was placed on the load cells in the greenhouse for several days , during which the weight variation was monitored . the data from the load cells with plants , wet wick and constant load were analysed by the following time - series analysis . weight readings were taken every 10 s. the load - cell reading stabilized after 2 s following stimulation by dropping a 70-g steel ball from a height of 700 mm ( manufacturer 's data ) . thus , a 10 s weight - sampling interval ensured that the maximum rate of weight decrease ( 0.5 g per 10 s ) was appropriately followed . the data analysis consisted of two steps : the first included averaging 10 s readings over 3 min periods . this stage was aimed at decreasing the number of data points in further data analysis ( second stage ) . the 3 min periods are clearly lower than the oscillation frequency ( 2040 min ) and higher than the nyquist frequency ( the highest frequency at which meaningful information can be obtained from a set of data ) . leaves were excised from each plant separately and their areas were measured using a leaf area scanner ( licor 3210 , lincoln , ne ) . the rate of water loss from the system , being the negative value of the wpt rate , was calculated by the first derivative of the measured weight time series(1)where wk and wk+1 are , respectively , the measured weights at time tk and the following time step tk+1 in general , differentiation acts as a high - pass filter , thereby significantly amplifying the high - frequency noise , and noises are amplified as the measurement ( sampling ) interval ( tk+1tk , equation 1 ) decreases . consequently , a signal treatment is essential when the noise associated with the transpiration - induced weight decrease is differentiated together with the high - frequency noises associated with the load - cell and data - acquisition systems , as both are embedded in the measured time series , owing to amplification of the noise introduced by the latter . in fact , measurement errors , which can never be avoided , complicate the differentiation , because these amplify the noise to such an extent that additional signal treatment is essential . the noise associated with the differentiation of noisy signals has stimulated a large number of investigations which have led to several solutions , in both the time and frequency domains ( savitzky and golay , 1964 ; cullum , 1971 ; anderssen and bloomfield , 1974a , b ; wahba , 1975 ; rice and rosenblatt , 1983 ; scott and scott , 1989 ) . noise can be reduced or eliminated by smoothing ( detrending ) the measured data ( time series ) so that the smoothed data becomes stationary prior to the subsequent stage of spectral analysis . many methods are used for smoothing noisy data in time - series analyses ( harvey and shephard , 1993 ) . these methods can be categorized as non - parametric smoothing [ e.g. moving average , savitzky - golay ( s - g ) , and fast fourier transform ( fft ) filtering ] and non - parametric regression ( fitting polynomials of various orders , exponential functions , symmetrical and asymmetrical - transition functions , etc to the measured data ] . smoothing should be performed with care since substantially different results can be obtained after differentiation , despite the high r values that are usually obtained for the different smoothing methods . the savitzky and golay ( 1964 ) method ( s - g ) was used in this study to smooth the measured time series . this smoothing method is based on least - squares quadratic polynomial fitting ( although higher orders can also be used ) across a moving window within the data ( press et al . , 1988 ) . the s - g method can be applied for various breadths of filtering windows , and it is considered a very good way of producing accurate smooth derivatives . the fit improves and includes fluctuations of higher frequencies and amplitudes with decreasing breadth of the filtering window . s - g with 30 data points was ultimately chosen for the window breadth as its time derivative provided a smooth ( but not too smooth ) pattern . it is assumed herein that the container weight - time series follows an additive model(2)where w is the value that the weight at time tk would have if it varied smoothly with time , and k is the deviation from that value . the system 's weight oscillations superimposed on the smoothed time series also constitute a time series , designated residual time series ( residuals are the differences between the measured data and the fitted curve ) . when the mean of the residual time series is zero , the trend of the measured time series has been properly removed . the residual time series k ( equation 2 ) is a superposition of two time series : one made up of residuals that originate from the noises related to data acquisition , and the other of residuals originating from either the ambient conditions and/or the intrinsic oscillations in wpt . the time series for the constant - weight , wet - wick , and whole - plant runs ( which were independently measured ) were used to examine its randomness ( white noise ) . the randomness of the noise originated from the data - acquisition system and the repeating patterns ( periodicity ) in the wet - wick and plant oscillations were examined by the autocorrelation function . this is a mathematical tool for finding repeating patterns , such as the presence of a periodic signal which has been buried under the noise function , by expressing the similarity between observations as a function of the time separation between them . if the noise is a white noise , the autocorrelation function should be near zero for any and all time - lag separations . if not a white noise , then one or more ( except at lag=0 ) of the autocorrelations will be significantly non - zero . the spectrum analysis of k was used to explore the existence of cyclical patterns by decomposing the complex time series with cyclical components into a few underlying sinusoidal ( sine and cosine ) functions of particular wavelengths . the characteristics of the phenomenon of interest become apparent when the important underlying cyclical components are identified . the spectrum analysis enabled us to uncover a few cycles of different lengths in the time series of interest , which at first looked more or less like random noise . the spectrum ( amplitudes versus frequencies ) of the residual time series was calculated by fast fourier transform ( fft ) , which decomposes a time - domain signal or time series into complex exponentials ( sines and cosines ) . the spectrum of the constant - load residual time series was used to determine the frequency threshold that would be used to filter out the high - frequency noise ( low - pass filter ) from the plant - weight residual time series . subsequently , the filtered spectrum was reconstructed back to a time series ( in the time domain ) by inverse fft . the calculations of the above were executed by tablecurve 2d ( systat software inc , ca ) . the time derivative of the reconstructed low - pass - filtered time series d(k)/dt ( ( k ) is the low - pass filter of k ) provided the oscillatory transpiration rate that superimposes the smoothed wpt rate . the evaporation rate ( smoothed and oscillatory ) from the wet wick integrates the effect of the ambient conditions on both the wick and the plant . a high correlation between the two time series is expected under optimal growth conditions , while deviations are likely to occur when the plants are exposed to stresses . the wet - wick evaporation rate - time series is regarded as the input to the plant system and the wpt rate - time series as the output . hence , a comparison between the patterns of smoothed and low - pass oscillations in wpt rate and wet - wick evaporation rate can reveal whether the synchronized oscillations in wpt rate are mainly driven by fluctuations in ambient conditions in the greenhouse , or if they tend to become autonomous under certain independent conditions ( e.g. water stress ) . the relationship between the two time series can be interpreted by quantitative and qualitative means . a qualitative estimation of the correlation between the transpiration- and evaporation - rate time series can be obtained by plotting the two time series on the same plot and a quantitative estimation is obtained by the cross - correlation function . a plot of the time series enables isolating periods throughout the day during which the two time series deviate , and relating these periods to the variation of the smoothed patterns of these variables or to other independent variables in the system . a cross - correlation function with a single and pronounced peak indicates that the transpiration and wet - wick evaporation oscillatory patterns are related , and that the oscillatory wpt rate is driven by the fluctuations in ambient conditions ( evaporative demand ) . a peak value close to 1 located at lag=0 indicates that the changes in wpt rate are fully and instantaneously driven by the fluctuations in evaporative demand , as reflected by the wet wick . as the wpt rate of a well - watered plant responds to environmental conditions , a good correlation coefficient is expected between well - watered plants and the wet wick , the latter closely simulating the evaporative demand . however , a perfect correlation coefficient was not anticipated as the boundary - layer resistances between the wet wick and the plant crown are dissimilar , the thermal properties and heat capacity of the two systems differ , and the plant can control its diffusive resistance by changing stomatal aperture . on the other hand , a rippled , low - value cross - correlation function , with no clear peak , can be interpreted as a lack of dependence between the two time series , namely , the oscillations in wpt rate are autonomous and self - regulated , independent of the fluctuations in ambient conditions . typical variations in weight during the night and subsequent daylight hours are shown in fig . as water was supplied to the plants in the late evening ( after 20.00 h ) , the weight decreased monotonically , albeit at different rates , during the night and daylight hours . the rate of the weight decrease ( transpiration rate increase ) intensified during the morning , stayed high during the noon and early afternoon hours , and weakened thereafter ( fig . the wpt - rate time series ( the negative values of the rate of weight decrease ) , calculated by the time derivative of the measured weight - time series with data points every 3 min ( equation 1 ) , is shown in fig . a noisy wpt rate was obtained despite what appeared to be a relatively smooth pattern of weight decrease ( fig . the amplitudes of the wpt rate were low during the night , early morning , and evening hours and large at the other times . a detailed pattern of the daily transpiration rate , especially when the ambient conditions varied with time , could barely be identified from this noisy time series . the smoothed wpt rate ( fig . 1b , solid line ) was obtained by differentiating a smoothed weight - time series obtained by applying the s - g smoothing method with a window breadth of 30 data points ( s - g 30 ) . the fluctuations around the smoothed transpiration - rate pattern are usually considered random noise ( white noise ) associated with the measurement system , and as such are generally ignored without further analysis . here , however , these fluctuations were further analysed in order to isolate the system - noise - induced fluctuations from those induced by the ambient and/or physiologically driven oscillations . time - course of transpiration rate calculated by numerical derivative of the measured and smoothed time - courses of the decrease in potted tomato weight ( b ) . the smoothed patterns of weight - variation rate ( smoothed by s - g 30 prior to time differentiation ) for a tomato plant , a wet wick , and a constant weight that was placed on a load cell are shown in fig . the whole - plant and wet - wick data were measured on 6 september 2006 , and the constant load on 9 september 2006 , all in the temperature - controlled greenhouse . 2a were measured for four neighbouring tomato plants that were grown together in the greenhouse ( data not shown ) with a similarly ample water supply . the residual time series ( difference between the measured and smoothed weight time series ) for the tomato plant , wet wick , and constant load are shown in fig . to exclude the possibility of the oscillations in wpt rate being simply system - related noise , these residuals were examined for randomness ( white noise ) by autocorrelation function ( fig . the autocorrelation function of the constant - weight residual time series , k ( fig . 2i ) was 1 at lag=0 and was close to zero for all other lags . by contrast , the autocorrelation functions of both the tomato plant and the wet wick ( fig . 2 g , h , respectively ) were periodic for the first 200 lags , with an approximate average 40 lag difference from peak to peak . the correlation between the fluctuations in plant weight around the smoothed weight variation and ambient conditions ( represented by the wet wick ) will be analysed further in the following . smoothed weight - variation - rate patterns in a whole potted tomato plant ( a ) , a wet wick ( b ) , and a constant load ( c ) . residual time series for the whole plant ( d ) , wet wick ( e ) , and constant load ( f ) . autocorrelation function of the residual time series for the whole plant ( g ) , wet wick ( h ) , and constant load ( i ) . the spectra for the tomato - plant , wet - wick , and constant - load residual time series are shown in fig . 3e ) provided an additional indication that its residual time series is practically a random signal ( white noise ) while those of the plant and wet wick are not . 3a ) had high amplitudes at f < 2.5 h and lower amplitudes at the higher frequencies . the wet - wick spectrum ( fig . 3c ) exhibited a similar pattern , i.e. high amplitudes ( although lower than those of the whole plant ) at f < 2 h and lower amplitudes at the higher frequencies . to filter out what was considered to be white noise , both spectra were low - pass filtered for f < 2 h prior to further analysis . the time derivative of the reconstructed low - pass - filtered residual time series provided oscillations in wpt and wet - wick evaporation rates , as well as in the constant - load weight . superpositions of these oscillations on the smoothed variation of these variables are shown in fig . spectra for the whole - plant ( a ) , wet - wick ( b ) , and constant - load ( c ) residual time series . time derivative of the low - pass - filtered residual time series superimposed on the time derivative of the smoothed weight variation with time for the whole plant ( d ) , wet wick ( e ) , and constant load ( f ) . to analyse the correlation between the fluctuations in wpt and wet - wick rates , the time derivatives of the reconstructed low - pass - filtered residual time series of these variables were plotted together in fig . 4a . the baseline in this figure ( weight loss=0 ) was the smoothed transpiration / evaporation rate . a quantitative measure of this correlation was obtained by the cross - correlation function ( fig . 4b ) , which is a standard method of estimating the degree to which two series are correlated . the cross - correlation between the two series was calculated for the morning ( 06.00 to 12.00 h ) and afternoon ( 12.00 to 18.00 h ) to examine the correlation between fluctuations in ambient conditions and the oscillations in wpt rate when transpiration rate increases ( morning hours ) and when it is already high and beginning to decrease ( afternoon hours ) . when the cross - correlation around lag=0 is higher than those at other lag values ( negative and positive ) , the oscillations in wpt rate can be more related to temporary variations ( fluctuations ) in ambient conditions . however , when low cross - correlation values are obtained at all lags , the two series are independent and the oscillations in wpt rate may be autonomous , putatively driven by independent physiological processes . the cross - correlation function for the morning hours had a slightly higher peak value than for the afternoon hours ( fig . the presence of multiple peaks indicated that the oscillations in wpt rate depend on the fluctuations in ambient conditions , with periods when the oscillations in wpt rate are independent of those of the wet wick , namely , ambient conditions . 4a ) revealed that they are temporarily out of phase between 07.30 and 10.00 h and between 13.30 and 15.30 h. from the smoothed transpiration pattern in fig . 3b , the shifts between the two oscillations coincided with periods of intensive change in transpiration rate be it increasing or decreasing . thus , it may be deduced that autonomous oscillations in wpt rate are associated with intensive variations in transpiration rate , even when water is fully available to the plant . oscillations in whole - plant transpiration ( wpt ) and wet - wick evaporation rates superimposed on the smoothed rates when the low - pass - filtered data are used to derive the transpiration and evaporation rates ( a ) . cross - correlation function between transpiration and evaporation rates in the morning and afternoon hours ( b ) . the effect of water - stress build - up on the momentary wpt rate ( smoothed and oscillatory ) is shown in fig . 5a and c is shown in fig . 5b and 5d , respectively , where the value of zero transpiration rate represents the smoothed transpiration rate in fig . 5a and 5c , the smoothed and superimposed oscillatory evaporation rates from the wet wick for the 5 d dehydration period are shown in fig . 5e and the isolated oscillations in evaporation rate ( the difference between the smoothed and oscillatory patterns ) for this period 5f . figure 5 indicates that ( i ) since these plants were grown in a temperature - controlled greenhouse with a repetitive pattern of daily ambient conditions ( demonstrated by the repetitive daily pattern in wet - wick evaporation rate , fig 5e ) , water can be assumed to be fully available to the plant as long as the transpiration - rate pattern is similar to the previous day . accordingly , water seems to be fully available to plant i during the first 2 d of dehydration , and to plant ii only on the first day ( fig . as opposed to plant i , whose transpiration rate decreased slightly on days 2 and 3 and substantially on days 4 and 5 , the transpiration rate of plant ii decreased substantially on day 2 and was markedly low during the last 3 d of dehydration . differences in size between the two plants led to a significant difference in the maximum transpiration rate on day 1 ( 90 g h versus 160 g h for plants i and ii , respectively ) and the amount of water remaining in the pots . ( ii ) similar to the transpiration - rate pattern in fig . 2a , the smoothed transpiration - rate patterns in fig . 5a and c have two daily peaks : a lower peak at noon followed by a higher one in the afternoon . note that the timing of the second daily peak coincides with the peak in daily evaporation rate . the two peaks in transpiration rate occurred despite the single daily peak in evaporation rate ( fig . furthermore , the two peaks in transpiration rate were accompanied by a change in the pattern of transpiration - rate oscillations : the oscillations were amplified while the transpiration rate increased toward the first and even the second peak , and were shifted from their concurrence with the oscillations in evaporation rate ( figs 2a , 5a the increase in oscillation amplitude during this period contrasted with the uniform distribution of oscillation amplitudes in evaporation rate ( fig . ( iii ) the ratio between the values of the two daily transpiration - rate peaks diminished with increasing water stress and was associated with the formation of a noticeable dip between the two peaks . ( iv ) the increase in oscillation amplitudes that was associated with transpiration - rate variation ( increase in the morning hours or decrease in the afternoon hours ) during the early stages of dehydration ( fig . 5a , c ) was also seen in the dip between the two daily transpiration - rate peaks as water stress progressed . smoothed and oscillatory whole - plant transpiration ( wpt ) rate for tomato plant i ( a ) , isolated oscillations in wpt rate for tomato plant i ( b ) , smoothed and oscillatory wpt rate for tomato plant ii ( c ) , isolated oscillations in wpt rate for tomato plant ii ( d ) , smoothed and superimposed oscillatory evaporation rates from the wet wick ( e ) , and isolated oscillations in wet - wick evaporation rate ( f ) , for five continuous days of dehydration . the oscillation amplitudes relative to the transpiration / evaporation rate ( hereafter designated relative oscillations ) are shown in fig . the relative oscillations are the ratio between the isolated oscillations in transpiration / evaporation rate and the smoothed transpiration / evaporation rate , respectively , in fig . 5 . the relative oscillations in evaporation rate are larger in the early morning hours when the evaporation rate is low ( fig . 5e ) , and then decrease as the evaporation rate increases and increase again in the late afternoon hours when the evaporation rate decreases again ( fig . figure 6a d shows that the relative oscillations in transpiration increase during the dehydration period together with the decrease in transpiration rate ( fig . the oscillations in wpt rate may be temporary , and even decrease to zero ( relative transpiration= 1 in fig . 6b ) under severe drought conditions when the transpiration rate is low . the daily pattern of relative oscillations in transpiration rate during the advanced dehydration stages ( e.g. fig . 6b , d ) indicates that they are relatively low in the morning , then intensify and stay high , even in the late afternoon hours . isolated oscillations in whole - plant transpiration ( wpt ) rate relative to the momentary values of the smoothed wpt rate for : plant i on day 1 ( a ) and day 4 ( b ) of dehydration , plant ii on day 1 ( c ) and day 4 ( d ) of dehydration . isolated oscillations in evaporation rate relative to the momentary values of the smoothed evaporation rate from the wet wick for day 1 ( e ) and day 4 ( f ) of dehydration . a comparison between the daily isolated oscillations in transpiration and wet - wick evaporation rates for dehydrating tomato plants i , together with the cross - correlation function for each dehydration day , are shown in fig . the cross - correlation function for each dehydration day for plant ii is shown ( hollow circles ) in fig . the cross - correlation function for plant i had an apparent single peak on day 1 ( fig . this high cross - correlation value indicates that the synchronized oscillations in wpt rate were mostly induced by the fluctuations in ambient conditions in the greenhouse . however , there were no evident peaks in the cross - correlation function as water stress increased , which indicates that the synchronized oscillations in transpiration rate became self - regulated and independent of the fluctuations in ambient conditions demonstrated by the pattern of wet - wick evaporation . the decrease in transpiration rate along with the development of self - regulated oscillations in transpiration rate took place while the gap between water demand and water availability broadened . note that the self - regulated oscillations in plant i appeared on days 2 and 3 of dehydration ( fig . 7d , f ) , while the transpiration rates were similar to that on day 1 ( fig . by contrast , the daily cross - correlation function for plant ii did not have any apparent single peak , not even on day 1 ( the hollow circles in fig . the differences in the cross - correlation functions between the two plants can be attributed to the high transpiration rate of plant ii on day 1 ( fig . plant ii was 6% heavier and had 7% higher leaf area than plant i. comparison between the isolated oscillations in tomato plant i transpiration and wet - wick evaporation rates and the respective cross - correlation function for these oscillations ( filled circles ) during the 5 d dehydration experiment : day 1 ( a and b , respectively ) , day 2 ( c and d , respectively ) , day 3 ( e and f , respectively ) , day 4 ( g and h , respectively ) , day 5 ( i and j , respectively ) . the cross - correlation function for plant ii during the 5 d dehydration experiment appear in b , d , f , h , and j , respectively as hollow circles . typical variations in weight during the night and subsequent daylight hours are shown in fig . as water was supplied to the plants in the late evening ( after 20.00 h ) , the weight decreased monotonically , albeit at different rates , during the night and daylight hours . the rate of the weight decrease ( transpiration rate increase ) intensified during the morning , stayed high during the noon and early afternoon hours , and weakened thereafter ( fig . the wpt - rate time series ( the negative values of the rate of weight decrease ) , calculated by the time derivative of the measured weight - time series with data points every 3 min ( equation 1 ) , is shown in fig . a noisy wpt rate was obtained despite what appeared to be a relatively smooth pattern of weight decrease ( fig . the amplitudes of the wpt rate were low during the night , early morning , and evening hours and large at the other times . a detailed pattern of the daily transpiration rate , especially when the ambient conditions varied with time , could barely be identified from this noisy time series . the smoothed wpt rate ( fig . 1b , solid line ) was obtained by differentiating a smoothed weight - time series obtained by applying the s - g smoothing method with a window breadth of 30 data points ( s - g 30 ) . the fluctuations around the smoothed transpiration - rate pattern are usually considered random noise ( white noise ) associated with the measurement system , and as such are generally ignored without further analysis . here , however , these fluctuations were further analysed in order to isolate the system - noise - induced fluctuations from those induced by the ambient and/or physiologically driven oscillations . time - course of transpiration rate calculated by numerical derivative of the measured and smoothed time - courses of the decrease in potted tomato weight ( b ) . the smoothed patterns of weight - variation rate ( smoothed by s - g 30 prior to time differentiation ) for a tomato plant , a wet wick , and a constant weight that was placed on a load cell are shown in fig . the whole - plant and wet - wick data were measured on 6 september 2006 , and the constant load on 9 september 2006 , all in the temperature - controlled greenhouse . 2a were measured for four neighbouring tomato plants that were grown together in the greenhouse ( data not shown ) with a similarly ample water supply . the residual time series ( difference between the measured and smoothed weight time series ) for the tomato plant , wet wick , and constant load are shown in fig . to exclude the possibility of the oscillations in wpt rate being simply system - related noise , these residuals were examined for randomness ( white noise ) by autocorrelation function ( fig . the autocorrelation function of the constant - weight residual time series , k ( fig . 2i ) was 1 at lag=0 and was close to zero for all other lags . by contrast , the autocorrelation functions of both the tomato plant and the wet wick ( fig . 2 g , h , respectively ) were periodic for the first 200 lags , with an approximate average 40 lag difference from peak to peak . the correlation between the fluctuations in plant weight around the smoothed weight variation and ambient conditions ( represented by the wet wick ) will be analysed further in the following . smoothed weight - variation - rate patterns in a whole potted tomato plant ( a ) , a wet wick ( b ) , and a constant load ( c ) . residual time series for the whole plant ( d ) , wet wick ( e ) , and constant load ( f ) . autocorrelation function of the residual time series for the whole plant ( g ) , wet wick ( h ) , and constant load ( i ) . the spectra for the tomato - plant , wet - wick , and constant - load residual time series are shown in fig . 3e ) provided an additional indication that its residual time series is practically a random signal ( white noise ) while those of the plant and wet wick are not . 3a ) had high amplitudes at f < 2.5 h and lower amplitudes at the higher frequencies . the wet - wick spectrum ( fig . 3c ) exhibited a similar pattern , i.e. high amplitudes ( although lower than those of the whole plant ) at f < 2 h and lower amplitudes at the higher frequencies . to filter out what was considered to be white noise , both spectra were low - pass filtered for f < 2 h prior to further analysis . the time derivative of the reconstructed low - pass - filtered residual time series provided oscillations in wpt and wet - wick evaporation rates , as well as in the constant - load weight . superpositions of these oscillations on the smoothed variation of these variables are shown in fig . spectra for the whole - plant ( a ) , wet - wick ( b ) , and constant - load ( c ) residual time series . time derivative of the low - pass - filtered residual time series superimposed on the time derivative of the smoothed weight variation with time for the whole plant ( d ) , wet wick ( e ) , and constant load ( f ) . to analyse the correlation between the fluctuations in wpt and wet - wick rates , the time derivatives of the reconstructed low - pass - filtered residual time series of these variables were plotted together in fig . 4a . the baseline in this figure ( weight loss=0 ) was the smoothed transpiration / evaporation rate . a quantitative measure of this correlation was obtained by the cross - correlation function ( fig . 4b ) , which is a standard method of estimating the degree to which two series are correlated . the cross - correlation between the two series was calculated for the morning ( 06.00 to 12.00 h ) and afternoon ( 12.00 to 18.00 h ) to examine the correlation between fluctuations in ambient conditions and the oscillations in wpt rate when transpiration rate increases ( morning hours ) and when it is already high and beginning to decrease ( afternoon hours ) . when the cross - correlation around lag=0 is higher than those at other lag values ( negative and positive ) , the oscillations in wpt rate can be more related to temporary variations ( fluctuations ) in ambient conditions . however , when low cross - correlation values are obtained at all lags , the two series are independent and the oscillations in wpt rate may be autonomous , putatively driven by independent physiological processes . the cross - correlation function for the morning hours had a slightly higher peak value than for the afternoon hours ( fig . the presence of multiple peaks indicated that the oscillations in wpt rate depend on the fluctuations in ambient conditions , with periods when the oscillations in wpt rate are independent of those of the wet wick , namely , ambient conditions . 4a ) revealed that they are temporarily out of phase between 07.30 and 10.00 h and between 13.30 and 15.30 h. from the smoothed transpiration pattern in fig . 3b , the shifts between the two oscillations coincided with periods of intensive change in transpiration rate be it increasing or decreasing . thus , it may be deduced that autonomous oscillations in wpt rate are associated with intensive variations in transpiration rate , even when water is fully available to the plant . oscillations in whole - plant transpiration ( wpt ) and wet - wick evaporation rates superimposed on the smoothed rates when the low - pass - filtered data are used to derive the transpiration and evaporation rates ( a ) . cross - correlation function between transpiration and evaporation rates in the morning and afternoon hours ( b ) . the effect of water - stress build - up on the momentary wpt rate ( smoothed and oscillatory ) is shown in fig . 5a and c is shown in fig . 5b and 5d , respectively , where the value of zero transpiration rate represents the smoothed transpiration rate in fig . 5a and 5c , the smoothed and superimposed oscillatory evaporation rates from the wet wick for the 5 d dehydration period are shown in fig . 5e and the isolated oscillations in evaporation rate ( the difference between the smoothed and oscillatory patterns ) for this period are shown in fig . figure 5 indicates that ( i ) since these plants were grown in a temperature - controlled greenhouse with a repetitive pattern of daily ambient conditions ( demonstrated by the repetitive daily pattern in wet - wick evaporation rate , fig 5e ) , water can be assumed to be fully available to the plant as long as the transpiration - rate pattern is similar to the previous day . accordingly , water seems to be fully available to plant i during the first 2 d of dehydration , and to plant ii only on the first day ( fig . as opposed to plant i , whose transpiration rate decreased slightly on days 2 and 3 and substantially on days 4 and 5 , the transpiration rate of plant ii decreased substantially on day 2 and was markedly low during the last 3 d of dehydration . differences in size between the two plants led to a significant difference in the maximum transpiration rate on day 1 ( 90 g h versus 160 g h for plants i and ii , respectively ) and the amount of water remaining in the pots . ( ii ) similar to the transpiration - rate pattern in fig . 2a , the smoothed transpiration - rate patterns in fig . 5a and c have two daily peaks : a lower peak at noon followed by a higher one in the afternoon . note that the timing of the second daily peak coincides with the peak in daily evaporation rate . the two peaks in transpiration rate occurred despite the single daily peak in evaporation rate ( fig . 5e ) . furthermore , the two peaks in transpiration rate were accompanied by a change in the pattern of transpiration - rate oscillations : the oscillations were amplified while the transpiration rate increased toward the first and even the second peak , and were shifted from their concurrence with the oscillations in evaporation rate ( figs 2a , 5a the increase in oscillation amplitude during this period contrasted with the uniform distribution of oscillation amplitudes in evaporation rate ( fig . ( iii ) the ratio between the values of the two daily transpiration - rate peaks diminished with increasing water stress and was associated with the formation of a noticeable dip between the two peaks . ( iv ) the increase in oscillation amplitudes that was associated with transpiration - rate variation ( increase in the morning hours or decrease in the afternoon hours ) during the early stages of dehydration ( fig . 5a , c ) was also seen in the dip between the two daily transpiration - rate peaks as water stress progressed . smoothed and oscillatory whole - plant transpiration ( wpt ) rate for tomato plant i ( a ) , isolated oscillations in wpt rate for tomato plant i ( b ) , smoothed and oscillatory wpt rate for tomato plant ii ( c ) , isolated oscillations in wpt rate for tomato plant ii ( d ) , smoothed and superimposed oscillatory evaporation rates from the wet wick ( e ) , and isolated oscillations in wet - wick evaporation rate ( f ) , for five continuous days of dehydration . the oscillation amplitudes relative to the transpiration / evaporation rate ( hereafter designated relative oscillations ) are shown in fig . the relative oscillations are the ratio between the isolated oscillations in transpiration / evaporation rate and the smoothed transpiration / evaporation rate , respectively , in fig . 5 . the relative oscillations in evaporation rate are larger in the early morning hours when the evaporation rate is low ( fig . 5e ) , and then decrease as the evaporation rate increases and increase again in the late afternoon hours when the evaporation rate decreases again ( fig . figure 6a d shows that the relative oscillations in transpiration increase during the dehydration period together with the decrease in transpiration rate ( fig . the oscillations in wpt rate may be temporary , and even decrease to zero ( relative transpiration= 1 in fig . 6b ) under severe drought conditions when the transpiration rate is low . the daily pattern of relative oscillations in transpiration rate during the advanced dehydration stages ( e.g. fig . 6b , d ) indicates that they are relatively low in the morning , then intensify and stay high , even in the late afternoon hours . isolated oscillations in whole - plant transpiration ( wpt ) rate relative to the momentary values of the smoothed wpt rate for : plant i on day 1 ( a ) and day 4 ( b ) of dehydration , plant ii on day 1 ( c ) and day 4 ( d ) of dehydration . isolated oscillations in evaporation rate relative to the momentary values of the smoothed evaporation rate from the wet wick for day 1 ( e ) and day 4 ( f ) of dehydration . a comparison between the daily isolated oscillations in transpiration and wet - wick evaporation rates for dehydrating tomato plants i , together with the cross - correlation function for each dehydration day , are shown in fig . the cross - correlation function for each dehydration day for plant ii is shown ( hollow circles ) in fig . the cross - correlation function for plant i had an apparent single peak on day 1 ( fig . this high cross - correlation value indicates that the synchronized oscillations in wpt rate were mostly induced by the fluctuations in ambient conditions in the greenhouse . however , there were no evident peaks in the cross - correlation function as water stress increased , which indicates that the synchronized oscillations in transpiration rate became self - regulated and independent of the fluctuations in ambient conditions demonstrated by the pattern of wet - wick evaporation . the decrease in transpiration rate along with the development of self - regulated oscillations in transpiration rate took place while the gap between water demand and water availability broadened . note that the self - regulated oscillations in plant i appeared on days 2 and 3 of dehydration ( fig . 7d , f ) , while the transpiration rates were similar to that on day 1 ( fig . by contrast , the daily cross - correlation function for plant ii did not have any apparent single peak , not even on day 1 ( the hollow circles in fig . the differences in the cross - correlation functions between the two plants can be attributed to the high transpiration rate of plant ii on day 1 ( fig . plant ii was 6% heavier and had 7% higher leaf area than plant i. comparison between the isolated oscillations in tomato plant i transpiration and wet - wick evaporation rates and the respective cross - correlation function for these oscillations ( filled circles ) during the 5 d dehydration experiment : day 1 ( a and b , respectively ) , day 2 ( c and d , respectively ) , day 3 ( e and f , respectively ) , day 4 ( g and h , respectively ) , day 5 ( i and j , respectively ) . the cross - correlation function for plant ii during the 5 d dehydration experiment appear in b , d , f , h , and j , respectively as hollow circles . the development of autonomous and self - regulated oscillations in wpt rate is postulated to be the plant response to the increasing gap between the evaporative demand and water availability . although the relative oscillations increased as water stress intensified ( fig . d ) , their short - term variation was associated with the temporal variations in the between evaporative - demand to water - availability balance . the relative oscillations were low during the early morning hours , then increased with the increase in evaporative demand , and remained high during the afternoon hours when the evaporative demand had already decreased ( fig . 6b , d ) . given that transpiration rate is highly dependent on temporal variation in soil water content ( kramer and boyer , 1995 ; ray and sinclair , 1998 ; doussan et al . , 2006 ; garrigues et al . , 2006 ) , the following scenario can be hypothesized to explain this irregular pattern of the relative oscillations . the moisture content around the plant root is usually lower at the end of the day and is gradually replenished during the night by mass flow from the wetter soil bulk . when the moisture content in the soil is high , this water flow from the bulk soil to the root vicinity also takes place during the daylight hours at a rate that enables the plant to catch up with the evaporative demand ( first 2 d in fig . when the transpiration rate follows the evaporative demand , soil water is considered fully available at the given evaporative demand . however , when soil water becomes depleted , the flow rate from the soil bulk to the root vicinity decreases owing to an increase in the soil 's resistance to flow , and the transpiration rate decreases despite the high evaporative demand ( last 3 d in fig . when the soil becomes dry , the only available water that can cope with the evaporative demand is the amount around the roots in the morning hours when the replenished water there can handle the concurrent low evaporative demand . 6b , d ) . however , when the evaporative demand increases and water content in the root vicinity is nearly depleted with no replenishment from the soil bulk , the relative oscillation amplitudes intensify with decreasing transpiration rate ( fig . plants are exposed to the risk of embolism under high evaporative demand , and particularly under drought stress owing to the increase in xylem tension , which is generally seen as a potentially catastrophic dysfunction of the axial water - conducting system ( tyree and sperry , 1989 ; tardieu and davies , 1993 ; hacke and sauter , 1995 ) . one interpretation of the observations in this study is that the autonomous and self - regulated oscillations in wpt rate with higher relative amplitudes are designed to diminish or even prevent the increase in xylem tension . ( 2002 ) in which the relationship between acoustic emission , transpiration rate , and water stress in tomato plants was measured . ( 2002 ) reported that under mild or no conditions of water stress , the acoustic emission ( representing the development of embolism ) significantly increases with transpiration rate , both positively and negatively . this observation agrees with our finding that temporary autonomous oscillations in wpt rate develop when the transpiration rate is sharply increasing in the morning hours ( and sharply decreasing in the afternoon ) ( figs 4a , 7a ) . ( 2002 ) showed that the acoustic emission increases with a decrease in the amount of soil water under mild or moderate conditions of water stress , and decreases with the decrease in the amount of soil water under severe water stress . other studies revealing a link between stomatal conductance , leaf water potential , and xylem embolism were performed by salleo et al . they suggested that xylem embolism may be a pre - signal for stomatal closure , thus representing an important step in the stomatal control of transpiration . the important role of the long ( root - to - shoot ) hydraulic signal in inducing stomatal closure under water stress has recently been reported ( christmann et al . , 2007 ) . the independent findings of a correlation between water stress and the formation of cavitation ( salleo et al . , 2000 ; qiu et al . , 2002 ; and others ) and the development of autonomous self - regulated oscillations in wpt rate under water stress suggest that these oscillations are related to the plant 's response to increasing xylem tension . consequently , it is postulated that the oscillations in wpt rate are driven by a rapid , root - induced hydraulic signal that spreads instantaneously throughout the entire vascular system and inter alia , regulates stomatal aperture . could the oscillations in wpt rate be related to short - term growth of the plant ? tomato plants maintain almost constant weight during the day , while their growth periods take place from the late afternoon to early morning ( menzel et al . tomato plants present ishohdric behaviour ( sade et al . , 2009 ) , namely , maintaining constant leaf relative water content during midday , under high available water conditions and drought condition alike . combining this intrinsic property with the observations that the autonomous and self - regulated oscillations in wpt appeared only under water stress , suggest that the measured oscillations were not induced by short - term oscillations in the plant 's growth . in conclusion , synchronized autonomous self - regulated oscillations in wpt rate with periods of 2050 min were measured for tomato plants under water stress . these oscillations were interpreted as changes in leaf conductance triggered by hydraulic signals spread instantaneously via the plant vascular system which resulted from drought - induced xylem tension . though the role of the oscillations is not yet clear , a plausible assumption is that they are a means used by the plant to control xylem tension while preventing the critical tension threshold that impels embolism .

abstractbackground : patients with heart disease experience limited activities of daily living ( adl ) . this is a cross - country comparison of the additive effects of socioeconomics , health behaviors , and the number of medical comorbidities on disability among patients with heart disease.methods:the current study used a cross - sectional design . data came from the research on early life and aging trends and effects ( relate ) . the current analysis utilized data on elderly individuals ( age 60 y ) from 13 countries . the outcome was any adl limitation ( i.e. bathing , dressing , using toilet , transferring , lifting heavy things , shopping , and eating meals ) . socioeconomics ( i.e. age , gender , education , and income ) , health behaviors ( i.e. exercise , smoking , and drinking ) , and number of chronic medical conditions ( i.e. hypertension , respiratory , arthritis , stroke , and diabetes ) were entered into country - specific logistic regressions , considering at least one limitation in adl as the main outcome.results:number of comorbid medical conditions and age were positively associated with disability in 85% of the countries . physical activity and drinking were linked to disability in 54%and 31% of countries , respectively . higher education and income were associated with lower disability in 31% and 23% of the countries , respectively . female gender was associated with higher disability only in 15% of the countries . smoking was not associated with disability , while the effects of socioeconomics , drinking , exercise , and medical comorbidities were controlled.conclusion:determinants of disability depend on the country ; accordingly , locally designed health promotion interventions may be superior to the universal interventions for patients with heart disease . medical comorbidities , however , should be universally diagnosed and treated .

functional limitation of patients with heart disease has been documented consistently by cross - sectional and longitudinal studies . in a recent study conducted in 15 countries , with no exception , heart disease was associated with poor subjective health , above and beyond the effect of socioeconomics . the study , however , showed cross - country differences in the interactions between socioeconomic factors and heart disease in shaping well - being of populations . heart disease had a larger effect on subjective health of the elderly in the u.s . and china , women in the u.s . , south africa , and india , low - income people in china and costa rica , and individuals with low education in uruguay and ghana . most of the research on the determinants of disability and the well - being of patients with heart disease has focused on either psychological , clinical , or behavioral characteristics . thus , less is known about the additive effects of social , behavioral , and comorbid conditions . multiple aspects of the daily life of patients with heart disease may be influenced by the condition . heart disease may be accompanied with a wide range of symptoms ( e.g. dyspnea , tiredness , and fatigue ) leading to functional limitation . patients with heart disease experience limitations in activities of daily living ( adl ) . impaired functional capacity and . additional research on the effect of the social determinants of the well - being of patients with heart disease is needed . old age is associated with limitation in function and impaired well - being , physical , and mental health . gender also influences perceived health , with women tending to report higher levels of disability and morbidity . education and income , the most commonly accepted proxies of socioeconomic position , are associated with subjective health , chronic disease , and mortality . physical activity and exercise reduce the likelihood of health - related disability , especially during old age , and improve health - related quality of life . drinking , smoking , and physically inactive life style carry individual risks to adl , especially later in life . chronic medical conditions associated with heart disease are also major causes of morbidity and mortality . most studies have documented lower health and well - being , functional status , and health - related quality of life in the presence of chronic medical conditions . although we already know that patients with heart disease experience and report functional limitations , the contribution of various determinants on disability may differ across countries . unfortunately , our information is very limited about cross - country differences in the additive effects of determinants of disability associated with heart disease . in response to the gap of knowledge on cross - country variations in the determinants of disability among patients with heart disease , we compared countries for the additive effects of social , behavioral , and medical determinants and disability among older adults with heart disease . we used publicly available data of the research on early life and aging trends and effects ( relate ) , a collection of multiple surveys from different countries across the world . the relate data are composed of the following surveys : 1 ) wisconsin longitudinal study ; 2 ) china health and nutrition study ; 3 ) chinese longitudinal healthy longevity survey ( clhls ) ; 4 ) costa rican study of longevity and healthy aging ; 5 ) puerto rican elderly : health conditions ; 6 ) study of aging survey on health and well being of elders ; and 7 ) who study on global ageing and adult health . the sample size distribution of each country in the publicly available data is presented in table 1 . sample size distribution of the participating countries in the relate data the original relate study enrolled more countries than were entered into the current analysis . this manuscript is limited to data from china , costa rica , puerto rico , mexico , barbados , brazil , chile , cuba , uruguay , india , ghana , south africa , and russia relate , research on early life and aging trends and effects ( relate ) ; creles , costa rican longevity and healthy aging study ; prehco , puerto rican elderly : health conditions ; sabe , survey on health , well - being , and aging in latin american and the caribbean ; who , world health organization ; sage , study on global ageing and adult health ; chns , china health and nutrition survey ; clhls , chinese longitudinal healthy longevity survey the participating countries represent a diverse range of national income levels and were selected from multiple continents . ghana represents low - income countries ; china and india represent lower middle - income countries ; cuba , uruguay , chile , costa rica , brazil , mexico , and russia represent upper middle - income countries ; and puerto rico and barbados represent high - income countries . although the original relate study included a few other countries as well , countries participating in the current analysis were limited to those with available data on our variables of interest and included china , costa rica , puerto rico , mexico , barbados , brazil , chile , cuba , uruguay , india , ghana , south africa , and russia . the presence of self - reported physician diagnosis of heart disease and age over 65 years were considered as eligibility criteria . self - reported data on physician - diagnosis of chronic medical conditions such as heart disease is valid and closely associated with the physician - diagnosis of heart disease and medical record data . the socioeconomic data included age ( continuous variable ) , gender ( dichotomous variable ) , education ( continuous variable ) , and income ( continuous variable ) . income in this study was per capita annual household income calculated as purchase power parity dollars ( ppp$ ) . to provide the ppp$ or international dollar , costs ( or incomes ) in local currency units an international dollar is a hypothetical currency that is used as a means of translating and comparing costs from one country to the other using a common reference point , the us dollar . the ppp$ exchange rates are provided by the world health organization . a ppp$ exchange rate can be defined as the number of units of a country 's currency required to buy the same amounts of goods and services in the domestic market as the u.s . the number of comorbid medical conditions was calculated based on the presence of self - reported physician diagnosis of diabetes , respiratory conditions , stroke , hypertension , and arthritis . self - reported data on chronic medical conditions are believed to be in agreement with physician diagnosis of conditions ( kappa : 0.740.92 ) . we approached physical disability from an operational point of view , focusing on limitations in adl . the adl items included in this study comprised bathing , getting dressed , going to the toilet , transferring , lifting heavy objects , shopping , and eating meals . informed consent was also provided by all the participants of all the surveys . for the statistical analyses , the statistical software spss version 20.0 for windows ( spss inc . , chicago , il ) was used . as weights were not applicable to surveys from china ( chns ) , we did not apply sampling weights . socioeconomic factors ( age , gender , education , and income ) , health behaviors ( exercise , smoking , drinking ) , and number of chronic medical conditions ( hypertension , respiratory , arthritis , stroke , and diabetes ) were entered into country - specific hierarchical logistic regressions . in the first step ( model i ) , we tested the main effects of socioeconomic factors . in the next step ( model ii ) , we also entered health behaviors . in the third step ( model iii ) , we also included the number of chronic medical conditions . odds ratios ( ors ) and 95% confidence intervals ( 95% ci ) were reported . the socioeconomic factors of the participants in each country have been reported elsewhere . in model i , high age was predictive of adl limitation in all the countries other than uruguay , ghana , and south africa . female gender was not associated with adl limitation in most countries , with the exception of mexico . in south africa , high income was linked to lower odds of adl limitation only in costa rica and puerto rico . in chile , higher education was associated with lower adl limitation in mexico , india , and russia . in chile , cross - country differences in associations between socioeconomic factors and disability among patients with heart disease as table 3 depicts , in model ii , only in 4 countries ( i.e. china , puerto rico , brazil , and cuba ) was exercise associated with lower adl limitation . in 3 countries ( i.e. india , costa rica , and mexico ) , the association between exercise and adl limitation was marginally significant . with a few exceptions ( i.e. china , brazil , chile , and uruguay ) , most countries did not show an association between drinking and adl limitation . smoking was not associated with adl limitation among individuals with heart disease , above and beyond the socioeconomic factors , exercise , and drinking . cross - country differences in associations between socioeconomic factors and health behaviors among patients with heart disease as table 4 demonstrates , in model iii , number of medical comorbidities was positively associated with odds of adl limitation in 10 countries . the number of medical comorbidities was marginally associated with adl limitation in one country ( barbados ) . cross - country differences in associations between socioeconomic factors , health behaviors , and medical conditions among patients with heart disease this study revealed major cross - country differences in the additive effects of socioeconomic , behavioral , and medical characteristics on disability among patients with heart disease . the number of medical comorbidities and age were predictive of disability in most countries , while gender and income were linked to disability in very few countries . surprisingly , smoking was not associated with disability in any of the countries , while socioeconomic factors and other health behaviors ( i.e. exercise and drinking ) were constant . to summarize , the number of comorbid medical conditions , age , physical activity , drinking , education , income , and gender were associated with disability in 85% , 85% , 54% , 31% , 31% , 23% , and 15% of the countries . there are very few previous studies to compare our findings with . based on a recent study that compared 15 countries , age in the u.s . and china ; gender in the u.s . , south africa , and india ; income in china and costa rica ; and education in uruguay and ghana modified the effect of heart disease on subjective health . in puerto rico , argentina , barbados , brazil , chile , cuba , and russia , the effect of heart disease on subjective health was above and beyond the influence of socioeconomic factors . the findings of a recent in press study revealed that countries largely vary in the contributors of adl limitation in the general population . the study particularly found considerable cross - country differences for the relationship between age and adl . the contribution of age and gender in explaining the variance of adl was very high in china and cuba , respectively . more variation was seen in the effect of education than income as a factor contributing to the adl across countries . health behaviors such as exercise and also chronic conditions ( in general ) consistently explained a significant portion of the variance of adl across all the 8 countries included in that study . based on our study , age was linked to disability among individuals with heart disease in 10 of the 13 countries . age is known to be positively associated with adl limitation . in almost all countries , individuals with diabetes are more likely to experience restrictions in adl , along with reduced mobility and role functioning . a recent study documented a significant correlation between the comorbidity score and all the measures of well - being among patients with ischemic heart disease . the comorbidity score was correlated with physical and mental quality of life , psychological distress , sleep quality , and dyadic adjustment . authors emphasized that primary health care physicians , family physicians , and cardiologists have a major role in identifying and treating comorbid somatic conditions among patients with ischemic heart disease . according to a cross - country study , in all countries and with no exception , heart disease was associated with higher odds of poor subjective health , above and beyond the effect of age , gender , education , and income . this is in line with previous studies suggesting the role of heart disease on well - being , quality of life , and disability . in a study , well - being was mostly affected by heart conditions , followed by asthma / chronic bronchitis , joint complaints , back problems , and diabetes . another study suggested that heart diseases , musculoskeletal diseases , lung diseases , neurological disorders , diabetes , and cancer may have more influence on disability at the population level , compared to other conditions . another study showed that patients with heart disease , as well as patients with hearing impairment , neurological disease , and vision impairment , report the highest levels of distress . a study also showed that after controlling the effect of age , sex , educational level , comorbidities , disability and pain , coronary artery disease and chronic hemodialysis were linked to the highest levels of depression . according to a cross - country study , heart disease was the only factor consistently associated with poor perceived health among individuals with diabetes . only in two countries , women report lower levels of quality of life , whereas men have lower mortality . in general the current study also documented cross - country differences in the association between education and income and adl limitation among elderly with heart disease . based on the current study , clinicians in different countries may need to consider different socioeconomic and behavioral factors to estimate or reduce disability ( adl limitation ) among patients with heart disease . based on our findings , locally designed health promotions may be superior to universal programs for patients with heart disease . in almost all countries , however , disability may be reduced if comorbid medical conditions are properly diagnosed and treated . that is , attention to comorbid conditions may be considered as a common component of disability prevention for patients with heart disease . similar to other studies , the current study is limited in several ways . due to the cross - sectional design , health behaviors such as smoking , drinking , and exercise were measured using single items . only a few comorbid medical conditions were included , and the type of conditions was not entered into the model . to conclude , there are major cross - country differences in the determinants of disability among patients with heart disease . the findings advocate designing and implementing country - specific programs to reduce disability among patients with heart disease .

the effects of whole body irradiation ( wbr ) on particle clearance and the development of pulmonary fibrosis have been investigated . using carbon , clearance is accomplished by polymorphonuclear leukocytes ( pmn ) and alveolar macrophages ( am ) , and only a few particles reach the interstitum . however , in preirradiated mice , the usual eflux of inflammatory cells is much delayed so that more free carbon remains in the alveoli , and by 1 week , many particles cross the epithelium to be phagocytized by interstitial macrophages . carbon is found in the peribronchiolar interstitium 6 months later with no evidence of fibrosis . in the present study , mice received 1 mg silica intratracheally 2 days after 6.5 gy wbr when the white blood cell count was low . a much - reduced am and pmn response was found in the following 2 weeks compared to the reaction to silica alone , and many silica particles reached interstitial macrophages . in this case , macrophage activation by silica was associated with fibroblast proliferation , and by 16 weeks , much more pulmonary fibrosis was produced than after silica or irradiation only . this was measured biochemically and correlated with a large increase in retained silica in the irradiation - silica group . the results indicate that radiation inhibits the inflammatory response to particle instillation , resulting in greater translocation of free particles to the pulmonary interstitium . in the case of silica , the greater , prolonged interaction with interstitial macrophages leads to a much exaggerated fibrotic reaction.imagesfigure 3.figure 4.figure 5.figure 6.figure 7 .

background the concept of osseointegration involves direct contact between titanium implant and bone . this transcutaneous prosthetic system for amputees is intended to assure stable long - term fixation . most metal transcutaneous implants have failed , primarily owing to infection.questions/purposeswe determined the frequency and describe the presentation of infectious complications with this novel method . we also evaluated the bacterial flora at the skin - penetration area and its relation to the development of local and implant - related infection.patients and methodswe prospectively followed 39 patients with arm and leg amputations fitted with transcutaneous osseointegrated titanium implants a mean of 56 months earlier ( range , 132133 months ) . there were 33 femoral , one tibial , four ulnar , four radial , and three humeral implants . patients were selected during a 6-month period in 2005 and identically reevaluated after 3 years . implant infection was defined as definite , probable , or possible based on clinical , radiologic , and microbiologic evidence.resultsthe frequency of implant infection was 5% at inclusion and 18% at followup . one patient with infection recovered owing to antibiotic treatment and another patient had the implant removed . most implant infections had low infectious activity , and in five of the seven patients with infections , prosthetic use was not affected . the most common bacteria in superficial and deep cultures were staphylococcus aureus and coagulase - negative staphylococci.conclusionsdespite frequent colonization around the skin - implant interface by potentially virulent bacteria such as staphylococcus aureus and bacteria associated with biomedical device infections such as coagulase - negative staphylococci , this titanium implant system for bone - anchored prostheses caused few infections leading to disability or implant removal.level of evidencelevel iv , therapeutic study . see the guidelines for authors for a complete description of levels of evidence .

in high - income countries , chronic vascular disease is the leading cause of limb loss . however , many previously healthy individuals undergo amputation secondary to trauma , neoplasia , infection , and arterial embolism . the conventional way to suspend a prosthetic limb to the body is with a prosthetic socket . users of socket prostheses commonly report impaired quality of life [ 8 , 13 , 26 , 27 ] and complications including dermatitis and infected sores [ 10 , 21 ] . our novel method is based on the principles of osseointegration ( fig . 1 ) , which has been in clinical use in prosthetic teeth replacement since 1965 . one report suggests a 15-year implant survival rate of approximately 90% in mandibular bone . with osseointegration , direct contact between fixture and bone tissue is intended , assuring a stable , long - term attachment for the external prosthesis . this technique results in easy prosthesis handling , improved limb control , and eliminates socket - impaired rom and socket - caused skin disorders [ 15 , 16 , 30 ] ( fig . 2 ) . during the years , various attempts to anchor prosthetic limbs with transcutaneous metal implants other than titanium [ 17 , 18 , 25 ] have failed , primarily owing to infection . currently , our center has treated more than 100 patients with femoral titanium implants ( fig . 3 ) , three with tibial implants , 15 with humeral implants , and 20 with ulnar and/or radial implants . treatment failures were more common before we introduced a standardized treatment protocol in 1999 [ 14 , 28].fig . 2this is a ventral view of the transcutaneous component ( abutment ) in a patient with a transfemoral amputation . ( published with permission from nigel jarvis , the university of gothenburg , the sahlgrenska academy , gothenburg , sweden.)fig . 3a lateral view is shown of a patient securing an external prosthesis to the abutment . ( published with permission from nigel jarvis , the university of gothenburg , the sahlgrenska academy , gothenburg , sweden . ) this schematic drawing shows the implant components and surrounding tissues . this is a ventral view of the transcutaneous component ( abutment ) in a patient with a transfemoral amputation . ( published with permission from nigel jarvis , the university of gothenburg , the sahlgrenska academy , gothenburg , sweden . ) a lateral view is shown of a patient securing an external prosthesis to the abutment . ( published with permission from nigel jarvis , the university of gothenburg , the sahlgrenska academy , gothenburg , sweden . ) clinical observations have led us to believe that poor primary osseointegration increases the risk of subsequent infection . however , experimental evidence supports antiinfectious properties of titanium compared with other biomaterials when implanted in bone and in soft tissues [ 2 , 7 ] . medical device infection is caused predominantly by staphylococci , with coagulase - negative staphylococci being the most common [ 32 , 33 ] . however , as the definitive diagnosis of infection is sometimes a problem , culture - based algorithms have been developed to increase diagnostic sensitivity [ 3 , 19 , 23 , 32 , 33 ] . we therefore base our definitions of infection on clinical , radiographic , and bacteriologic findings . although current titanium transcutaneous implants are intended to reduce infections compared with previously used metals and transcutaneous techniques , there are little data regarding infections with these implants . in the related but incomparable setting of dental osseointegration , infection is well characterized , but less has been published regarding infection in bone - anchored hearing aids . our aims were ( 1 ) to describe the frequency and ( 2 ) the bacterial flora at the skin - penetration area and its relation to the development of local and implant - related infection in this novel method . we prospectively studied bacterial colonization and infectious complications in 39 patients with arm and leg amputations previously treated with 45 transcutaneous osseointegrated titanium implants . all patients attending the osseointegration outpatient clinic at sahlgrenska university hospital , gteborg , sweden , for scheduled or emergency visits between january and june 2005 were included . at least 3 months had elapsed since the second surgical procedure ( abutment insertion ) . this cohort was followed longitudinally for an average of 3 years to identify implant infections and cross sectionally surveyed twice ( at inclusion and after approximately 3 years ) for bacterial presence , local infection , and antibiotic use . all skin - penetrating loci were subjected to separate clinical , bacteriologic , and radiographic assessment . there were 18 women and 21 men with a mean age of 49.3 years ( range , 2874 years ) . thirty - three of the implants were femoral ( bilateral in one ) , four each were ulnar and radial ( bilateral in one ) , three were humeral , and one was tibial . the patients had been living with the implant(s ) a mean of 54 months ( range , 3132 months ) . indications for these implants were severe discomfort when using conventional socket prostheses or poor stump conditions . is inserted into the residual bone and allowed to integrate for 6 months before the skin - penetrating extension ( abutment ) is inserted . based on the experience of successful skin - penetrating implants in the head and neck regions , the skin is attached directly to the distal end of the residual bone to reduce soft tissue mobility and risk of infection . we consider the implant osseointegrated when it is stable on clinical examination , pain free when loaded , and there are no signs of loosening seen on radiographs , ie , no radiolucent zone around the implant . postoperative followup includes clinical examination ( pain evaluation , implant stability , skin and soft tissue condition ) , a rehabilitation protocol , and radiographs at 6 months and 1 , 2 , 3 , 5 , 7 , 10 , and 15 years after surgery . we used the following five definitions for implant infection and required all stated criteria for the given diagnosis : ( 1 ) definite implant infection : ( a ) clinical symptoms of implant - related infection , ( b ) radiographic signs consistent with periimplant bone infection , and ( c ) at least three of five intraoperatively obtained cultures yielding identical pathogens ; ( 2 ) probable implant infection : ( a ) clinical symptoms of implant - related infection , ( b ) radiographic signs consistent with periimplant bone infection , and ( c ) positive relevant culture not as defined previously ; ( 3 ) possible implant infection : ( a ) clinical symptoms of implant - related infection , ( b ) radiographic signs consistent with periimplant bone infection , and ( c ) no relevant cultures ; ( 4 ) local infection in the skin penetration area : ( a ) local signs / symptoms of infection ( inflammation with or without secretion ) in the skin penetration area but no symptoms of deep infection , ( b ) no radiographic signs consistent with periimplant bone infection , and ( c ) with or without relevant cultures ; ( 5 ) bacterial colonization around the skin - implant interface : ( a ) neither local inflammatory signs nor other clinical symptoms of infection , with or without secretion , at the skin - implant interface , ( b ) no radiographic signs consistent with periimplant bone infection , and ( c ) positive bacterial culture from the skin - implant interface . we considered radiographic evidence of osteolysis with or without periosteal sclerosis around a previously integrated implant to be consistent with implant infection . the definitions also were based on previously proposed culture diagnostics in infected hip and knee arthroplasties [ 19 , 23 ] . at inclusion patients were examined clinically and asked to answer a questionnaire ( appendix 1 ) regarding infectious complications and antibiotic use during the 6 months preceding the visit . without any preparation of the surrounding skin , samples were taken from the skin - implant interface with a sterile cotton swab , transported in a coal - based medium , and cultured on routine agar plates for at least 2 days . a second set of bacterial cultures patients who were not scheduled for a visit at this time collected bacterial samples ( cotton swabs ) themselves according to careful written instructions and , if necessary , with the assistance of their attending clinic . these patients were experienced in handling the skin - implant area and we anticipated no methodologic problems with the patients performing their own cultures . cultures and questionnaires were sent to sahlgrenska university hospital by mail . sampled cultures transported in a coal - based medium the number of colony forming units ( cfu ) was defined as + + + ( > 100 cfu ) , + + ( 10100 cfu ) , or + ( < 10 cfu ) . a routine disk method was used to determine antibiotic resistance . the latest scheduled or symptom - prompted radiographs at inclusion and followup were checked for signs of bone infection . two already had osteomyelitic changes at the beginning of the study , four were excluded , and two were without infectious symptoms . the implant was extracted owing to mechanical loosening in a previously radiated femur ( october 2006 ) . in another patient , two patients ( one swedish ) did not complete the followup protocol for nonmedical reasons . the frequencies for patients with definite / probable / possible implant infections were 5% ( two of 39 ) at inclusion and 18% ( seven of 39 ) at followup ( table 1 ) . seven patients had a history of local infection at the skin penetration area during the 6-month period before inclusion and 11 patients had local infections during the 6-month period before followup ( table 1 ) . four and six patients , respectively , had been treated with short - term oral antibiotics . the secretion was purulent.table 1bacterial colonization and infection at the beginning of the study and at followuptype of infectioninitial assessmentfollowup ( 2 - 3 years)possible / probable / definite implant infection26local soft tissue infection in the skin penetration area*711superficial colonization without signs of infection23 ( 24)16 ( 17)no growth of bacteria or signs of infection7 ( 12)2 ( 4)lost for followup4 * including all episodes of infection during 6 months before first and second assessments ; figures in parentheses correspond to total number of clinical sites , which exceed the number of patients ; three patients had more than one implant ; total number of implant infections during the 3-year study was seven ; one patient had been treated successfully at followup ; contact with admitting clinic indicated no implant infection . bacterial colonization and infection at the beginning of the study and at followup * including all episodes of infection during 6 months before first and second assessments ; figures in parentheses correspond to total number of clinical sites , which exceed the number of patients ; three patients had more than one implant ; total number of implant infections during the 3-year study was seven ; one patient had been treated successfully at followup ; contact with admitting clinic indicated no implant infection . the most common bacteria found around the skin - implant interface were staphylococcus aureus , coagulase - negative staphylococci , and streptococci group a , b , or g. in three of seven patients with implant infections at followup , inclusion cultures from the skin - implant interface yielded the same species as the suspected infectious agent ( table 2 ) . staphylococcus aureus was cultured from specimens from 16 patients ( 17 implants ) , coagulase - negative staphylococci from 10 , streptococcus group b , g , or nontypable from nine , beta - hemolytic streptococcus group a from one , enterococcus sp . from three , alfa streptococci from one , and gram - negative rods from four ( table 2 ) at inclusion . no staphylococcus aureus strain was methicillin-resistant.table 2bacterial findings at the skin - implant interface at first observation and second observations 2.5 to 3 years later*bacteriafirst observation ( n = 39)second observation ( n = 30)not quantifiednumber of patients+++bacterial coloniesnumber of patients+++bacterial colonies++++++s . streptococci101011coryneforms000011000no growth80excluded / no culture9 * several patients had more than one bacterial species isolated ; one patient had a positive s. aureus culture from two loci ; cons = coagulase - negative staphylococci ; several patients had growth of two or more bacteria at the same site . bacterial findings at the skin - implant interface at first observation and second observations 2.5 to 3 years later * * several patients had more than one bacterial species isolated ; one patient had a positive s. aureus culture from two loci ; cons = coagulase - negative staphylococci ; several patients had growth of two or more bacteria at the same site . followup culture specimens were obtained from 30 patients , as five patients did not send specimens as instructed . staphylococcus aureus was still the most common finding ( 19 ) followed by coagulase - negative staphylococci ( 11 ) , streptococcus group b ( 3 ) , serratia sp . ( 2 ) enterococcus sp . , alfa streptococcus , coryneforms , and pseudomonas aeruginosa ( one each ) ( table 2 ) . eight of 13 patients with recultured specimens with staphylococcus aureus at inclusion also had staphylococcus aureus isolated at followup . the clinical presentation varied . two patients had chronic skin fistulas to the implant with occasional secretion but without pain , fever , or implant loosening . fistulas were present more than 5 years before inclusion and were not treated with antibiotics during the observation time . it was verified by biopsies of the culture specimens and treated with ciprofloxacin for 6 months . distal infection involving bone and soft tissue was seen in two patients with good primary osseointegration . the etiology was mixed with staphylococcus aureus / coagulase - negative staphylococci and staphylococcus aureus / enterococcus faecalis being suspected . acute osteomyelitis occurred in one patient who had no fever but had acute pain develop in an osseointegrated femur . intraoperative cultures yielded staphylococcus aureus and coagulase - negative staphylococci ( table 3 ) . in two of seven patients with implant infections at followup , prosthetic use was not affected at any time , three patients were affected only briefly during the time around surgical intervention , and for the patient with acute osteomyelitis , the treatment outcome is still pending.table 3etiology and outcome of patients with implant infections at followupage of patient ( years)months since surgical session 2 at inclusionmonths to infection after surgical session 2locussuspected bacterial etiology*culture at skin penetration areatreatment given at followupoutcome36335femurs . aureusno treatmentongoing , fistulas * cultures taken from tissue specimens or from fistulas ; bacterial swab taken at initial assessment from the area where the implant system penetrates the skin ; cons = coagulase - negative staphylococci ; gbs = group b streptococci . etiology and outcome of patients with implant infections at followup * cultures taken from tissue specimens or from fistulas ; bacterial swab taken at initial assessment from the area where the implant system penetrates the skin ; cons = coagulase - negative staphylococci ; gbs = group b streptococci . transcutaneous osseointegrated titanium implants for prosthetic systems in patients with amputations provide improved performance and less socket - caused complications in selected patients [ 15 , 16 ] . our aim was to describe the frequency , clinical presentation , and bacterial occurrence in local and implant - related infections . first is the relatively short followup , although some patients had been living with the implant for more than 10 years . most patients were young or middle - aged and probably will require use of their implants or other prostheses for numerous decades . rather , we studied a subgroup of 39 patients recruited during a 6-month period . as all patients attending ( regular and emergency visits ) the clinic during the inclusion period agreed to participate , we presume this cohort represents the entire population . a retrospective analysis of implant infection and risk factors in 100 patients treated with femoral osseointegrated implants and a prospective analysis of patients treated with the refined protocol are in progress . skin - penetrating implants were long regarded as unattainable owing to the high rates of failure caused by infection . seven of 39 ( 18% ) patients either had an implant infection or one developed within 3 years . the implant - infection / implant - year ratio ( 7/135 ) is slightly less ( 5% ) than comparable results from queen mary s hospital in london where four of 16 patients treated with the same method had an implant - related infection develop during a cumulative 67-year period ( 6% ) ( unpublished data , sooriakumaran s , robinson kp , ward da , darcy r , chittoor sn , written communication from 12th ispo congress in vancouver , canada , july 29 , 2007 ) . in our cohort , only one of seven patients was diagnosed and treated for an implant - related infection earlier than 31 months ( mean , 34 months ) after the surgical procedure . with arthroplasty , late infection ( > 24 months ) is considered more indicative of hematogenous seeding compared with early ( < 3 months ) or delayed ( 324 months ) infections . in our four cases with more virulent bacteria , late onset of symptoms clearly indicates that intraoperative contamination was not the cause of infection . however , we can not know whether some of the infections were either of hematogenous or new origin , rather than as a result of residual surgical contamination . we assessed the presence of local skin infections twice , with a greater frequency at followup ( table 2 ) . these adverse effects must be compared with those of conventional socket prostheses . for patients using conventional prostheses , socket - related problems such as ulcers and rashes on the residual limb and restricted movement dermatologic problems were reported to occur among 34% to 41% of those using socket prostheses [ 10 , 21 ] . , no socket is needed to suspend the artificial limb and consequently no patient in our study had ulcers or contact dermatitis on the residual limb . although various microorganisms may cause foreign body - related infection , staphylococci are the most frequently isolated pathogens [ 11 , 32 ] . in our study , approximately of the patients were colonized with potentially virulent staphylococcus aureus in the skin penetration area . despite this , only three patients had a staphylococcus aureus implant infection . the ability to adhere to the implant material and to promote biofilm formation are important pathogenic properties for staphylococci and other bacteria . the biocompatibility is greater with titanium [ oxide ] compared with stainless steel and cobalt - chrome alloys [ 2 , 7 ] . in osseointegration , the tight junction formed between the titanium and the bone tissue might prevent adhesion , colonization , and subsequent biofilm formation . osseointegration also can be established and maintained in aggressive inflammatory environments such as experimental arthritis in a rabbit knee model and in patients with rheumatoid arthritis . the osseointegration might explain why some patients with implant infections have such slow , or even no , progressive destruction of the bone tissue with subsequent loosening . it also may explain why patients with poor primary osseointegration are more susceptible to infectious complications . we found the osseointegration method with titanium implants and skin penetration of the titanium system in patients with leg and arm amputations caused few severe infectious complications . however , infectious complications occur in approximately two - fifths of the patients during a 3-year period , mostly as local infections in the skin penetration area and more rarely as low - activity implant - associated infections . staphylococcus aureus and coagulase - negative staphylococci were the most commonly cultured bacteria in the skin penetration area . in our opinion , the frequency and severity of infections do not appear to be obstacles for use of this method . however , future studies with longer followup will address risk factors and specific infectious complications in an attempt to reduce morbidity and increase usefulness of this novel treatment .

[ purpose ] disabilities after stroke are known to have adverse effects on the quality of life . this study investigated the relationship between communication activities of daily living ( c - adl ) and quality life of elderly stroke patients to provide basic data for use in enhancing the quality of life . [ subjects and methods ] one hundred sixty five elderly over the age of 60 , who were diagnosed as having stroke and receiving treatment in rehabilitation departments of general hospitals were surveyed . stroke patients basic communication ability to survive in daily living was measured using the c - adl second edition , and stroke patients quality of life was measured with the stroke - specific quality of life ( ssqol ) . the relationship between c - adl and ssqol was analyzed using multiple regression analysis . [ results ] c - adl had a significant positive relationship with ssqol . [ conclusion ] this result implies that it is necessary to enhance stroke patients communication ability in daily living in order to raise their quality of life .

in line with the worldwide aging trend , korea became an aging society in 2000 and is expected to become an aged society with an elderly population over the age of 65 of 14% of total population1 . in addition , the average life span of koreans was 79.6 years in 2010 , 15 years longer than that of 19801 . despite increases in social demands by the elderly for high - quality and satisfactory life , with the increase in the elderly population and their extended average life span , there is a high possibility that the elderly will lives accompanied by more diseases because of the degree of prolongation of their life span2 . especially , cerebrovascular diseases are known as typical geriatric diseases that threaten the health of the elderly . according to a survey by the korea center for disease control and prevention , 7.2% of the elderly population over the age of 65 had experience of stroke in 2013 , which is an 5.3% increase from 20013 . stroke is a generic term for both cerebral infarction caused by the blockage of a blood vessel in the brain , and cerebral hemorrhage caused by the rupture of a blood vessel in the brain . stroke is not only the most common of disease , but it also has enormous social costs , such as medical costs . medicine costs , loss of productivity , and the socio - economic cost of stroke in korea were estimated to amount to u$ 3.5 billion as of 20104 . although stroke occurs in all age groups , the elderly are the most susceptible group , since its prevalence rate increases rapidly with every 10 years of age5 . twenty - five percent of the stroke patients die within 1 month of stroke onset , and even after survival , it has been reported that 60% of the surviving patients suffer from various disabilities such as paralysis , motor disorder , cognitive impairment and speech disorders , which leave them unable to lead independent daily lives without the help of others . moreover , 70% of survivors suffer from communication disorders such as dysarthria and aphasia6 , and among the disabilities resulting from stroke , communication problems frequently occur . according to the american heart association and american stroke association , among 2 million stroke survivors in 2005 , approximately 1 million had aphasia7 . disabilities after stroke are known to have adverse effects on the quality of life8 , and among them , communication disorders place burdens not only on patients but on their families as well9 . as the majority of stroke patients have to lead their lives with disabilities , the quality of life including psychological and social aspects should be given consideration . especially , communication activities of daily living ( c - adl ) is important , because it examines the basic communication ability necessary for survival in daily living such as social interactions and non - verbal communication10 . similarly , although communication is an important factor in terms of quality of life , studies on communication of stroke patients remain limited to the understanding of the linguistic abilities of stroke patients , and there is a lack of studies on the theme of communication . while studies of stroke patients activities of daily living ( adl ) have been regularly conducted , studies of c - adl are very rare . this study investigated the relationship between c - adl and the quality of elderly stroke patients lives to provide basic data for use in enhancing the quality of life . the present study conducted convenience sampling 4 times from august through december 2014 , and surveyed 165 elderly over the age of 60 , who were diagnosed as having stroke and receiving treatment in rehabilitation departments of general hospitals located in seoul , inchon , daejeon and gwangju . one hundred fifty - eight fully completed questionnaires were anaylzed ; 7 questionnaires had incomplete answers . as the required minimum number of samples was 88 patients to obtain a significance level of =0.05 , and an effect size of 0.5 , with a statistical power of ( 1-)=0.95 according to the g - power 3.0 program , the number of subjects in this study was more than sufficient . the subjects were given sufficient explanation regarding the purpose and experimental method of this study before participation and gave their voluntary consent . the study protocol was approved by the institutional review board of n university and was conducted in accordance with the ethical principles of the declaration of helsinki . together with the questionnaire on communication activities of daily living , a survey of quality of life was conducted via individual interviews . the inclusion criteria for subjects were : literate patients with consciousness , within 1 year after the onset of stroke , who had no accompanying neurological diseases such as dementia or parkinson s disease , or apraxia or agnosia . stroke patients basic communication ability to survive in daily living was measured with the communication activities of daily living - second edition ( c - adl-2)11 . c - adl-2 is composed of a total of 50 questions with responses scored on a 3-point scale ( 0 point : wrong answer , 1 point : proper answer , 2 points : correct answer ) and higher scores indicate higher communication ability in daily living . test questions are classified into following 7 categories : ( 1 ) reading , writing or using numbers , ( 2 ) social interactions , ( 3 ) divergent communication , ( 4 ) contextual communication , ( 5 ) nonverbal communication , ( 6 ) sequential relationships , and ( 7 ) humor , metaphor , absurdity . among them , divergent communication includes deciding lunch time and choosing menus , and contextual communication contains interpretation of danger signs while sequential relationships includes understanding of speed limits and buying things in a supermarket . stroke patients quality of life was assessed using the stroke - specific quality of life ( ssqol)12 developed by williams et al . ssqol is an index of stroke patients characteristics and comprises 49 questions in 12 areas including movement abilities ( 6 questions ) , functions of upper extremity ( 5 questions ) , linguistic function ( 5 questions ) , thinking abilities ( 3 questions ) , visual functions ( 3 questions ) , self - care ( 5 questions ) , emotional state ( 5 questions ) , personality ( 3 questions ) , physical stamina ( 3 questions ) , role in the family ( 3 questions ) , social role ( 5 questions ) and productive capability ( 3 questions ) . each response is scored on a 5-point scale and higher scores indicate higher levels of quality of life . covariate variables were age , sex , final education ( middle school and lower , high school or above ) , the average monthly income of households ( less than 2 million won , 24 million won , more than 4 million won ) , marital status ( living with spouse , living without spouse , unmarried person ) , drinking ( non - drinker , former drinker , current drinker ) , smoking ( non - smoker , past smoker , current smoker ) , stroke type ( cerebral hemorrhage , cerebral infarction ) , time since onset of stroke ( less than 3 months , 36 months , 612 months ) , depressive symptoms in the last 1 months ( yes , no ) . first , for subjects general characteristics , means and percentages were calculated and are presented as descriptive statistics . second , in order to examine the differences in ssqol based on subjects c - adl characteristics , the independent t - test and one - way anova were performed . finally , the relationship between c - adl and ssqol was analyzed using multiple regression analysis . the average age of the subjects was 67.5 ( standard deviation 6.8 years ) ; 53.8% of the subjects were males , 88.5% were married , 63.6% were middle school graduates or above , 67.0% had cerebral infarction , 61.9% had a monthly household income of less than 2 million won , 26.5% were , past smokers , 38.5% were past drinkers , 68.0% had experience of depression symptoms in the last 1 month , and the majority of the subjects were 612 months since the onset of stroke . subjects mean c - adl was 62.3 points ( standard deviation 16.5 ) and mean ssqol was 149.9 points ( standard deviation 35.7 ) . in the multiple regression analysis , the r of the final model was 0.23 and the explanatory power of the model was 23% with the variance inflation factors ( vif ) being all less than 10 , proving that there was no problem with multi - collinearity among the independent variables . in the bivariate analysis , a significant positive relationship was found for c - adl with ssqol ( b=0.54 , t=13.28 , p<0.05 ) . in model 1 , after adjusting for age , sex , final education , the average monthly income of households , marital status , drinking and smoking , the relationship of c - adl with ssqol was maintained ( b=0.48 , t=3.32 , p<0.05 ) . in model 2 , after adjusting for all the compounding variables , c - adl had a significant positive relationship with ssqol ( b=0.46 , t=3.11 , p<0.05 ) . although communication problems frequently occur due to the after - effects of stroke , there is a lack of studies of stroke patients communication abilities in daily life . the present study investigated the relationship between elderly stroke patients c - adl and quality of life , and found that c - adl had a significant positive relationship with quality of life . numerous studies have reported a relationship between adl and quality of life . according to these studies , stroke patients adl is a significant variable predicting stroke patients quality of life13 , and the higher the functions of adl , the higher the quality of life14 . these results are similar to the results of the present study that higher stroke patients adl indicates higher quality of life . as disabilities from stroke become chronic , inability to independently perform adl , such as putting on clothes and eating for an extended period of time , not only causes helplessness and depression in stroke patients14 , but also inflicts emotional pain , such as intellectual regression , despair and anxiety10 . that is , functional disorders in daily life cause stroke patients to experience psychological pain and loss of social functions , and if these problems are prolonged , they are likely to cause deterioration in their quality of life and maladjustment in social relationships , changes in role and economic difficulties . although it is difficult to directly compare the results of preceding studies10 , 13 , 14 with those of the present study , communication is likely to have a significant effect on quality of life , considering that it is an ability necessary for instrumental daily life . especially , stroke patients experience deterioration of social functions due to limitation of communication , and when they have difficulty in understanding the meaning of what another party says , or in producing speech , even when they have clear consciousness , it is highly possible that they feel extreme frustration and depression15 . nevertheless , therapeutic rehabilitation for stroke mainly focuses on physical rehabilitation , while linguistic , emotional and social rehabilitation tends to be ignored . as communication problems are likely to lower the quality of life by leading stroke patients to give up their will to live16 , in order to enhance stroke patients quality of life , it is necessary to consistently improve communication functions from the early stage after onset of stroke , and to extend support to reduce stroke patients difficulties in leading their daily lives . a limitation of this study was the exclusion of patients with severe aphasia , for whom test could not be conducted , therefore , caution should be taken in generalizing the results of this study . also , since this study was a cross - sectional study , the results can not be interpreted in causal relationships . a significant positive relationship with ssqol was found for c - adl in this study . this result implies that it is necessary to enhance stroke patients communication ability in daily living in order to raise their quality of life .

the objectives of this study were ( i ) to develop a screening - level quantitative property - property relationship ( qppr ) for intrinsic clearance ( clint ) obtained from in vivo animal studies and ( ii ) to incorporate it with human physiology in a pbpk model for predicting the inhalation pharmacokinetics of vocs . clint , calculated as the ratio of the in vivo vmax ( mol / h / kg bw rat ) to the km ( m ) , was obtained for 26 vocs from the literature . the qppr model resulting from stepwise linear regression analysis passed the validation step ( r2 = 0.8 ; leave - one - out cross - validation q2 = 0.75 ) for clint normalized to the phospholipid ( pl ) affinity of the vocs . the qppr facilitated the calculation of clint ( l pl / h / kg bw rat ) from the input data on log pow , log blood : water pc and ionization potential . the predictions of the qppr as lower and upper bounds of the 95% mean confidence intervals ( lmci and umci , resp . ) were then integrated within a human pbpk model . the ratio of the maximum ( using lmci for clint ) to minimum ( using umci for clint ) auc predicted by the qppr - pbpk model was 1.36 0.4 and ranged from 1.06 ( 1,1-dichloroethylene ) to 2.8 ( isoprene ) . overall , the integrated qppr - pbpk modeling method developed in this study is a pragmatic way of characterizing the impact of the lack of knowledge of clint in predicting human pharmacokinetics of vocs , as well as the impact of prediction uncertainty of clint on human pharmacokinetics of vocs .

the evolving scientific and regulatory activities in europe and north america emphasize the need for the development of tools that refine , replace , or reduce the use of animals and human volunteers in pharmacokinetic and toxicity tests [ 13 ] . the ability to base the toxic responses on the target tissue dose or internal concentration of the toxic moiety of the chemicals is key to the predictive tools reflective of the current state of science . therefore , physiologically based pharmacokinetic ( pbpk ) models that are capable of providing a priori prediction of the time course of chemicals in blood and tissues is of tremendous interest . pbpk models are mechanistically based mathematical descriptions of the absorption , distribution , metabolism , and excretion of chemicals or pharmaceutical compounds . in pbpk models , the organism is represented as a set of several tissue compartments interconnected by blood flows . in these models , the internal dose measures ( e.g. , blood or tissue concentrations , amount metabolized ) of a chemical are described on the basis of mass - balance differential equations requiring species - specific properties ( e.g. , alveolar ventilation rate , cardiac output , regional blood flows , and tissue volumes ) and chemical - specific input parameters ( e.g. , partition coefficients and metabolic constants ) . although the species - specific values of several physiological parameters are available in the literature [ 46 ] , the partition coefficients ( pcs ) and metabolic constants need to be determined experimentally or calculated by using animal - replacement methods for each chemical individually . the values of tissue : blood or tissue : plasma partition coefficients essential for developing pbpk models have been estimated for a wide range of chemicals and chemical classes , including drugs , with the use of tissue composition - based algorithms or qsar methods ( e.g. , [ 819 ] ) . regarding the metabolism parameters ( i.e. , hepatic clearance , intrinsic clearance , vmax , km , kcat , free energy of binding , energy of activation , or activation enthalpy ) , some studies have developed 2-d and 3-d qsars but with a specific focus on either a single isozyme , a single reaction or a single class of substances [ 8 , 2038 ] . none of these past efforts succeeded in predicting both vmax and km ( or clint ) of environmental chemicals for direct incorporation within animal or human pbpk models . alternatively , few studies utilized the group contribution method of gao [ 3943 ] , to predict metabolic rates for pbpk models . in this method , the chemical is decomposed into different structural fragments or groups , the contributions of which are obtained by regression analysis . accordingly , these publications demonstrated the feasibility of developing structure - property relationships for the metabolism rates . the group contribution method was successfully used to develop quantitative structure - property relationships ( qsprs ) for the tissue : air partition coefficients as well as intrinsic ( clint ) and hepatic clearance ( clh for a group of low - molecular - weight volatile organic chemicals ( vocs ) in rats [ 41 , 42 ] . these qspr models , in turn , were incorporated within pbpk models to predict reasonably well the blood kinetics of inhaled vocs in rats . as these qsprs are species specific , they could not be used to conduct interspecies extrapolations . to overcome this limitation , bliveau et al . developed biologically based algorithms for pcs and clh to conduct rat to human extrapolations of the inhalation toxicokinetics of vocs . in this study , qsprs based on the group contribution method were developed for the chemical - specific input parameters of the biological algorithms for pcs ( i.e. , oil : air , water : air , and blood protein : air ) and clint ( intrinsic clearance normalized for cytochrome p450 2e1 content ) . more recently , qsprs were developed for the metabolic constants vmax ( maximum velocity of reaction ) and km ( michaelis constant ) and were further incorporated within a rat pbpk model to predict the toxicokinetics of mixtures of vocs . despite the successful use of the group contribution method in qspr modeling of metabolism rates , their principal limitation relates to the fact that the chemical space they cover is extremely limited ( low - molecular - weight vocs containing one or more of the following fragments : ch3 , ch2 , ch , c , c = c , h , br , cl , f , benzene ring , and h on benzene ring ) . more experimental data on diverse chemicals would be needed to determine the contributions of other molecular fragments , as has been done with pow ( e.g. , estimation of the contribution of 130 fragments ( i.e. , groups ) required 1200 measurements of pow ) . to extend the currently available qspr for clint to cover more diverse fragments and at the same time respect a reasonable ratio of the number of parameters to the number of observations , since the critical limitation in the construction of pbpk models for new substances continues to be the metabolism rate , a pragmatic approach particularly for inhaled vocs is to evaluate the maximum and minimum possible blood concentration profiles in exposed individuals . thus , using a hepatic extraction ratio ( e ) of 0 and 1 in the pbpk models , poulin and krishnan obtained simulations of the physiological limits ( i.e. , maximal and minimal blood concentration profiles ) for inhaled vocs in humans . assuming the conceptual pbpk model and the values of its physiological parameters are reliable , the real answer , that is , the actual concentrations and kinetic curve , would be somewhere in between the theoretical limits simulated with these pbpk models . the uncertainty associated with these theoretical bounds can be reduced by developing better estimates of the metabolism constants . this could be done , at a practical level , by developing in silico tools that provide a range of plausible values , in lieu of a single accurate point estimate . such a tool might be of use for the toxicokinetic screening of substances , until the time when the chemical - specific measurements are obtained in vivo , in vitro , or with a highly precise mechanistic in silico method . since human exposures to environmental contaminants in most cases do not attain levels that approach or exceed saturation , it is not crucial to predict vmax and km separately , particularly for simulating kinetics in humans exposed to low atmospheric concentrations of vocs . therefore , the availability of in silico approaches based on easily available parameters to predict plausible range of clint would be desirable as a screening - level tool . the objective of this study was therefore to develop a quantitative property - property relationship ( qppr ) model of animal data to generate initial estimates ( or bounds ) of intrinsic clearance of vocs , for eventual incorporation within a human pbpk model to simulate blood concentration profiles associated with inhalation exposures . in this regard , we focused on evaluating the impact of the uncertainty associated with qppr predictions of clint on the blood kinetics of vocs in humans , relative to that of the uncertainty associated with the total lack of knowledge of the metabolic rate in humans . furthermore , the reliability of applying the qppr to predict the area under the blood concentration versus time curve ( auc ) of parent chemicals was evaluated , as a function of the sensitivity of the metabolism parameter in the pbpk model and the prediction uncertainty of qppr model . the qppr predictions were then compared with experimental data for several vocs and the pharmacokinetics in humans were simulated using integrated qppr - pbpk models for these 26 vocs . the predictions of qppr were evaluated further with an external data set of clint for 11 vocs . the development of a global qppr model for metabolism was initially undertaken using experimental data on the in vivo intrinsic clearance of 26 vocs in rats , collated and evaluated in previous studies by bliveau et al . [ 40 , 41 ] ( 1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; 1,1,2-trichloroethane ; 1,1-dichloroethane ; 1,1-dichloroethylene ; 1,2-dichloroethane ; benzene ; bromochloromethane ; bromodichloromethane ; carbon tetrachloride ; chloroethane ; chloroform ; cis-1,2-dichloroethylene ; dibromomethane ; dichloromethane ; ethylbenzene ; hexachloroethane ; isoprene ; methyl chloride ; m - xylene ; n - hexane ; pentachloroethane ; styrene ; toluene ; trichloroethylene ; vinyl chloride ) [ 24 , 4653 ] . subsequently , the resulting qppr model was evaluated with experimental in vivo data on clint for 11 additional vocs in rats ( 1,1,1-trichloroethane ; 1,2,4-trimethylbenzene ; bromoform ; dibromochloromethane ; furan ; halothane ; o - xylene ; trans-1,2-dichloroethylene ; tetrachloroethylene ; propylene ; ethylene ) [ 46 , 48 , 5461 ] . these 11 chemicals outside the calibration set were also lipophilic , low - molecular - weight vocs and likely substrates of cytochrome p450 2e1 [ 32 , 62 ] . moreover except for halothane and 1,2,4-trimethylbenzene , the chemicals of the evaluation dataset possess values of pow , ionization potential , and blood : water pc within the range of values for the chemicals in the qppr calibration set . for qppr modeling , clint ( expressed in units of l blood , clintblood , or l phospholipids , clintpl ) initially , clintblood ( l blood / h / kg ) for all the studied chemicals was computed as allometrically scaled vmax ( mol / h / kg)/km ( mol / l blood ) . since cyps are located in the endoplasmic reticulum embedded in the phospholipidic bilayer , the clintpl values reflecting chemical affinity for the phospholipids ( pl ) were subsequently computed . the values of clintpl ( l phospholipid / h / kg ) were obtained by dividing vmax ( mol / h / kg ) with km expressed as mol / l pl . the km values in m of pl were obtained by multiplying the values of km expressed as mol / l blood with the chemical - specific phospholipid : blood partition coefficients ( pplb ) calculated as follows : ( 1)pplb=0.3poa+0.7pwapba , where poa is the n - octanol : air pc , pwa the water : air pc , and pba the blood : air pc . the above equation computes pplb as the ratio of phospholipid : air to blood : air pcs of the vocs , based on poulin and krishnan [ 10 , 12 ] . the input parameters required for converting the clint obtained from the literature were poa , pwa , and pba . ( 1 ) poa and pwathe n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . the n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . ( 2 ) pbaexperimental values were used for rat blood : air [ 54 , 56 , 59 , 6468 ] . the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1 . experimental values were used for rat blood : air [ 54 , 56 , 59 , 6468 ] . the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1 . the rate and affinity for p450-mediated metabolism would appear to be related to the size , shape , charge , and energy of the substrate ; therefore variables that reflect these properties were chosen for the qppr analysis [ 21 , 23 , 27 , 28 , 32 , 6971 ] . the descriptors of the size and shape of the molecule were the molecular length , width , depth , volume , surface , and the kappa 2 index , as well as two descriptors used in the work of lewis et al . , namely , the ratio of the molecular length to the molecular width ( l / w ) and the ratio of the area of the molecule ( i.e. , length times width ) to the square of the depth ( a / d ) . the dipole moment and ionization potential ( ip ) were used as measure of the charge disposition and the energy in the molecule , respectively . the values of all the previously cited descriptors were calculated using commercially available software ( molecular modeling pro , chem sw , fairfield , ca ) . before calculating the molecular descriptors with molecular modeling pro , the 3d molecules were drawn and minimized using the full mm2 ( molecular mechanics program ) method provided in the software . the dipole moment and the ionization potential hydrophobic descriptors such as log pow ( log of the n - octanol : water pc ) that reflect hydrogen bonding and - stacking have already been correlated to the values of metabolic constants [ 6971 ] . in this study , the following physicochemical parameters were chosen to describe the relative solubility and partitioning into diverse biological media : log pow , log phospholipid : water pc ( log pplw ) ; log blood : water pc ( log pbw ) , and log water : air pc ( log pwa ) . the blood : water and phospholipid : water pcs were obtained by dividing the blood : air and phospholipid : air pcs values by the water : air pc values . the values of pow , pwa , blood : air , and phospholipid : air pcs were obtained as described for the calculation of pplb ( 1 ) . multilinear regression analysis approach was chosen for the qppr analysis of clint because linear regression models are simple , transparent , and easy to reproduce . stepwise regression analysis was performed to select the qpprs based on the most statistically significant independent variable(s ) from an a priori list ( see section 2.1.4 ) . the coefficient of determination r , the adjusted r ( radj ; adjusted for number of variables ) , the standard error of the estimate s , and the value and significance of the f statistic were calculated . the normality of the residuals was checked visually on normal probability plots of the standardized residuals ( i.e. , expected normal cumulative probability versus observed cumulative probability ) . leave - one - out cross - validation was conducted and the results were expressed in terms of q , a measure of precision error of the model . the q was computed as follows : ( 2)q2=1pressssy , where press is that predicted residual sum of squares and ssy the sum of squares of the response values . the statistical significance ( p < 0.05 ) of the regression coefficients was estimated by a t statistic test . multicollinearity refers to the occurrence of correlation between two independent variables in the multiple linear regression model . multicollinearity of the variables in the model was assessed by calculating the variance inflation factor ( vif ) for all independent variables . the value of vif was calculated as follows : ( 3)vifi=11ri2 , where vifi is the variance inflation factor of the independent variable i in the multilinear regression model and ri the coefficient of determination of the regression between the independent variable i and the other independent variables in the multilinear regression model . for each model , the application domain was documented by reporting the ranges of values of the descriptors , the modeled response , and the endpoint . a qppr model was considered adequate when : the values of r and radj were 0.6 , the value of q was 0.6 , and the independent variables were not highly correlated ( i.e. , vif < 4 ) . the predictions of the qppr model were obtained in terms of lower and upper bounds of the 95% mean confidence intervals ( lmci and umci , resp . ) in order to represent the uncertainty associated with the mean predicted value . the lmci and umci for the 11 vocs , not in the qspr calibration dataset , were obtained by adding them in the spss file containing the data used for the qppr , along with the values of their independent variables only . in the pbpk model , the value of intrinsic clearance was calculated as the product of the qppr value of clintpl ( l of pl / h / kg ) and the phospholipid : blood pc ( values of pplb in table 1 ) . the intrinsic clearance ( l blood / h / kg ) was used within the human pbpk models to compute the hepatic clearance . the rate of metabolism was calculated on the basis of hepatic clearance ( i.e. , hepatic clearance times the arterial concentration ) [ 4 , 40 , 41 , 45 ] . for chloroethane , dichloromethane , vinyl chloride , and dibromomethane a first - order constant ( 1 , 2 , 1 , and 0.7 h , resp . ) was included in the calculation of the hepatic clearance , clh ( l / h ) : ( 4)clh = qle , where e = ( clint + kf vl)/((clint + kf vl ) + ql ) , ql is the blood flow through the liver ( l / h ) , clint the intrinsic clearance ( l blood / h ) , kf the first order metabolic constant ( h ) , and vl the liver volume ( l ) . the qppr values of clint were included in a human pbpk model for inhaled vocs . briefly , the pbpk model consisted in four tissue compartments ( i.e. , liver , fat , richly , and poorly perfused tissues ) and a gas exchange lung , which were interconnected by blood flows . the distribution of vocs into tissue compartments was described as perfusion limited , and the metabolism was limited to liver . to evaluate the impact of uncertainty on the metabolic rate , for all the chemicals , pbpk simulations were also conducted by setting the value of e to 0.999 ( emax ) and then to 0.001 ( emin ) , respectively . the human physiological parameters of the pbpk model ( i.e. , body weight = 70 kg ; cardiac output = 18 l / h / kg ; alveolar ventilation = 18 l / h / kg ; tissue compartment volumes , fraction of body weight : liver = 0.026 ; richly perfused tissues = 0.05 ; poorly perfused tissues = 0.62 ; fat = 0.19 ; perfusion of the tissue compartments , fraction of cardiac output : liver = 0.26 ; richly perfused tissues = 0.44 ; poorly perfused tissues = 0.25 ; fat = 0.05 ) were obtained from tardif et al . . table 1 presents the value of the partition coefficients used in the pbpk model ( i.e. , blood : air , tissue : blood , and phospholipid : blood pcs ) . the phospholipid : blood pc was calculated using ( 1 ) , whereas the blood : air pc and tissue : blood pcs were gathered from the literature [ 48 , 50 , 52 , 54 , 5659 , 61 , 66 , 67 , 7780 ] . the pbpk model ( differential and algebraic mass - balance equations , physiological parameters , qspr equations for metabolic constants , and pcs ) was written in acsl ( acslx , version 2.5 , aegis technologies group , inc , huntsville , al ) . the model code is included in the supplementary data available online at doi:10.1155/2012/286079 . to compare the impact of different ( uncertain ) scenarios of rate of metabolism on the pharmacokinetics in human , simulations were carried out by setting ( i ) the value of clint equal to the lower and upper bound of the qppr predicted mean 95% confidence interval , or ( ii ) the liver extraction ratio to 0.001 ( no metabolism ) and 0.999 ( maximum extraction ) . the 24 h venous blood kinetics corresponding to the four scenarios of metabolism were simulated for an 8 h exposure to 1 ppm of each voc . the 24 h area under the curve ( auc24 ) of the venous blood kinetics was also calculated to compare the four scenarios of metabolism simulated with pbpk models . additionally , the venous blood kinetics of m - xylene , toluene , ethylbenzene , dichloromethane , styrene , 1,2,4-trimethylbenzene , and 1,1,1-trichloroethane were compared to experimental data [ 61 , 67 , 8183 ] . the applicability of the qppr model was evaluated on the basis of the level of uncertainty in the qppr estimate and the impact ( sensitivity ) of metabolism on the auc24 . figure 1 illustrates the role of uncertainty and sensitivity in the reliability of the qppr - pbpk modeling framework , based on reference . the sensitivity of the metabolism to the auc was estimated by the ratio of the auc24 obtained with no metabolism ( emin ) to that obtained with the maximum theoretical metabolism ( emax ) . the sensitivity of auc24 to metabolism was considered to be low , medium , or high if the ratio ( aucemin/aucemax ) was within a factor of 2 , within an order of magnitude , or greater . the uncertainty in the qppr prediction was evaluated by comparing it to the experimental data . the prediction uncertainty was considered to be low , medium or high if the prediction was within a factor of two , within an order of magnitude and above 10-fold of the experimental data , respectively . this approach was applied to evaluate the reliability of applying the qppr within the pbpk model for two situations : ( i ) for the calibration set of chemicals , for which the uncertainty of the qppr was evaluated by comparing the predictions of clintpl with the experimentally derived clintpl values and ( ii ) for chemicals in the evaluation dataset , for which the uncertainty in the qppr prediction was considered to be high , to replicate the data poor situations with new or tested chemicals with unknown experimental clint values . the initial effort to develop a qppr model for metabolism rate ( expressed as clintblood , in units of l blood / hr ) , based on a stepwise analysis of its relationship to various molecular descriptors and physicochemical properties , was not successful ( not shown ) . same analysis , repeated for clint expressed in units of l pl / h ( clintpl ) , yielded a qppr that consisted of log pplw , log pbw , and ip ( ionization potential , ev ) as input parameters . this model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.802 ; radj = 0.775 ) , leave - one - out cross validation ( q = 0.755 ) , and multicollinearity ( vifs : log pplw = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . the values of the regression coefficients were significant ( p value < 0.001 for the constant , log pplw and log pbw , and 0.007 for ip ) . however , as the value of log pow can be obtained more readily than log pplw , the regression analysis was repeated by using log pow , log pbw , and calculated ip , and it yielded the following qppr : ( 5)log clintpl=5.63(1.187)1.287(0.149)log pow + 1.08(0.233)log pbw 0.328(0.111)ip . this qppr model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.796 ; radj = 0.768 ) , leave - one - out cross validation ( q = 0.748 ) , and multicollinearity ( vifs : logpow = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . the application domain of the model can be described with [ min ; max ] as follows : log pow = [ 1.09 ; 4.03 ] ; logpbw [ 0.16 ; 2.49 ] ; calculated ionization potential [ 9.13;11.28 ] . the qppr ( 5 ) was subsequently applied to calculate the clintpl of the vocs in the calibration set . table 2 presents the values of the input parameters , along with the experimental data for the 26 vocs used in qppr development . figure 2 illustrates the comparison of the predicted values of clintpl ( lmci and umci ) and the experimental data . the uncertainty in the predicted log clintpl can be characterized by the difference between the umci and the lmci ; this value ranged from 0.37 ( 1,1-dichloroethane ) to 1.23 ( n - hexane ) with a mean of 0.54 and a standard deviation of 0.18 . the nearest confidence bounds of the predicted log clintpl were higher than 5-fold of the experimental value ( exp . ) for three substances ( cis-1,2-dichloroethylene , lmci = 0.55 versus exp. = 0.09 ; styrene , lmci = 0.45 versus exp. = 0.09 ; and 1,1,2-trichloroethane , umci = 0.46 versus exp. = 0.02 ) . the impact of the imprecision of these qppr predictions of the metabolic constants on the pharmacokinetics in humans was then evaluated by pbpk modeling . figure 3 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 26 vocs used in the qppr analysis . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . overall , the envelope of the concentrations predicted using the qppr predictions reduced the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. , ranging from 0 to 1 ) . the average ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations ( i.e. , cmax ) obtained with emin and emax was 4.19 1.81 . the lowest and highest ratios , based on the theoretical bounds of hepatic extraction ( i.e. , emin and emax ) , were observed for isoprene ( 1.63 ) and 1,1,2,2-tetrachloroethane ( 8.05 ) , respectively . however , the average ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations , based on qppr - generated bounds ( lmci , umci ) , was 1.29 0.27 . this ratio was the highest for hexachloroethane ( 2.39 ) and the lowest for 1,1-dichloroethylene ( 1.06 ) . for the 26 vocs used in the development of the qppr , the values of auc24s for a 1 ppm continuous exposure the ratio of the highest to the lowest auc predicted with emin and emax was 4.3 1.94 ranging from 1.63 ( isoprene ) to 8.7 ( 1,1,2,2-tetrachloroethane ) . the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.36 0.4 ranging from 1.06 ( 1,1-dichloroethylene ) to 2.8 ( isoprene ) . figure 4 illustrates the range of predictions of venous blood pharmacokinetics compared to experimental data [ 67 , 81 , 82 ] . overall , the predicted envelope of concentrations approximated reasonably the experimental data for dichloromethane , ethylbenzene , styrene , toluene , and m - xylene . the reliability of applying the qppr within the pbpk model was assessed for the 26 vocs in the calibration dataset ( table 4 ) . the uncertainty of the qppr prediction was estimated as the ratio of predicted clintpl to experimental clintpl . for 3 vocs ( isoprene , 1,1-dichloroethylene , and vinyl chloride ) the sensitivity of auc to clint was low ( ratio of aucs < 2 ) whereas uncertainty of the clint qppr was low for isoprene and vinyl chloride and medium for 1,1-dichloroethylene . for the other 23 vocs , the ratio of aucs was between 2 and 5 . for 16 of the later 23 vocs ( benzene ; bromochloromethane ; bromodichloromethane ; chloroform ; dibromomethane ; 1,2-dichloroethane ; hexachloroethane ; n - hexane ; pentachloroethane ; styrene ; 1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; toluene ; 1,1,2-trichloroethane ; trichloroethylene ; m - xylene ) the prediction uncertainty was low , thus the confidence in using the qppr in the pbpk model is high for these compounds . the uncertainty was medium for the prediction of clintpl for 7 vocs ( carbon tetrachloride ; chloroethane ; 1,1-dichloroethane ; cis-1,2-dichloroethylene ; dichloromethane ; ethylbenzene ; methyl chloride ) . therefore , for these chemicals , the confidence in using the qppr in an inhalation pbpk model to evaluate the auc is medium . the qppr model was applied to predict the clintpl of 11 vocs that were not in the calibration dataset . table 5 presents the values of the input parameters along with the experimental data for the 11 vocs used in qppr evaluation . figure 5 illustrates the comparison of the predicted values of clintpl ( lmci and umci ) and the experimental data . the average difference ( standard deviation ) between the umci and the lmci was 0.57 0.11 ranging from 0.46 ( bromoform ) to 0.84 ( 1,2,4-trimethylbenzene ) . the highest umci - lmci ranges were obtained for furan ( 0.62 ) , tetrachloroethylene ( 0.63 ) , and 1,2,4-trimethylbenzene ( 0.84 ) . the nearest predicted values of umci and lmci on log clintpl were greater than 5-fold of the experimental data for tetrachloroethylene ( lmci = 0.02 versus exp = 1.8 ) . as in the qppr development section , the impact of the imprecision on these log clint predictions on the pharmacokinetics in humans was evaluated by pbpk modeling . figure 6 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 11 vocs used in the qppr evaluation . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . the reduction of the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. , ranging from 0 to 1 ) by the envelope of the concentrations predicted using the qppr predictions was observed for the 11 vocs . the mean ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations obtained with emin and emax was 3.92 2.13 ranging from 1.42 ( ethylene ) to 7.45 ( bromoform ) . however , the same average ratio ( standard deviation ) of pbpk simulated blood concentrations , based on qppr - generated bounds ( lmci and umci ) was 1.2 0.1 , ranging from 1.07 ( ethylene ) to 1.33 ( bromoform ) . table 6 presents the values of the auc24s ( mg / l - h ) for the 11 vocs used in the evaluation of the qppr . the average ratio of the highest to lowest auc predicted using emin and emax was 4.08 2.31 ( mean sd ) . the lowest and highest ratios , based on the theoretical bounds of hepatic extraction ( i.e. , e = 0.001 or 0.999 ) , the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.2 0.1 , ranging from 1.07 ( propylene ) to 1.33 ( dibromochloromethane ) . figure 7 illustrates the range of predictions for two of the chemicals in the external dataset ( 1,2,4-trimethylbenzene and 1,1,1-trichloroethane ) venous blood pharmacokinetics compared to experimental data [ 61 , 85 ] . envelope of concentrations simulated reasonably the experimental data for 1,2,4-trimethylbenzene whereas the blood concentrations of 1,1,1-trichloroethane were underestimated by about 30% . the reliability of applying the qppr within the pbpk model was assessed for the 11 vocs in the evaluation dataset , using the framework shown in figure 1 . considering that the experimental data of clintpl for new or untested chemicals will be essentially unknown , it is realistic to consider the uncertainty of the qppr prediction of clintpl to be high for all chemicals in the evaluation dataset . the results of the analysis of applicability for the chemicals in the evaluation dataset are reported in table 7 . for 3 vocs ( ethylene ; propylene ; 1,1,1-trichloroethane ) the sensitivity was low ( ratio of aucs < 2 ) thus the reliability of using their clint qppr in the pbpk was considered high . for the other 8 vocs ( bromoform ; dibromochloromethane ; trans-1,2-dichloroethylene ; furan ; halothane ; tetrachloroethylene ; 1,2,4-trimethylbenzene ; o - xylene ) , the ratio of the maximum to the minimum possible aucs was between 2 and 5 , such that the confidence in using the qppr in an inhalation pbpk model to evaluate the aucs is medium for these chemicals . sars , qsar , qsprs , and qpprs have been developed for various toxicological and chemical properties but only very few studies have focused on developing such models to parameterize pbpk models [ 8 , 86 ] . a limitation in developing pbpk models relates to the availability of the metabolic constants ( clint , vmax , and km ) . quantitative relationships between structure and metabolism rates have been investigated for a limited number of closely related compounds , even though their applicability to pbpk modeling has not been demonstrated ( e.g. , qspr models for kcat and 1/km ) . other works in this area relate to the development of quantum chemical or quantum dynamic methods for prediction of activation energy or enthalpy of activation of p450 mediated reactions [ 20 , 25 , 26 , 31 , 36 , 38 , 8891 ] , which have not been used to derive metabolism constants for direct incorporation within rodent or human pbpk models . the use of the group contribution method to develop qsprs for integration within pbpk models has been successfully demonstrated , particularly for the inhalation toxicokinetics of vocs [ 4043 ] . this approach however is limited to vocs containing one or more of the molecular groups or fragments for which the contribution has been evaluated ( i.e. , ch3 , ch2 , ch , c , c = c , benzene ring , h on benzene ring , and halogens ) . in order to extend the applicability domain then , it is important to investigate the feasibility of developing qsprs based on more global , physicochemical properties . in this regard , the present study investigated the development of a qppr , that used chemical properties rather than chemical structure as input , and it was calibrated to predict clint expressed in terms of chemical affinity to phospholipids in the endoplasmic reticulum in which cyp enzymes are embedded . this logical transformation of clint data , reported here for the first time in literature , facilitated the development of more adequate qppr than the conventional clint based on blood concentrations . all efforts to develop qpprs for predicting clint based on blood concentrations were unsuccessful . the qppr based chemical affinity to phospholipids obtained in this study should be regarded as a screening level tool to provide plausible range of metabolism rates in order to facilitate a first - cut evaluation of the blood concentration of inhaled vocs in humans . the uncertainty associated with this qppr tool should be evaluated along with the sensitivity of clint on the dose metrics of the chemical of interest , in the perspective of intended precision . accordingly , if the dose metric is highly sensitive to clint and the qppr predictions of clint are highly uncertain , then the present tool is of limited use even for screening purposes . in such cases , then in vivo or in vitro studies can be undertaken to get chemical - specific estimates of clint. the qppr predictions were reasonably in accordance with experimental values for most but not all chemicals in the calibration and evaluation datasets . for some chemicals , the predicted values of log clint for 1,1,1-trichloroethane ( figure 5(a ) ) and tetrachloroethylene ( figure 5(k ) ) exceeded the experimental values by two orders of magnitude . the qspr for rat hepatic clearance developed by bliveau and colleagues also overestimated the metabolic rate of these two vocs . however the pbpk model for 1,1,1-trichlorethane indicated that the auc of parent chemical in venous blood is not sensitive to vmax and km [ 92 , 93 ] . this was demonstrated in figure 6(a ) , showing that qppr - overestimation of clintpl of 1,1,1-trichloroethane led only to a minimal impact , in terms of the underestimation of the venous blood concentration . in the case of tetrachloroethylene , a poorly metabolized halogenated voc , the overestimation of the clintpl led to a 3-fold underestimation of the cmax ( figure 6(k ) ) or a 4 - 5-fold underestimation of the auc24 ( table 7 ) . if this magnitude of error is not acceptable for screening - level evaluation , then the metabolic rate should be experimentally determined . the combined assessment of the uncertainty / sensitivity of metabolic constants in pbpk models would facilitate the determination of the applicability of the qppr model , given the level of precision need for an application ( figure 1 ) the qppr developed in this study is a generic tool to provide initial estimates of clint of vocs metabolized by hepatic cyp . it does not take into account stereochemistry or other pathway - specific rates and processes , which may be important for some chemicals ( e.g. , predicted values of clint are almost identical for 1,1-dichloroethylene and cis-1,2-dichloroethylene but experimental values vary by log units of 1.06 ) . therefore , predictions of clint based on generic considerations are likely to be inaccurate for specific chemicals but are of limited use in that the estimates ( along with the bounds , representing the level of uncertainty ) can be integrated with human physiology to provide a first - cut view of the plausible kinetic profiles . the utility of the qppr models depends , in part , on the ability to reproducibly calculate the descriptors . hence , in this study , the descriptors that could be easily calculated and interpreted were chosen and obtained using episuite ( for log pow and pwa ) and mmpro ( for the ionization potential ) . however , the blood solubility parameter ( i.e. , blood : air pc ) is additionally required and this can either be obtained experimentally in vitro or using other qsars that account for protein ( i.e. , haemoglobin and plasma protein ) binding in addition to solubility considerations . there are some algorithms and qsars available in this regard , but further development is necessary to adequately account for the protein binding phenomena in human blood for various classes of chemicals . the qppr developed in this study computes clintpl , which can then be converted to clintblood for use in pbpk modeling . in an effort to evaluate whether the same input parameters can be used to relate to clintblood these yielded the following equation ( significant terms only ) : ( 6)log clintblood=5.1170.305logpow0.324ip . even though ( 5 ) and ( 6 ) give almost identical results ( one for clintpl and the other for clintblood ) despite the differing r values ( 0.796 versus 0.402 ) , it should be noted that ( 5 ) was obtained based on statistical analysis of calibration dataset ( i.e. , modeling ) whereas ( 6 ) was derived simply by fitting clintblood to the specific input parameters . further rearrangements and simplifications of the qppr , as well as the loss of accuracy associated with such attempts , were not performed in the current study . the output of the qppr developed in the present study is log clint , which is useful for simulating pharmacokinetics in humans of chemicals at low levels of exposure . clint is applicable to first - order situations ( i.e. , when blood levels in humans are much lower than the km for metabolizing enzyme ) and is derived by dividing the vmax ( i.e. , the enzyme turn - over ) with km ( representing the affinity of the substrate for the enzyme ) . the input parameters of the qppr , namely , log pow and log pbw , are estimates of the relative solubility in octanol , water , and blood . then , an interpretation of the model for clint could be that the binding to the p450 enzyme is a result of hydrophobic interactions which , in turn , can be estimated with parameters reflective of the solubility in n - octanol and blood . the solubility in blood is the sum of the solubility in its components ( water , phospholipids , neutral lipids , and proteins ) . most of the studied vocs are likely to bind to hemoglobin because of their lipophilicity ( log pow value above 1 ) and low molecular volume . the pbw , thus , is likely an indicator of the binding to proteins , whereas the log pow reflects more the affinity for biotic lipids in the metabolism microenvironment . similar to log pow , the ionization potential has already been correlated with metabolic rates , namely , the vmax and vmax/km , as this latter parameter could be correlated with the energy needed to break a covalent bond for the oxidation of the substrate . the qspr model for clint developed in this study has a defined theoretical endpoint , is nonambiguous , has a defined domain of application , was analyzed using appropriate goodness - of - fit ( r ) and robustness ( q ) , and has an attempt of mechanistic interpretation . the in vivo dataset on 26 vocs used for the qppr calibration was chosen because it was previously collated and used in qspr analyses [ 40 , 41 ] . the qspr analysis was also attempted with the entire dataset of 37 vocs ( calibration + external dataset ) but it did not improve the goodness - of - fit statistics ( not shown ) . the predicted bounds of the 95% confidence interval of intrinsic clearance were incorporated within a pbpk model to predict the blood toxicokinetics of vocs . the simulations of blood kinetics were comparable to experimental data for 6 vocs ( toluene , m - xylene , ethylbenzene , styrene , dichloromethane,1,2,4-trimethylbenzene , and 1,1,1-trichloroethane , figures 4 and 7 ) . the simulations obtained in the present study , using lower and upper confidence intervals on the mean predicted clint , reduced clearly the uncertainty bounds associated with the total lack of knowledge ( i.e. , e ranging anywhere between 0 and 1 ) . furthermore , the present study incorporated the qppr predictions of clint along with physiological parameters , such that impact on in vivo kinetics could be simulated . in effect , in some cases where the uncertainty on clint predictions was high , it did not translate into a proportionate error on the predictions of kinetics , due to the additional consideration of physiological constraints , and such observations are critical in data - poor situations for designing focused studies to generate chemical - specific data in vitro or in vivo . the qppr developed in this study approximated the experimental rat metabolic constants for the various low - molecular - weight vocs ; and it was used along with the human physiology to generate initial or screening level values of clint to construct human pbpk models that could be of potential use to interpret data such as measured biomarker levels or for designing kinetic studies to reduce database uncertainty . as shown with some vocs ( e.g. , figure 3 : 1,1,1,2-tetrachloroethane , hexachloroethane , and n - hexane ) , the blood concentration profile is extremely influenced by clint , such that metabolism can not be neglected in simulating or interpreting human exposure data . and in such cases , the ability to generate at least a range of plausible values of clint , as done in the present study , would facilitate first in - human simulations of pharmacokinetics of parent chemicals . integrating information on the impact of metabolism on dose metrics ( i.e. , auc ) along with prediction uncertainty of the qppr facilitates the determination of the level of confidence in using this screening level tool . depending upon the overall confidence in the qppr application for predicting dose metrics ( low , medium , and high ) relative to the use purposes , overall , the qppr developed in the present study allows to predict the clint of vocs on the basis of generic molecular descriptors rather than with fragment constants as done previously . the chemical concentration in phospholipids , for the first time , was found to be a dose metric amenable to qppr analysis . the qppr was then used to generate range of values of clint ; the level of confidence in these estimates was assessed by considering the impact of clint on the simulated dose metrics ( i.e. , auc of parent chemical in venous blood ) . for other dose metrics and situations , a more robust qppr needs to be developed , and such efforts can be based on the methodological developments accomplished in this study . the qppr - based simulation of pharmacokinetics reduced the range of uncertainty for few substances relative to complete lack of knowledge of the clint , but it needs to be evaluated / refined with much larger dataset should this screening - level approach be adopted for providing more precise estimates of metabolism rates . overall , the integrated qppr - pbpk model developed in this study is a potentially useful tool for characterizing and reducing the uncertainty associated with the complete lack of knowledge of clint in predicting human pharmacokinetics of inhaled vocs .

diabetes mellitus , especially type 2 diabetes ( t2 dm ) , is one of the most common chronic diseases and continues to increase in numbers with large proportion of health care budget being used . many animal models have been established in order to investigate the mechanisms and pathophysiologic progress of t2 dm and find effective treatments for its complications . on the basis of their strains , features , advantages , and disadvantages , various types of animal models of t2 dm can be divided into spontaneously diabetic models , artificially induced diabetic models , and transgenic / knockout diabetic models . among these models , the spontaneous rodent models are used more frequently because many of them can closely describe the characteristic features of t2 dm , especially obesity and insulin resistance . in this paper , we aim to investigate the current available spontaneous rodent models for t2 dm with regard to their characteristic features , advantages , and disadvantages , and especially to describe appropriate selection and usefulness of different spontaneous rodent models in testing of various new antidiabetic drugs for the treatment of type 2 diabetes .

in nearly all countries of the world , diabetes mellitus is one of the most common chronic diseases and continues to increase in numbers and significance , because of the reduced physical activity and increased obesity caused by changing lifestyles . it is estimated that the global figure of diabetes patients is about 250 million currently , and the number is expected to be 380 million by 2025 . between 2010 and 2030 , there will be a 69% increase in number of adults with diabetes in developing countries and a 20% increase in developed countries . in north america , about 9095% of all cases of diabetes are type 2 diabetes mellitus ( t2 dm ) , and the population over 65 with t2 dm is about 20% . t2 dm may result in severe complications , including renal failure , blindness , slow healing wounds , and arterial diseases . about 510% of the total health care budget has been used for t2 dm in many countries . in a general classification , diabetes mellitus is divided into two groups : type 1 diabetes mellitus ( t1 dm ) and t2 dm . t1 dm or insulin dependent diabetes mellitus ( iddm ) is an autoimmune disease that leads to destruction of islet beta cells in the pancreas , which results in a complete halt of insulin production , while t2 dm , also called noninsulin dependent diabetes mellitus ( niddm ) , is a heterogeneous disorder characterized by a progressive decline in insulin action ( insulin resistance ) in liver and peripheral tissues , accompanied by the inability of beta cells to compensate for insulin resistance ( pancreatic beta cell dysfunction ) leading to overt hyperglycemia . insulin resistance , characterized by reduced responsiveness to normal circulating concentrations of insulin , is a common feature of almost all patients with t2 dm , and it plays a key role at the beginning and in the development of whole process of t2 dm . the presence of insulin resistance leads to increased beta cell insulin secretion with compensatory hyperinsulinemia . impaired function of beta cell will cause deterioration in glucose homeostasis , at this point , insulin secretion can not keep pace with the underlying insulin resistance and glucose intolerance , then t2 dm occurs . in patients destined to develop t2 dm , the beta cell compensatory response declines , and then develop relative or absolute insulin insufficiency . previous studies have demonstrated that t2 dm is a multifactorial disease in which genetic factors consisting of multiple susceptibility genes and environmental factors contribute to the disease development , and the pathogenesis of t2 dm is still unclear now . because of ethical considerations , clinical studies of human diabetes , especially disease pathology research is constrained to a certain extent , thus using various animal models can induce the disease by a different mechanism but with the similar or same results and offer promise of new insights into human diabetes , which is advantageous in biomedical studies owing to the uncertain etiology of t2 dm and its causes are multifarious . rodent seems to be the most suitable model for the study of t2 dm because of the small size , short generation interval , easy availability , and economic considerations . in this study , we aim to review the current available spontaneous rodent models for t2 dm with regard to their characteristic features , advantages , and disadvantages in order to investigate whether they can replicate the pathophysiological process of t2 dm . right now , there are lots of rodent models available for the study of t2 dm , but some of them may not always be satisfactory to simulate human t2 dm totally due to the large heterogeneity in the latter . obviously , no single rodent model can represent the onset and development of human t2 dm in all details . taken together , the existing rodent models provide a rich array of opportunities for investigators to study the complex pathogenesis and pathophysiological process of t2 dm . there is no fully unified classification criteria for rodent models of t2 dm , and in this paper various types of rodent models of t2 dm will be divided into spontaneously diabetic rodent models , artificially induced diabetic rodent models , and transgenic / knockout diabetic rodent models on the basis of their strains , features , advantages , and disadvantages . among these large number of animal models of t2 dm , the spontaneous type 2 diabetic rodent models are considered the most outstanding and most useful . we will respectively introduce several spontaneous rodent models of t2 dm in order to investigate the process of insulin resistance of t2 dm in human in the following part . obesity and the consequent insulin resistance are major features of t2 dm in human beings , and they play a key role at the beginning and in the development of the whole process of t2 dm . consequently , obese animal models of t2 dm have been used to simulate the complex pathogenesis and pathophysiological process of human disease and to gain insights into the human condition . the ob / ob mouse , db / db mouse , and zucker fatty ( fa / fa ) rat are the most typical examples of t2 dm obese models with monogenic background . on the other hand , the kk mouse , the nzo mouse , the oletf rat , and the nsy mouse are the representation of obesity - induced diabetes models with polygenic background . since these models show severe obesity , hyperinsulinemia , and insulin resistance throughout their lives , the agents like insulin sensitizers , antiobesity , and some other antihyperglycemia agents , which decrease the body weight and improve peripheral insulin sensitivity , have been largely tested by making use of these obese rodent models [ 16 , 17 ] . leptin , the product of the ob gene , may be a partial factor contributing to insulin resistance . the ob / ob mouse strain , from the bar harbor - jackson laboratory , has a well - known feature of leptin deficiency because of the mutation identified in leptin gene leading to severe insulin resistance . the ob gene was transferred from the stock of origin onto the b/6 genomic background and was located on chromosome 6 . in early 1970s , these ob / ob mice were used to investigate the pathogenesis of insulin resistance as the first rodent model . these ob / ob mice exhibit rapid gain in body weight and may reach three times of the normal weight of wild - type controls . in addition to obesity , early in the life of these mice , insulin resistance and hyperinsulinemia occur , which are out of proportion to their adiposity at early stages . in the ob / ob model , hyperinsulinemia manifests at 3 to 4 weeks of age together with hyperphagia and insulin resistance . on the contrary , leptin treatment in ob / ob mice causes decrease of both glucose and insulin levels within hours of administration before either food intake or body weight changes . similarly , in normal rodents leptin also has a clear insulin - sensitizing effect acutely and after chronic administration . in many rodent models of obesity , increased glucocorticoids also can mediate both the hyperphagia and insulin resistance due to leptin deficiency or resistance . in ob / ob mice , at least in rodents , suppressing hypothalamic - pituitary - adreno - cortical ( hpa ) axis by leptin replacement may be an important component of leptin action on insulin sensitivity . the primary defect in the ob / ob mice is from neuroendocrine origin , but the exact aberration and its site are not yet fully known . its expression is a lack of satiety control at the hypothalamic and/or pituitary level . the major action site of leptin is hypothalamus , where neurons are directly regulated by leptin reside . neuropeptide y ( npy ) , produced by hypothalamus , is a neuromodulator implicated in the control of energy balance and in the hypothalamus of ob / ob mice . overproduced as a central effector of leptin deficiency , npy is associated with obesity , a typical symptom of t2 dm and infertility , and eventually plays a major role on insulin action or secretion regulation in central nervous system ( cns ) . the symptom of t2 dm of ob / ob mice attenuates with age , being manifested by the continuous decline of plasma insulin levels in the second year of life , with a consequent effect of glucose tolerance and insulin resistance improvement and a loss of adipose body weight . it is interesting to notice that ob / ob mice are not known to develop major diabetic complications despite the marked insulinemia in contrast to other species . the diabetes db gene mutation occurred spontaneously in the leptin - receptor - deficient c57bl / ksj strain of mice and is originally derived from autosomal recessive mutation on chromosome 4 with complete penetrance , originating from bar harbor , maine . the db / db mouse can be considered as having a natural history that closely parallels to human beings . it becomes hyperphagic , hyperinsulinemic , and insulin resistant early in life ( within 2 weeks of age ) , then develops obesity at the age of 3 to 4 weeks . the hyperglycemia becomes manifest at the age of 4 to 8 weeks due to beta cell failure . at this time , the insulin secretion of pancreatic beta cell depletes and the hyperinsulinemia recedes , the mouse exhibits ketosis , gradual body weight loss , and deceases no longer than 8 to 10 months . in view of this , the sequence of events in this model appears to mimic human t2 dm , but it seems not compatible to investigate the complication of t2 dm because of the short life span . however , it is reported that the db / db mouse was used to investigate the renal and microvascular diabetic complications [ 29 , 30 ] . leptin controls food intake and neuroendocrine function by activating the long form of leptin receptor and ultimately regulates adiposity which influences insulin sensitivity . it also regulates glucose homeostasis independent on the energy balance in the same way . owing to alternative splicing , the leptin receptor which belongs to the cytokine receptor class i superfamily has several well - known isoforms in mouse , while the long form lepr b isoform ( lepr - b ) is the only stat3 signaling - competent isoform . compared with ob / ob mouse , the db / db mouse represents similar hypothalamic disturbances and npy abnormalities as a result of lack of leptin receptors . at the same time , the leptin is still produced but it can not activate the leptin receptors and restrain obesity and hyperinsulinemia induced by npy . in order to manifest this viewpoint , some investigators treated db / db mouse with exogenous leptin , the results showed that leptin administration has no effect on food intake and body weight gaining . the kk mouse originating from japan is a polygenic model of obesity and t2 dm . it was crossed with the bar harbor c57bl/6j mouse , and the body size of original strain of this mouse was large . in 1957 , kondo et al . reported the kk mouse at first , which spontaneously exhibited distinct adiposity , hyperglycemia , and hyperinsulinemia . at about 2 months of age , the kk mouse manifested moderate obesity due to hyperphagic , which was associated with insulin resistance , compensatory hyperinsulinemia , and islet cell hyperplasia . the insulin resistance and hyperinsulinemia reached to the peak at about 5 months and returned to normal at 9 to 12 months due to beta cell failure . the kk mouse bred several substrains throughout the world , and they vary both genetically and phenotypically from each other . among these substrains , kk / ay mouse , also named as yellow kk mouse , developed as a result of the dominant mutation of yellow agouti ( ay ) gene in japanese kk . homozygous kk / ay mouse will die before implantation or shortly thereafter , whereas heterozygous kk / ay mouse can grow up representing severe obesity , insulin resistance , hyperglycemia , and hyperinsulinemia at the age of about 8 weeks . the hyperglycemia and hyperinsulinemia of kk / ay mouse continue to develop along with the age and it has no need to maintain a high energy diet in order to become diabetic . for the past many years , the kk / ay mouse was used as mild hyperglycemia model for studying the treatment to prevention and intervention with obesity . zucker fatty ( fa / fa ) rat , along with finding of the spontaneous mutation ( fatty ) in the rat stock of sherman and merck , was found by zucker in harriet bird memorial laboratory during 1974 and 1975 . on chromosome 5 of the zucker fatty ( fa / fa ) rat , the simple autosomal recessive ( fa ) gene is mutant spontaneously , which results in hyperphagia and early onset of obesity . at the age of 4 weeks , the zucker fatty ( fa / fa ) rat from the obese strain gained weight more rapidly because of the increased growth of subcutaneous fat depot , and it had a considerably higher body weight at about the age of 9 weeks [ 38 , 39 ] . like the db / db mouse model , the hyperphagia and obesity in the zucker fatty ( fa / fa ) rat are attributed to hypothalamic defect in leptin receptor signaling , which is also associated with mild hyperglycemia , insulin resistance , mild glucose intolerance , hyperlipidemia , hyperinsulinemia , and moderate hypertension . the zucker diabetic fatty ( zdf ) rat was an inbred line of zucker fatty rat developing into a genetic model , which was established in 1985 and in 1991 . compared with zucker fatty rats , male zdf rats become less obese but more insulin resistant , and then rapidly progress to frank diabetes because of the lack of sufficient insulin secretion required adequate compensation for the insulin resistance . the male zdf rat represents hyperglycemia at about 7 weeks of age , and fully diabetic at 12 weeks . the serum insulin levels of male zdf rat reach the peak at about 7 to 10 weeks , but as soon as the beta cells of pancreas can not respond to glucose stimulus , the insulin levels drop subsequently as pancreatic cease to . the female zdf rat develops diabetes just on a diabetogenic diet unlike male zdf rat . it has been reported that in the progression of zdf rat , the decrease of beta cell glucose transporter 2 ( glut-2 ) membrane receptors and the concomitant loss of muscle glucose transporter 4 ( glut-4 ) transporters are responsible for the impaired insulin secretion and subsequent hyperglycemia . the activity of glut-4 receptors decreased in adipose tissue and skeletal muscles of zdf rat , which leads to the decreased beta cell transport ability together with the peripheral insulin resistance . the decrease of glut-2 membrane receptors coupled with insulin resistance and beta cell dysfunction in zdf rat can be commonly used to simulate the mechanism of human t2 dm and test insulin sensitizers and other various agents . the new zealand strain of obese mice , a model of polygenic obesity , which was obtained by selective inbreeding from a stock colony in the hugh adam department of cancer , considerably gains weight during the first 10 weeks of life as a result of hyperphagia and reachs to peak at about 12 months , coupled with the corresponding hyperglycemia and hyperinsulinemia . unlike other obese models , the nzo mouse manifests insulin resistance at an early age and gradually represents hyperleptinemia together with leptin resistance . with the growth of nzo mouse , hyperglycemia and impaired glucose tolerance increase continuously , and the level of blood glucose reaches 300400 mg / dl at the age of 20 to 24 weeks . in nzo mouse , the hyperglycemia occupies a leading position despite the fact that the level of insulin is lower than that in other obese models because of the distinct peripheral insulin resistance and increased gluconeogenesis . although nzo mouse is a rare preferred model , it becomes a useful model for studying the relationship between autoimmunity , obesity , and diabetes , and its new recombinant congenic strains are used to study the the otsuka long - evans tokushima fatty ( oletf ) rat was obtained by selectively inbreeding from an outbred colony of long - evans rats purchased from charles river , canada , develops mild to moderate obesity early at about 4 weeks with hyperglycemia later in life at around 18 to 25 weeks age . oletf rats exhibit the characteristics of innate polyphagia , mild obesity , late onset of hyperglycemia , hyperinsulinemia , hypertriglyceridemia , hypercholesterolemia , chronic course disease , and clinical onset of diabetes mostly in males , which are similar to the pathophysiologic progress of human t2 dm . many recessive genes on several chromosomes including the x chromosome are involved in the induction of diabetes in oletf rats . it is also observed that the oletf rats carry a null allele for the cholecystokinin a receptor which may be related to the regulation of food intake [ 50 , 51 ] . the pancreatic islets of oletf rats change progressively , and less than 9 weeks of age , the islets just exhibit mild lymphocyte infiltration , then show hyperplastic alterations and fibrosis in or around islets at around 10 to 40 weeks of age , finally more than 40 weeks , represent atrophy of islets . with oletf rats , many investigators carried out some researches to test exogenous insulin and antidiabetic drugs such as cilnidipine , acarbose , pioglitazone , and so on . the nagoya - shibata - yasuda ( nsy ) mouse , an inbred polygenic animal model of t2 dm , was established by selective inbreeding from the jc1 : icr mouse , from which the nod mouse was also derived . the nsy mouse closely imitates human t2 dm in which the characteristics of nsy mouse are mild obesity with abdominal and visceral fat accumulation , accompanied by impaired insulin secretion and moderate insulin resistance contributing to diabetes development in an age - dependent manner . for nsy mice , there is a marked gender difference that almost all males develop diabetes , while the percentage for females is only about 30% . the nsy mouse is also a polygenic model of t2 dm , and three major susceptibility loci ( nidd1n , nidd2n , and nidd3n ) have been mapped on chromosomes 11 , 14 , and 6 . the nsy mouse is particularly useful for studying the age - related damages and phenotypes of t2 dm , as well as the undiscovered genetic differentiations and correlations between t1 dm and t2 dm . in 1992 , the tsumura suzuki obese diabetes ( tsod ) mouse , accompanied with the other strain named tsumara suzuki non - obese ( tsno ) , was established by tsumura and suzuki through repeatedly selective inbreeding of obese male mice of ddy strain . differing from the tsno mouse that does not become obese , the tsod mouse exhibits polygenic obesity and insulin resistant at about 2 months old , which results in hyperinsulinemia and hyperglycemia , but just in males . in tsod mouse , pancreatic islets are hypertrophic and -cell mass increases , which controls the blood glucose levels so that the severe diabetes does not develop . the impaired glut4 translocation in both skeletal muscle and adipocytes of tsod mouse is one of the important reasons for the reduced insulin sensitivity and insulin resistance . it is investigated that many susceptibility loci have been mapped on chromosomes 11 , 1 , and 2 , which are closely related to obesity , hyperglycemia , and hyperinsulinemia of tsod mouse , and the combination of these genetic loci may lead to the symptoms of tsod mouse similar to human t2 dm . m16 mice , a unique line of mice created through election for 3 to 6 weeks weight gain for many generations from an outbred icr base stock in institute of cancer research in london , are a new animal model to simulate human obesity and t2 dm . compared with icr , both males and females of m16 mice gain weight at early age and maintain moderate obesity at all ages because of hyperphagia , accompanied by increased body fat percentage , fat cell size and numbers , and organ weights . at 8 weeks of age , all m16 mice exhibit hyperglycemic , hyperinsulinemic , and hypercholesterolemic relative to icr , but the fasted blood glucose levels are , respectively , 56 percent higher in males and 22 percent higher in females . the spontaneously hypertensive obese rat ( shr / ncp ) , an obese model of t2 dm with hypertension , part of the resultant descendants exhibits obesity with normotensive or mildly hypertensive , which is determined by an autosomal recessive trait . the characteristics of shr / ncp rats are hyperphagia and early onset of obesity , insulin resistance , impaired glucose tolerance , hyperinsulinemic , and normal or slight hyperglycemia . a new model of obese type 2 diabetes , spontaneously diabetic torii ( sdt ) fatty ( fa / fa ) rat , was established by introducing the fa allele of the zucker fatty rat into the spontaneously diabetic torii ( sdt ) rat genome via the speed congenic method . compared to male sdt rats , sdt fatty ( fa / fa ) rats showed overt obesity , and hyperglycemia and hyperlipidemia at younger ages ( 5~6 weeks ) , which is associated with hyperphagia by an induced disorder of leptin action , and plasma triglyceride ( tg ) and total cholesterol ( tc ) levels in sdt fatty ( fa / fa ) rats were significantly higher than those in original sdt rats . furthermore , with an early incidence of diabetes mellitus , diabetes - associated complications ( such as renal lesions and cataract ) in the sdt fatty ( fa / fa ) rat are seen at younger ages than in the sdt rats [ 6971 ] . the pharmacological effects of antidiabetic drugs , such as metformin , pioglitazone , and dipeptidyl peptidase-4 inhibitor , have been tested on sdt fatty ( fa / fa ) rats . sdt fatty rat is expected to be a useful model for analysis of diabetic complications and the evaluation of drugs used for metabolic disease . it is demonstrated that human t2 dm can also exist in the absence of obesity . thus , it is necessary for developing nonobese models to study this condition of the t2 dm . the gk ( goto - kakizaki ) rat , cohen diabetic rat , and spontaneously diabetic torii ( sdt ) rat are the typical examples of nonobese models of t2 dm . these nonobese rodent models are very useful for studying the mechanisms of diabetes complications ( e.g. , renal , retinal , and peripheral nerves lesions ) . however , because of their absence of obesity and hypoinsulinemia , only very few studies on drug testing using these models have been reported in the literature in 1973 , goto and his collaborators in japan developed the goto kakizaki ( gk ) rat , a polygenic nonobese model of t2 dm with early and relatively stable hyperglycemia , hyperinsulinemia , and insulin resistance , through selective inbreeding of nondiabetic wistar rats with mild glucose intolerance over many generations . for gk rats , because of the reduced number of islets at birth , both insulin resistance and impaired insulin secretion are present at their adult lives , coupled with moderate but stable fasting hyperglycemia which is present at the end of the first 2 weeks . and after 8 weeks , hyperglycemia degenerates and insulin secretion of the islets stimulated by glucose is more severely impaired , but generally , during its lifetime , fasting glucose remains mild and stable and rises only after challenge with glucose . for gk rats , 60% of pancreatic -cell mass decreased along with the distinct defects on -cell function , because of the secondary loss of -cell differentiation manifested by the three genetic loci correlated with impaired insulin secretion and glucotoxicity . in the pancreatic islets of gk rats at 2 months of age , the deposition of fibrous and nonamyloid material bring about the result that the islets become transmuted in shape but do not increase in total area . with the exception of the defects of pancreatic -cell mass , the decrease of insulin sensitivity in peripheral tissues and hepatic glucose overproduction are also mechanisms of development of hyperglycemia and insulin resistance . for gk rat , developing some features of complications of diabetes can be used to study the diabetic complications seen in humans , including glomerulopathy , peripheral neuropathy , and retinopathy . the gk rat is one of the useful animal models for studying the defects of pancreatic -cell mass and pathophysiologic progress of t2 dm and its complications . cohen diabetic rat , an exceptional genetical model derived from diet - induced t2 dm model by placing the rat on a synthetic 72% sucrose - copper - poor diet for 2 months , displays many features of the human t2 dm . meanwhile , the cohen diabetic rat expresses genetic susceptibility ( sensitivity and resistance ) to a carbohydrate - rich diet , a central feature of t2 dm in human beings . nevertheless , the cohen diabetic rat has many shortcomings , a major of which is that it has never been systematically characterized in terms of phenotype or genotype . for this reason , the cohen diabetic rat model has been studied until recently only to a limited extent after being established nearly 30 years ago . spontaneously diabetic torii ( sdt ) rat is a new spontaneously nonobese diabetic strain derived from the sprague - dawley rat in 1997 at torii pharmaceutical co. , ltd . it has been found that the distinct characteristics of this model are glucose intolerance , hyperglycemia , hypoinsulinemia , and hypertriglyceridemia . it was reported that there is a major cumulative incidence of diabetes in male rats ( 100% by 40 weeks of age ) than in female rats ( 33.3% even by 65 weeks of age ) . because of the chronic severe hyperglycemia , sdt rats develop diabetic retinopathy ( dr ) [ 8284 ] , diabetic peripheral neuropathy [ 85 , 86 ] , and diabetic nephropathy . with worldwide rises of t2 dm incidences , many of the animal models have been established by different mechanisms to describe similar characteristic features of this human disease and new therapeutic methods still need to be found . among these large numbers of animal models of t2 dm , the spontaneous rodent models are the most outstanding , distinctive , and useful one , because of the small size , short generation interval , easy availability and economic considerations . additionally , many of the spontaneous animal models can commendably simulate the characteristics of t2 dm , such as obesity , insulin resistance , hyperinsulinemia , hyperglycemia , and hyperlipemia . however , every spontaneous rodent model of t2 dm has either advantages or disadvantages and each of them can not replicate all characteristic features of t2 dm because of the complex , heterogeneous , multifactorial syndrome of this disease . so , in the future , more promising animal models that closely simulate human t2 dm regarding all aspects of t2 dm are needed urgently .

we describe a 24-year - old woman with anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis that developed 3 weeks after normal delivery . she was treated with methylprednisolone , intravenous immunoglobulin , and plasmapheresis , in addition to teratoma excision . however , her recovery was slow , and dysmnesia and mental juvenility persisted even two years after onset . to date , five patients with postpartum anti - nmdar encephalitis have been reported . all of those patients showed psychotic symptoms and were suspected of having postpartum psychosis in the early period of the encephalitis . changes in hormonal factors , modification of immune tolerance , or retrograde infection of the ovary may be contributing factors for postpartum anti - nmdar encephalitis .

anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis is an autoimmune limbic encephalitis induced by antibodies directed against the nr1 subunit of the nmdar ( 1 ) . typical patients with anti - nmdar encephalitis show nonspecific prodromal symptoms , such as a fever and headache , followed by symptoms resembling schizophrenia , and then develop generalized seizure , altered mental status , hypoventilation , autonomic instability , and characteristic movement disorders , such as orofacial - limb dyskinesia and catatonia ( 2 - 4 ) . anti - nmdar encephalitis was originally reported as a paraneoplastic syndrome associated with ovarian teratoma ( 2 ) . however , it is now acknowledged that the spectrum of this encephalitis is much broader , as there have been many cases in women without ovarian teratoma , men , and children ( 5 ) . there is also a possibility that pregnancy and/or delivery could trigger anti - nmdar encephalitis , as several patients developed this disorder during pregnancy or in the postpartum period ( 6 - 19 ) . we herein report a japanese patient who developed severe anti - nmdar encephalitis three weeks after normal delivery and discuss the pathophysiology of postpartum anti - nmdar encephalitis . the patient was a 24-year - old primiparous japanese woman with no significant medical history . she had no complications during the course of the pregnancy and gave birth to a healthy baby girl via vaginal delivery . one week later , she presented with auditory hallucination and abnormal behavior and was mandatorily hospitalized in the department of psychiatry of a general hospital . she was diagnosed with postpartum psychosis and treated with antipsychotic drugs . on the second hospital day , she presented with somnolence and unstable breathing followed by generalized seizure . on the third hospital day , she developed status epilepticus and hyperthermia and was transferred to the intensive care unit . generalized seizure was difficult to control despite treatment with propofol and antiepileptic drugs , and respiratory depression led to tracheal intubation and artificial ventilation . she was treated with methylprednisolone ( mpsl ) pulse therapy at a dose of 1 g for 3 days and intravenous immunoglobulin therapy ( ivig ) at a dose of 0.4 g / kg for 5 days ( fig . 1 ) . however , her symptoms deteriorated gradually and she developed involuntary movements in the face and right upper limb . simv : synchronized intermittent mandatory ventilation , ps : pressure support , cpap : continuous positive airway pressure , ivig : intravenous immunoglobulin , pe : plasma exchange , dfpp : double filtration plasmapheresis , mpsl : methylprednisolone , dvt : deep vein thrombosis on admission , her body temperature was 38.5c . a neurological examination showed orofacial dyskinesia and athetoid movement in the right hand even under deep sedation with propofol . laboratory tests revealed inflammatory reaction ( white blood cell , 13,350/l ; c reactive protein , 4.31 mg / dl ) and mild liver dysfunction ( aspartate aminotransferase , 37 iu / l ; alanine aminotransferase , 103 tests for herpes simplex , herpes zoster , and epstein - barr virus were negative . autoantibodies were all negative , except for anti - thyroglobulin antibody and anti - thyroperoxidase antibody . the results of a cerebrospinal fluid ( csf ) analysis showed lymphocytic pleocytosis ( 82/l , mononuclear cells 77/l ) , a slightly elevated protein level ( 51 mg / dl ) , and a normal glucose level ( 73 mg / dl ) . anti - nmdar antibody was positive ( 20 , examined by cosmic corporation , tokyo , japan ) in the csf . electroencephalogram ( eeg ) demonstrated diffuse beta activity superimposed on frontally dominant high - voltage rhythmic delta bursts , consistent with 2 ) . brain magnetic resonance imaging ( mri ) showed slightly increased signal intensity with swelling in the bilateral medial temporal lobes on t2 and flair imaging ( fig . abdominal computed tomography ( ct ) revealed a right ovarian cystic tumor with small calcifications ( fig . 4a ) . based on the characteristic clinical findings and positivity for anti - nmdar antibody , a diagnosis of anti - diffuse beta activity superimposed on frontally dominant high - voltage rhythmic delta bursts was observed . brain magnetic resonance image ( mri ) of the patient ( flair image , axial view ) . ( a ) mri performed at 1 month after onset . high signal intensity and slight swelling were seen in the bilateral medial temporal lobes . 4b ) consisting of stratified squamous epithelium with cutaneous appendage , neural tissue with choroid plexus , adipose tissue , bone , cartilage , and intestinal structure with goblet cells . in addition to resection of the teratoma , she was treated with ivig , mpsl pulse therapy , plasma exchange ( pe ) , and double filtration plasmapheresis ( dfpp ) ; however , her involuntary movements , higher brain dysfunction , and autonomic dysfunction were prolonged . we could not perform further intensive immunosuppressive therapy , such as rituximab and cyclophosphamide , due to repetitive severe infections , including pneumonia and sepsis ( fig . after the fifth ivig , her involuntary movements and respiratory failure improved gradually , and she was transferred to rehabilitation hospital eight months after onset ( fig . 1 ) . at the time of transfer , she was still in a bedridden state and had severe cognitive dysfunction . brain mri 8 months after onset showed an almost normal appearance with no abnormal signals or brain atrophy . her neurological condition continued to improve after transfer , and she achieved independent gait and verbal communication at 12 months after onset . neither brain atrophy nor abnormal signals were revealed on mri ( fig . 3b ) , but her memory disturbance and mental juvenility persisted even two years after onset . ( a ) computed tomography of the pelvis showed a cystic tumor ( 1.5 cm in maximal diameter ) with small calcifications adjacent to the right ovary . ovarian teratoma is the most common associated tumor of anti - nmdar encephalitis in female patients and contains antigenic neural tissue . in addition , the majority of patients have a history of prodromal flu - like symptoms , including headache , fever , nausea , vomiting , diarrhea , or upper respiratory tract symptoms . therefore , the combination of ectopic expression of nmdar , especially the nr1 subunit contained in the teratoma , and the adjuvant effect of the prodromal flu - like syndrome is thought to contribute to initiation of the immune response and production of pathogenic antibodies ( 3 ) . the present case involved an ovarian teratoma with the development of anti - nmdar encephalitis three weeks after delivery without prodromal flu - like symptoms . therefore , anti - nmdar encephalitis may have been triggered by normal vaginal delivery in our patient . with regard to the relationship between anti - nmdar encephalitis and pregnancy / delivery , 10 patients developed anti - nmdar encephalitis during pregnancy ( 6 - 15 ) , and 5 patients developed the disease during the postpartum period ( 16 - 19 ) . one of the major immunological modifications during pregnancy is the th1/th2 shift , due to the progressive increases in levels of progesterone and estrogen during pregnancy , which suppress th1 cytokines and stimulate th2-mediated immunological responses as well as antibody production ( 22 ) . therefore , th1-mediated diseases , such as rheumatoid arthritis and multiple sclerosis , tend to improve during pregnancy and worsen during the postpartum period ( 23,24 ) . in contrast , th2-mediated diseases , such as systemic lupus erythematosus , tend to worsen during pregnancy ( 25 ) . however , recent studies have shown that annualized relapse rate of neuromyelitis optica ( nmo ) tends to decrease in the first half of pregnancy and increase after the pregnancy ( 26,27 ) , although nmo is a th2-mediated disease . in myasthenia gravis , worsening of symptoms is more likely during the first half of pregnancy and postpartum ( 28 ) . therefore , the influence of pregnancy and delivery on patients with immunological disorders should be analyzed with respect to each disease . with regard to anti - nmdar encephalitis , the precise immunopathogenesis remains to be elucidated , although b cells , but not t cells , have been proposed to be involved ( 29 ) . therefore , further investigations regarding the immunopathogenesis of the disease , including the th1/th2 balance and cytokine dynamics , and large - scale epidemiological surveys are necessary to determine the effects of pregnancy and delivery on anti - nmdar encephalitis . several hypotheses have been proposed to explain the pathomechanism of anti - nmdar encephalitis associated with pregnancy and/or delivery . it was demonstrated that markedly increased estrogen promotes formation of autoreactive lymphocytes through rapid maturation of b cells and secretion of interleukin-10 during pregnancy ( 30,31 ) , which may facilitate the production of anti - nmdar antibody and/or its crossing of the blood - brain barrier , thereby contributing to the development of anti - nmdar encephalitis ( 3 ) . indeed , symptomatic recovery seemed to accelerate after giving birth in three patients with anti - nmdar encephalitis ( 11 ) . in contrast , the maternal immune system is modified to allow immune tolerance to fetal antigens during pregnancy . yu et al . hypothesized that pregnancy - induced modulation of the immune system may contribute to the occurrence of postpartum anti - nmdar encephalitis by a breakdown in tolerance to self - antigens ( 16 ) . table shows the clinical characteristics of patients with anti - nmdar encephalitis that developed during the postpartum period ( 16 - 19 ) . all of the patients showed psychotic symptoms , including anxiety , delusions , bizarre behavior , insomnia , agitation , irritability , hallucinations , psychomotor excitement , confusion , and depression , and were suspected of having postpartum psychosis in the early period of the encephalitis . the patient reported by koksal et al . showed dramatic improvement shortly after teratoma resection ( 17 ) . in contrast , the patient reported by yu et al . showed little improvement following tumor resection , mpsl , and pe , but improved dramatically shortly after rituximab administration ( 16 ) . rituximab was also very effective in a patient with postpartum anti - nmdar encephalitis without teratoma ( 18 ) . our patient showed very slow improvement after tumor resection , which may have been caused by the delay in surgical resection and hesitation to perform aggressive immunosuppressive treatment , such as rituximab , due to recurrent severe bacterial infections . severe bacterial infections and deep vein thrombosis are complications which occur frequently in patients with severe anti - nmdar encephalitis . management of these complications is crucial and has a considerable effect on the prognosis of anti - nmdar encephalitis . ivm : involuntary movement , gtcs : generalized tonic - clonic seizure , rt . : right , mpsl : methylprednisolone therapy , pe : plasma exchange , ivig : intravenous immunoglobulin therapy , dfpp : double filtration plasmapheresis to our knowledge , there are no obvious differences in the clinical characteristics of patients between anti - nmdar encephalitis associated with pregnancy , associated with delivery , and with no pregnancy / delivery association . recently screened 96 consecutive patients with postpartum psychosis and 64 healthy postpartum women and found 2 patients with postpartum psychosis positive for anti - nmdar antibody ( 19 ) . both patients recovered after treatment with lithium , lorazepam , and antipsychotic agents , and remission was sustained , despite the absence of any immunosuppressive treatment ( 19 ) , suggesting that mild anti - nmdar antibody encephalitis may be underdiagnosed among a cohort of postpartum psychosis patients .

genome - wide mrna transcription profiles reveal widespread molecular sexual dimorphism or sex - biased gene expression , yet the relationship between molecular and phenotypic sexual dimorphism remains unclear . a major unresolved question is whether sex - biased genes typically perform male- and female - specific functions ( whether these genes have sex - biased phenotypic or fitness consequences ) or have similar functional importance for both sexes . to elucidate the relationship between sex - biased transcription and sex - biased fitness consequences , we analyzed a large data set of lethal , visible , and sterile mutations that have been mapped to the drosophila melanogaster genome . the data permitted us to classify genes according to their sex - specific mutational effects and to infer the relationship between sex - biased transcription level and sex - specific fitness consequences . we find that mutations in female - biased genes are ( on average ) more deleterious to females than to males and that mutations in male - biased genes tend to be more deleterious to males than to females . nevertheless , mutations in most sex - biased genes have similar phenotypic consequences for both sexes , which suggests that sex - biased transcription is not necessarily associated with functional genetic differentiation between males and females . these results have interesting implications for the evolution of sexual dimorphism and sex - specific adaptation .

to identify genes with sex - limited and nonsex - limited functions , we searched flybase ( tweedie et al . 2009 ) for mutations within the following phenotypic categories : visible , lethal , semilethal , sterile , male sterile , and female sterile ( searches used the termlink section ; http://flybase.org/static_pages/termlink/termlink.html ) . we trimmed the data set to include alleles associated with specific genes ( although many alleles have been mapped to specific chromosomes and/or cytological bands , the mapping resolution for these cases was generally insufficient to be included within the final data set ) . individual genes can potentially have multiple alleles within the data set ( though the majority of drosophila genes were not associated with any alleles ) . we therefore classified each gene according to its range of mutant allele phenotypes , which fall between entirely female - specific to entirely male - specific . the genes were classified as follows : genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type;genes with female - biased fitness effects contain female - sterile alleles and any combination of visible , lethal , and semilethal alleles;genes with male - limited fitness effects contain male - sterile alleles and no other allele type;genes with male - biased fitness effects contain male - sterile alleles and any combination of visible , lethal , and semilethal alleles;genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles , visible alleles , lethal alleles , and/or semilethal alleles . genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type ; genes with female - biased fitness effects contain female - sterile alleles and any combination of visible , lethal , and semilethal alleles ; genes with male - limited fitness effects contain male - sterile alleles and no other allele type ; genes with male - biased fitness effects contain male - sterile alleles and any combination of visible , lethal , and semilethal alleles ; genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles , visible alleles , lethal alleles , and/or semilethal alleles . genes associated with sterility , but with neither sex specified ( the underlying allelic data did not provide information about sex ) , were considered ambiguous and excluded from the analysis . the sample of sterile alleles that were included in the analysis is potentially heterogeneous because some studies examine fertility in only one sex rather than both . nevertheless , the proportion of sex - limited and nonsex - limited steriles in our data set is consistent with independent experimental results that explicitly test male and female fertility ( alleles associated with sex - specific sterility are roughly three times as common as alleles associated with sterility in both sexes ; see lindsley and lifschytz 1972 ; ashburner et al . this suggests that most genes and alleles classified as sex - limited are in fact associated with sex - limited sterility . molecular expression profiles were obtained from the sex bias database ( sebida version 2.0 : http://141.61.102.16:8080/sebida/index.php ; gnad and parsch 2006 ) . we downloaded male versus female expression ratios ( m / f ) from 15 different microarray studies ( data were originally reported in : parisi et al . ; gibson et al . 2004 ; stolc et al . 2004 ; mcintyre et al . 2006 ; goldman and arbeitman 2007 ; ayroles et al . 2009 ) and m / f ratios from a meta - analysis of several studies ( details of the meta - analysis are described at sebida ) . m / f ratios can potentially range from zero to infinity , with male - biased transcription for m / f > 1 and female - biased transcription for m / f < 1 . to impose symmetry on sex - biased expression levels , we rescaled the data using an index of sex - biased expression : x = m/(m + f ) . this variable ranges between zero and one , with female - biased transcription for x < 0.5 and male - biased transcription for x > 0.5 . the final data set included 2,433 genes with m / f expression information from at least 1 of the 15 studies . within the final data set , there were 1,955 genes with similar mutational effects on both sexes , 298 genes with female - biased fitness effects , 43 female - limited genes , 87 genes with male - biased fitness effects , and 50 male - limited genes ( an additional 53 genes had ambiguous sex - specific sterility phenotypes ) . supplementary table s1 ( supplementary material online ) provides a breakdown of the data set into phenotypic subcategories , including the mean and median number of alleles per gene , per phenotypic category . whitney u tests ( implemented in r ; r development core team 2005 ) were used to assess whether the distribution of sex - biased transcription levels differs between phenotypically defined gene categories . to examine whether different categories of sex - biased transcription have different compositions of phenotypes , we subdivided the data set into five expression categories , each with equal range : ( 1 ) 0 < x < 0.2 ; ( 2 ) 0.2 < x < 0.4 ; ( 3 ) 0.4 < x < 0.6 ; ( 4 ) 0.6 < x < 0.8 ; and ( 5 ) 0.8 < x < 1.0 . two - tailed fisher s exact tests were used to examine whether female - biased transcription categories ( 1 , 2 ) were enriched for genes with female - specific phenotypes and whether male - biased transcription categories ( 4 , 5 ) were enriched for genes with male - specific phenotypes . the results presented below use meta - analysis expression profiles ( from sebida ; see above ) to transcriptionally categorize genes . the meta - analysis data set represents a composite of several independent microarray studies , which minimizes the likelihood of sex - biased transcription misclassification for each gene ( compared with classifications based on single studies ) . the meta - analysis also includes data for a high proportion of the 2,433 genes ( compared with single studies ) , which maximizes statistical power . the results are consistent across studies , though the statistical power is often lower , due to decreased gene representation . results for each platform are presented within the supplementary figs . s1 and s2 ( supplementary material online ) . to identify genes with sex - limited and nonsex - limited functions , we searched flybase ( tweedie et al . 2009 ) for mutations within the following phenotypic categories : visible , lethal , semilethal , sterile , male sterile , and female sterile ( searches used the termlink section ; http://flybase.org/static_pages/termlink/termlink.html ) . we trimmed the data set to include alleles associated with specific genes ( although many alleles have been mapped to specific chromosomes and/or cytological bands , the mapping resolution for these cases was generally insufficient to be included within the final data set ) . individual genes can potentially have multiple alleles within the data set ( though the majority of drosophila genes were not associated with any alleles ) . we therefore classified each gene according to its range of mutant allele phenotypes , which fall between entirely female - specific to entirely male - specific . the genes were classified as follows : genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type;genes with female - biased fitness effects contain female - sterile alleles and any combination of visible , lethal , and semilethal alleles;genes with male - limited fitness effects contain male - sterile alleles and no other allele type;genes with male - biased fitness effects contain male - sterile alleles and any combination of visible , lethal , and semilethal alleles;genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles , visible alleles , lethal alleles , and/or semilethal alleles . genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type ; genes with female - biased fitness effects contain female - sterile alleles and any combination of visible , lethal , and semilethal alleles ; genes with male - limited fitness effects contain male - sterile alleles and no other allele type ; genes with male - biased fitness effects contain male - sterile alleles and any combination of visible , lethal , and semilethal alleles ; genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles , visible alleles , lethal alleles , and/or semilethal alleles . genes associated with sterility , but with neither sex specified ( the underlying allelic data did not provide information about sex ) , were considered ambiguous and excluded from the analysis . the sample of sterile alleles that were included in the analysis is potentially heterogeneous because some studies examine fertility in only one sex rather than both . nevertheless , the proportion of sex - limited and nonsex - limited steriles in our data set is consistent with independent experimental results that explicitly test male and female fertility ( alleles associated with sex - specific sterility are roughly three times as common as alleles associated with sterility in both sexes ; see lindsley and lifschytz 1972 ; ashburner et al . this suggests that most genes and alleles classified as sex - limited are in fact associated with sex - limited sterility . molecular expression profiles were obtained from the sex bias database ( sebida version 2.0 : http://141.61.102.16:8080/sebida/index.php ; gnad and parsch 2006 ) . we downloaded male versus female expression ratios ( m / f ) from 15 different microarray studies ( data were originally reported in : parisi et al . ; gibson et al . 2004 ; stolc et al . 2004 ; mcintyre et al . 2006 ; goldman and arbeitman 2007 ; ayroles et al . 2009 ) and m / f ratios from a meta - analysis of several studies ( details of the meta - analysis are described at sebida ) . m / f ratios can potentially range from zero to infinity , with male - biased transcription for m / f > 1 and female - biased transcription for m / f < 1 . to impose symmetry on sex - biased expression levels , we rescaled the data using an index of sex - biased expression : x = m/(m + f ) . this variable ranges between zero and one , with female - biased transcription for x < 0.5 and male - biased transcription for x > 0.5 . the final data set included 2,433 genes with m / f expression information from at least 1 of the 15 studies . within the final data set , there were 1,955 genes with similar mutational effects on both sexes , 298 genes with female - biased fitness effects , 43 female - limited genes , 87 genes with male - biased fitness effects , and 50 male - limited genes ( an additional 53 genes had ambiguous sex - specific sterility phenotypes ) . supplementary table s1 ( supplementary material online ) provides a breakdown of the data set into phenotypic subcategories , including the mean and median number of alleles per gene , per phenotypic category . two - tailed mann whitney u tests ( implemented in r ; r development core team 2005 ) were used to assess whether the distribution of sex - biased transcription levels differs between phenotypically defined gene categories . to examine whether different categories of sex - biased transcription have different compositions of phenotypes , we subdivided the data set into five expression categories , each with equal range : ( 1 ) 0 < x < 0.2 ; ( 2 ) 0.2 < x < 0.4 ; ( 3 ) 0.4 < x < 0.6 ; ( 4 ) 0.6 < x < 0.8 ; and ( 5 ) 0.8 < x < 1.0 . two - tailed fisher s exact tests were used to examine whether female - biased transcription categories ( 1 , 2 ) were enriched for genes with female - specific phenotypes and whether male - biased transcription categories ( 4 , 5 ) were enriched for genes with male - specific phenotypes . the results presented below use meta - analysis expression profiles ( from sebida ; see above ) to transcriptionally categorize genes . the meta - analysis data set represents a composite of several independent microarray studies , which minimizes the likelihood of sex - biased transcription misclassification for each gene ( compared with classifications based on single studies ) . the meta - analysis also includes data for a high proportion of the 2,433 genes ( compared with single studies ) , which maximizes statistical power . the results are consistent across studies , though the statistical power is often lower , due to decreased gene representation . results for each platform are presented within the supplementary figs . s1 and s2 ( supplementary material online ) . genes with sexually dimorphic phenotypic effects tend to have relatively sex - biased mrna expression levels ( fig . 1 ; supplementary figs . s1 and s2 , supplementary material online ) . compared with genes with similar phenotypic effects in both sexes ( those with visible , lethal , semilethal , and/or alleles for sterility in both sexes ) , genes with female - limited and female - biased fitness effects have higher mrna expression in females ( mann whitney u tests : female - limited effects genes associated with male - limited and male - biased fitness effects have higher mrna expression in males ( male - limited effects these results largely extend to comparisons between individual phenotypic categories and the distribution of sex - biased expression throughout the entire drosophila genome . genes with greater phenotypic effects in females show greater female - biased mrna expression ( female - limited effects : p = 4.38 10 ; female - biased fitness effects : p < 2.2 10 ) . genes with male - limited phenotypic effects are more male biased in transcription ( p = 6.78 10 ) , though genes with male - biased fitness effects do not differ from the genome - wide distribution ( p = 0.228 ) . unexpectedly , genes that similarly affect both sexes ( e.g. , lethal , visible , male , and female sterile ) are relatively female biased ( transcriptionally ) compared with the genomic distribution ( fig . each of these results is highly consistent across individual microarray studies ( supplementary figs . s1 and s2 , supplementary material online ) . genes with sexually dimorphic mrna transcription levels are associated with sex - biased fitness effects . the upper panel shows the cumulative distribution [ pr(x < x ) ] for sex - biased transcription among five phenotypic categories ( color coded ) and for the entire genome ( the black curve ) . each phenotypic category differs significantly from the genome - wide distribution ( mann whitney u ; p < 10 ) except for genes with male - biased fitness effects ( p = 0.228 ) ; each category significantly differed from the nonsex - biased phenotypic class of genes ( partially male - limited : p = 0.0105 ; other categories : p < 0.00001 ) . the lower panel shows the proportion of each phenotypic class within five sex - biased transcription categories . two - tailed fisher exact tests ( * * * p < 0.001 ; * * * * p < 0.0001 ) were used to determine : 1 ) whether female - biased genes ( 0 < x < 0.2 ; 0.2 < x < 0.4 ) were enriched for female - biased or female - limited phenotypic effects and 2 ) whether male - biased genes were enriched for male - biased or male - limited phenotypic effects ( 0.6 < x < although these patterns validate the intuition that male - biased genes should be more important for male fitness and female - biased genes should be more important for female fitness , such associations are far from absolute . genes with highly dimorphic transcription often have similar mutational effects on both sexes ( fig . 1 , within the upper and lower 20 percent tails for x : 60 percent of female - biased genes and 38 percent of male - biased genes had roughly equal fitness effects in both sexes ; also see supplementary fig . genes with more moderate sex - biased transcription patterns are even less likely to have sex - limited fitness consequences . for example , among genes with 1.5-fold to 4-fold differential expression between the sexes ( ranges 0.2 < x < 0.4 and 0.6 < x < 0.8 ) , roughly 80 percent had similar mutational effects in each sex . thus , although genes with male - biased and female - biased transcription are statistically associated with male - biased and female - biased fitness effects ( respectively ) , the vast majority of sex - biased genes appear to be functionally important for both sexes ( genome - wide , over 90 percent of genes fall within the range 0.2 < x < 0.8 ) . there are several evolutionary routes that may lead to sex - biased gene expression ( ellegren and parsch 2007 ) , yet only some are expected to generate associations between the relative transcription level of a gene and its fitness consequences in each sex . sex - biased gene expression may be directly selected for when males and females have different gene expression optima . because direct selection for expression dimorphism is not necessarily expected to alter the relative importance of the gene for either sex , sex - biased mutational effects may not accompany sex - biased transcription . alternatively , the evolution of sex - biased expression may coincide with sex - specific selection for novel protein coding sequences or the differential incorporation of proteins into sex - specific genetic interaction networks ( arnold et al . 2009 ) . this may involve sex - specific expression of previously noncoding sequence , gene duplication and sex - specific cooption of paralogs , or a change in a gene s function ( or subfunction ) within one sex but not the other . any of these processes can generate an association between sex - biased expression and sex - biased function and phenotypic consequence . the results indicate that male - biased and female - biased transcription does not necessarily equate with sex - specific importance or function . phenotypically characterized and mapped mutations tend to similarly affect both sexes , despite their variable associations with sexually dimorphic gene transcription . female - biased genes can even produce alleles that are more harmful to males than to females and vice versa , although such cases are rare . however , despite this broad pattern of shared importance , there is also a clear statistical association between transcriptional dimorphism and the probability that a gene has a sex - limited or sex - biased phenotypic effect . this implies that a fraction of male- and female - biased genes adopt sex - specific functions or become incorporated into sex - specific molecular pathways . this pattern is particularly striking when one considers the coarseness of the transcriptional data for each gene . the ratio of male to female gene expression is a composite signal from multiple tissues during the adult life - history stage and ignores the fine - scale spatial and temporal resolution that defines the process of drosophila development ( meisel 2011 ) . the association between sexually dimorphic gene expression and sex - biased phenotypic effects proves that such transcriptional assays represent a biologically and evolutionarily meaningful measurement . these patterns may also have implications for debates over the population genetic consequences of sex - specific selection ( e.g. , chippindale et al . 2008 ; whitlock and agrawal 2009 ; agrawal 2011 ; connallon et al . 2010 ; mallet and chippindale 2011 ) . theory predicts that stronger selection in males than females will enhance purifying selection and reduce the mutational load of females ( whitlock and agrawal 2009 ) , whereas stronger purifying selection in females and sexually antagonistic selection between the sexes increases the female mutational load ( day and bonduriansky 2004 ; bonduriansky and chenoweth 2009 ) . considering patterns of selection across the genome , net fitness costs to females can potentially emerge when female - biased purifying selection or sexual antagonism operates across a relatively small fraction of the genome ( connallon et al . the results presented here suggest that , while a proportion of the genome is subject to much stronger selection in females than males , most genes with female - biased expression experience selection in males . this may either mitigate the female genetic load or expand the sequence space that can experience sexually antagonistic selection . molecular evolutionary contrasts between genes in different phenotypic categories , including selection parameter estimates from resequencing data ( using approaches described by keightley and eyre - walker 2010 ) , may shed additional light on the population genetic consequences of sex - specific selection . one surprising observation is that alleles with similar effects on both sexes are associated with female - biased transcription , compared with the genomic distribution ( fig . 1 ; fig . s1 , supplementary material online ) . this phenotypic category is dominated by lethal alleles , which suggests that female - biased genes ( at least those with moderately higher expression in females than males ) have a higher proportion of essential functions than male - biased genes . these relatively severe mutational effects might partially explain why male - biased genes evolve more rapidly than female - biased genes ( e.g. , zhang et al . 2006 ; clark et al . 2007 ; ellegren and parsch 2007 ; larracuente et al . 2008 ) . female - biased genes might have a smaller fraction of effectively neutral mutations or a higher degree of pleiotropy ( mank et al . 2008 ; mank and ellegren 2009 ; meisel 2011 ) , leading to decreased opportunities for neutral and adaptive evolution ( e.g. , fisher 1958 ; kimura 1983 ) . these conclusions should be considered tentative , as they may be sensitive to characteristics of the available data . lethal and sterile alleles reduce fitness to zero , whereas visible and semilethal alleles may have similarly strong effects on total fitness ( e.g. , due to strong mate discrimination against carriers of visible and/or subviable alleles ; grossfield 1975 ; hollis et al . 2009 ) . because mutations with small fitness effects preclude direct laboratory measurement , a broader analysis of the sex - specific fitness consequences of sex - biased genes will require a different empirical approach perhaps one that combines gene functional assays with molecular population genetics . . finally , phenotypic assays of mutations are not systematic , as they represent a collection of alleles characterized during the history of drosophila genetics research . future studies that systematically characterize genes and mutant phenotypes using targeted mutagenesis ( e.g. , p element insertions ) or deletion mapping will further illuminate the connection between molecular and phenotypic sexual dimorphism . supplementary figures s1s2 and table s1 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .

morvan 's syndrome is a rare autoimmune disorder characterized by triad of peripheral nerve hyperexcitability , autonomic dysfunction , and central nervous system symptoms . antibodies against contactin - associated protein - like 2 ( caspr2 ) , a subtype of voltage - gated potassium channel ( vgkc ) complex , are found in a significant proportion of patients with morvan 's syndrome and are thought to play a key role in peripheral as well as central clinical manifestations . we report a patient of morvan 's syndrome with positive caspr2anti - vgkc antibody having syndrome of inappropriate antidiuretic hormone as a cause of persistent hyponatremia .

morvan 's syndrome is a rare autoimmune disorder characterized by peripheral nerve hyperexcitability , autonomic dysfunction , and central nervous system symptoms . the syndrome of muscle twitching , dysautonomia , insomnia , and delirium was first reported by morvan by the name of la choree fibrillare in 1890 . we report a rare case of contactin - associated protein - like 2 ( caspr2 ) , a subtype of voltage - gated potassium channel ( vgkc ) complex antibody positive morvan 's syndrome , with syndrome of inappropriate antidiuretic hormone secretion ( siadh ) . a 45-year - old male presented with 4-month duration of nonradiating mild back pain , followed a month later by burning sensation in palms and soles with nocturnal exacerbations . he became aggressive , over - talkative , and insomniac over 15 days before presentation . electromyography showed spontaneous activity including myokymic discharges [ figure 1 ] , doublets , and triplets in both upper and lower limb muscles . the patient had positive serum anti - caspr2 antibody , a subtype of vgkc complex detected by immunofluorescence method . his cerebrospinal fluid examination showed raised proteins 76 mg / dl ( normal : 2040 mg / dl ) , with normal cell count ( cells : 3/mm , all lymphocytes ) . computed tomography ( ct ) of the chest showed no evidence of thymoma . the patient was diagnosed as morvan 's syndrome with positive anti - caspr2 vgkc antibody . spontaneous activity in right tibialis anterior showing myokymic discharges ( sweep speed : 0.1 ms and sensitivity : 50 v ) there was persistent low serum sodium in the range of 125130 meq / l , for which patient was evaluated . his urinary osmolarity was raised ( 216.36 mosm / kg , normal < 100 mosm / kg ) and random urinary sodium was increased ( 42 mmol / l , normal < 30 mmol / l ) . the serum osmolarity was decreased ( 271.5 mosm / kg ) and urinary specific gravity was 1.010 . the patient was treated with intravenous immunoglobulin ( iv ig ) 2 g / kg in 5 divided doses . he was given phenytoin at dose of 100 mg three times a day for symptomatic relief for twitching , which acts as membrane stabilizer . the patient was started on oral prednisone ( 1 mg / kg ) and fluid restriction was advised . he had marked improvement in muscle twitching and was able to sleep properly with immunotherapy . electromyography done 2 weeks after the course of iv ig showed decrease in spontaneous activity ; occasional fasciculations were seen . his hyponatremia was also corrected . on follow - up , after 3 months , the patient was completely normal and electromyography showed no spontaneous activity . oral prednisone was given 1 mg / kg for 3 months and later tapered gradually over next 2 months . morvan 's syndrome is characterized by myokymia associated with muscle pain , excessive sweating , weight loss , hallucinations , sleep disorders , and behavioral abnormality . the basic mechanism for both presentations is same , that is anti - vgkc antibody , acts at different levels of neuraxis , both at central and peripheral level . caspr2 antibodies positive cases but common in anti - leucine - rich glioma inactivated-1 ( lgi-1 ) antibodies positive cases . lgi-1 antibodies are usually associated with hyponatremia , and caspr2 antibodies are usually associated with thymomas which carry poor prognosis . caspr2 antibodies mostly bind the neuropil , whereas antibodies to lgi-1 bound to neuronal cell bodies including the antidiuretic hormone - secreting and orexin - secreting hypothalamic neurons present in hypothalamus , raphe nucleus , and locus coeruleus . hyponatremia is not commonly reported in morvan 's syndrome although it is present in half of the patient in lgi-1 antibodies positive limbic encephalitis . in addition , fasciculation , doublets , triplets , multiplets , and positive sharp waves are also present . this is a rare case report of morvan 's syndrome with siadh having anti - caspr2vgkc antibodies that responded to immunosuppression .

benzophenone-4 ( bz4 ) was separated from surfactants , dyes , preservatives , and other components of hair shampoos by thin - layer chromatography on silica gel 60 stationary phase , with ethyl acetate - ethanol - water - ph 6 phosphate buffer ( 15 : 7 : 5 : 1 v / v / v / v ) as mobile phase . densitometry scanning of chromatograms was performed at 285 nm . the densitometric calibration curve for bz4 was nonlinear ( second - degree polynomial ) , with r > 0.999 . the limits of detection and quantification were ca . 0.03 and ca . 0.1 g spot1 , respectively . the results obtained by hptlc - densitometry were compared to those obtained by zero and 2nd derivative uv spectrophotometry . in the case of spectrophotometric methods , calibration curves were linear with r > 0.9998 . the chromatographic method was fully validated .

benzophenone ( bz4 , figure 1 ) is a water - soluble uvb filter ( max in methanol 285 nm ) manufactured by basf under the trademark uvinul ms40 and used in cosmetic formulations at concentrations up to 5% ( as free acid ) . because of its highly acidic character , it must be neutralized with sodium hydroxide or triethanolamine prior to addition to a cosmetic formulation . due to its good water and alcohol solubility , it is commonly used in aqueous preparations ( shampoos , oil - free sunscreen sprays ) or to protect cosmetic products including perfumes against photodegradation . benzophenone-4 was quantified in cosmetic products mainly by rp - hplc [ 29 ] or , less frequently , si ( sequential injection ) analysis or ce ( capillary electrophoresis ) [ 11 , 12 ] . liquid chromatography was also used in bz4 quantification in environmental samples [ 13 , 14 ] or in skin permeation studies . in such studies , a radioanalytical method involving bz4 labeled with radioisotope c ( at carbonyl group ) was also applied . the objective of this study was to propose a cheap and convenient method of quantification of benzophenone-4 in aqueous cosmetic preparations by hydrophilic interaction thin - layer chromatography followed by densitometry and , alternatively , by uv spectrophotometry . ethyl acetate , ethanol , methanol , potassium phosphate monobasic , and sodium phosphate dibasic were from polskie odczynniki chemiczne ( poch ) , gliwice , poland . hair shampoos containing benzophenone-4 and benzophenone - free ( blank shampoo ) were purchased locally . all shampoos analyzed throughout this study were preserved with sodium benzoate , bronopol , or thiazolinone derivatives and contained sodium laureth sulfate , cocamide dea , dyes ( ci 19140 , ci 42080 , or ci 17200 ) , vitamins , fragrances , and plant extracts ( chamomile , lavender , cornflower , and wild rose ) . uvinul ms40 , 500 mg , was weighed accurately into a 100 ml volumetric flask , dissolved in 16.2 ml of 0.1 mol l aqueous naoh , and diluted to volume with water to give a stock solution i of the concentration 5 mg ml . the appropriate volumes of bz4 stock solution were pipetted to 25 ml volumetric flasks , ph 6 phosphate buffer was added ( 2 ml ) , and water was added to volume to furnish standard solutions of bz4 ( 0.1 , 0.2 , 0.4 , 0.6 , 0.8 , 1.0 , 1.2 , 1.4 , 1.6 , 1.8 , and 2.0 mg ml , bz4 expressed as free acid ) . 2 ml of bz4 stock solution i was diluted to 100 ml with water to furnish a stock solution ii ( 0.1 mg ml ) . the appropriate volumes of stock solution ii were transferred to 25 ml volumetric flasks , ph 6 phosphate buffer ( 5 ml ) was added , and the solutions were diluted to volume with water to furnish solutions of bz4 of concentrations 0.004 , 0.008 , 0.016 , 0.024 , 0.032 , and 0.040 mg ml ( bz4 expressed as free acid ) . 15 mg bz4 was weighed accurately into 10 ml volumetric flasks and diluted with ph 6 phosphate buffer ( 1 ml ) and water to volume . 0.52.5 mg bz4 was weighed accurately into 100 ml flasks and diluted with ph 6 phosphate buffer ( 5 ml ) and water to volume . thin - layer chromatography was performed on 10 10 cm hp quality silica gel 60 plates ( layer thickness 0.2 mm ) from merck or on 10 20 cm standard quality silica gel 60 plates ( layer thickness 0.25 mm ) , also from merck . plates were developed with methanol - dichloromethane 1 : 1 ( v / v ) and dried at room temperature overnight prior to use . standard solutions prepared for method i ( section 2.1 ) were spotted with the desaga as30 sampler equipped with a 10 l syringe ( 1 l spot ) , 15 mm from the from the plate bottom edge , and at 8 mm intervals , starting 10 mm from the plate edge and developed with ethyl acetate - ethanol - water - ph 6 phosphate buffer 15 : 7 : 5 : 1 ( v / v / v / v ) as mobile phase . plates were developed in a vertical chromatographic chamber lined with filter paper and previously saturated with the mobile phase vapor for 20 min . after development , plates were dried at room temperature ( 20c ) , scanned , and analyzed in reflectance mode with the desaga cd 60 densitometer at 285 nm ( rf = 0.78 ) . spectroscopic measurements were performed with lambda 25 uv / vis spectrophotometer , perkin - elmer . ( method ii ) were placed in 1 cm quartz glass cuvettes and scanned over the wavelength range 200420 nm . the shampoo solutions in buffered water ( method i ) prepared as described above ( section 2.2 ) were spotted on silica gel 60 hptlc plates ( 110 l spot depending on the expected bz4 concentration ) . the plates were then chromatographed as described above for bz4 standards ( section 2.3 ) . the shampoo solutions in buffered water ( method ii ) prepared as described above ( section 2.2 ) were placed in 1 cm quartz glass cuvettes and scanned with the lambda 25 uv / vis spectrophotometer as described above ( section 2.4 . ) . the sun - care preparations analyzed in this study contained benzophenone-4 and surfactants , plant extracts , preservatives , and other ingredients that may potentially absorb light within the same range as bz4 . on the basis of our earlier research , it was decided that silica gel 60 is the stationary phase of choice and the most effective mobile phase capable of bz4 separation from other cosmetic ingredients was ethyl acetate - ethanol - water - ph 6 phosphate buffer 14 : 7 : 5 : 1 ( v / v / v / v ) . analytical wavelength suitable for bz4 analysis ( 285 nm ) was selected on the basis of multiwavelength scans obtained for this sunscreen ( figure 2 ) . the cosmetic preparations under investigation contained raw materials other than bz4 ( such as cosmetic dyes ) that may absorb uv light within the same range as bz4 , thus influencing the accuracy of the analysis ( see figure 3 , curves 1 and 2 , solutions of a typical shampoo matrix containing 0% bz4 ) . one of the most convenient methods used to reduce matrix effects in uv / vis spectrophotometry is derivative spectroscopy with the 2nd derivative being the most effective tool capable of matrix effects suppression . after the analysis of 2nd derivative spectra for bz4 standards and the shampoo matrix ( figure 4 ) , it was decided that the analytical wavelength of choice is in this case 285 nm ( at this wavelength , the 2nd derivative of absorbance for the shampoo matrix is very close to 0 even at higher concentrations and for bz4 samples and standards there is a clear maximum of the da / d curve ) . identity of chromatographic spots for bz4 isolated from the shampoo by thin - layer chromatography was tested by comparison of their densitograms with those obtained for the bz4 standard solutions ( figure 5 ) . further investigations of the identity and purity of spots were based on the analysis of the uv spectra of bz4 isolated from the shampoo and the bz4 standards . spectra were collected directly from the chromatographic plates in the reflectance mode ( for each spot three spectra were collected , for the spot center and both edges ; in figure 6 only two spectra are shown for each spot for the sake of clarity ) . the densitometric calibration plot was obtained by plotting peak areas against the amount of bz4 over the range 0.12.0 g spot . the densitometric calibration plot was finally generated in the form of the second - degree polynomial ( table 1 ) and its quality was assessed by means of r values and nonnumerical analysis of residues according to ( figure 7 ) . the spectrophotometric calibration plots were linear within the studied concentration range ( table 1 ) . the general method for linearity testing proposed in this reference is based on the analysis of the ratio of the analytical signal ( y ) to the analyte concentration ( x ) . if the signal - concentration relationship is linear , y / x is constant ( within an acceptable range , usually 5% ) . when this approach was applied to the linear calibration plots generated during this study , all values of signal - to - concentration ratio were within acceptable limits for particular plots ( 45.54 3.3% absorbance and 23.85 2.2% for the 2nd derivative of absorbance , resp . ) . repeatability of chromatographic method was tested according to [ 19 , 20 ] by replicating the entire method ( section 2.5 ) on the same day , using the same cosmetic preparations , batches of solvents , and chromatographic plates , by the same analyst ( day 1 , analysis a and analysis b ) . intermediate precision was verified according to [ 19 , 20 ] by repeating the procedure on the same cosmetic preparations but on a different day , by a different analyst , using other batches of solvents and chromatographic plates ( day 2 ) . the results of these experiments ( table 2 ) prove that the methods precision is sufficient for routine product analysis . the instrumental detection limits ( idl ) and instrumental quantification limits ( iql ) for chromatographic method were determined experimentally on the basis of signal - to - noise ( s / n ) ratio according to . blank solutions of ph 6 phosphate buffer containing zero concentration of bz4 were analyzed in triplicate along with the series of bz4 standards according to the procedure described in section 2.3 . the idl and iql were calculated as the concentrations that yielded peaks with s / n of 3 and 10 , respectively . the results of these determinations expressed as the amount of bz4 per spot are given in table 1 . in order to estimate the method loq , the minimum concentration of bz4 that can be realistically quantified in the actual cosmetic preparations , the blank shampoo samples were fortified with bz4 at the following concentrations : 0.01 , 0.02 , 0.04 , and 0.06% ( w / w ) , diluted with ph 6 phosphate buffer and analyzed in triplicate as described in section 2.5 . in order to avoid excessive viscosity and foaming of the sample solutions , their maximum concentration was 100 mg ml and due to the capacity of the applicator syringe , the maximum application volume was 10 l . as the result of these experiments , it was estimated that the lower quantification limit for bz4 in shampoos by thin - layer chromatography / densitometry without samples preconcentration is ca . instrumental limits of detection / quantification ( idl and iql ) were calculated from the signal - to - noise ratio ( table 1 ) and the limit of quantification ( loq ) for the entire 2nd derivative spectroscopic method was studied on the basis of the same shampoo samples containing 0.01 , 0.02 , 0.04 , and 0.06% bz4 ( w / w ) . samples were diluted with ph 6 phosphate buffer and water ( 10 mg ml , higher concentrations should be avoided because of increasing matrix effects ) and the estimated loq in shampoos was ca . after due consideration of factors that can influence the analysis results , it was concluded that the critical points are the quality of chromatographic plates ( hptlc versus tlc ) , the method of spotting , and the ph of the mobile phase . the same cosmetic preparations were analyzed on hptlc silica gel 60 chromatographic plates with automatic spotting and on standard tlc silica gel 60 plates with manual spotting with a hamilton microsyringe . the mobile phase ethyl acetate - ethanol - water - ph 6 phosphate buffer 15 : 7 : 5 : 1 ( v / v / v / v ) was replaced with ethyl acetate - ethanol - water 15 : 7 : 6 ( v / v / v ) . the results of these analyses ( table 2 ) are similar but coefficients of variations are , as it may have been expected , slightly higher for manual spotting . blank shampoo was spiked with bz4 at five concentrations : 0.5 , 1.0 , 1.5 , 2.0 , and 5.0% ( w / w ) corresponding to 0.25 , 0.50 , 0.75 , and 1.0 g spot ( 1 l spot of the shampoo solution prepared according to section 2.2 , 50 mg of shampoo per 1 ml of sample solution ) and 1.25 g spot ( 1 l spot of the shampoo solution prepared according to section 2.2 , 25 mg of shampoo per 1 ml of sample solution ) , respectively . the chromatographic procedure described in section 2 was performed on the samples and the recoveries are presented in table 3 . spectrophotometric determination of bz4 in the same spiked shampoos was performed according to section 2.6 ( sample solutions were prepared according to section 2.3 , 2.5 or 1.0 mg of shampoo per 1 ml of sample solution ) and the results are given in table 3 . standard solutions of bz4 used in this investigation were refrigerated between the experiments and not exposed to light except for time needed for plates spotting . the stability of all solutions was in these conditions excellent over at least two weeks . benzophenone-4 may be effectively separated from other cosmetic ingredients ( dyes , preservatives , vitamins , and plant actives ) by hydrophilic interaction high performance thin - layer chromatography on silica gel 60 with ethyl acetate - ethanol - water - ph 6 phosphate buffer as mobile phase . densitometric quantification of bz4 at 285 nm is fast , reliable , and cost - effective and it may be recommended for routine analysis of shampoos containing bz4 at different levels of concentrations ( concentrations of the shampoo solutions and volumes per spot may be adjusted to the needs over a relatively broad range of bz4 concentration in a cosmetic formulation ) . the results of chromatographic / densitometric determination ( precision , accuracy ) are superior to those of spectrophotometric analysis by zero derivative spectroscopy and similar to those obtained by 2nd derivative spectroscopic method but the flexibility of the chromatographic method is higher ; the matrix effects do not pose a problem and the limit of quantification is slightly lower than that of the spectrophotometric analysis .

background . exercise has an anti - inflammatory effect against , and immune cells play critical roles in the development , of insulin resistance and atherosclerotic vascular disease ( avd ) . thus , the goal of this study was to determine whether exercise improves insulin sensitivity in insulin - resistant subjects by downregulating proinflammatory signaling in immune cells . methods . seventeen lean , 8 obese nondiabetic , and 11 obese type 2 diabetic individuals underwent an aerobic exercise program for 15 days and an insulin clamp before and after exercise . peripheral mononuclear cells ( pmnc ) were obtained for determination of toll - like receptor ( tlr ) 2 and 4 protein content and mitogen - activated protein kinase phosphorylation . results . compared with that in lean individuals , tlr4 protein content was increased by 4.2-fold in diabetic subjects . this increase in tlr4 content was accompanied by a 3.0-fold increase in extracellular signal - regulated kinase ( erk ) phosphorylation . exercise improved insulin sensitivity in the lean , obese , and type 2 diabetes groups . however , exercise did not affect tlr content or erk phosphorylation . conclusions . tlr4 content and erk phosphorylation are increased in pmnc of type 2 diabetic individuals . while exercise improves insulin sensitivity , this effect is not related to changes in tlr2/tlr4 content or erk phosphorylation in pmnc of type 2 diabetic individuals .

regular exercise is fundamental for the prevention and management of insulin - resistant disorders such as type 2 diabetes mellitus ( t2 dm ) and avd . while exercise affects numerous organ systems , there is increasing evidence that acute and chronic exercises have important immunomodulatory properties which may contribute to the beneficial effects of exercise on metabolism and cardiovascular health . in addition , aerobic exercise can alter the number and the function of immune cells in innate and adaptive immunities [ 46 ] and reduce the levels of proinflammatory markers . numerous reports have demonstrated that immune cells play a critical role in the development of insulin resistance . hevener et al . showed that deletion of peroxisome proliferator - activated receptor gamma ( ppar ) in monocytes results in glucose intolerance and decreased insulin signaling in the skeletal muscle , liver , and adipose tissue of mice . another study demonstrated that regulatory cd4 t cells expressing the transcription factor foxp3 ( treg cells ) are predominately found in the abdominal fat of normal mice but are reduced in the abdominal fat in mouse models of insulin resistance . this study also showed that interleukin-10 ( il-10 ) , produced by these foxp3 treg cells , inhibited tumor necrosis factor - alpha ( tnf)-induced expression of proinflammatory mediators and insulin resistance in 3t3-l1 adipocytes . taken together , these findings suggest that immune cells have the potential to affect whole body glucose homeostasis and insulin action . in addition to their role in insulin resistance , immune cells are also involved in the development of atherosclerosis . in t2 dm patients , atherosclerosis is systemic , rather than localized to the heart , and also affects the microvasculature of the limbs , kidneys , eyes , and other tissues . monocytes play crucial roles in the early events that lead to this systemic atherosclerosis . yet , the molecular mechanisms that promote accelerated atherosclerosis in type 2 diabetic subjects are not fully understood . accumulating evidence implicates toll - like receptor ( tlr ) 2 and 4 in the pathogenesis of atherosclerosis and insulin resistance [ 1316 ] . for example , monocytes from type 2 diabetic subjects have elevated surface protein content of tlr2 and tlr4 . moreover , deletion of tlr2 or tlr4 in rodents attenuates atherosclerosis and insulin resistance [ 14 , 15 , 18 , 19 ] . in view that immune cells have a critical role in the pathophysiology of insulin resistance and avd , in this study , we examined the molecular mechanism underlying the anti - inflammatory effect of aerobic exercise on immune cells of type 2 diabetic individuals . although some studies have shown that either aerobic or resistance exercise induces a decrease in tlr4 expression in monocytes from healthy normal subjects [ 4 , 20 , 21 ] , it is unclear whether aerobic exercise can modulate proinflammatory signaling pathways in immune cells of insulin - resistant subjects . it is well documented that aerobic exercise can improve insulin sensitivity in type 2 diabetic patients . however , no study has described whether the improvement of insulin sensitivity is due to a decrease in proinflammatory signaling pathways in immune cells of type 2 diabetic subjects . we focused our analysis on pmnc because of the considerable evidence indicating that these inflammatory cells infiltrate insulin - sensitive tissues ( skeletal muscle , adipocytes , and liver ) where they promote insulin resistance [ 2224 ] and are also involved in the pathophysiology of avd . therefore , we hypothesized that aerobic exercise downregulates proinflammatory signaling pathways in pmnc of insulin - resistant individuals . we studied 17 lean , 8 obese nondiabetic , and 11 obese type 2 diabetic participants . three t2 dm subjects were newly diagnosed , and one t2 dm subject was diagnosed with diabetes 2 months prior to the study . two t2 dm subjects took a sulfonylurea , which was stopped 2 days before any study procedure . all participants were sedentary ( zero or one exercise bout per week ) and had stable body weight ( 1 kg ) for three months before the study . each participant underwent a medical history , physical examination , and a 75 g oral glucose tolerance test ( ogtt ) . lean and obese participants did not have a family history of type 2 diabetes and were normal glucose tolerant . no participant was taking any medication known to affect glucose metabolism ( other than sulfonylureas ) . the study was approved by the institutional review board of the university of texas health science center at san antonio , and all participants gave written consent . plasma glucose , insulin , and nonesterified fatty acids ( nefa ) were measured 30 , 15 , and 0 minutes before and every 15 min for 2 h after the ingestion of 75 g of glucose . within 37 days after the ogtt , vo2peak was determined using a cycle ergometer and a metabolic measurement system ( sensormedics , savi park , ca , usa ) as previously described . within 1 - 2 weeks after the baseline vo2peak measurement , participants returned to the clinical research center at 07:00 am to undergo a 180 min euglycemic - hyperinsulinemic ( 160 mu m min ) clamp study as previously described . within one week of the insulin clamp , participants undertook a supervised exercise program of cycle ergometer exercise for 40 min per day , for 15 consecutive days . the 40 min of exercise consisted of 4 identical 10 min periods comprised of 8 min of exercise at 70% vo2peak followed by 2 min at 90% of vo2peak . each of these 10 min sets was followed by 2 min of complete rest . we employed this program because , during pilot experiments , we observed increases in vo2peak of ~20% . blood samples were collected immediately before and at the end of the pre- and postexercise insulin clamps for isolation of pmnc . louis , mo , usa ) density gradient , followed by centrifugation at 800 g for 30 min at room temperature . mononuclear cell ( 1 10cells ) samples were lysed in ice - cold lysis buffer ( ph 7.4 ) , and western blot analyses were performed as previously described . after blocking with bovine serum albumin , the membranes were incubated overnight with primary antibody against tlr4 ( santa cruz biotechnology , santa cruz , ca , usa ) and tlr2 ( ebioscience , san diego , ca , usa ) , which are cell surface receptors that signal through erk and c - jun amino - terminal kinase ( jnk ) . we also performed western blotting for phosphorylated erk ( invitrogen , carlsbad , ca , usa ) , erk ( cell signaling , danvers , ma , usa ) , phosphorylated jnk ( cell signaling , beverly , ma , usa ) , and jnk ( cell signaling , beverly , ma , usa ) . bound primary antibodies were detected with a secondary antibody ( anti - rabbit immunoglobulin - horseradish peroxidase - linked antibody ) using enhanced chemiluminescence reagents . bands were quantitated with imagequant ( ge healthcare , piscataway , nj , usa ) . nfb p65 binding was measured using an elisa kit ( active motif , carlsbad , ca , usa ) , as previously described . plasma insulin ( diagnostic products , los angeles , ca , usa ) was measured by radioimmunoassay . glucose was measured by the glucose oxidase method using a beckman glucose oxidase analyzer , and hemoglobin a1c ( hba1c ) was measured using a dca2000 analyzer ( bayer , tarrytown , ny , usa ) . plasma nefa concentrations were determined using a colorimetric method ( wako , richmond , va , usa ) . high - sensitivity - c - reactive protein ( hs - crp ) was measured by elisa ( alpco diagnostics , salem , nh , usa ) . endothelin-1 and soluble intercellular adhesion molecule-1 ( sicam-1 ) plasma levels were measured by elisa ( r&d systems , minneapolis , mn , usa ) . baseline comparisons between groups were done using one - way anova , and the effect of exercise training and insulin was analyzed using anova with repeated measures followed by the tukey test . sigmastat version 3.5 ( systat software , san jose , ca , usa ) was used for statistical analysis . table 1 shows the characteristics of study participants before and after the 15-day aerobic exercise training program . compared with the lean group , the obese and type 2 diabetic subjects had higher body mass index ( bmi ) and body weight . the obese group tended to have elevated plasma nefa concentrations compared with the lean group ( p = 0.056 ) . both diabetic and obese subjects had higher fasting insulin levels when compared with the lean group . the type 2 diabetic patients had higher levels of the inflammatory and endothelial markers hs - crp , endothelin-1 , and sicam-1 . diabetic and obese individuals were insulin resistant compared to the lean controls , based on the lower total glucose disposal during the insulin clamp . after the exercise training , the insulin - resistant individuals ( obese and t2 dm groups ) did not show a significant decrease in bmi or weight . similarly , there was no change in hba1c , nefa , glucose , and insulin plasma levels in the obese and diabetic groups . exercise also did not decrease the plasma levels of hs - crp , endothelin-1 , and sicam-1 . after exercise , vo2peak increased by 26% ( p < 0.05 ) , 14% ( p = 0.08 ) , and 8% ( p the baseline m levels were 10.9 0.7 , 7.8 0.8 , and 4.2 0.6 mg / kgmin in lean , obese , and type 2 diabetic individuals , respectively . after exercise , insulin sensitivity ( m values ) increased by 11% ( p < 0.05 ) in lean , 15% ( p < 0.05 ) in obese , and 32% ( p < 0.05 ) in type 2 diabetes groups . compared with lean participants , baseline tlr4 protein content was significantly increased in the pmnc of diabetic subjects by 4.2-fold ( p < 0.05 ) ( figure 1 ) . in obese individuals , tlr4 also tended to be elevated by 2.7-fold ( p = 0.07 ) compared to that of the individuals in the lean group ( figure 1 ) . in contrast to tlr4 , the protein content of tlr2 was not different in lean , obese , and diabetic subjects ( figure 2 ) . as shown in figure 3 , baseline erk phosphorylation was significantly elevated in the diabetic subjects . the increase in erk phosphorylation was mainly observed with erk2 ( 3.0-fold versus the lean group ; p < 0.05 ) . erk2 phosphorylation tended to be decreased in type 2 diabetic subjects after exercise ( p = ns ) . we performed these measurements just before the end of the 180 min clamp , based on previous studies that showed that insulin activation of erk occurs as late as 100240 min during a clamp [ 27 , 28 ] . it is possible , however , that insulin could have activated erk if measurements had been done at earlier time points . nfb p65 dna binding was not different between lean , obese , and type 2 diabetic participants , and neither exercise nor insulin affected nfb dna binding ( figure 5 ) . exercise training studies and cross - sectional comparisons between physically active and inactive individuals have shown a reduction in the inflammatory response to lps and lower tlr4 cell surface expression in monocytes from physically active subjects [ 20 , 21 ] . because immune cells play a role in the regulation of glucose metabolism [ 8 , 29 ] , we examined whether exercise improves insulin sensitivity by inducing modifications in the pro - inflammatory signaling pathways of the pmnc of insulin - resistant subjects . we anticipated that short - term training would improve insulin sensitivity in insulin - resistant individuals and that this would then be associated with a decrease in tlr4 protein content and erk signaling in pmnc . yet , acute aerobic exercise , which improved insulin sensitivity , did not affect tlr4 protein content , although it tended to attenuate erk2 phosphorylation . it is possible , however , that the 15-day intervention was not sufficient to significantly alter tlr4 protein content and erk phosphorylation . on the other hand , others have reported that a single session of cycle exercise ( 1.52 h long ) is sufficient to decrease tlr4 cell surface expression in monocytes from healthy individuals , although tlr - downstream signaling ( erk ) was not examined in that study . the apparently discrepant results between the present and previous studies may be related to differences in the duration of training , the type of training being performed ( aerobic , resistance , or combination ) , the intensity of the training ( moderate versus high ) , and/or the population examined . timmerman et al . tested the effect of combined aerobic and resistance training on monocyte subpopulations and found that tlr4 expression was reduced in proinflammatory ( cd1416 ) but not in classical proinflammatory monocytes adhere more avidly to activated endothelial cells and are precursors to the cd16 macrophages which are distributed throughout atherosclerotic lesions . we can not rule out that the exercise intervention employed in the present study could have led to changes in tlr4 protein content and erk signaling in specific monocyte subpopulations . in this study , we found that insulin - resistant individuals have elevated tlr4 protein content in pmnc . our findings are in agreement with previous studies where tlr levels were found to be increased in inflammatory cells from type 1 and type 2 diabetic subjects , although , in contrast to the present study , subjects had increased levels of both tlr2 and tlr4 [ 17 , 33 ] . the physiologic relevance of the increased tlr4 protein content is underscored by studies in which tlr4 was ablated specifically in hematopoietic cells from mice , an intervention which reduced inflammatory markers in liver and adipose tissue and ameliorated high - fat diet - induced insulin resistance . moreover , our results also demonstrate that the upregulation in tlr4 protein levels is accompanied by an increase in erk phosphorylation , especially erk2 . studies conducted in ob / ob - erk1 double knockout mice showed that erk deletion improves insulin sensitivity in skeletal muscle and reduced liver fat content . defining the role of erk2 on insulin resistance future studies of tissue - specific erk2 ablation will help to clarify its role in the pathogenesis of insulin resistance and type 2 diabetes . avd is the major cause of mortality in subjects with type 2 diabetes and insulin resistance . atherosclerosis occurs earlier in the natural course of diabetes and is more severe and generalized than in nondiabetic patients . to test whether tlr4 has a direct role in the pathogenesis of atherosclerosis , michelsen et al . crossed apolipoprotein e ( apoe ) deficient mice , which are prone to develop atherosclerotic lesions , with mice that are null for tlr4 . this group demonstrated that mice deficient in both tlr4 and apoe have a significant reduction in the size of the atherosclerotic lesions , as well as a lower number of macrophages infiltrating these lesions , compared with mice deficient with apoe only . more recently , it was reported that hematopoietic cell ablation of tlr4 reduces arterial wall macrophage infiltration and atherosclerotic lesion areas . erk also has a crucial role in the early steps in the pathogenesis of atherosclerosis because activation of erk leads to increased monocyte proliferation , migration , and differentiation into macrophages and mediates monocyte chemotactic protein-1-induced monocyte adhesion . indeed , ( monocyte - derived ) dendritic cells from patients with acute coronary syndrome in addition , a recent study demonstrated that hypertensive patients have increased erk phosphorylation in isolated white blood cells compared to healthy normotensive individuals , suggesting that erk activation in leukocytes may serve as a biomarker for hypertension . collectively , the results from these studies and our present findings strongly suggest that enhanced signaling through tlr4 and erk , in inflammatory cells ( monocytes / macrophages ) , plays an important role in the pathogenesis of atherosclerosis . tlr4 protein content and erk signaling were elevated in the type 2 diabetes group , which were slightly but significantly older than the lean and obese groups . based on previous reports from human and animal studies [ 21 , 43 , 44 ] , we believe that the increase in tlr4 protein content and erk signaling is not due to the small difference in age . in fact , stewart et al . compared tlr2 and tlr4 cell surface expression on monocytes from older adults ( age 6085 ) with that from young adults ( age 1835 ) and found no independent effect of age on tlr2 and tlr4 levels and lps - induced cytokine production . furthermore , animal studies evaluating tlr levels and signaling in macrophages of old mice have yielded inconsistent findings . a study performed in young and old mice found that aged mouse macrophages have lower tlr4 protein . in contrast , others have not observed differences in tlr4 cell surface expression in macrophages from aged versus young mice . in this study , we did not observe differences in jnk phosphorylation between groups . in contrast , studies in mice suggest that jnk plays an important role in the pathogenesis of obesity and insulin resistance . to the best of our knowledge , most of the human studies supporting the role of jnk in insulin resistance have examined adipose tissue [ 4648 ] . moreover , our finding is in line with that of a previous study showing that jnk activity in pmnc was not associated with impaired insulin action , whereas increased jnk activity in adipose tissue directly correlated with measurements of insulin resistance . studies of nfb activity in inflammatory cells from insulin - resistant subjects have yielded contrasting results . in line with a study by patel et al . , we did not observe differences in nfb activity in pmnc in insulin - resistant subjects . in contrast , ghanim et al . and dasu et al . reported increased nfb activity in pmnc from obese individuals and monocytes ( a subpopulation of pmnc ) from type 2 diabetic subjects , respectively [ 17 , 50 ] . studies carried out in larger cohorts of subjects might help to clarify whether nfb signaling is up regulated in pmnc from obese and type 2 diabetic individuals and whether this alteration is specific to monocytes . in summary , acute aerobic exercise improved insulin sensitivity and cardiovascular fitness in insulin - resistant subjects . however , exercise was not associated with changes in tlr4 protein content and erk signaling in immune cells . it remains to be determined whether longer training protocols or different modes of exercise ( i.e. , resistance training or combination of aerobic and resistance training , as well as moderate- versus high - intensity exercise program ) affect tlr4 expression and erk signaling in immune cells of insulin - resistant individuals .

a 73-year - old man who had been transferred to our emergency room due to sudden chest pain was diagnosed with st - segment elevation myocardial infarction ( stemi ) . primary percutaneous coronary intervention was performed . a long , white object which looked like a parasitic worm was retrieved via intracoronary aspiration and revascularization was successfully completed . contrast computed tomography revealed a huge 76 cm mass in the right upper pulmonary lobe with direct pulmonary vein invasion . histopathologic examination of the aspirated coronary object revealed pleomorphic lung carcinoma . this is an unusual case of stemi caused by lung tumor embolization via direct pulmonary vein invasion to the left side of the heart .

the taccalonolides are microtubule stabilizers isolated from plants of the genus tacca . taccalonolide af is 231 times more potent than the major metabolite taccalonolide a and differs only by the oxidation of the c-22,23 double bond in a to an epoxy group in af . in the current study , 10 other rare natural taccalonolides were epoxidized and in each case epoxidation improved potency . the epoxidation products of taccalonolide t and ai were the most potent , with ic50 values of 0.43 and 0.88 nm , respectively . these potent taccalonolides retained microtubule stabilizing effects , and t - epoxide demonstrated antitumor effects in a xenograft model of breast cancer . additional reactions demonstrated that reduction of the c-6 ketone resulted in an inactive taccalonolide and that c-22,23 epoxidation restored its activity . these studies confirm the value of c-22,23 epoxidation as an effective strategy for increasing the potency of a wide range of structurally diverse taccalonolide microtubule stabilizers .

paclitaxel , the first microtubule stabilizer identified from the taxus brevifolia , is one of the most successful anticancer drugs currently used in the clinic . members of a second class of plant - derived microtubule stabilizers , the taccalonolides , have been isolated from a variety of tacca species . the taccalonolides are highly acetylated hexacyclic steroid lactones that exhibit effects similar to other microtubule stabilizers in that they increase the density of cellular microtubules , interrupt mitotic progression , and consequentially lead to the apoptosis of cancer cells . despite their low antiproliferative potencies in vitro , taccalonolides a and e were found to be potent and effective antitumor agents in vivo(13,14 ) with the ability to circumvent multiple mechanisms of drug resistance , including mutations in the taxane binding site and the expression of mrp7 , iii - tubulin , and p - glycoprotein ( pgp ) . the recent isolation of taccalonolides with low nanomolar potency facilitated biochemical and structural studies demonstrating that the taccalonolides bind to tubulin covalently and impart unique interprotofilament stability to microtubules . the most potent of these rare natural taccalonolides , af ( 1 ) , contains an epoxy group at c-22,23 that results in a 231-fold increase in potency compared with taccalonolide a ( 2 ) , which contains a double bond at this site . taccalonolide af is an effective antitumor agent that causes tumor regression in the mda - mb-231 breast cancer xenograft model , albeit with a narrow therapeutic window . a simple and efficient method was developed to semisynthetically epoxidize the c-22,23 double bond in taccalonolides a and b ( 3 ) to generate taccalonolides af and aj ( 4 ) , respectively . taccalonolide aj has an ic50 value of 4.2 nm , which is 734-fold more potent than the parent molecule , taccalonolide b , suggesting that epoxidation of the c-22,23 double bond is an effective way to increase the potency of this class of molecules . in the current study , the c-22,23 double bond in an additional 10 rare natural microtubule stabilizing taccalonolides was epoxidized , including the newly isolated taccalonolide ai ( 5 ) . our results demonstrate that this modification increases the potency of each taccalonolide , in some cases leading to subnanomolar potency . y , were previously isolated from various tacca sp . by multiple investigators . we have continued to search for additional rare natural taccalonolides and conduct semisynthetic reactions to fully understand the sar of this class of compounds with the goal of identifying taccalonolides with optimal properties for consideration for clinical development . the potent semisynthetic 4 is the c-22,23 epoxidation product of the naturally occurring 3 . in the process of isolating highly purified 3 from the roots and rhizomes of t. chantrieri its molecular formula ( c35h48o11 ) was determined by high resolution electrospray ionization mass spectroscopy ( hresims ) , 645.3268 [ m + h ] ( calcd for c35h49o11 645.3269 ) , and nmr data . the h nmr spectrum of 5 showed characteristics of the taccalonolide backbone , including four methyl singlets at 1.67 ( s , 3h ) , 1.36 ( s , 3h ) , 0.82 ( s , 3h ) , and 0.76 ( s , 3h ) , one methyl doublet at 0.95 ( d , j = 7.0 hz , 3h , 21-ch3 ) , one acetyl at 2.08 ( s , 3h ) , epoxyl signals at 3.55 ( t , j = 4.0 hz , 1h , h-2 ) and 3.40 ( br , 1h , h-3 ) , and an olefin singlet at 5.02 ( br , 1h , h-22 ) . the upfield shift of h-15 at 4.38 ( ddd , j = 9.5 , 8.3 , 2.5 hz , 1h ) indicated a 15-oh similar to 3 and 6 . the only acetoxy group was assigned to c-12 due to the chemical shift of h-12 at 4.99 ( t , j = 2.8 hz , 1h ) and the hmbc correlation between h-12 and the acetoxy carbon at 169.3 . the downfield chemical shift of h-1 at 4.59 ( d , j = 5.2 hz , 1h ) required an acyloxy substitution at c-1 . this acyloxy group was determined to be an isovaleryloxy by the h nmr signals at 2.18 ( m , 2h , h-2 ) , 2.14 ( m , 1h , h-3 ) , 1.01 ( d , j = 7.0 hz , 3h , h-4 ) , 1.00 ( d , j = 6.0 hz , 3h , h-5 ) , and 2d nmr correlations . the hmbc correlations between h-1 and the carbonyl carbon of the isovaleryloxy at 171.8 confirmed this substitution . structures of taccalonolides af , a , b , aj , ai , and n. compound 5 retains the microtubule stabilizing activity of other taccalonolides and has antiproliferative activity with an ic50 of 47 nm in hela cells , which makes it one of the most potent natural taccalonolides isolated to date ( table 1 ) . the potency of this taccalonolide is consistent with previously determined sar , which indicates that a large bulky group at c-1 is optimal for activity . the bulky isovaleryloxy group at c-1 is the only difference between 5 and 6 , which has an acetoxy at this site and is approximately 200-fold less potent ( table 1 ) . additionally , the only difference between 5 and taccalonolide am is the absence of the c-5 hydroxyl in 5 , which confers a 42-fold increase in potency ( table 1 ) . therefore , the combination of modifications at c-1 and c-5 appear to be important for the potency of 5 . considering the multiple fragile functional groups present in the taccalonolides and small quantity of natural taccalonolides available ( less than 1 mg in some cases ) , a mild and efficient epoxidation method was needed . dimethyldioxirane ( dmdo ) can rapidly epoxidize alkenes under neutral and mild conditions , and it is also well suited for the synthesis of sensitive epoxides of enol esters and enol lactones , as are present in the taccalonolides . the reaction is highly efficient , generally furnishing the desired epoxides in almost quantitative yield . it is also very convenient , and in most cases pure products are obtained after evaporation of the solvent . due to the dramatic increases in potency conferred by the epoxidation of the c-22,23 double bond in 2 and 3 to generate 1 and 4 , we applied this method to epoxidize a wide variety of other natural taccalonolides : e ( 7 , 1.15 mg ) , n ( 6 , 1.75 mg ) , r ( 8 , 1.44 mg ) , t ( 9 , 2.1 mg ) , z ( 10 , 0.74 mg ) , aa ( 11 , 1.0 mg ) , ab ( 12 , 0.94 mg ) , ad ( 13 , 1.28 mg ) , ai ( 5 , 0.62 mg ) , and an ( 14 , 0.65 mg ) ( see scheme 1 ) . most of these natural taccalonolides are very rare with estimated content in the plant at ppm levels or less . only the epoxy was obtained as determined by the small coupling constant between h20/h22 . we hypothesize that this is a result of spatial strains of the , axial orientation of the 27-ch3 . the antiproliferative potency of each c-22,c23 epoxy - taccalonolide was evaluated in hela cells and compared with the ic50 of the parent compound ( table 1 ) . in each case , c-22,23 epoxidation resulted in an increase in potency with an over 200-fold improvement in potency observed for 5 of the 10 new epoxy - taccalonolides ( table 1 ) . remarkably , the c-22,23 epoxidation of 9 and 5 resulted in the generation of the first ever compounds of this class with subnanomolar potencies of 0.45 and 0.88 nm , respectively . this makes 18 and 23 more potent than paclitaxel , which had an ic50 of 1.2 nm in this assay . the fold increase in potency achieved by c-22,23 expoxidation is shown in brackets in the last column . in addition to their potent antiproliferative effects , each epoxidized taccalonolide caused interphase microtubule bundling in hela cells . these effects are depicted in figure 2 for the two most potent taccalonolides , 18 and 23 , and representative images of the cellular microtubule stabilizing effects of other , less potent epoxy taccalonolides are shown in the supporting information . in addition to their microtubule stabilizing effects in cells , both 18 and 23 enhanced the polymerization of purified porcine brain tubulin in turbidimetric assays ( figure 3 ) . similarly to other potent taccalonolides , including 1 and 4 , they enhanced the extent of tubulin polymerization compared with vehicle controls without affecting the time required to initiate tubulin polymerization , a feature that makes this class of microtubule stabilizers distinct from other microtubule stabilizers that bind to the paclitaxel or laulimalide binding sites . microtubules were visualized by immunofluorescence using a -tubulin antibody after treatment of hela cells for 18 h with ( a ) vehicle ( etoh ) , ( b ) 4 nm 18 , or ( c ) 10 nm 23 . purified porcine brain tubulin was incubated with 6 m 23 , 0.7 m 18 , or etoh vehicle , and microtubule polymerization was monitored turbidimetrically after shift to 37 c . despite the fact that 9 and 5 are rare natural products , both semisynthetic c-22,23 epoxidation products 23 and 18 antitumor studies could be performed with small quantities of material due to the exquisite potency of the taccalonolides in vivo.(13,14,16 ) the ability of taccalonolides to covalently bind to microtubules likely contributes to their in vivo potency . the low doses of taccalonolides needed to observe effects in vivo allow them to be diluted in aqueous solvents , in this case less than 10% etoh in phosphate buffered saline ( pbs ) . the antitumor efficacies of 18 and 23 were compared with nontreated tumors as a negative control and three doses of 15 mg / kg paclitaxel administered on days 0 , 3 , and 7 as a positive control for antitumor activity . individual doses of 0.25 mg / kg 18 were administered twice in the first week ( days 0 , 3 ) based on preliminary dose tolerance studies that showed a total dose of 0.5 mg / kg was acceptable . no additional drug was administered due to an average 10% body weight loss observed on day 7 . despite the low total dose administered ( 0.5 mg / kg ) , tumor growth was completely inhibited through day 7 ( figure 4 ) . during days 714 , the mice gradually recovered to 4% body weight loss while significant antitumor effects were sustained for over a week after the final dose was administered ( figure 4 ) . by day 17 , the mice had fully recovered from drug - induced weight loss and some antitumor effects persisted . one lethality was encountered on day 21 , which was 18 days after the final dose and after a full recovery of body weight loss ; it is therefore unclear whether this was a drug - related toxicity . as has been noted for other taccalonolides , 18 has a narrow therapeutic window based on a concurrent study where two doses of 0.375 mg / kg administered on days 0 and 3 ( 0.75 total dose ) resulted in an lc40 with 2 of the 5 mice succumbing 811 days after the final dose . despite this narrow therapeutic window , the ability of a total dose of 0.5 mg / kg 18 to produce antitumor effects highlights the exceptional in vivo potency of the taccalonolides . taccalonolide 23 was administered at 0.75 mg / kg on days 0 , 3 , and 7 for a total dose of 2.25 mg / kg . only slight antitumor effects were observed , but dosing could not be increased due to a limited amount of material , which was fully expended on day 7 . although 23 was only 2-fold less potent than 18 in vitro , no significant antitumor effects or weight loss were observed with 23 at a dose 3-fold higher than the dose of 18 that produced antitumor effects . although we can not rule out the possibility that 23 may have antitumor efficacy at higher concentrations , these results highlight that in vitro potency can not be used as a sole predictor of in vivo efficacy , even when drugs of the same class are being compared . these results are consistent with studies showing that 1 had excellent antitumor effects while 4 did not . in vivo efficacy of a cumulative total dose of 2.25 mg / kg 23 or 0.5 mg / kg 18 in an mda - mb-231 xenograft model of triple negative breast cancer compared with a cumulative total dose of 45 mg / kg paclitaxel . the role of this carbonyl group in the microtubule stabilizing activity of the taccalonolides is unknown . reduction of this carbonyl group with nabh4 resulted in two new semisynthetic products , 25 and 26 , that were isolated with respective 2% and 9% yields after hplc purification ( scheme 2 ) . due to the spatial strain of the orientation of both 18-ch3 and 7-oh , we hypothesize that the hydride attached to the carbonyl from the face resulting in the 6 reduction products of 25 and 26 . compound 25 was obtained as white powder , and a molecular formula of c34h46o13 was deduced from the hrms , 663.3021 ( calcd for c34h47o13 663.3011 ) . the h nmr showed signals only for three acetyl methyl groups , suggesting the loss of one acetyl group . the chemical shift of h-15 at 4.39 ( t , j = 8.7 hz ) , ca . 1.1 ppm higher than that of taccalonolide a , indicated that the acetyl group at 15-oh was lost . this is consistent with previous studies showing that the c-15 acetoxy can easily be hydrolyzed . the c-6 carbonyl signal was also lost , and instead , signals for a hydroxymethine at h 3.80 ( br , h-6 ) and c 73.5 ( c-6 ) were observed , indicating the successful reduction of the c-6 carbonyl group . the orientation of the 6-oh was determined to be ( equatorial ) due to the small coupling constant of h-6 . thus , 25 is a product of the reduction of the c-6 carbonyl and the hydrolysis of the c-15 acetoxyl group . the molecular formula of c36h48o14 was determined by hrms , 705.3137 ( calcd for c36h49o14 705.3168 ) , corresponding to the reduction of a carbonyl group . resonance at 4.37 , suggested the hydrolysis of this acetyl group to give 15-oh . interestingly , this acetyl group appears to shift to the newly generated c-6 hydroxyl group based on the h 5.07 ( br , h-6 ) , c 76.7 ( c-6 ) , and cosy correlations between h-6/h-5 ( 2.05 , m ) , h-6/h-7 ( 3.79 , dd , j = 9.5 , 2.5 hz ) , and h-7/h-8 ( 1.94 , m ) . the configuration of 6-oac was determined to be ( equatorial ) due to the small coupling constant of h-6 . we hypothesize that the migration of the acetyl group from 15-oh to 6-oh might have occurred through two steps ( see scheme 3 ) . the 15-acetyl group could have first migrated to 7-oh since they are very close and the 15-acetoxy is prone to loss of the acetyl group as demonstrated previously . then the newly formed alkoxide from the reduction of the 6-ketone could have attached to the carbonyl carbon of the 7-oac group to yield 26 , the 6-oac product . both 25 and 26 were found to have no antiproliferative effects at concentrations up to 50 m , suggesting the importance of the c-6 ketone for activity . similarly to the results observed in table 1 , c-22,23 epoxidation of 26 resulted in an over 300-fold improvement of potency to generate an active taccalonolide , 27 , with an ic50 of 163 10 nm . the newly isolated taccalonolide 5 and the known taccalonolide 9 showed relatively potent antiproliferative activities with ic50 values of 47 and 335 nm , respectively , which are 181- and 39-fold more potent than 6 and 8 . the only difference between taccalonolides 5/6 and 9/8 is that both 5 and 9 have an isovaleryloxy group at c-1 , while 6 and 8 have an acetyloxy group at c-1 . this result supports previous assertions that a bulky substitution at c-1 is preferred for the antiproliferative activity of the taccalonolides . epoxidation of the c-22,23 double bond significantly increased the potency of every taccalonolide analyzed . taking into account 1 and 4 ( the epoxy products of 2 and 3 , respectively ) , epoxidation of the c-22,23 double bond resulted in an over 200-fold increase in potency for 7 of the 12 natural taccalonolides tested ( table 1 ) . although these data show that a c-22,23 epoxide is optimal for the potency of a wide range of taccalonolides , further information can be gleaned from the relative effect that this epoxidation has on activity . two taccalonolides , 10 and 11 , showed relatively modest 7- and 2-fold increases in potency , respectively , after epoxidation . however , it is important to note that these two taccalonolides were relatively potent before epoxidation ( 120 and 32 nm , respectively ) and that their epoxidized forms are some of the most potent taccalonolides identified to date ( table 1 ) . therefore , it appears that there may be a maximum potency that can be conferred to some taccalonolides by epoxidation . the notable outliers to this are the epoxidized forms of 9 and 5 , which each contain a bulky isovaleryloxy group at c-1 and are the first taccalonolides to show subnanomolar potency . together , these results suggest that the combination of a c-22,23 epoxide with a bulky c-1 modification is optimal for taccalonolide potency . taccalonolides 13 and 14 also showed only modest increases of 24-fold in potency after c-22,23 epoxidation . these compounds differ from those previously mentioned in that even the epoxidized forms , 22 and 24 , have only moderate potencies , between 685820 nm . therefore , although epoxidation increased the potency of these taccalonolides , the resulting activities remained modest . we hypothesize that these nonoptimal substituents on c-6 and c-7 limit the potency of 13 even when epoxidized at c-22,23 . a similar limit in potency was conferred by the hydrolysis of the c-1 group in 14 . both c-6 reductive products of the major metabolite taccalonolide a , 25 and 26 , exhibited decreased potency , further demonstrating the importance of the c-6 ketone for activity . however , the finding that c-22,23 epoxidation of 26 leads to an over 300-fold improvement in potency demonstrates that this epoxidation can dramatically improve the activity even of taccalonolides that otherwise have no detectable antiproliferative effects . together , these data provide more extensive sar for the taccalonolides , including the critical importance of c-22,23 , c-1 , and c-6 modifications as well as the interplay between these substituents . each of the 11 taccalonolides epoxidized at c-22,23 in this study exhibited increased potency compared with their precursors , the majority showing over 200-fold improvement in activity . two of these products , 18 and 23 , are the first taccalonolides identified with subnanomolar potency . these two epoxidized taccalonolides have potent microtubule stabilizing activities in cells and with purified tubulin , but only 18 demonstrated antitumor efficacy at the dose and schedule tested . these results demonstrate that a c-22,23 epoxy combined with a bulky c-1 isovaleryloxy group facilitates optimal potency for the taccalonolide class of microtubule stabilizers and further enhances our understanding of the structure while both c-22,23 epoxidated and nonepoxidated taccalonolides cause microtubule stabilization in cells , the recent finding that some epoxy taccalonolides can covalently bind to microtubules leads to a hypothesis that the c-22,23 epoxide may facilitate their irreversible binding , which would be consistent with the increased potency afforded by this modification . additional studies nmr spectra were acquired on bruker avance 500 , 600 , or 700 mhz instruments equipped with cryoprobes using cdcl3 as solvent . all spectra were measured and reported in ppm using trimethylsilane as an internal standard . the hrms data were obtained on an aglient technologies 6224 toflc / ms mass spectrometer . lc / ms was performed with a waters alliance 2695 hplc module , 996 photodiode array detector , and micromass quattro triple quadrupole mass spectrometer equipped with esi under the positive mode . tlc was performed on aluminum sheets ( silica gel 60 f254 , merck kgaa , germany ) . spots were visualized by spraying with 20% sulfuric acid in ethanol followed by heating . taccalonolides e ( 7 ) , n ( 6 ) , r ( 8) , t ( 9 ) , z ( 10 ) , aa ( 11 ) , ab ( 12 ) , ad ( 13 ) , and an ( 14 ) were obtained previously . a taccalonolide b ( 3 ) enriched fraction was further purified by reversed phase hplc ( phenomenex luna , 5 m c18 250 21.2 mm column ) eluting with a gradient of 40100% acetonitrile in h2o in 50 min to yield highly pure 3 and the minor product , taccalonolide ai ( 5 ) . white powder . hrms ( m / z ) calcd for c35h49o11 [ m + h ] 645.3269 , found 645.3268 ; h nmr ( 500 mhz , cdcl3 ) 5.23 ( d , j = 2.6 hz , 1h , oh-15 ) , 5.02 ( br , 1h , h-22 ) , 4.99 ( t , j = 2.8 hz , 1h , h-12 ) , 4.72 ( s , 1h , oh-25 ) , 4.59 ( d , j = 5.2 hz , 1h , h-1 ) , 4.45 ( br , 1h , oh-7 ) , 4.38 ( ddd , j = 9.5 , 8.3 , 2.5 hz , 1h , h-15 ) , 4.01 ( d , j = 10.3 hz , 1h , h-7 ) , 3.55 ( t , j = 4.0 hz , 1h , h-2 ) , 3.40 ( br , 1h , h-3 ) , 2.70 ( dd , j = 11.3 , 4.5 hz , 1h , h-5 ) , 2.39 ( dd , j = 13.1 , 10.9 hz , 1h , h-16 ) , 2.26 ( dd , j = 16.1 , 4.5 hz , 1h , h-4a ) , 2.19 ( m , 1h , h-20 ) , 2.18 ( m , 2h , h-2 ) , 2.15 ( m , 1h , h-9 ) , 2.14 ( m , 1h , h-3 ) , 2.12 ( m , 1h , h-4b ) , 2.08 ( s , 3h , 12ococh3 ) , 2.07 ( m , 1h , h-14 ) , 1.98 ( dd , j = 13.2 , 10.1 hz , 1h , h-17 ) , 1.71 ( m , 1h , h-8 ) , 1.69 ( m , 2h , h-11 ) , 1.67 ( s , 3h , h-28 ) , 1.36 ( s , 3h , h-27 ) , 1.01 ( d , j = 7.0 hz , 3h , h-4 ) , 1.00 ( d , j = 6.0 hz , 3h , h-5 ) , 0.95 ( d , j = 7.0 hz , 3h , h-21 ) , 0.82 ( s , 3h , h-19 ) , 0.76 ( s , 3h , h-18 ) . c nmr ( 125 mhz , cdcl3 ) 209.1 ( c-6 ) , 175.4 ( c-26 ) , 171.8 ( c-1 ) , 169.3 ( 12-ococh3 ) , 154.9 ( c-23 ) , 110.4 ( c-22 ) , 78.9 ( c-25 ) , 77.5 ( c-7 ) , 73.5 ( c-12 ) , 71.8 ( c-15 ) , 70.4 ( c-1 ) , 57.3 ( c-14 ) , 52.5 ( c-3 ) , 50.8 ( c-24 ) , 49.9 ( c-16 ) , 49.3 ( c-2 ) , 47.7 ( c-17 ) , 43.8 ( c-13 ) , 43.4 ( c-5 ) , 42.6 ( c-2 ) , 42.2 ( c-8 ) , 41.2 ( c-10 ) , 36.1 ( c-9 ) , 30.8 ( c-20 ) , 25.1 ( c-27 ) , 25.4 ( c-3 ) , 24.7 ( c-11 ) , 21.9 ( c-4,5 ) , 21.7 ( c-28 ) , 20.9 ( c-4 ) , 20.7 ( 12-ococh3 ) , 19.3 ( c-21 ) , 12.9 ( c-18 ) , 12.3 ( c-19 ) . dimethyldioxirane was prepared by reaction of oxone with acetone , and the concentration of dimethyldioxirane was determined by uv . 13 mol ) was dissolved in 500 l of ch2cl2 and cooled to 20 c . dmdo ( 23 equiv ) was added , and the mixture was stirred at room temperature until the taccalonolide was completely epoxidized ( 14 h ) . the epoxides were obtained after removal of the solvents and reagent with no further purification required . white powder . hrms ( m / z ) calcd for c34h45o13 [ m + h ] 661.2854 , found 661.2851 ; h nmr ( 500 mhz , cdcl3 ) 5.47 ( t , j = 9.1 hz , 1h , h-15 ) , 4.91 ( d , j = 3.0 hz , 1h , h-12 ) , 4.60 ( d , j = 5.3 hz , 1h , h-1 ) , 3.86 ( dd , j = 10.1 , 3.7 hz , 1h , h-7 ) , 3.83 ( br , 7-oh ) , 3.51 ( t , j = 4.7 hz , 1h , h-2 ) , 3.39 ( br , 1h , h-3 ) , 3.30 ( s , 1h , h-22 ) , 2.67 ( m , 2h , h-5 , 25-oh ) , 2.32 ( dd , j = 10.9 , 8.6 hz , 1h , h-14 ) , 2.262.03 ( m , 6h ) , 2.12 ( s , 3h , 1-ococh3 ) , 2.10 ( s , 3h , 12-ococh3 ) , 2.01 ( s , 3h , 15-ococh3 ) , 1.76 ( s , 3h , h-28 ) , 1.751.60 ( m , 4h ) , 1.35 ( s , 3h , h-27 ) , 1.08 ( d , j = 7.4 hz , 3h , h-21 ) , 0.75 ( s , 3h , h-19 ) , 0.70 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c32h43o12 [ m + h ] 619.2749 , found 619.2757 ; h nmr ( 500 mhz , cdcl3 ) 5.25 ( d , j = 3.1 hz , 1h , h-12 ) , 5.08 ( br , 1h , oh ) , 4.88 ( d , j = 3.0 hz , 1h , oh ) , 4.59 ( d , j = 5.4 hz , 1h , h-1 ) , 4.44 ( br , 1h , oh ) , 4.30 ( t , j = 10.0 hz , 1h , h-15 ) , 4.01 ( d , j = 10.4 hz , 1h , h-7 ) , 3.53 ( t , j = 4.3 hz , 1h , h-2 ) , 3.41 ( br , 1h , h-3 ) , 3.28 ( s , 1h , h-22 ) , 2.71 ( dd , j = 11.7 , 4.7 hz , 1h , h-5 ) , 2.312.03 ( m , 6h ) , 2.10 ( s , 3h , 1-ococh3 ) , 2.09 ( s , 3h , 12-ococh3 ) , 1.76 ( s , 3h , h-28 ) , 1.751.67 ( m , 4h ) , 1.37 ( s , 3h , h-27 ) , 1.07 ( d , j = 7.3 hz , 3h , h-21 ) , 0.74 ( s , 3h , h-19 ) , 0.71 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c36h47o15 [ m + h ] 719.2910 , found 719.2907 ; h nmr ( 500 mhz , cdcl3 ) 5.55 ( d , j = 10.8 hz , 1h , h-7 ) , 5.50 ( t , j = 8.6 hz , 1h , h-15 ) , 4.88 ( br , 1h , h-12 ) , 4.81 ( d , j = 5.1 hz , 1h , h-1 ) , 3.70 ( t , j = 4.5 hz , 1h , h-2 ) , 3.58 ( d , j = 3.8 hz , 1h , h-3 ) , 3.31 ( s , 1h , 5-oh ) , 3.29 ( s , 1h , h-22 ) , 2.82 ( td , j = 10.8 , 7.1 hz , 1h , h-9 ) , 2.70 ( s , 1h , 25-oh ) , 2.542.47 ( m , 2h , h-4a,14 ) , 2.261.91 ( m , 6h ) , 2.16 ( s , 3h , 1-ococh3 ) , 2.14 ( s , 3h , 7-ococh3 ) , 2.12 ( s , 3h , 12-ococh3 ) , 1.97 ( s , 3h , 15-ococh3 ) , 1.73 ( s , 3h , h-28 ) , 1.67 ( m , 3h , h2 - 11 , h-20 ) , 1.33 ( s , 3h , h-27 ) , 0.80 ( d , j = 7.4 hz , 3h , h-21 ) , 0.78 ( s , 3h , h-19 ) , 0.70 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c39h53o15 [ m + h ] 761.3379 , found 761.3362 ; h nmr ( 600 mhz , cdcl3 ) 5.55 ( d , j = 10.7 hz , 1h , h-7 ) , 5.50 ( t , j = 8.7 hz , 1h , h-15 ) , 4.88 ( t , j = 3.2 hz , 1h , h-12 ) , 4.79 ( d , j = 5.1 hz , 1h , h-1 ) , 3.72 ( t , j = 4.4 hz , 1h , h-2 ) , 3.57 ( br , 1h , h-3 ) , 3.30 ( br , 1h , 5-oh ) , 3.29 ( br , 1h , h-22 ) , 2.82 ( dt , j = 11.6 , 8.5 hz , 1h , h-9 ) , 2.72 ( s , 1h , 25-oh ) , 2.50 ( m , 2h , h-4a,14 ) , 2.252.16 ( m , 5h , h4b , h-17 , h2 - 2 , h-3 ) , 2.16 ( s , 3h , 7-ococh3 ) , 2.13 ( s , 3h , 12-ococh3 ) , 1.97 ( s , 3h , 15-ococh3 ) , 1.95 ( m , 1h , h-8 ) , 2.02 ( dd , j = 13.9 , 9.2 hz , 1h , h-16 ) , 1.73 ( s , 3h , h-28 ) , 1.67 ( m , 3h , h2 - 11 , h-20 ) , 1.32 ( s , 3h , h-27 ) , 1.07 ( d , j = 7.3 hz , 3h , h-21 ) , 1.01 ( t , j = 6.6 hz , 6h , h-4,5 ) , 0.77 ( s , 3h , h-19 ) , 0.70 ( s , 3h , h-18 ) . c nmr ( 150 mhz , cdcl3 ) 201.5 ( c-6 ) , 177.9 ( c-26 ) , 172.1 ( 1-ococh3 ) , 172.3 ( 15-ococh3 ) , 171.4 ( 7-ococh3 ) , 169.7 ( 12-ococh3 ) , 92.8 ( c-23 ) , 79.9 ( c-5 ) , 79.6 ( c-25 ) , 76.4 ( c-7 ) , 74.5 ( c-12 ) , 72.5 ( c-1 ) , 72.0 ( c-15 ) , 66.2 ( c-22 ) , 55.0 ( c-3 ) , 54.4 ( c-14 ) , 50.8 ( c-2 ) , 48.5 ( c-16 ) , 46.9 ( c-24 ) , 45.1 ( c-17 ) , 44.9 ( c-13 ) , 44.7 ( c-10 ) , 43.4 ( c-2 ) , 34.4 ( c-9 ) , 32.0 ( c-20 ) , 27.3 ( c-4 ) , 26.6 ( c-3 ) , 26.4 ( c-11 ) , 23.9 ( c-28 ) , 23.4 ( 15-ococh3 ) , 22.9 ( c-4,5 ) , 21.9 ( 7,12-ococh3 ) , 20.0 ( c-27 ) , 18.9 ( c-21 ) , 15.1 ( c-18 ) , 13.9 ( c-19 ) . white powder . hrms ( m / z ) calcd for c36h47o16 [ m + h ] 735.2859 , found 735.2867 ; h nmr ( 500 mhz , cdcl3 ) 5.53 ( t , j = 8.9 hz , 1h , h-15 ) , 5.19 ( brd , j = 12.2 hz , 1h , h-11 ) , 5.16 ( br , 1h , h-12 ) , 4.85 ( d , j = 5.3 hz , 1h , h-1 ) , 4.71 ( dd , j = 10.3 , 5.2 hz , 1h , h-7 ) , 3.74 ( t , j = 4.7 hz , 1h , h-2 ) , 3.64 ( br , 1h , 5-oh ) , 3.61 ( br , 1h , h-3 ) , 3.47 ( d , j = 4.9 hz , 1h , 7-oh ) , 3.27 ( s , 1h , h-22 ) , 3.16 ( t , j = 11.5 hz , 1h , h-9 ) , 2.66 ( s , 1h , 25-oh ) , 2.57 ( d , j = 16.5 hz , 1h , h-4a ) , 2.50 ( t , j = 10.0 hz , 1h , h-14 ) , 2.23 ( d , j = 16.5 hz , 1h , h-4b ) , 2.17 ( s , 3h , 1-ococh3 ) , 2.16 ( s , 3h , 12-ococh3 ) , 2.152.03 ( m , 2h , h-16,17 ) , 2.09 ( s , 3h,11-ococh3 ) , 1.98 ( s , 3h , 15-ococh3 ) , 1.76 ( s , 3h , h-28 ) , 1.58 ( m , 1h , h-20 ) , 1.34 ( s , 3h , h-27 ) , 1.01 ( d , j = 7.4 hz , 3h , h-21 ) , 0.88 ( s , 3h , h-19 ) , 0.72 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c38h49o17 [ m + h ] 777.2964 , found 777.2947 ; h nmr ( 500 mhz , cdcl3 ) 5.70 ( d , j = 10.9 hz , 1h , h-7 ) , 5.53 ( t , j = 8.3 hz , 1h , h-15 ) , 5.31 ( br , 1h , 5-oh ) , 5.19 ( d , j = 12.6 hz , 1h , h-11 ) , 5.18 ( br , 1h , h-12 ) , 4.90 ( d , j = 5.2 hz , 1h , h-1 ) , 3.72 ( t , j = 4.8 hz , 1h , h-2 ) , 3.59 ( br , 1h , h-3 ) , 3.28 ( m , 2h , h-9,22 ) , 2.69 ( s , 1h , 25-oh ) , 2.60 ( t , j = 10.2 hz , 1h , h-14 ) , 2.56 ( d , j = 17.1 hz , 1h , h-4a ) , 2.152.00 ( m , 3h , h-4b , 8,16 ) , 2.20 ( s , 3h , 1-ococh3 ) , 2.17 ( s , 3h , 7-ococh3 ) , 2.16 ( s , 3h , 12-ococh3 ) , 1.99 ( s , 6h , 11,15-ococh3 ) , 1.75 ( s , 3h , h-28 ) , 1.62 ( m , 2h , h-17,20 ) , 1.32 ( s , 3h , h-27 ) , 1.09 ( d , j = 7.3 hz , 1h ) , 1.02 ( d , j = 7.3 hz , 3h , h-21 ) , 0.93 ( s , 3h , h-19 ) , 0.71 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c34h45o15 [ m + h ] 693.2753 , found 693.2771 ; h nmr ( 500 mhz , cdcl3 ) 5.30 ( s , 1h , 25-oh ) , 5.22 ( dd , j = 12.0 , 2.5 hz , 1h , h-11 ) , 5.13 ( d , j = 2.4 hz , 1h , h-12 ) , 4.96 ( br , 2h , 15,25-oh ) , 4.91 ( dd , j = 10.6 , 4.5 hz , 1h , h-7 ) , 4.81 ( d , j = 5.4 hz , 1h , h-1 ) , 4.38 ( td , j = 9.1 , 3.7 hz , 1h , h-15 ) , 4.03 ( d , j = 4.5 hz , 1h , 7-oh ) , 3.75 ( t , j = 4.5 hz , 1h , h-2 ) , 3.68 ( s , 1h , 5-oh ) , 3.62 ( br , 1h , h-3 ) , 3.25 ( s , 1h , h-22 ) , 3.15 ( t , j = 11.6 hz , 1h , h-9 ) , 2.54 ( dd , j = 16.6 , 2.1 hz , 1h , h-4a ) , 2.27 ( d , j = 16.6 , hz , 1h , h-4b ) , 2.21 ( m , 1h , h-14 ) , 2.17 ( s , 3h , 1-ococh3 ) , 2.15 ( s , 3h , 12-ococh3 ) , 2.09 ( m , 2h , h-16,17 ) , 1.98 ( s , 3h , 11-ococh3 ) , 1.77 ( s , 3h , h-28 ) , 1.61 ( m , 2h , h-8,20 ) , 1.36 ( s , 3h , h-27 ) , 0.99 ( d , j = 7.4 hz , 3h , h-21 ) , 0.85 ( s , 3h , h-19 ) , 0.76 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c36h45o15 [ m + h ] 717.2753 , found 717.2738 ; h nmr ( 500 mhz , cdcl3 ) 6.19 ( s , 1h , 6-oh ) , 5.64 ( t , j = 8.5 hz , 1h , h-15 ) , 5.43 ( dd , j = 11.3 , 2.3 hz , 1h , h-11 ) , 5.26 ( d , j = 2.3 hz , 1h , h-12 ) , 4.90 ( d , j = 5.5 hz , 1h , h-1 ) , 3.55 ( t , j = 4.2 hz , 1h , h-2 ) , 3.41 ( t , j = 3.7 hz , 1h , h-3 ) , 3.38 ( d , j = 19.7 hz , 1h , h-4a ) , 3.28 ( br , 1h , h-22 ) , 2.83 ( t , j = 12.3 hz , 1h , h-11 ) , 2.74 ( s , 1h , 25-oh ) , 2.62 ( m , 3h , h-4b,8,14 ) , 2.44 ( dd , j = 11.4 , 8.0 hz , 1h , h-16 ) , 2.18 ( s , 3h , 15-ococh3 ) , 2.11 ( s , 3h , 1-ococh3 ) , 2.10 ( s , 3h , 11-ococh3 ) , 2.00 ( s , 3h , 12-ococh3 ) , 1.75 ( m , 2h , h-17,20 ) , 1.71 ( s , 3h , h-28 ) , 1.31 ( s , 3h , h-27 ) , 1.21 ( s , 3h , h-18 ) , 1.07 ( d , j = 6.8 hz , 3h , h-21 ) , 0.92 ( s , 3h , h-19 ) . white powder . hrms ( m / z ) calcd for c35h49o12 [ m + h ] 661.3219 , found 661.3211 ; h nmr ( 500 mhz , cdcl3 ) 5.26 ( d , j = 3.0 hz , 1h , 15-oh ) , 5.08 ( s , 1h , 25-oh ) , 4.90 ( t , j = 2.7 hz , 1h , h-12 ) , 4.57 ( d , j = 5.3 hz , 1h , h-1 ) , 4.44 ( d , j = 3.1 hz , 1h , 7-oh ) , 4.29 ( t , j = 9.5 hz , 1h , h-15 ) , 4.01 ( d , j = 10.3 hz , 1h , h-7 ) , 3.55 ( t , j = 4.4 hz , 1h , h-2 ) , 3.40 ( d , j = 3.2 hz , 1h , h-3 ) , 3.27 ( s , 1h , h-22 ) , 2.69 ( dd , j = 11.4 , 4.8 hz , 1h , h-5 ) , 2.27 ( d , j = 16.7 , 5.0 hz , 1h , h-4a ) , 2.242.02 ( m , 7h , h-4b,9,14,16,3 , h22 ) , 2.10 ( s , 3h , 12-ococh3 ) , 1.67 ( s , 3h , h2 - 11 , h-20 ) , 1.76 ( s , 3h , h-28 ) , 1.37 ( s , 3h , h-27 ) , 1.06 ( d , j = 7.4 hz , 3h , h-21 ) , 1.02 ( dd , j = 6.2 , 3.6 hz , 6h , h-4,5 ) , 0.75 ( s , 3h , h-19 ) , 0.71 ( s , 3h , h-18 ) . white powder . hrms ( m / z ) calcd for c30h41o11 [ m + h ] 577.2643 , found 577.2657 ; h nmr ( 500 mhz , cdcl3 ) 5.18 ( d , j = 3.1 hz , 1h , 15-oh ) , 5.08 ( s , 1h , h-12 ) , 4.94 ( br , 1h , 25-oh ) , 4.30 ( t , j = 9.9 hz , 1h , h-15 ) , 4.35 ( br , 1h , 7-oh ) , 3.98 ( d , 1h , j = 10.3 hz , 1h , h-7 ) , 3.71 ( dd , j = 8.4 , 5.7 hz , 1h , h-1 ) , 3.55 ( br , 1h , h-3 ) , 3.42 ( t , j = 5.1 hz , 1h , h-2 ) , 3.28 ( br , 1h , h-22 ) , 2.51 ( dd , j = 11.1 , 5.1 hz , 1h , h-5 ) , 2.282.11 ( m , 4h , h2 - 4 , h-9,16 ) , 2.10 ( s , 3h , 12-ococh3 ) , 2.082.02 ( m , 2h , h-14,17 ) , 1.76 ( s , 3h , h-28 ) , 1.701.58 ( m , 2h , h-8,11 ) , 1.37 ( s , 3h , h-17 ) , 1.08 ( d , j = 7.2 hz , 1h , h-21 ) , 0.72 ( s , 3h , h-19 ) , 0.63 ( s , 3h , h-18 ) . compound 2 ( 10.2 mg ) was dissolved in 2 ml of meoh and cooled in a sodium chloride ice bath . the ch2cl2 soluble material was subjected to hplc separation to yield compounds 25 ( 0.15 mg ) and 26 ( 0.93 mg ) . white powder . hrms ( m / z ) calcd for c34h47o13 [ m + h ] 663.3011 , found 663.3021 ; h nmr ( 600 mhz , cdcl3 ) 5.40 ( d , j = 2.9 hz , 1h , 15-oh ) , 5.35 ( dd , j = 11.7 , 2.8 hz , 1h , h-11 ) , 5.23 ( d , j = 2.8 hz , 1h , h-12 ) , 5.01 ( s , 1h , h-22 ) , 4.64 ( s , 25-oh ) , 4.56 ( d , j = 5.4 hz , 1h , h-1 ) , 4.38 ( t , j = 8.9 hz , 1h , h-15 ) , 3.80 ( s , 1h , h-6 ) , 3.51 ( m , 1h , h-7 ) , 3.47 ( m , 1h , h-2 ) , 3.39 ( s , 1h , h-3 ) , 2.39 ( dd , j = 13.4 , 10.6 hz , 1h , h-16 ) , 2.25 ( t , j = 13.8 hz , 1h , h-4a ) , 2.15 ( m , 2h , h-9,20 ) , 2.11 ( s , 3h , 1-ococh3 ) , 2.07 ( s , 3h , 12-ococh3 ) , 1.95 ( s , 3h , 11-ococh3 ) , 2.00 ( m , 1h , h-8 ) , 1.97 ( m , 1h , h-14 ) , 1.94 ( m , 1h , h-4b ) , 1.70 ( m , 2h , h-5,17 ) , 1.66 ( s , 3h , h-28 ) , 1.34 ( s , 3h , h-27 ) , 1.24 ( s , 1h ) , 1.02 ( s , 3h , h-18 ) , 0.99 ( s , 3h , h-19 ) , 0.91 ( d , j = 7.1 hz , 3h , h-21 ) . c nmr ( 150 mhz , cdcl3 ) 176.2 ( c-26 ) , 171.3 ( 11-ococh3 ) , 170.2 ( 1-ococh3 ) , 169.8 ( 12-ococh3 ) , 154.8 ( c-23 ) , 111.2 ( c-22 ) , 78.9 ( c-25 ) , 74.2 ( h-11 ) , 74.1 ( 2xc , c-1,7 ) , 73.5 ( c-6 ) , 72.0 ( c-15 ) , 71.0 ( c-12 ) , 56.3 ( c-14 ) , 50.9 ( c-24 ) , 48.2 ( c-17 ) , 44.6 ( c-13 ) , 40.5 ( c-10 ) , 37.5 ( c-9 ) , 35.3 ( c-8 ) , 33.4 ( c-5 ) , 31.4 ( c-20 ) , 26.0 ( c-4 ) , 25.6 ( c-27 ) , 22.0 ( c-28 ) , 21.7 ( 11-ococh3 ) , 21.3 ( 12-ococh3 ) , 21.0 ( 1-ococh3 ) , 20.5 ( c-21 ) , 14.6 ( c-18 ) , 13.7 ( c-19 ) . white powder . hrms ( m / z ) calcd for c36h49o14 [ m + h ] 705.3168 , found 705.3137 ; h nmr ( 500 mhz , cdcl3 ) 5.47 ( s , 1h , 15-oh ) , 5.36 ( dd , j = 11.7 , 2.6 hz , 1h , h-11 ) , 5.23 ( d , j = 2.6 hz , 1h , h-12 ) , 5.06 ( t , j = 2.7 hz , 1h , h-6 ) , 4.98 ( d , j = 1.1 hz , 1h , h-22 ) , 4.74 ( br , 1h , oh ) , 4.64 ( d , j = 5.5 hz , 1h , h-1 ) , 4.36 ( t , j = 9.2 hz , 1h , h-15 ) , 3.78 ( d , j = 7.0 hz , 1h , h-7 ) , 3.59 ( s , 1h , oh ) , 3.46 ( dd , j = 5.1 , 3.9 hz , 1h , h-2 ) , 3.34 ( d , j = 3.3 hz , 1h , h-3 ) , 2.37 ( dd , j = 13.3 , 10.6 hz , 1h , h-16 ) , 2.22 ( m , 1h , h-9 ) , 2.21 ( s , 3h , 6-ococh3 ) , 2.17 ( m , 1h , h-20 ) , 2.12 ( s , 3h , 1-ococh3 ) , 2.06 ( s , 3h , 12-ococh3 ) , 2.05 ( m , 1h , h-5 ) , 1.97 ( s , 3h , 11-ococh3 ) , 1.981.92 ( m , 4h , h-8,14 , h2 - 4 ) , 1.80 ( dd , j = 13.4 , 9.8 hz , 1h , h-17 ) , 1.65 ( s , 3h , h-28 ) , 1.32 ( s , 1h , h-27 ) , 1.03 ( s , 1h , h-18 ) , 1.01 ( s , 1h , h-19 ) , 0.90 ( d , j = 7.0 hz , 3h , h-21 ) . c nmr ( 125 mhz , cdcl3 ) 175.5 ( c-26 ) , 173.7 ( 6-ococh3 ) , 170.8 ( 11-ococh3 ) , 169.6 ( 1-ococh3 ) , 169.4 ( 12-ococh3 ) , 154.5 ( c-23 ) , 110.4 ( c-22 ) , 78.9 ( c-25 ) , 76.2 ( c-6 ) , 74.0 ( c-7 ) , 73.6 ( c-12 ) , 73.4 ( c-1 ) , 71.2 ( c-15 ) , 70.5 ( c-11 ) , 56.1 ( c-14 ) , 52.0 ( c-3 ) , 51.2 ( c-16 ) , 50.7 ( c-24 ) , 49.9 ( c-2 ) , 47.6 ( c-17 ) , 44.2 ( c-13 ) , 39.9 ( c-9 ) , 37.0 ( c-10 ) , 34.8 ( c-8 ) , 31.7 ( c-5 ) , 30.8 ( c-20 ) , 25.2 ( 2xc , c-4,27 ) , 21.6 ( c-28 ) , 21.0 ( 11-ococh3 ) , 20.7 ( 1,6-ococh3 ) , 20.4 ( 12-ococh3 ) , 20.1 ( c-21 ) , 13.9 ( c-18 ) , 13.3 ( c-19 ) . white powder . hrms ( m / z ) calcd for c36h49o15 [ m + h ] 721.3066 , found 721.3043 ; h nmr ( 600 mhz , cdcl3 ) 5.53 ( d , j = 2.8 hz , 1h , 15-oh ) , 5.32 ( dd , j = 11.8 , 2.9 hz , 1h , h-11 ) , 5.15 ( d , j = 2.9 hz , 1h , h-12 ) , 5.11 ( s , 1h , oh ) , 5.05 ( t , j = 2.8 hz , 1h , h-6 ) , 4.63 ( d , j = 5.6 hz , 1h , h-1 ) , 4.27 ( brt , j = 7.2 hz , 1h , h-15 ) , 3.79 ( brd , j = 9.5 hz , 1h , h-7 ) , 3.53 ( s , 1h , oh ) , 3.46 ( m , j = 4.7 hz , 1h , h-2 ) , 3.33 ( br , 1h , h-3 ) , 3.23 ( s , 1h , h-22 ) , 2.21 ( s , 3h , 6-ococh3 ) , 2.261.87 ( m , 9h , h2 - 4 , h-5,8,9,14,16,17,20 ) , 2.12 ( s , 3h , 1-ococh3 ) , 2.08 ( s , 3h , 12-ococh3 ) , 1.96 ( s , 3h , 11-ococh3 ) , 1.73 ( s , 3h , h-28 ) , 1.32 ( s , 3h , h-27 ) , 1.02 ( s , 3h , h-18 ) , 1.01 ( d , j = 7.4 hz , 3h , h-21 ) , 0.90 ( s , 3h , h-19 ) . the antiproliferative activities and ic50 values of the taccalonolides were determined using the sulforhodamine b assay in hela cells as previously described . the data are from an average of three experiments , each performed in triplicate , with standard deviation . microtubules in interphase hela cells were visualized by immunofluorescence using a -tubulin antibody ( sigma no . images were acquired 18 h after indicated drug or vehicle treatment using a nikon eclipse 80i fluorescence microscope and nis elements ar 3.0 software . the ability of the taccalonolides 23 and 18 to enhance tubulin polymerization was determined as previously described . briefly , 1 l of a 100 stock solution of each compound in etoh was added to a final volume of 100 l containing 2 mg / ml purified porcine brain tubulin ( cytoskeleton ) in gpem buffer ( 80 mm na - pipes , ph 6.9 , 1 mm egta , 1 mm mgcl2 , 1 mm gtp , and 10% glycerol ) , and microtubule formation was monitored turbidimetrically at od340 on a spectramax 96-well plate reader . the antitumor efficacy of the two most potent taccalonolides , 23 and 18 , was evaluated in a mda - mb-231 triple negative breast cancer xenograft model . tumor fragments were bilaterally implanted in female nude ( nu / nu ) mice . mice were randomly placed into separate treatment groups ( n = 5 ) , and dosing was initiated when the average tumor size was 134 mm . taccalonolides 23 and 18 were solubilized in 100% etoh at 1 and 1.25 mg / ml , respectively , while paclitaxel stocks were solubilized in 50% cremophor/50% etoh at 15 mg / ml . drug stocks were diluted a minimum of 1:10 in 200 l of phosphate buffered saline immediately prior to intraperitoneal injection . dose and schedule taccalonolide 23 was administered at a concentration of 0.7 mg / kg on days 0 , 3 , and 7 after which dosing was halted due to expenditure of all available material . taccalonolide 18 was administered at a concentration of 0.25 mg / kg on days 0 and 3 , after which dosing was halted due to an average 10% weight loss . paclitaxel was administered at 15 mg / kg on days 0 , 3 , and 7 . during the period of drug administration , mice were monitored daily , and weight and tumor measurements were taken 23 times weekly . control treated mice were sacrificed on day 17 due to large tumor size ; all other mice were sacrificed on day 21 . the mice were purchased from harlan laboratories , housed in an aalac - approved facility under fully licensed veterinary care , and provided water and food ad libitum .

here , we report the synthesis and characterization of size - controllable and stimuli - responsive dna nanohydrogels as effective targeted gene delivery vectors . dna nanohydrogels were created through a self - assembly process using three kinds of building units , respectively termed y - shaped monomer a with three sticky ends ( yma ) , y - shaped monomer b with one sticky end ( ymb ) , and dna linker ( lk ) with two sticky ends . hybridization at the sticky ends of monomers and lk leads to nanohydrogel formation . dna nanohydrogels are size - controllable by varying the ratio of yma to ymb . by incorporating different functional elements , such as aptamers , disulfide linkages , and therapeutic genes into different building units , the synthesized aptamer - based nanohydrogels ( y - gel - apt ) can be used for targeted and stimuli - responsive gene therapy . y - gel - apt strongly inhibited cell proliferation and migration in target a549 cells , but not in control cells . by taking advantage of facile modular design and assembly , efficient cellular uptake , and superior biocompatibility , this y - gel - apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy .

a common complaint of older listeners is that they can hear speech , yet can not understand it , especially when listening to speech in a background noise . when target and competing speech signals are concurrently presented , a difference in the fundamental frequency ( f0 ) between competing speech signals , which determines the pitch of voice , can be an important and commonly occurring cue to facilitate the separation of the target message from the interfering message , consequently improving intelligibility of the target message . to address the question of whether the older listeners have reduced ability to use f0 and how the age - related deficits in the processing of f0 are theoretically explained , this paper is divided into three parts . the first part of this article summarizes how the speech - communication difficulties that older listeners have are theoretically explained . in the second part , literatures on the perceptual benefits from f0 and the age - related deficits on the use of f0 are reviewed . as a final part , three theoretical models explaining the general processing of f0 are compared to discuss which better explains the age - related deficits in the processing of f0 .

understanding a spoken message in the presence of background noise or competing speech is a common listening problem for many older adults . the recognition process in the presence of competing signals is naturally complicated since it includes the encoding of each input signal at the peripheral level , followed by processing of the central auditory and cognitive systems . when age - related internal changes occur in peripheral , central , and general cognitive functions , any one of these changes can be sufficient to affect the understanding of competing speech signals , but older adults often experience changes at multiple levels of processing . when the target and competing voices are presented together , young adults are able to use differences in the fundamental frequency ( f0 ) of competing speech signals in order to better identify the target message against the interfering message . however , this important cue may not be ideally processed for older listeners , revealed by numerous studies that reported the age - related disadvantage in the processing of f0 for the intelligibility of competing signals . thus , it is important to determine any negative impact of aging on the use of f0 cues between two competing speech signals , and various theoretical models which describe possible reasons of the age - related deficits above . this article addresses three issues , 1 ) theoretical hypotheses about age - related deficits on general speech communication , 2 ) previous findings on the perceptual benefits of f0 for competing speech signals and the age - related deficits on these benefits , and 3 ) theoretical models on the processing of f0 in order to compare which better explains the age - related disadvantage in the processing of f0 . a working group of the committee on hearing and bioacoustics and biomechanics of the national research council reviewed the issues of the speech communication problems in older adults . as a possible explanation of age - related deficits on speech communication , the sensori - neural hearing loss and cochlear pathology of older listeners can result in difficulties for the peripheral encoding of input sounds , and the age - related decline in central - auditory and general cognitive functions can also adversely influence processing of deteriorated input signals.1 ) when the age - related peripheral deficit in hearing sensitivity , often referred to as presbycusis , is the sole deficit , the listener 's understanding of the target message is primarily affected by the inaudibility of the target speech arising from the presence of cochlear pathology.2 - 6 ) because this age - related hearing loss rarely results in total deafness , most of older adults with presbycusis can still hear speech , but often have difficulty understanding speech . given the normal involvement of processing at peripheral , central - auditory , and cognitive levels in speech understanding , three hypotheses ( i.e. , auditory peripheral hypothesis , central - auditory hypothesis , and general cognitive hypothesis ) have been used to explain the speech - understanding difficulties of the elderly adults with impaired hearing.1,7 ) age - related structural and functional changes in the auditory periphery have been assumed to directly affect the audibility and the processing of speech sounds in the peripheral hypothesis . the age - related peripheral hearing loss often starts in the high frequencies , with a progressive deterioration at high frequencies that is faster than at low frequencies . due to the high - frequency hearing loss , some high - frequency , low - intensity speech sounds , usually consonantal sounds such as " t , f , s , th " , can become inaudible even when a single talker speaks at typical conversational levels ( 60 - 65 db spl ) in quiet conditions.8 ) many previous studies have consistently found that older individuals ' speech - understanding performance is well - explained by the audibility of speech , estimated from hearing thresholds , when a single talker speaks in quiet or in a steady - state background noise.3,7 - 11 ) when a target speech signal is presented with a background noise that fluctuates like speech or with competing speech , individual differences in the speech - understanding performance of older individuals are not predicted by audibility alone . rather , age - related deficits often emerge as primary or strong secondary predictors.11,12 ) the role of age - related deficits in central - auditory and general cognitive process has also been confirmed in several studies.2,11 - 16 ) once the reduced speech audibility of the target message has been alleviated by clinical amplification or laboratory spectral shaping , other factors associated with central and cognitive deficits emerge , especially while listening to speech with competing speech in the background . when the target and interfering messages are presented concurrently , spectral and temporal features of both competing speech signals are encoded and contrasted in order to segregate the target from the competing source . for the procedure of identification for multiple speech streams , listeners need to divide their attention to monitor multiple conversations , and then selectively attend to one speech stream while inhibiting the competing information.15 ) the age deficits in cognitive processing also contribute to speech - understanding difficulties in a multitalker conversation , possibly influenced by declined attentional capacity and inefficient allocation of attentional resources . as described above , all the age - related vascular , metabolic , or other systemic factors can cause detrimental changes to peripheral , central - auditory , or cognitive processes , yet various acoustic external factors such as fundamental frequency differences between competing voices can also impact the auditory segregation of collocated competing signals . the fundamental frequency ( f0 ) is critical to speech perception especially in noise since f0 provides an important and robust cue to segregate multiple speech streams . the fundamental frequency means the frequency at which the vocal folds vibrate for making voiced speech sounds , and the vibration generates a periodic fluctuation of air pressure . f0 is calculated based on the number of vibrations per second and this is generally expressed in units of hertz ( hz ) . f0 is inversely proportional to the vibrating mass and directly proportional to the tension ( stiffness ) of the vocal folds . depending on the features of mass or tension , a slow or fast vibration of vocal folds yields a low or high f0 value , consequently eliciting a low- or high - pitched sound . for example , the f0 value of men is typically an octave lower than that of women due to the longer and heavier vocal folds , eliciting a low - pitch sensation ( e.g. , f0 of 132 hz for men and 224 hz for women).17 ) f0 can be raised when a speaker increases tension to the vocal folds . the f0 value conveys not only the acoustic cues to vowel identity , intonation , and talker 's gender , but also the cues on the speakers ' age and even emotional state.16 ) when listeners simultaneously hear two speech signals with different f0s , it is easier to separate one from the other sound source . there is ample evidence that f0 separation remarkably improves performance for concurrent speech sounds overlapping in frequency and time , when the test materials were competing vowels,18 - 22 ) competing non - sense syllables,23 ) and competing sentences.16,24 - 26 ) a doubling of sound frequency is known as an octave . the octave interval is divided into twelve semitones ( sts ) , which results in one st corresponding to the 12th root of 2 ( i.e.,1 st=1.0595 ) . when concurrent vowels have been studied , often using a method referred to as the " double - vowel paradigm " , a small difference in f0 of less than 1 st dramatically enhanced identification accuracy or segregation perception . with f0 greater than 2 st , performance often reached an asymptote for normal - hearing ( nh ) listeners27 - 29 ) or both nh and hearing - impaired ( hi ) listeners.30 ) in contrast to the asymptote in the double - vowel paradigm , the young adults ' identification of competing sentences received a gradual and progressive benefit from f0 shifts beyond 2 st , regardless of whether the natural variation of f0 over time was preserved or artificially removed.24,25,31,32 ) besides the robust benefit from f0 separation in young normal hearers , f0 benefit has also been found in hi listeners with cochlear pathology and in older adults.18,19,22,30,33 - 35 ) for example , the ability of 8 nh ( age range : 21 - 30 yrs ) and 9 hi ( age range : 36 - 70 yrs ) listeners to utilize f0s was compared in a monotonic double - vowel paradigm.18 ) in this experimental design , each vowel was made sufficiently audible to the hi group by a presentation level of at least 90 db spl . overall , nh participants outperformed the hi group in vowel identification , yet the amount of benefit from a f0 of 2 st was similar to each group ( about 17 - 18 percentage points of improvement ) . the f0s greater than 2 st did not progressively enhance the vowel identification of either group . results of masked vowel thresholds showed that both nh and hi groups could identify the weaker - intensity target vowel against the more intense masker vowel , regardless of f0 separation between vowel pairs . taken together , the poorer identification and greater masking in the hi group ( compared to the nh group ) led the authors to speculate that both would lead to less efficient use of f0 cues by the hi . as a series of studies,33 ) the double vowels were spectrally adjusted by introducing 25 db of gain at frequencies above 1000 hz and additionally tested six hi listeners ( 1 hi : 38 yrs , 5 hi : 53 - 69 yrs ) who participated in the earlier study.18 ) despite high - frequency amplification ensuring the audibility of frequencies corresponding to the second and higher formants of vowels , the authors failed to observe a significant improvement in the overall identification performance of the hi group compared to that achieved without amplification . this suggests that the suprathreshold deficits in spectro - temporal processing , rather than speech audibility alone , contribute to the double - vowel performance of hi listeners . a follow - up study19 ) measured the ability to detect the presence of two different vowels and identify target words in young nh listeners and hi individuals of various ages ( age range : 19 - 76 yrs ) . in this double - vowel paradigm , two experiments were conducted where f0 , presentation mode , and target - to - masker ratio ( tmr ) at which listeners could just identify target vowels in the presence of a masking vowel were varied . results of the first experiment showed that the hi listeners were less accurate at identifying the target vowels than the nh group across five tmrs , although a strong benefit from f0 of 2 st was observed in both groups . in the second experiment , both nh and hi groups were superior at using f0 cues in the dichotic condition compared to the monaural condition , with a greater dichotic benefit in the hi group than in the nh group . given the reduced overall ability of hi group in vowel identification of two experiments , the authors conjectured that the increased susceptibility to peripheral masking from broadened auditory filters would degrade the internal representations of competing vowels and consequently yield poorer double - vowel perception for the hi group . in another study,30 ) competing vowels were separated by f0 values from 0 to 9 st and also the vowels ' harmonic structures were manipulated in order to explore whether the use of f0 depends on the disruption of harmonic structures in the low- or high - frequency regions . when the harmonic structures of five synthesized vowels were not disrupted , a f0 of 0.5 st improved the vowel - identification performance of the nh group by 33 percentage points and the same f0 separation benefited the hi group by 29 percentage points . neither group received additional benefit from f0 values beyond 1 st , which was consistent with the asymptote in other double - vowel studies . when the harmonic structures of double vowels were manipulated , the nh and hi groups were both effective at utilizing f0 cues in the lower - formant ( f1 ) frequency region . however , the hi listeners inefficiently derived f0 information from higher - order harmonics ( f2-f5 ) . their findings supported a lack of negative effects of hearing loss when making use of the f0 cues , at least , in the low - frequency region , and thus suggested an association between the degraded use of f0 in the hi listeners and their reduced frequency selectivity at high frequencies . the previous studies described above18,19,30,33 ) only focused on the use of f0 in a double - vowel paradigm . very few studies measured identification of both concurrent vowels and sentences . for example , the performance of middle - aged and older listeners with nh ( age range : 49 - 74 yrs ) or with hearing impairment ( age range : 59 - 77 yrs ) was measured on three tasks.35 ) the three tasks tested were f0 discrimination of a single vowel and the identification of competing vowels and sentences . the results for the difference limen of f0 ( f0 dl ) across five steady - state synthetic vowels revealed a greater sensitivity of nh listeners to f0 difference ( 0.5 - 2 hz for f0 dl ) compared to the hi listeners ( 1 - 4 hz for f0 dl ) . when the subjects identified concurrent vowels , the f0 shift from 0 to 4 st provided the nh group with a perceptual benefit of 20 percentage points while the same f0 increased the mean identification accuracy of the hi group by only 8 percentage points . the authors reported not only the slightly less f0 benefit in the hi group compared to the nh group on the double vowels and double sentences , but also larger individual differences in performance of hi listeners . interestingly , a different factors appeared to be related to the large intersubject variability of hi listeners for three - task performances.35 ) hearing sensitivity of the hi individuals at 2000 hz was predictive of their sensitivity to the f0 differences on the discrimination task . in contrast , chronological age best accounted for the amount of f0 benefit in the competing vowel or sentence paradigms , indicating an association between age - related internal changes among the middle - aged and elderly participants and the higher - level processing required for the identification of competing speech signals . a weak link between double - vowel and double - sentence identification was also found , and this emphasized a possibility of overgeneralization of results from competing - vowel studies to competing - sentence performance , at least for the middle - aged and older listeners . as noted , several previous studies18,19,30 ) agreed on the deleterious effect of cochlear hearing loss on performance in a double - vowel paradigm . in addition , there were often substantial age differences between the nh and hi groups . for example , the abilities between younger adults with normal hearing ( ynh ) and eldely listeners with normal hearing ( enh ) compared to demonstrate the age - related deficits in the use of f0 separation in a double - vowel paradigm.22 ) in the double - vowel paradigm , f0 dl measures using a synthesized vowel and vowel - identification performance using f0-separated double vowels were examined . when the f0 of the vowel /a/ was increased in steps of 0.1 hz under monaural presentation , the enh listeners were three times less sensitive to f0 compared to the ynh . here , individual differences in f0 dl were significantly correlated with age among the older adults . when the concurrent vowels were identified , mean identification accuracy of both listener groups improved markedly as f0 increased from 0 to 0.5 st in f0 , yet not beyond a 0.5 st shift . moreover , the enh group was , on average , less accurate than the ynh group in identifying both vowels , yet the vowel identification of three enh individuals was comparable to the mean identification accuracy of the ynh group . analyses of error responses revealed that incorrect responses of both the ynh and the enh groups occurred more from the misidentification of one vowel than from both vowels , suggesting a presence of vowel dominance . based on the results and the association between synchrony coding and benefit from f0 argued by de cheveign,36 ) the authors concluded that an age - related loss of neural synchrony increasing temporal jitter would evoke a declined periodicity coding and consequently a degraded benefit from f0 for concurrent speech signals . a recent study16 ) examined the negative effects of both hearing loss and age - related declines on the use of f0 by comparing four listener groups differing in hearing status and age . results showed that f0 cues were beneficial to both cue - word detection and color - number identification performance . elderly adults with impaired hearing had the greatest difficulty with the identification task despite the application of spectral shaping to restore the audibility of the speech stimuli . also the authors concluded that the reduced audibility or aging alone is not the cause for the poor performance of the elderly hi listeners , indicating a combined contribution of hearing loss and aging on the reduced f0 benefit . as discussed above , age - related declines in the use of f0 appeared to be clear in numerous studies . can any hearing assistive devices help old people better access to f0 cues ? recently , in order to address whether electro - acoustic and cochlear - implant hearing can enhance processing of f0 , both young and older listeners ' use of f0 cues was compared in simulations of electro - acoustic and cochlear - implant hearing.37 ) although the electric and acoustic stimulation ( eas ) is known to allow the listeners to combine residual low - frequency hearing with electrical hearing , expecting a greater benefit from f0s , some studies reported that old listeners may not benefit from eas devices as much as young listeners . thus , researchers37 ) focused on the ability of aged listeners in the processing of f0 cues using eas and cochlear - implant hearing simulations . the age - related deficits in the perception of vowel multiplicity were found in terms of amount of f0 benefit as well as overall identification . however , eas simulation provided a large inter - subject variability in f0 benefit of older individuals , consequently requiring more research on the effectiveness of eas stimulus to the old individuals in using f0 cues and other various acoustic cues . to understand the mechanism underlying the age - related degradation in the use of f0 or periodicity coding , it is critical to review the different aspects of three models , spectral , spectro - temporal , and temporal models , that can account for the f0 benefit observed for concurrent speech signals . in the spectral model , based on a place theory , auditory peripheral filtering is used to allocate frequency components corresponding to each competing vowel . according to the place theory of pitch , different pure tones produce maximal activity at different places with maximal activity near the base of the cochlea for high frequencies and near the apical part in the cochlea for low frequencies . these different spatial patterns of mechanical activity along the basilar membrane lead to activity in different auditory nerve fibers distributed along the length of the cochlea , producing the tonotopic responses of the auditory nerve . fig . 1 displays schematic magnitude spectra of mixtures of two different vowels , /i/ , /a/ , and mixed vowel of /i/ and /a/. as reported,38 ) the spectral envelope of the mixed vowels with different f0s shows peaks of each harmonic series at different frequencies corresponding to the first - formant ( f1 ) frequency of each vowel , allowing a better resolution of individual harmonic structures . in contrast , the peaks in the spectral envelope of the mixed vowels with the same f0 are not separable as depicted by filled circles in fig . 1 , suggesting that information on the individual harmonic series of each vowel would be poorly resolved for the competing vowels with the same f0 . that is , in the spectral model based on the peripheral filtering , the degree of spectral resolution of two competing vowels was assumed to directly affect the identification accuracy of competing vowels , or the perceptual benefit of f0 in a double - vowel paradigm . in the spectro - temporal model , the information of each vowel is analyzed in a series of auditory peripheral channels as in the spectral model . yet , a subset of auditory channels responds to the period of one vowel and to the period of the other vowel . in this way , the periodicity corresponding to each vowel can be determined based on the spectro - temporal analysis over time even when the double vowels share the same auditory channel . when waveform interaction or beating is the basis of f0 benefit in the spectro - temporal model,21,28,39 ) a remarkable f0 benefit is possible even at small f0 separations between double vowels as long as the periodicity of the waveforms or the harmonicity of the speech signals is decoded.40 ) when there is a small f0 between two vowels , the f0 or corresponding harmonics of each vowel can be closely located in terms of frequency and possibly excite the same region of the basilar membrane . those two closely - spaced spectral or harmonic components might generate beating as a product of waveform interactions . 2a shows that a mixture of 125-hz and 128-hz pure tones produces rising and falling amplitude patterns or spectral envelopes with a repetition period of 3 hz , which is a relatively slow modulation . whereas , a mixture of 125-hz and 200-hz pure - tone ( fig . when the temporal - envelope fluctuation of 3 hz is added to the dynamic fluctuation of two competing sounds , this might allow the listeners to glimpse the target signal against the masker one . better identification of longer - duration vowel pairs relative to shorter vowel pairs20,41,42 ) may be evidence of the spectro - temporal analyses since a longer duration might allow better encoding of a full modulation cycle in beating . as spectro - temporal model , an array of autocorrelation functions or autocorrelated temporal patterns between signals was also used to explain f0 benefit between concurrent vowels.43 ) in this spectro - temporal model based on autocorrelation segregation , autocorrelation functions of individual peripheral channel outputs are summed across channels . in a double - vowel paradigm , the largest peak within a summary of distributed autocorrelation function is considered a dominant periodicity , and all the channels with the dominant periodicity are selected to segregate a target vowel from a competing vowel . in other words , the auditory channels responding to the period of the dominant vowel are selected and partitioned from other channels , producing a strong f0 benefit in vowel identification . since the autocorrelation function determines the dominant periodicity or temporal pattern over the others , the requirement in this model is that each of the individual peripheral channels should be active for the different periodicities of each competing vowel , and the masker vowel should not dominate all the auditory channels . overall , the spectro - temporal models based on time - domain periodicity have been better than the spectral model in explaining the significant role of a very small f0 shift in the double - vowel paradigm , especially when the level of the target vowel is at or above that of the masker vowel . however , both the spectral model and the spectro - temporal model fail to explain how the f0 separation becomes beneficial to vowel identification even when the level of the target vowel is weaker , by 10 or 20 db , than that of the interfering vowel,19,36,44 ) presumably making all the auditory channels dominated by the more intense vowel and the individual harmonics of the target vowel unresolved . a temporal model based on a neural cancellation mechanism was proposed to explain the salient f0 benefit at negative tmrs more effectively.36 ) the temporal model based on the neural cancellation filter employs the temporal discharge pattern of auditory nerve fibers occurring within a channel , rather than requiring harmonic resolution across channels . fig . 3 demonstrates how the neural cancellation filter works by displaying a discharge distribution of an auditory never fiber in response to a 100-hz pure - tone pulse.36 ) the auditory - nerve fiber response to a single vowel with f0 of 100 hz is represented as a simple half - wave rectified sine wave of 100 hz in fig . 3a , and this serves as the input to the neural cancellation filter to the vowel . the neural cancellation filter calculates a delay that corresponds to the periodicity within input spikes and cancels the input spikes corresponding to the delay pattern , seen as the output of the cancellation filter in fig . 3c is another input consisting of a half - wave rectified sum of two sine waves , 100 hz ( target ) and 80 hz ( competing ) , corresponding to a mixture of 100-hz target vowel and 80-hz masker vowel with a 10-db greater intensity . with competing vowels with different f0 values , the neural cancellation filter calculates a delay according to different periodicity of each vowel and is tuned to cancel the period of the masker vowel , discarding information about the periodicity of the masker vowel , as displayed in fig . the temporal model is based on the periodicity calculation , and this thus supposes that auditory system can derive pitch information from the period of discharges of auditory nerve fibers that do not resolve individual harmonics of the speech signal , even though the target vowel is presented with the more intense masker vowel . in other words , the temporal model selects the auditory channels based on the neural cancellation filters , not based on the outputs of auditory peripheral channels . this also appears consistent with some behavioral findings of a strong f0 benefit at unfavorable tmrs.19,44 ) using the guinea pig , the distribution of neural synchrony was examined across the population of auditory nerve fibers.45,46 ) by counting the spikes in bins synchronized with the double vowels , a histogram was constructed determining interspike interval distributions of the auditory nerve to the mixed vowel . the data were well fit via temporal analysis , compared to fits by spectral or spectro - temporal analyses . other animal studies , based on a wide variety of measurements , have observed an age - related reduction in the synchrony of auditory nerve fiber responses.47 - 51 ) as noted above , among the three types of model that have been developed , the temporal model provides the most favorable explanation for f0 benefit at low tmr values and for the age - related reduction in the f0 benefit for the vowel identification . if the older listeners have reduced neural activity or a loss of neural synchrony , this would result in a decline in periodicity coding and an inefficient neural cancellation of competing speech signals . however , this does not necessarily mean that the deleterious effect of cochlear hearing loss would be unrelated to the outputs of the neural cancellation operation . although a broadening of auditory filters due to cochlear pathology would not directly damage the process of neural cancellation operations per se , the reduced input information due to the reduced audibility would , at least partially , degrade the input into the neural cancellation filter , possibly limiting the effectiveness of the neural cancellation filter . the primary objective of this study was to provide information on the age - related declines in the use of f0s between competing voices and the controversial theoretical explanations on this issue . as reviewed , aging adversely affected the identification of the target speech , either vowel or sentence , against interfering competing speech . as a possible explanation , aging can negatively influence neural encoding of the temporal properties in competing sounds , consequently resulting in a declined periodicity coding and an inefficient neural cancellation of competing speech signals . this was better supported by temporal model based on the periodicity calculation compared to other theoretical models . in order to improve the ability of older adults to utilize f0s between competing voices , future studies are needed to investigate innovative technical development of the hearing assistive devices focusing on better access to the f0 segregation cues and also deriving greater f0 benefits to the older listeners .

purposeto evaluate the clinical outcome and mri findings after carotid artery stenting ( cas ) without post-dilatation.methodsbetween may 2005 and april 2012 , a total of 169 consecutive patients ( 61.4% symptomatic ) underwent 176 cas procedures performed with an embolic protection device ( guardwire , n=116 ; filterwire ez , n=60 ) . all stents were deployed without post - dilatation . periprocedural complications and mid - term outcomes were analyzed.resultsthe stroke rate was 2.3% within 30 days post - cas ( asymptomatic patients 1.5% ; symptomatic patients 2.8% ) . cerebral infarction occurred in one asymptomatic patient ( 1.5% ) and one symptomatic patient ( 0.9% ) . intracranial hemorrhage occurred in two symptomatic patients ( 1.9% ) . post - cas diffusion - weighted imaging ( dwi ) revealed a high - intensity area in 26 of 176 procedures ( 14.8% ) . ipsilateral stroke after 31 days occurred in two patients ( 1.1% ) and restenosis occurred in six ( 3.4% ) . a post - cas comparison of the embolic protection devices revealed no difference in stroke incidence within 30 days and in dwi high - intensity area.conclusionsour cas procedure without post - dilatation is feasible , safe and associated with a low incidence of stroke and restenosis .

carotid artery stenting ( cas ) is the standard modality for patients with severe carotid artery stenosis at high risk for carotid endarterectomy . however , a major drawback of cas is the potential risk of embolic stroke caused by the dislodgement of atheromatous plaque during the procedure . the traditional cas procedure involves initial vascular balloon dilatation ( pre - dilatation ) to achieve access for other devices , followed by optional placement of an embolic protection device ( epd ) , positioning of a stent across the stenosis and , finally , post - stenting balloon dilatation ( post - dilatation ) to achieve maximum vessel expansion . emboli can arise in each procedural step , but most emboli are produced during the post - dilatation stage , which might occur if the balloon pushes the stent struts against the atheromatous plaque . post - dilatation is therefore considered to be the riskiest part of the procedure.1 2 to minimize procedural thromboembolic complications , we sometimes perform cas without post - dilatation ; the aim of this study was to evaluate the efficacy and safety of our technical strategy for cas . a retrospective registry between may 2005 and april 2012 was accessed to recover 171 consecutive patients with 178 carotid artery stenoses who underwent cas with an epd at either the national hospital organization mito medical center ( ibaraki , japan ) or an affiliated hospital . cas was indicated in symptomatic or asymptomatic patients by a carotid artery stenosis of > 50% or > 80% , respectively , according to north american symptomatic carotid endarterectomy trial ( nascet ) criteria.3 our study included 169 patients ( 148 men , 21 women ) with 176 carotid artery stenoses who underwent cas without post - dilatation . a total of 108 lesions ( 61.4% ) were symptomatic and 68 ( 38.6% ) were asymptomatic . patients received two of the three following antiplatelet agents : aspirin ( 100 mg ) , clopidogrel ( 75 mg ) and cilostazol ( 200 mg ) for at least 1 week before the procedure . systemic heparinization was administered following insertion of a sheath , and an activated clotting time of 300 s was maintained during the procedure . a guiding catheter or long sheath was advanced into the common carotid artery . under roadmap guidance , the stenotic lesion was crossed with an epd which was navigated distal to the stenotic lesion . all 107 cas procedures through may 2010 were performed using the guardwire temporary occlusion and aspiration system ( medtronic , santa rosa , california , usa ) . after june 2010 we primarily performed cas using the filterwire ez embolic protection system ( boston scientific , natick , massachusetts , usa ) . cas was performed using the guardwire system for patients with soft plaque deposits , as identified by a black blood mri indicating high - intensity and long lesions ( > 25 mm ; figure 1 ) . after june 2010 we performed cas using the filterwire ez system for 60 lesions and the guardwire system for nine lesions . pre - stenting balloon dilatation was subsequently applied to the stenotic lesions after epd placement and was usually performed using a 4.05.5 mm30 mm angioplasty balloon . the most commonly used stents were wallstents ( boston scientific , natick , massachusetts , usa ) , which were applied in 174 lesions ( 98.9% ) , and precise stents ( cordis , johnson & johnson , miami , florida , usa ) , which were applied in two lesions . angiographic views of the head and neck were obtained at the onset of the procedure and after successful stent placement . moreover , 16 patients with preoperative hemodynamic compromise or postoperative angiographic hyperemia of the lenticulostriate arteries underwent intravenous deep sedation for 24 h following the procedure . all the patients underwent a clinical examination ( including a neurological examination ) and mri including dwi both before and 24 h after the procedure . two antiplatelet agents were administered for a minimum of 3 months after surgery , after which one of the agents was discontinued . following discharge , the patients were examined and ultrasound follow - up studies were performed at 30 days , 3 months and then once every 3 months indefinitely . an octogenarian with symptomatic carotid artery stenosis who underwent carotid artery stenting using the guardwire system . ( a ) black blood t1-weighted mri showing a high - intensity plaque lesion in the right internal carotid artery ( white arrow ) . ( b ) angiogram of the right common carotid artery showing severe stenosis of the internal carotid artery with a long lesion . ( c ) post - procedural angiogram of the right common carotid artery ( pre - dilatation with 430 mm balloon and stent deployment with 1024 mm carotid wallstent ) showing improvement of the internal carotid artery stenosis . a major adverse event was defined as any incidence of stroke , myocardial infarction or death within 30 days after cas . stroke was defined as a neurological deficit that persisted for > 24 h , whereas a minor stroke was defined as a new neurological deficit that resolved completely or returned to baseline within 30 days . a transient ischemic attack ( tia ) was defined as a new neurological deficit that persisted for < 24 h and completely resolved or returned to baseline . statistical analysis was performed to evaluate the efficacy of our cas procedure according to epd as well as the proportion of residual stenosis immediately after the procedure . nominal data were analyzed using the fisher exact test and numerical data were analyzed using the t test for parametric values or the mann a retrospective registry between may 2005 and april 2012 was accessed to recover 171 consecutive patients with 178 carotid artery stenoses who underwent cas with an epd at either the national hospital organization mito medical center ( ibaraki , japan ) or an affiliated hospital . cas was indicated in symptomatic or asymptomatic patients by a carotid artery stenosis of > 50% or > 80% , respectively , according to north american symptomatic carotid endarterectomy trial ( nascet ) criteria.3 our study included 169 patients ( 148 men , 21 women ) with 176 carotid artery stenoses who underwent cas without post - dilatation . a total of 108 lesions ( 61.4% ) were symptomatic and 68 ( 38.6% ) were asymptomatic . patients received two of the three following antiplatelet agents : aspirin ( 100 mg ) , clopidogrel ( 75 mg ) and cilostazol ( 200 mg ) for at least 1 week before the procedure . systemic heparinization was administered following insertion of a sheath , and an activated clotting time of 300 s was maintained during the procedure . a guiding catheter or long sheath was advanced into the common carotid artery . under roadmap guidance , the stenotic lesion was crossed with an epd which was navigated distal to the stenotic lesion . all 107 cas procedures through may 2010 were performed using the guardwire temporary occlusion and aspiration system ( medtronic , santa rosa , california , usa ) . after june 2010 we primarily performed cas using the filterwire ez embolic protection system ( boston scientific , natick , massachusetts , usa ) . cas was performed using the guardwire system for patients with soft plaque deposits , as identified by a black blood mri indicating high - intensity and long lesions ( > 25 mm ; figure 1 ) . after june 2010 we performed cas using the filterwire ez system for 60 lesions and the guardwire system for nine lesions . pre - stenting balloon dilatation was subsequently applied to the stenotic lesions after epd placement and was usually performed using a 4.05.5 mm30 mm angioplasty balloon . the most commonly used stents were wallstents ( boston scientific , natick , massachusetts , usa ) , which were applied in 174 lesions ( 98.9% ) , and precise stents ( cordis , johnson & johnson , miami , florida , usa ) , which were applied in two lesions . angiographic views of the head and neck were obtained at the onset of the procedure and after successful stent placement . moreover , 16 patients with preoperative hemodynamic compromise or postoperative angiographic hyperemia of the lenticulostriate arteries underwent intravenous deep sedation for 24 h following the procedure . all the patients underwent a clinical examination ( including a neurological examination ) and mri including dwi both before and 24 h after the procedure . two antiplatelet agents were administered for a minimum of 3 months after surgery , after which one of the agents was discontinued . following discharge , the patients were examined and ultrasound follow - up studies were performed at 30 days , 3 months and then once every 3 months indefinitely . an octogenarian with symptomatic carotid artery stenosis who underwent carotid artery stenting using the guardwire system . ( a ) black blood t1-weighted mri showing a high - intensity plaque lesion in the right internal carotid artery ( white arrow ) . ( b ) angiogram of the right common carotid artery showing severe stenosis of the internal carotid artery with a long lesion . ( c ) post - procedural angiogram of the right common carotid artery ( pre - dilatation with 430 mm balloon and stent deployment with 1024 mm carotid wallstent ) showing improvement of the internal carotid artery stenosis . a major adverse event was defined as any incidence of stroke , myocardial infarction or death within 30 days after cas . stroke was defined as a neurological deficit that persisted for > 24 h , whereas a minor stroke was defined as a new neurological deficit that resolved completely or returned to baseline within 30 days . a transient ischemic attack ( tia ) was defined as a new neurological deficit that persisted for < 24 h and completely resolved or returned to baseline . statistical analysis was performed to evaluate the efficacy of our cas procedure according to epd as well as the proportion of residual stenosis immediately after the procedure . nominal data were analyzed using the fisher exact test and numerical data were analyzed using the t test for parametric values or the mann cas was performed successfully for all lesions , although major adverse events occurred in four patients ( 2.3% ) . the overall stroke rate was 2.3% within 30 days after cas ; none of the patients experienced myocardial infarction and there were no deaths . cerebral infarction and intracranial hemorrhage occurred in two patients each ( 1.1% ) and tia occurred in three patients ( 1.7% ) . dwi obtained after cas revealed a high - intensity area in 26 of 176 procedures ( 14.8% ) . in 68 asymptomatic patients , one minor stroke ( 1.5% ) occurred because of subacute instent thrombosis , one tia ( 1.5% ) occurred within 30 days after cas and dwi obtained 24 h after cas revealed a high - intensity area in seven procedures ( 10.3% ) . in 108 symptomatic patients , one minor stroke ( 0.9% ) occurred because of thromboembolic complications . two intracranial hemorrhages and two tias ( 1.9% ) occurred within 30 days after cas , and dwi obtained 24 h after cas revealed a high - intensity area in 19 ( 17.6% ) procedures ( table 1 ) . subacute in - stent thrombosis complicated with tia developed in one of the symptomatic patients . follow - up data beyond 1 year for all 176 lesions included two cases of symptomatic restenosis . the ipsilateral stroke rate was 1.1% ( symptomatic 1.9% ; asymptomatic 0% ) after 31 days . four asymptomatic restenosis cases were diagnosed during follow - up ultrasound examinations ; subsequently , three of these patients underwent angioplasty and the overall restenosis rate was 3.4% . the mean stenosis diameter according to the nascet criteria3 was reduced from 75.011.8% to 17.311.2% . the immediate angiographic outcomes after cas were as follows : residual stenosis 10% ( n=54 , 30.7% ) ; residual stenosis > 10% and 20% ( n=52 , 29.5% ) ; residual stenosis > 20% and 30% ( 30% and 40% ( n=22 , 12.5% ) ; and residual stenosis > 40% ( n=5 , 2.8% ) . restenosis or subacute in - stent thrombosis after cas was observed in eight patients . the mean stenosis rate immediately after cas was 32.1% in this group , compared with 16.9% in the group without restenosis or subacute in - stent thrombosis . thus , the mean stenosis rate immediately after cas was significantly higher in the group with restenosis or subacute in - stent thrombosis ( p=0.0004 ) . five of 27 patients ( 18.5% ) with residual stenosis immediately after cas > 30% suffered restenosis or in - stent thrombosis and three of 149 patients ( 2.0% ) without residual stenosis immediately after cas > 30% suffered restenosis or in - stent thrombosis . the rate of restenosis or in - stent thrombosis was significantly higher in the group with residual stenosis immediately after cas > 30% ( table 2 ) . comparison of the procedural outcomes according to the residual stenosis immediately after carotid artery stenting ( cas ) a comparison of epd ( table 3 ) revealed no difference in the incidence of stroke within 30 days after cas and high intensity areas on dwi obtained 24 h after cas . comparison of the procedural outcomes between patients treated with the guardwire ( gw ) and filterwire ez ( fw ) systems dwi , diffusion - weighted imaging . strokes arising from carotid stenosis are most often due to atheroembolisms.46 during carotid endarterectomy the plaque is completely removed ; however , with carotid stenting , the plaque remains contained between the stent and the vessel wall . stroke occurring after cas is probably caused by the release of fractured plaque deposits through the struts of the stent . in the carotid revascularization endarterectomy versus stenting trial,7 the periprocedural stroke rate was significantly higher in the stenting group than in the endarterectomy group ( 4.1% vs 2.3% , p=0.01 ) . transcranial doppler studies have demonstrated the generation of emboli with each passage across a stenosis with a guidewire , epd , balloon or stent,8 9 with the highest potential for embolization occurring during post - dilatation when the balloon crushes friable plaque against the metal stent struts.8 9 although the clinical significance of microemboli is unclear , ackerstaff et al10 demonstrated in a study of 550 patients that multiple microemboli ( > 5 showers ) at post - dilatation were independently associated with cerebral deficits . the traditional cas technique includes initial balloon pre - dilatation during which the epd or stent is accessible distal to the stenosis , subsequent optional placement of the epd followed by deployment of a stent covering the stenosis , and finally post - dilatation to achieve maximum vessel expansion after stent deployment . we performed cas without post - dilatation to minimize plaque disruption and the incidence of periprocedural stroke . the results of this study revealed a relatively low incidence of complications within 30 days after the procedure ( 2.3% overall stroke , death or myocardial infarction ) . symptomatic patients are known to have emboligenic plaque.11 in the carotid revascularization endarterectomy versus stenting trial , the rate of any post - procedural stroke within 30 days was 5.5% in symptomatic patients treated with cas and 3.2% in symptomatic patients treated with carotid endarterectomy.7 in this study , the rate of any post - procedural stroke within 30 days was 1.5% in asymptomatic patients and 2.8% in symptomatic patients . in symptomatic patients , the incidence of periprocedural stroke was relatively low in this study because the omission of post - dilatation might have minimized plaque disruption and reduced thromboembolic complications . most embolic complications occur after the procedure and are probably caused by late emboli through the struts of the stent , as described above.11 a closed cell stent has smaller struts than an open cell stent.12 bosiers et al11 reported that the rate of post - procedural events was higher when using the open cell stent than when using the closed cell stent . these differences were highly pronounced among symptomatic patients.11 wallstents , the most commonly used stents in this study , were applied in 174 lesions ( 98.9% ) . in addition to the omission of post - dilatation , the use of a wallstent might also reduce thromboembolic complications . the closed cell stent has a lower radial force than an open cell stent,12 and poor stent expansion immediately after the procedure was associated with a higher risk of restenosis.13 furthermore , omission of post - dilatation and the use of a closed cell stent might cause restenosis . a systematic review by grschel et al14 reported a restenosis rate with conventional cas of 46% at 1 year ; the rate of restenosis with our cas protocol ( 3.4% ) was therefore lower than that previously reported . in our procedure , the goal of pre - dilatation was to enlarge the stenotic lumen suboptimally with only the balloon . therefore , pre - dilatation alone without post - dilatation can achieve a sufficient luminal opening to prevent restenosis . on the other hand , arterial injury with both balloons and stents can initiate an inflammatory response and activate a proliferative repair process , the end result of which can lead to luminal narrowing and in - stent restenosis.1519 in fact , omission of post - dilatation and the use of a closed cell stent might be beneficial to minimize such restenosis incidents . on the other hand , the rate of restenosis or in - stent thrombosis was significantly higher in the patient group with > 30% residual stenosis immediately after cas in the present study . post - dilatation might therefore be added to avoid restenosis and in - stent thrombosis in patients with > 30% residual stenosis . nevertheless , additional post - dilatation might induce thromboembolic complications , particularly in symptomatic patients with unstable risky plaque deposits . in cases with > 30% residual stenosis immediately after cas , we should consider whether or not to add post - dilatation to the procedure for symptomatic patients and decide how much post - dilatation to add . in a previous study to evaluate microembolic signals detected by transcranial doppler during cas , the amount of microembolic signals was significantly higher using the filterwire system than with the guardwire system.20 the distal occlusion device is thought to be more effective than the filter in reducing distal embolization.20 however , balloon occlusion ( whether proximal or distal ) is associated with cerebral intolerance in 510% of cases.21 22 on the other hand , the main advantage of filters is the maintenance of blood flow to the brain throughout the procedure . a comparison of epds revealed no difference in the incidence of stroke within 30 days after cas and the high - intensity area on dwi obtained 24 h after cas in the present study . cas without post - dilatation can take advantage of filter protection because of the reduction in distal embolisms . second , we could not observe any advantage of omission of post - dilatation using the open cell stent , although closed cell stents were used in 174 lesions ( 98.9% ) while open cell stents were used in only two lesions . lastly , this analysis could be improved by a longer radiological and clinical follow - up period to further evaluate the clinical significance . carotid stenting without post - dilatation might achieve minimal plaque disruption and reduction of thromboembolic complications . our cas procedure is effective , particularly in symptomatic patients . although it might be possible to employ new types of stents and epds in the future , concepts to minimize plaque disruption are important because plaque deposits remain contained in the vessel wall in carotid stenting procedures

backgroundprevious studies reported that the mild hypothermia therapy ( mht ) could significantly improve the clinical outcomes for patients with hypertensive intracerebral hemorrhage ( hich ) . therefore , this meta - analysis was conducted to systematically assess whether the addition of local mht ( lmht ) could significantly improve the efficacy of minimally invasive surgery ( mis ) in treating hich.methodsrandomized clinical trials on the combined application of mis and lmht ( mis+lmht ) vs mis alone for treating hich were searched up to september 2016 in databases . response rate and mortality rate were the primary outcomes , and the neurologic function and barthel index were the secondary outcomes . side effects were also analyzed.resultstotally , 28 studies composed of 2,325 patients were included to compare the efficacy of mis+lmht to mis alone . the therapeutic effects of mis+lmht were significantly better than mis alone . the pooled odds ratio of response rate and mortality rate was 2.68 ( 95% confidence interval [ ci]=2.223.24 ) and 0.43 ( 95% ci=0.320.57 ) , respectively . in addition , the mis+lmht led to a significantly better improvement in the neurologic function and activities of daily living . the incidence of pneumonia was similar between the two treatment methods.conclusionthese results indicated that compared to mis alone , the mis+lmht could be more effective for the acute treatment of patients with hich . this treatment modality should be further explored and optimized .

intracerebral hemorrhage ( ich ) is a common cerebrovascular disease with a high mortality and poor outcomes in clinical practice , and also the leading cause of death and disability in elderly patients.1 it was reported to account for about 10% of all strokes and 30% of all cerebrovascular diseases.2,3 the mortality rate could be up to 50% , and the mortality rate in one month could be up to 40%.3,4 moreover , there could be up to 40% of severely disabled survivors.3 normally , the edema and hematoma expansion are the two major factors contributing to worsened outcomes and secondary damage.5 many factors could lead to ich , but hypertension is the major cause of ich , and the hypertensive ich ( hich ) is a common neurological disease . to date , there are still no effective drug therapies for hich.6 moreover , hich is characterized by high incidence , high mortality and high morbidity , which seriously endangers the health of patients and causes substantial economic burden for families and society . therefore , it is urgently necessary to develop an effective therapy for this disease . during the treatment , the key point is to quickly clear the hematoma and reduce the intracranial pressure.7 along with minimally invasive technique , minimally invasive surgery ( mis ) for hich is gaining increasing attention . it could effectively decrease the mortality rate in the acute treatment of patients with hich.8 additionally , some previous studies reported that the mild hypothermia therapy ( mht ) could be useful for neuroprotection and be used to treat cerebral edema after acute brain injury.911 previous study reported that the addition of mht could significantly improve the efficacy of mis in treating hich.12 therefore , the aim of this study was to review the available published studies and conduct a meta - analysis to systematically assess whether the combined application of mis and local mht ( lmht ) ( mis+lmht ) could result in a better efficacy than mis alone . the first step of this meta - analysis was to obtain the eligible clinical trials . we conducted electronic searches in the following databases : cochrane controlled trails register , pubmed , embase , web of science , chinese biomedical literature database on disc and chinese national knowledge infrastructure ( dated up to september 2016 ) . the search terms that we used were hypothermia , minimally invasive and hypertensive cerebral hemorrhage . the inclusion criteria included the following items : 1 ) randomized clinical trials comparing the mis+lmht with mis alone ; 2 ) patients with hypertensive cerebral hemorrhage over 18 years of age ; 3 ) the outcomes including at least one of these three indexes : response rate , mortality rate and neurologic function ; and 4 ) patients could provide informed consent . the exclusion criteria included the following items : 1 ) no control group or not used mis as the control group and 2 ) case reports , reviews and duplicate studies . the neurologic function , barthel index and side effects were chosen as the secondary outcomes . the barthel index was used to assess the activities of daily living ( adl ) after treatment . because pneumonia might be the main side effect caused by hypothermia,13 we only analyzed the incidence of pneumonia to assess the acceptability of these treatment methods . in order to ensure the high accuracy of the extracted data , two authors ( yu han and ke sheng ) were arranged to independently screen the potential studies according to the aforementioned inclusion / exclusion criteria and extract the data . the extracted data included the demographic data of the patients , the parameters of the mis and lmht , the first author and outcomes ( primary and secondary outcomes ) . good - faith efforts were made to obtain the data which were not available from the included studies . revman 5.1 software was used to conduct the meta - analysis . for discontinuous data , the summary odds ratio ( or ) was used as the effect size ; for continuous data , the weighted mean difference ( wmd ) was used as the effect size . the chi - square test and i index were used to assess the heterogeneity.14 if the corresponding p - value was more than 0.10 and i was less than 50% , then the mantel haenszel fixed - effects model was used ; otherwise , the random - effects model was used . the first step of this meta - analysis was to obtain the eligible clinical trials . we conducted electronic searches in the following databases : cochrane controlled trails register , pubmed , embase , web of science , chinese biomedical literature database on disc and chinese national knowledge infrastructure ( dated up to september 2016 ) . the search terms that we used were hypothermia , minimally invasive and hypertensive cerebral hemorrhage . the inclusion criteria included the following items : 1 ) randomized clinical trials comparing the mis+lmht with mis alone ; 2 ) patients with hypertensive cerebral hemorrhage over 18 years of age ; 3 ) the outcomes including at least one of these three indexes : response rate , mortality rate and neurologic function ; and 4 ) patients could provide informed consent . the exclusion criteria included the following items : 1 ) no control group or not used mis as the control group and 2 ) case reports , reviews and duplicate studies . the response rate was defined according to the criteria of the included studies . the neurologic function , barthel index and side effects were chosen as the secondary outcomes . the barthel index was used to assess the activities of daily living ( adl ) after treatment . because pneumonia might be the main side effect caused by hypothermia,13 we only analyzed the incidence of pneumonia to assess the acceptability of these treatment methods . in order to ensure the high accuracy of the extracted data , two authors ( yu han and ke sheng ) were arranged to independently screen the potential studies according to the aforementioned inclusion / exclusion criteria and extract the data . the extracted data included the demographic data of the patients , the parameters of the mis and lmht , the first author and outcomes ( primary and secondary outcomes ) . good - faith efforts were made to obtain the data which were not available from the included studies . discontinuous data , the summary odds ratio ( or ) was used as the effect size ; for continuous data , the weighted mean difference ( wmd ) was used as the effect size . the chi - square test and i index were used to assess the heterogeneity.14 if the corresponding p - value was more than 0.10 and i was less than 50% , then the mantel haenszel fixed - effects model was used ; otherwise , the random - effects model was used . based on the inclusion and exclusion criteria , 129 studies were excluded . the reasons for exclusion included the following : 1 ) no randomization , 2 ) no control group , 3 ) compared the mis+lmht with the conservative treatment , 4 ) no available data , 5 ) did not use mis and 6 ) being a retrospective study . finally , 28 clinical trials composed of 2,325 adult patients with hypertensive cerebral hemorrhage were included in this meta - analysis.1643 the matched demographic data were observed in the included studies ( table 1 ) . more than half of the included studies used cooling blanket as the device of lmht . overall , 916 of 1,156 ( 79.2% ) patients receiving the mis+lmht and 673 of 1,129 ( 59.6% ) patients receiving mis alone were classified as responders . the pooled or was 2.68 with 95% confidence interval ( ci)=2.223.24 , which indicated that the mis+lmht could yield a higher response rate than mis alone . overall , 79 of 883 ( 8.9% ) patients receiving the mis+lmht and 162 of 868 ( 18.7% ) patients receiving mis alone died after treatment . the pooled or was 0.43 with 95% ci=0.320.57 , which indicated that the mis+lmht could yield a lower mortality rate than mis alone . the funnel plot showed that there was no publication bias ( figure 2 ) . among the included studies , 11 studies used css to assess the neurologic function of patients ( figure 4 ) . eight of 11 studies assessed the neurologic function one month after treatment ; two studies conducted the assessment 21 days later and one study 14 days later . the pooled wmd was 5.83 with 95% ci=7.18 to 4.47 , which indicated that the mis+lmht could be more effective than mis alone in improving the neurologic function of patients . seven studies assessed adl using barthel index one month after treatment ( figure 5a ) . heterogeneity ( i=67% , p=0.006 ) existed . ten studies assessed adl three months after treatment ( figure 5b ) . heterogeneity ( i=87% , p<0.00001 ) existed . these results indicated that the mis+lmht could be more effective than mis alone in improving the adl of patients . overall , 17 of 77 patients receiving the mis+lmht and 16 of 76 patients receiving the mis alone experienced pneumonia . the pooled or was 1.05 with 95% ci=0.432.59 , which indicated that the incidence of pneumonia was similar between these two treatment methods . based on the inclusion and exclusion criteria , 129 studies were excluded . the reasons for exclusion included the following : 1 ) no randomization , 2 ) no control group , 3 ) compared the mis+lmht with the conservative treatment , 4 ) no available data , 5 ) did not use mis and 6 ) being a retrospective study . finally , 28 clinical trials composed of 2,325 adult patients with hypertensive cerebral hemorrhage were included in this meta - analysis.1643 the matched demographic data were observed in the included studies ( table 1 ) . more than half of the included studies used cooling blanket as the device of lmht . overall , 916 of 1,156 ( 79.2% ) patients receiving the mis+lmht and 673 of 1,129 ( 59.6% ) patients receiving mis alone were classified as responders . the pooled or was 2.68 with 95% confidence interval ( ci)=2.223.24 , which indicated that the mis+lmht could yield a higher response rate than mis alone . overall , 79 of 883 ( 8.9% ) patients receiving the mis+lmht and 162 of 868 ( 18.7% ) patients receiving mis alone died after treatment . the pooled or was 0.43 with 95% ci=0.320.57 , which indicated that the mis+lmht could yield a lower mortality rate than mis alone . among the included studies , 11 studies used css to assess the neurologic function of patients ( figure 4 ) . eight of 11 studies assessed the neurologic function one month after treatment ; two studies conducted the assessment 21 days later and one study 14 days later . heterogeneity ( i=91% , p<0.0001 ) existed . the pooled wmd was 5.83 with 95% ci=7.18 to 4.47 , which indicated that the mis+lmht could be more effective than mis alone in improving the neurologic function of patients . seven studies assessed adl using barthel index one month after treatment ( figure 5a ) . heterogeneity ( i=67% , p=0.006 ) existed . ten studies assessed adl three months after treatment ( figure 5b ) . heterogeneity ( i=87% , p<0.00001 ) existed . these results indicated that the mis+lmht could be more effective than mis alone in improving the adl of patients . overall , 17 of 77 patients receiving the mis+lmht and 16 of 76 patients receiving the mis alone experienced pneumonia . the pooled or was 1.05 with 95% ci=0.432.59 , which indicated that the incidence of pneumonia was similar between these two treatment methods . we conducted this meta - analysis of 28 randomized clinical trials to compare the efficacy of mis+lmht with mis alone in the treatment of patients with hich . the results showed that the mis+lmht could yield higher response rate ( or=2.68 ) and lower mortality rate ( or=0.43 ) . moreover , this treatment modality could significantly improve the neurologic function and adl of patients . with respect to the analysis of side effects , only three studies reported the number of patients with pneumonia , which was insufficient to make a robust conclusion on the safety of the mis+lmht . however , these results demonstrated that the lmht could be an effective augmentation strategy for mis in treating patients with hich . in this study , almost all relevant randomized clinical trials were included , but some trials might have been missed , partly because these were published in some journals that are not indexed by international databases . fortunately , it is likely that these trials are of low quality , and could not significantly affect the results.44 additionally , there was one trial without the needed data,45 and despite our best efforts , we could not obtain them . however , this trial concluded that the mis+lmht could be good at protecting nerve cell and promoting neurofunctional rehabilitation . therefore , this trial would not affect our conclusion . under conditions of mild hypothermia , the oxygen consumption and metabolic rate in brain tissue are decreased , the production of free radicals is reduced and the synthesis of xanthine oxidase is slowed down and then the tissue damage could be alleviated . meanwhile , the abnormal sodium calcium exchange between cell membrane and sarcoplasmic reticulum is suppressed , which could alleviate the calcium overload.46 under normal conditions , vasoactive substances , such as endothelin and vascular vasopressin , are in dynamic equilibrium to maintain the systolic and diastolic function of blood vessels.47 the acute period of ich is usually accompanied by the disorder of vasoactive substances . previous studies reported that the mild hypothermia could reduce the level of endothelin and vascular vasopressin.40,48 previous studies on ischemic and hemorrhagic stroke subtypes showed that the mild hypothermia ( body temperature reduced by 3c5c ) was neuroprotective.49,50 but one point should be noticed in clinical practice : systemic mild hypothermia is difficult to perform because of its possible side effects.10,11 alternatively , local mild hypothermia could quickly obtain the target temperature and overcome the potential side effects.51 therefore , compared to systemic mild hypothermia , the local mild hypothermia could be more effective in treating patients with hich . first , all of the included studies were from the people s republic of china , which might limit the applicability of our findings.52 second , only three studies were used to analyze the side effects ; hence , future studies are needed to further assess the safety of the mis+lmht . third , the target temperature and treatment time of lmht were not consistent , but there were also the general problems for meta - studies to solve.53,54 by pooling analysis of 28 randomized clinical trials , we found that the addition of lmht could significantly improve the efficacy of mis in the treatment of patients with hich . the or of response rate and mortality rate was 2.68 and 0.43 , respectively , which was in favor of the mis+lmht . the clinical applicability of this modality showed greater promise and should be further explored and optimized .

objectivefibroblast growth factor ( fgf)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models , while human studies revealed increased fgf-21 levels in obesity and type 2 diabetes . given that fgf-21 has been suggested to be a peroxisome proliferator activator receptor ( ppar ) dependent regulator of fasting metabolism , we hypothesized that free fatty acids ( ffas ) , natural agonists of ppar , might modify fgf-21 levels.research design and methodsthe effect of fatty acids on fgf-21 was investigated in vitro in hepg2 cells . within a randomized controlled trial , the effects of elevated ffas were studied in 21 healthy subjects ( 13 women and 8 men ) . within a clinical trial including 17 individuals , the effect of insulin was analyzed using an hyperinsulinemic - euglycemic clamp and the effect of ppar activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks.resultsoleate and linoleate increased fgf-21 expression and secretion in a ppar-dependent fashion , as demonstrated by small - interfering rna induced ppar knockdown , while palmitate had no effect . in vivo , lipid infusion induced an increase of circulating fgf-21 in humans , and a strong correlation between the change in fgf-21 levels and the change in ffas was observed . an artificial hyperinsulinemia , which was induced to delineate the potential interaction between elevated ffas and hyperinsulinemia , revealed that hyperinsulinemia also increased fgf-21 levels in vivo , while rosiglitazone treatment had no effect.conclusionsthe results presented here offer a mechanism explaining the induction of the metabolic regulator fgf-21 in the fasting situation but also in type 2 diabetes and obesity .

a total of 13 women and 8 men participated in the first study ( lipid study ) . briefly , the individuals were normal weight ( bmi 23.7 0.8 kg / m ) , were aged 26.4 0.8 years , had a waist - to - hip ratio ( whr ) of 0.83 0.02 , and had basal lipid parameters in the normal range ( total cholesterol 4.53 0.17 mmol / l , hdl cholesterol 1.44 0.03 mol / l , ldl cholesterol 2.64 0.14 mmol / l , triacylglycerols 1.01 0.08 mmol / l , and ffas 0.68 0.12 none of the participants had taken any medication for at least 3 months before the study . all participants were initially screened for any systemic disease or biochemical evidence of impaired hepatic or renal function . subjects with a history of hypertension , type 2 diabetes , renal or liver disease , dyslipidemia , heart failure , or a family history of diabetes or any other endocrine disorder were excluded from this study . body weight of the participants was stable for at least 2 months before the study . informed written consent was obtained from each participant . for the second human trial ( ppar study ) 7 male and 10 female volunteers with impaired glucose tolerance the individuals were overweight ( bmi 32.8 2.2 kg / m ) , were aged 59.1 1.6 years , and had a whr of 0.88 0.03 . all participants were initially screened for any systemic disease or biochemical evidence of impaired hepatic or renal function . patients with heart failure , impaired hepatic or renal function , anemia , disturbed coagulation , or any other endocrine disorder were excluded . informed written consent was obtained from all subjects after explanation of the nature , purpose , and potential risks of the studies . the patients were instructed not to modify their way of living ( diet and exercise ) during the treatment period . all study protocols were approved by the institutional review board of the charit medical school , campus benjamin franklin . hepg2 cells were grown in eagle 's minimum essential medium with 10% fcs and 1% nea ; 24 h before analysis , hepg2 cells were incubated with basal serum - free media . to analyze the effect of ffas , palmitic , oleic , or linoleic acid ( sigma - aldrich , seelze , germany ) or a mixture of these fatty acids ( one - third of each ) were dissolved in water with 125 mol / l naoh and 10 mol / l fatty acid free bovine serum albumin ( bsa ) ( calbiochem , germany ) in a concentration of 160 mol / l ( ratio 16:1 ) and cells were incubated within that medium for 1 , 2 , 4 , 8 , and 24 h. time course and concentration - dependent effects of ffas on fgf-21 secretion and expression were analyzed . therefore , primary human myoblasts were prepared from human skeletal muscle of healthy individuals und cultured in skeletal muscle cell growth medium ( promocell , heidelberg , germany ) containing 10% fetal bovine serum and 1.5% glutamine and gentamicin . after the first passage , myoblasts were separated from fibroblasts by neural cell adhesion molecule labeled magnetic beads ( miltenyi biotech , bergisch - gladbach , germany ) . to analyze the effect of lecithin and glycerol on fgf-21 secretion , hepg2 cells were incubated for 4 h using a concentration of 2.5 mg / ml lecithin ( fluka ; sigma - aldrich ) and 1 mmol / l glycerol ( sigma - aldrich ) . small - interfering rna ( sirna ) experiments were performed to investigate whether those effects depend on ppar. therefore , sirna for ppar ( nos . a total of 100 pmol / well of each sirna and electroporation with amaxa nucleofector ii ( lonza cologne , cologne , germany ) was used for transfection . the fgf-21 levels were measured in the supernatant by radioimmunoassay ( phoenix europe , karlsruhe , germany ) using 125 i labeled fgf-21 as a tracer and normalized on total protein levels . protein quantification was performed using the roti - nanoquant ( karl - roth , karlsruhe , germany ) . for rt - pcr 20 ng of total rna per well were used and fgf-21 rna was analyzed according to the manufacturer 's the instructions of the iscript sybr green rt - pcr kit ( biorad , hercules , ca ) . real - time quantitative pcr was performed using an abi prism 7300 real - time pcr system ( applied biosystems , foster city , ca ) . primer sequences will be sent upon request . the ddct - method ( change in dct [ = ct of the target gene minus ct of the housekeeping gene ] ) eligible participants were randomly assigned to receive either lipid / heparin infusion ( lhi ) or saline / heparin infusion ( shi ) in a cross - over design for the lipid study . the study was performed during the early follicular phase of two subsequent menstrual cycles ( days 46 ) . to avoid interactions between the study procedures , the study was performed at intervals of at least 2 days in men . following a 10-h overnight fast , a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling . a 0.9% saline infusion plus heparin ( 0.4 units kg min ) or a lipid infusion ( abbolipid 20% [ contents in 1.000 ml : 100 g soybean oil , 100 g safflower oil , 25.0 g glycerol , and 12.0 g egg phospholipids ] , abbott , wiesbaden , germany ; or lipovens 20% [ contents in 1.000 ml : 200 g soybean oil , 25.0 g glycerol , 12.0 g egg phospholipids , and 0.3 goleate ] , fresenius kabi , bad homburg , germany ) plus heparin ( 0.4 units kg min ) was infused at a constant rate of 1.5 ml / min for 330 min . four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp ( 14 ) was started in six subjects . each subject was studied before and after an 8-week rosiglitazone treatment ( 2 8 mg / day ; glaxosmithkline , mnchen , germany ) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously ( 16 ) . in brief , 40 miu / m per min human insulin ( actrapid ; novo nordisk , bagsvaard , denmark ) and a variable infusion of 10% glucose ( serag wiessner , naila , germany ) was used , and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 mmol / l via variation of the glucose infusion rate . insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate ( mg glucose / min ) during the steady state of the clamp by the body weight . plasma fgf-21 levels were measured before and during lhi shi , and steady state at the end of the hyperinsulinemic - euglycemic clamp . metabolic parameters , including ffas , triacylglycerols , total cholesterol , ldl cholesterol , hdl cholesterol , glucose , and insulin were determined as described previously ( 7 ) . serum fgf-21 levels were determined by radioimmunoassay ( phoenix europe ) using 125 i labeled fgf-21 as tracer . the linear range of the assay was 0.58.5 ng / ml , and the standard range was 0.23430 ng / ml . the interassay and intra - assay coefficients of variation were 5.0 and 14% , respectively . all statistical procedures were performed using spss version 15.0 ( spss , chicago , il ) . profiles of ffas , insulin , glucose , triacylglycerols , and fgf-21 were compared by repeated - measures anova with treatment and time as within - subject factors and the huynh - feldt- procedure as correction factor , which corrects for a positive biased f test for within - subject factors . the area under the concentration time curve ( auc ) was calculated by using the trapezoidal integration . the change in fgf-21 , ffas , glucose , triacylglycerols and insulin between baseline and after lhi and shi was calculated by subtracting the baseline value from the value 4 h after each infusion procedure . data were compared by paired student 's t test for normally distributed data and wilcoxon test for skewed data . the correlations between the values were estimated by pearson 's correlation test and adjusted for age , sex bmi , and change in insulin . the unpaired t test or mann - whitney test results were considered to be significant if the two - sided was < 0.05 . hepg2 cells were grown in eagle 's minimum essential medium with 10% fcs and 1% nea ; 24 h before analysis , hepg2 cells were incubated with basal serum - free media . to analyze the effect of ffas , palmitic , oleic , or linoleic acid ( sigma - aldrich , seelze , germany ) or a mixture of these fatty acids ( one - third of each ) were dissolved in water with 125 mol / l naoh and 10 mol / l fatty acid free bovine serum albumin ( bsa ) ( calbiochem , germany ) in a concentration of 160 mol / l ( ratio 16:1 ) and cells were incubated within that medium for 1 , 2 , 4 , 8 , and 24 h. time course and concentration - dependent effects of ffas on fgf-21 secretion and expression were analyzed . therefore , primary human myoblasts were prepared from human skeletal muscle of healthy individuals und cultured in skeletal muscle cell growth medium ( promocell , heidelberg , germany ) containing 10% fetal bovine serum and 1.5% glutamine and gentamicin . after the first passage , myoblasts were separated from fibroblasts by neural cell adhesion molecule labeled magnetic beads ( miltenyi biotech , bergisch - gladbach , germany ) . to analyze the effect of lecithin and glycerol on fgf-21 secretion , hepg2 cells were incubated for 4 h using a concentration of 2.5 mg / ml lecithin ( fluka ; sigma - aldrich ) and 1 mmol / l glycerol ( sigma - aldrich ) . the fgf-21 levels were measured in the supernatant and normalized to total protein levels . small - interfering rna ( sirna ) experiments were performed to investigate whether those effects depend on ppar. therefore , sirna for ppar ( nos . a total of 100 pmol / well of each sirna and electroporation with amaxa nucleofector ii ( lonza cologne , cologne , germany ) was used for transfection . the fgf-21 levels were measured in the supernatant by radioimmunoassay ( phoenix europe , karlsruhe , germany ) using 125 i labeled fgf-21 as a tracer and normalized on total protein levels . protein quantification was performed using the roti - nanoquant ( karl - roth , karlsruhe , germany ) . for rt - pcr 20 ng of total rna per well were used and fgf-21 rna was analyzed according to the manufacturer 's the instructions of the iscript sybr green rt - pcr kit ( biorad , hercules , ca ) . real - time quantitative pcr was performed using an abi prism 7300 real - time pcr system ( applied biosystems , foster city , ca ) . the ddct - method ( change in dct [ = ct of the target gene minus ct of the housekeeping gene ] ) was used for relative quantification . fold changes in expression eligible participants were randomly assigned to receive either lipid / heparin infusion ( lhi ) or saline / heparin infusion ( shi ) in a cross - over design for the lipid study . the study was performed during the early follicular phase of two subsequent menstrual cycles ( days 46 ) . to avoid interactions between the study procedures , the study was performed at intervals of at least 2 days in men . following a 10-h overnight fast , a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling . a 0.9% saline infusion plus heparin ( 0.4 units kg min ) or a lipid infusion ( abbolipid 20% [ contents in 1.000 ml : 100 g soybean oil , 100 g safflower oil , 25.0 g glycerol , and 12.0 g egg phospholipids ] , abbott , wiesbaden , germany ; or lipovens 20% [ contents in 1.000 ml : 200 g soybean oil , 25.0 g glycerol , 12.0 g egg phospholipids , and 0.3 goleate ] , fresenius kabi , bad homburg , germany ) plus heparin ( 0.4 units kg min ) was infused at a constant rate of 1.5 ml / min for 330 min . four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp ( 14 ) was started in six subjects . each subject was studied before and after an 8-week rosiglitazone treatment ( 2 8 mg / day ; glaxosmithkline , mnchen , germany ) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously ( 16 ) . in brief , 40 miu / m per min human insulin ( actrapid ; novo nordisk , bagsvaard , denmark ) and a variable infusion of 10% glucose ( serag wiessner , naila , germany ) was used , and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate ( mg glucose / min ) during the steady state of the clamp by the body weight . eligible participants were randomly assigned to receive either lipid / heparin infusion ( lhi ) or saline / heparin infusion ( shi ) in a cross - over design for the lipid study . the study was performed during the early follicular phase of two subsequent menstrual cycles ( days 46 ) . to avoid interactions between the study procedures , the study was performed at intervals of at least 2 days in men . following a 10-h overnight fast , a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling . a 0.9% saline infusion plus heparin ( 0.4 units kg min ) or a lipid infusion ( abbolipid 20% [ contents in 1.000 ml : 100 g soybean oil , 100 g safflower oil , 25.0 g glycerol , and 12.0 g egg phospholipids ] , abbott , wiesbaden , germany ; or lipovens 20% [ contents in 1.000 ml : 200 g soybean oil , 25.0 g glycerol , 12.0 g egg phospholipids , and 0.3 goleate ] , fresenius kabi , bad homburg , germany ) plus heparin ( 0.4 units kg min ) was infused at a constant rate of 1.5 ml / min for 330 min . four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp ( 14 ) was started in six subjects . each subject was studied before and after an 8-week rosiglitazone treatment ( 2 8 mg / day ; glaxosmithkline , mnchen , germany ) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously ( 16 ) . in brief , 40 miu / m per min human insulin ( actrapid ; novo nordisk , bagsvaard , denmark ) and a variable infusion of 10% glucose ( serag wiessner , naila , germany ) was used , and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate ( mg glucose / min ) during the steady state of the clamp by the body weight . plasma fgf-21 levels were measured before and during lhi shi , and steady state at the end of the hyperinsulinemic - euglycemic clamp . metabolic parameters , including ffas , triacylglycerols , total cholesterol , ldl cholesterol , hdl cholesterol , glucose , and insulin were determined as described previously ( 7 ) . serum fgf-21 levels were determined by radioimmunoassay ( phoenix europe ) using 125 i labeled fgf-21 as tracer . the linear range of the assay was 0.58.5 ng / ml , and the standard range was 0.23430 ng / ml . the interassay and intra - assay coefficients of variation were 5.0 and 14% , respectively . all statistical procedures were performed using spss version 15.0 ( spss , chicago , il ) . profiles of ffas , insulin , glucose , triacylglycerols , and fgf-21 were compared by repeated - measures anova with treatment and time as within - subject factors and the huynh - feldt- procedure as correction factor , which corrects for a positive biased f test for within - subject factors . the area under the concentration time curve ( auc ) was calculated by using the trapezoidal integration . the change in fgf-21 , ffas , glucose , triacylglycerols and insulin between baseline and after lhi and shi was calculated by subtracting the baseline value from the value 4 h after each infusion procedure . data were compared by paired student 's t test for normally distributed data and wilcoxon test for skewed data . the correlations between the values were estimated by pearson 's correlation test and adjusted for age , sex bmi , and change in insulin . the unpaired t test or mann - whitney test was used for group comparison , as appropriate . results were considered to be significant if the two - sided was < 0.05 . incubation of hepg2 cells with a mixture of palmitic , linoleic , or oleic acid induced an increase in fgf expression and secretion compared with the bsa control . the analysis of the individual fatty acids revealed that this increase was not detectable for palmitic acid , while stimulation with oleic or linoleic acid resulted in a significant elevation of fgf-21 secretion . within those experiments , d ) , and we analyzed time course dependent changes in mrna expression ( fig . the maximum of those effects was observed at 24 h for fgf-21 expression and 48 h for protein secretion , a time course in line with our in vivo experiments . furthermore , the dose - response experiments using different oleate - to - bsa ratios ( control vs. 1:1 , 2:1 , 4:1 , 8:1 , and 16:1 ) suggested an increase in fgf-21 secretion in a dose - dependent manner ( 6.61 0.81 vs. 6.93 1.25 , 7.36 1.18 , 8.98 1.18 , 9.66 1.65 , and 10.47 1.10 ng / ml ; p < 0.05 for control vs. 4:1 , 8:1 , and 16:1 ; p < 0.05 for 1:1 vs. 8:1 and 16:1 ; and p < 0.05 for 2:1 vs. 16:1 ) . protein levels of fgf-21 in the supernatant of hepg2 cells after stimulation with palmitate ( a ) , oleate ( b ) , linoleate ( c ) , and an ffa mixture ( d ) for 1 , 2 , 4 , 8 , and 24 h. results are expressed as means se . * p < 0.05 vs. bsa at the same time point , p = 0.072 for fgf-21 protein levels during linoleate stimulation at 2 h vs. bsa , p = 0.069 for fgf-21 protein levels during linoleate stimulation at 8 h vs. bsa , respectively . mrna expression of fgf-21 in hepg2 cells after stimulation with linoleate for 1 , 2 , 4 , and 8 h. results are expressed as means se . * p < 0.05 vs. bsa at the same time point . to evaluate whether these effects were mediated via ppar the ppar-specific sirna induced a knockdown of ppar of 60% , although the expression of the ppar target gene cpt1a was reduced by only 35% . despite the incomplete knockdown of ppar , the increase in fgf-21 secretion and mrna expression during stimulation with linoleate and ffa mixture was not further observed . thus , results at mrna and protein levels indicate that the ffa - induced effects on fgf-21 depend on activation of ppar ( fig . 3 ) . a : gene expression of fgf-21 , cpt1a , and ppar in hepg2 cells in bsa medium after sirna knockdown of ppar compared with control sirna ( 100% ) . results are expressed as means se . * p < 0.05 vs. expression during control sirna , respectively . b : fgf-21 mrna expression in hepg2 cells during linoleate or ffa mixture stimulation after sirna knockdown of ppar ( compared with control sirna and bsa stimulation [ 100% ] ) . results are expressed as means se . * p < 0.05 vs. expression during control sirna , respectively . c : concentration of fgf-21 in the supernatant of hepg2 cells after sirna knockdown of ppar and subsequent stimulation by linoleate or ffa mixture compared with control sirna and bsa medium . * p < 0.05 vs. control sirna and bsa medium . as lipid infusion contains a mixture of different fatty acids , glycerol , and lecithin , we analyzed the effect of lecithin and glycerol on fgf-21 secretion in hepg2 cells . compared with controls , no significant effect of those substances was detectable in hepg2 cells . to evaluate the regulation of fgf-21 expression in human muscle actually , no fgf-21 expression was detectable in these experiments , despite accurate positive controls in hepg2 cells . those data suggest that the primary human myoblasts used here do not express fgf-21 ; therefore , we did not analyze the effects of ffas in those cells . there was no baseline difference of ffas before lhi and shi ( p = ns ) . as expected , ffa levels increased during lhi , whereas only a small increase was seen during shi , resulting in significantly higher ffa levels during lhi compared with shi ( auc lhi vs. shi : 1,183.5 66.7 vs. 228.8 17.9 nmol p < 0.001 ; treatment - vs .- time interaction : p < 0.001 ) ( fig . 4a ) . accordingly , triglyceride levels rose during lhi ( p < 0.05 ) , while a moderate decrease was observed during shi ( p < 0.05 ) , finally also resulting in significantly higher triglyceride levels during lhi ( auc lhi vs. shi : 1,360.6 172.0 vs. 134.0 12.5 nmol p < 0.05 , p < 0.01 ; treatment - vs .- time interaction : p < 0.001 ) ( fig . concentrations of ffas ( a ) , triacylglycerols ( b ) , and fgf-21 ( c ) during shi ( ) and lhi ( ) . results are expressed as means se . * * p < 0.01 ; * p < 0.05 . glucose levels were unchanged during both lhi and shi ( baseline : 4.91 0.80 vs. 4.84 0.54 mmol / l , treatment - vs .- time interaction : p = ns ; auc lhi vs. shi : 1,130.0 24.9 vs. 1,110.1 17.6 mmol no change of insulin levels was observed during lhi compared with baseline ( baseline : 5.17 0.53 vs. 4 h : 4.97 0.62 mu / l ; p = ns ) , while a small , but progressive , decline in insulin concentration was detected during shi ( baseline : 5.80 0.76 vs. 4 h : 2.98 0.32 mu this decline resulted in lower insulin values during shi compared with lhi ( auc lhi vs. shi : 1,384.2 132.5 vs. 1,021.8 109.8 mu p < 0.001 ; treatment - vs .- time interaction : p < 0.001 ) . while no change of fgf-21 levels was detected during shi ( p = ns ) , an increase in fgf-21 levels thus , fgf-21 levels were significantly higher during lhi compared with shi ( treatment - vs .- time interaction : p < 0.05 . ; auc : 1,191 91 vs. 1,090 86 ng ml min ; p < 0.05 ) ( fig . notably , we found at 300 min a progressive and highly significant increase of fgf-21 in the remaining 15 participants , who had an isolated lipid infusion over a total of 300 min ( p < 0.001 ; data not shown ) interestingly , the change of fgf-21 levels compared with baseline was positively correlated to the change of ffa levels during both lhi and shi ( r = 0.528 , p < 0.001 ) ( fig . 5 ) . given the circadian changes in the control group , we also included the saline group in the correlation analysis between changes of ffas and fgf-21 . most importantly , no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi , while the correlation between ffas and fgf-21 was robust , even after adjustment for additional confounders ( age , sex bmi , and insulin ) ( r = 0.474 , p < 0.005 ) . correlation between changes of fgf-21 and ffas during lhi and shi . the saline group ( ) and the lipid group ( ) were included in the correlation analysis . correlation after adjustment for sex , age , bmi , and change in insulin levels ( r = 0.474 , p < 0.005 ) . as in some participants , the lipid infusion induced a reduction in fgf-21 concentrations , and we separated those participants according to the change in fgf-21 levels during lhi . however , no significant difference was found between both groups concerning their sex , age bmi whr , baseline levels of blood glucose , fasting fgf-21 , fasting ffas , and homeostasis model assessment values . to delineate a potential interaction of lipid- and insulin - induced effects on fgf-21 , we additionally analyzed hyperinsulinemic - euglycemic clamps , which were performed at baseline in a rosiglitazone treatment trial in 17 subjects with impaired glucose tolerance . steady - state blood samples were taken not less than 3 h after start of insulin infusion . an 700% increase of insulin levels was observed during this protocol ( p < 0.005 ) . under those conditions , a small but significant increase in fgf-21 levels during hyperinsulinemia was observed in these subjects ( fig . results are expressed as means se . as expected and described in numerous studies , the glucose response during the oral glucose tolerance test and insulin sensitivity . effect of ppar stimulation by rosiglitazone treatment on fgf-21 levels in subjects with impaired glucose tolerance . the accuracy of the fgf-21 assay was investigated by adding various amounts of lipid solution ( lipovens 20% ; fresenius kabi , bad homburg , germany ) ( 0 , 0.5 , 1.5 , and 2.5% vol / vol ) to human plasma samples . this ex vivo addition of lipid infusion resulted in measured triglyceride concentrations of 1.28 , 3.56 , 8.09 , and 12.69 mmol / l . we next added 6 mmol / l oleic acid or linoleic acid to these 0 , 0.5 , 1.5 , and 2.5% vol / vol triglyceride dilutions , resulting in ffa concentrations comparable with those in plasma samples during lipid infusion ( 4.577.31 the subsequently measured fgf-21 values were not significantly different , neither for oleic acid nor for linoleic acid . taken together , neither triglyceride nor ffa concentrations similar to those observed in vivo in the present study interfered with the measured concentrations of fgf-21 . incubation of hepg2 cells with a mixture of palmitic , linoleic , or oleic acid induced an increase in fgf expression and secretion compared with the bsa control . the analysis of the individual fatty acids revealed that this increase was not detectable for palmitic acid , while stimulation with oleic or linoleic acid resulted in a significant elevation of fgf-21 secretion . within those experiments , d ) , and we analyzed time course dependent changes in mrna expression ( fig . the maximum of those effects was observed at 24 h for fgf-21 expression and 48 h for protein secretion , a time course in line with our in vivo experiments . furthermore , the dose - response experiments using different oleate - to - bsa ratios ( control vs. 1:1 , 2:1 , 4:1 , 8:1 , and 16:1 ) suggested an increase in fgf-21 secretion in a dose - dependent manner ( 6.61 0.81 vs. 6.93 1.25 , 7.36 1.18 , 8.98 1.18 , 9.66 1.65 , and 10.47 1.10 ng / ml ; p < 0.05 for control vs. 4:1 , 8:1 , and 16:1 ; p < 0.05 for 1:1 vs. 8:1 and 16:1 ; and p < 0.05 for 2:1 vs. 16:1 ) . protein levels of fgf-21 in the supernatant of hepg2 cells after stimulation with palmitate ( a ) , oleate ( b ) , linoleate ( c ) , and an ffa mixture ( d ) for 1 , 2 , 4 , 8 , and 24 h. results are expressed as means se . * p < 0.05 vs. bsa at the same time point , p = 0.072 for fgf-21 protein levels during linoleate stimulation at 2 h vs. bsa , p = 0.069 for fgf-21 protein levels during linoleate stimulation at 8 h vs. bsa , respectively . mrna expression of fgf-21 in hepg2 cells after stimulation with linoleate for 1 , 2 , 4 , and 8 h. results are expressed as means se . * p < 0.05 vs. bsa at the same time point . to evaluate whether these effects were mediated via ppar the ppar-specific sirna induced a knockdown of ppar of 60% , although the expression of the ppar target gene cpt1a was reduced by only 35% . despite the incomplete knockdown of ppar , the increase in fgf-21 secretion and mrna expression during stimulation with linoleate and ffa mixture was not further observed . thus , results at mrna and protein levels indicate that the ffa - induced effects on fgf-21 depend on activation of ppar ( fig . 3 ) . a : gene expression of fgf-21 , cpt1a , and ppar in hepg2 cells in bsa medium after sirna knockdown of ppar compared with control sirna ( 100% ) . results are expressed as means se . * p < 0.05 vs. expression during control sirna , respectively . b : fgf-21 mrna expression in hepg2 cells during linoleate or ffa mixture stimulation after sirna knockdown of ppar ( compared with control sirna and bsa stimulation [ 100% ] ) . results are expressed as means se . * p < 0.05 vs. expression during control sirna , respectively . c : concentration of fgf-21 in the supernatant of hepg2 cells after sirna knockdown of ppar and subsequent stimulation by linoleate or ffa mixture compared with control sirna and bsa medium . * p < 0.05 vs. control sirna and bsa medium . as lipid infusion contains a mixture of different fatty acids , glycerol , and lecithin , we analyzed the effect of lecithin and glycerol on fgf-21 secretion in hepg2 cells . compared with controls , no significant effect of those substances was detectable in hepg2 cells . to evaluate the regulation of fgf-21 expression in human muscle actually , no fgf-21 expression was detectable in these experiments , despite accurate positive controls in hepg2 cells . those data suggest that the primary human myoblasts used here do not express fgf-21 ; therefore , we did not analyze the effects of ffas in those cells . there was no baseline difference of ffas before lhi and shi ( p = ns ) . as expected , ffa levels increased during lhi , whereas only a small increase was seen during shi , resulting in significantly higher ffa levels during lhi compared with shi ( auc lhi vs. shi : 1,183.5 66.7 vs. 228.8 17.9 nmol p < 0.001 ; treatment - vs .- time interaction : p < 0.001 ) ( fig . 4a ) . accordingly , triglyceride levels rose during lhi ( p < 0.05 ) , while a moderate decrease was observed during shi ( p < 0.05 ) , finally also resulting in significantly higher triglyceride levels during lhi ( auc lhi vs. shi : 1,360.6 172.0 vs. 134.0 12.5 nmol p < 0.05 , p < 0.01 ; treatment - vs .- time interaction : p < 0.001 ) ( fig . concentrations of ffas ( a ) , triacylglycerols ( b ) , and fgf-21 ( c ) during shi ( ) and lhi ( ) . results are expressed as means se . * * p < 0.01 ; * p < 0.05 . glucose levels were unchanged during both lhi and shi ( baseline : 4.91 0.80 vs. 4.84 0.54 mmol / l , treatment - vs .- time interaction : p = ns ; auc lhi vs. shi : 1,130.0 24.9 vs. 1,110.1 17.6 mmol no change of insulin levels was observed during lhi compared with baseline ( baseline : 5.17 0.53 vs. 4 h : 4.97 0.62 mu / l ; p = ns ) , while a small , but progressive , decline in insulin concentration was detected during shi ( baseline : 5.80 0.76 vs. 4 h : 2.98 0.32 this decline resulted in lower insulin values during shi compared with lhi ( auc lhi vs. shi : 1,384.2 132.5 vs. 1,021.8 109.8 mu p < 0.001 ; treatment - vs .- time interaction : p < 0.001 ) . while no change of fgf-21 levels was detected during shi ( p = ns ) , an increase in fgf-21 levels was found during lhi ( p < 0.05 ) . thus , fgf-21 levels were significantly higher during lhi compared with shi ( treatment - vs .- time interaction : p < 0.05 . ; auc : 1,191 91 vs. 1,090 86 ng ml min ; p < 0.05 ) ( fig . notably , we found at 300 min a progressive and highly significant increase of fgf-21 in the remaining 15 participants , who had an isolated lipid infusion over a total of 300 min ( p < 0.001 ; data not shown ) . interestingly , the change of fgf-21 levels compared with baseline was positively correlated to the change of ffa levels during both lhi and shi ( r = 0.528 , p < 0.001 ) ( fig . 5 ) . given the circadian changes in the control group , we also included the saline group in the correlation analysis between changes of ffas and fgf-21 . most importantly , no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi , while the correlation between ffas and fgf-21 was robust , even after adjustment for additional confounders ( age , sex bmi , and insulin ) ( r = 0.474 , p < 0.005 ) . the saline group ( ) and the lipid group ( ) were included in the correlation analysis . correlation after adjustment for sex , age , bmi , and change in insulin levels ( r = 0.474 , p < 0.005 ) . as in some participants , the lipid infusion induced a reduction in fgf-21 concentrations , and we separated those participants according to the change in fgf-21 levels during lhi . however , no significant difference was found between both groups concerning their sex , age bmi whr , baseline levels of blood glucose , fasting fgf-21 , fasting ffas , and homeostasis model assessment values . to delineate a potential interaction of lipid- and insulin - induced effects on fgf-21 , we additionally analyzed hyperinsulinemic - euglycemic clamps , which were performed at baseline in a rosiglitazone treatment trial in 17 subjects with impaired glucose tolerance . steady - state blood samples were taken not less than 3 h after start of insulin infusion . an 700% increase of insulin levels was observed during this protocol ( p < 0.005 ) . under those conditions , a small but significant increase in fgf-21 levels during hyperinsulinemia was observed in these subjects ( fig . as expected and described in numerous studies , the glucose response during the oral glucose tolerance test and insulin sensitivity were improved after 8 weeks of rosiglitazone treatment . effect of ppar stimulation by rosiglitazone treatment on fgf-21 levels in subjects with impaired glucose tolerance . the accuracy of the fgf-21 assay was investigated by adding various amounts of lipid solution ( lipovens 20% ; fresenius kabi , bad homburg , germany ) ( 0 , 0.5 , 1.5 , and 2.5% vol / vol ) to human plasma samples . this ex vivo addition of lipid infusion resulted in measured triglyceride concentrations of 1.28 , 3.56 , 8.09 , and 12.69 mmol / l . we next added 6 mmol / l oleic acid or linoleic acid to these 0 , 0.5 , 1.5 , and 2.5% vol / vol triglyceride dilutions , resulting in ffa concentrations comparable with those in plasma samples during lipid infusion ( 4.577.31 the subsequently measured fgf-21 values were not significantly different , neither for oleic acid nor for linoleic acid . taken together , neither triglyceride nor ffa concentrations similar to those observed in vivo in the present study interfered with the measured concentrations of fgf-21 . various recent cross - sectional studies ( 5,6,11 ) described a positive correlation between fgf-21 levels and parameters of lipid metabolism , specifically ffas and triacylglycerols . however , existing experimental data regarding fgf-21 did not yet elucidate the direction of that relationship . both , the experimental data presented here and the results of the controlled , randomized cross - over human trial strongly suggest a direct regulation of circulating fgf-21 levels in humans by ffas . interestingly , insulin appears to have an independent effect on circulating fgf-21 , although our data , respective of that effect , are not based on a controlled trial and future studies are required to further strengthen that finding . in terms of physiology , the ffa - induced increase of fgf-21 might contribute to the adaption of the organism to starvation . in response to fasting , fgf-21 is known to be stimulated by activation of the ppar , a nuclear receptor , which is itself activated by fatty acids ( 2,17 ) . ffas usually increase during fasting , suggesting that a stimulation of fgf-21 secretion may be induced by increased ffas directly via ppar activation , an assumption that is strongly underlined by our data . the relevance of ppar in the regulation of fgf-21 was supported by recent data ( 3 ) showing increased fgf-21 levels after pharmacological ppar activation using fibrates . fgf-21 increased insulin sensitivity in animal experiments and attenuated the hormone - stimulated lipolysis in human adipocytes probably due to reduced expression of perilipin , a phosphoprotein that is thought to recruit several lipases to the surface of the lipid droplets for subsequent triglyceride hydrolysis ( 18 ) . these biological properties suggest that fgf-21 may contribute to the anabolic switch of the organism under conditions of starvation and the mechanism presented here might explain the previously described elevated fgf-21 levels in patients with prevalent lipid disorders , obesity , or diabetes . the insulin - sensitizing effect of fgf-21 has been convincingly demonstrated within animal experiments . as current data suggest differential effects of saturated and unsaturated fatty acids , and especially polyunsaturated fatty acids on glucose metabolism ( 19 ) , it is tempting to speculate that some of these effects might be mediated by fgf-21 . indeed unsaturated fatty acids , and specifically the polyunsaturated linoleic acid , stimulated fgf-21 secretion , while the saturated fatty acid palmitic had no effect on fgf-21 secretion in hepg2 cells . various studies suggested that the ffa - induced activation of ppar depends on the degree of saturation of those ffas ( 20 ) . therefore , the different effects on fgf-21 secretion may be caused by different ppar binding of saturated and unsaturated fatty acids . specific effects regarding the development of metabolic disorders remains to be elucidated . a negative correlation between fasting insulin and fgf-21 levels was recently described in diabetic subjects ( 6,11 ) , while this association was reported to be positive in obese individuals ( 5 ) . since we observed slightly higher insulin levels during lhi compared with shi in our participants , hyperinsulinemic - euglycemic clamps were performed for an in - depth analysis of the effects of insulin on fgf-21 levels . the slight increase of fgf-21 by hyperinsulinemia supports an independent effect of insulin on the regulation of fgf-21 . given a well - known reduction of ffas during hyperinsulinemia , this effect may have been even underestimated . ( 21 ) , showing that activation of akt contributes to the expression of fgf-21 . thus , hyperinsulinemia and elevated levels of ffas might contribute together to the elevated fgf-21 levels seen in diabetic patients ( 5,6 ) . nevertheless , both in vivo and in vitro data support a direct effect of ffas on fgf-21 expression and secretion , suggesting that the effect of ffas seen in our lipid trial would be at least in part independent of the moderate hyperinsulinemia , which occurs in the time frame presented here compared with the control group . most importantly , no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi , a fact that is in line with an independent effect of ffas on fgf-21 levels . the ffa - induced effect in liver cells seems to depend on ppar stimulation , which is strongly supported by the in vitro data presented here . however , it should be mentioned in that context , other tissues like adipose tissue or muscle might also contribute to the ffa - induced increase in fgf-21 in vivo . we do not feel that the fact of absent fgf-21 expression in primary human myoblasts contradicts a substantial release of fgf-21 from muscle cells in vivo . our cells were predominantly undifferentiated cells , and the degree of differentiation might contribute to the expression pattern respective of fgf-21 . a potential role of muscle cells was clearly supported by the study of izumiya et al . ( 17 ) , although the relevance of muscle cell differentiation might require furthers studies . even if recent animal data suggested a regulation of fgf-21 also by ppar ( 12 ) , no change of fgf-21 levels was detected during insulin - sensitizing treatment with ppar stimulation in the human trial in subjects with impaired glucose tolerance ( fig . although ppar agonists may recruit different nuclear cofactors , which may explain different biological effects , our data do not support ( but also not entirely exclude ) that the observed effects on fgf-21 levels during lhi were mediated in part by ppar stimulation . limitations of the present study need to be mentioned . in a recent study ( 3 ) although small effects may have been underestimated in the abovementioned study , it is noteworthy that the increase of ffas during fasting is substantially lower in comparison to changes observed during lipid infusion . as such , although the intervention performed here is a well - established model investigating metabolic effects of hyperlipidemia ( 2226 ) , supraphysiological levels of ffas , as observed in the present study , may have reduced the period potentially required to observe effects of more moderate ffa elevations on fgf-21 . in addition , ffas and triacylglycerols are both increased by lhi ( 2729 ) , and the respective effects are difficult to dissociate . although the correlation between changes in ffas and fgf-21 during lhi suggests that ffas contribute to the effect on fgf-21 , we can not entirely exclude a role of triacylglycerols or other compounds within the applied infusions . notably , no effect of glycerol or lecithin was detectable in hepg2 cells , suggesting that those substances have no substancial effect on fgf-21 secretion . recent human data ( 6 ) indicate a specific role of ffas on fgf-21 secretion by showing a positive correlation between both parameters in healthy subjects . furthermore , dostalova et al . ( 30 ) observed not only reduced fgf-21 levels but also lower ffas in anorectic women compared with control subjects , while no difference in insulin , triacylglycerols , and fasting glucose was detected between anorectic and healthy women in this study . those data do support a model of ffas as major regulators of fgf-21 levels . in summary , we demonstrated in vitro that ffas stimulate fgf-21 expression and secretion in a human hepatic carcinoma cell line in a ppar-dependent fashion . the in vivo relevance of this finding was confirmed in a controlled , randomized cross - over trial in humans . within that trial , acutely increased levels of fgf-21 were observed . most interestingly , hyperinsulinemia also induced a small but significant increase of fgf-21 , suggesting that both ffas and insulin regulate fgf-21 , which may explain elevated levels of fgf-21 in obese and diabetic patients but offers also a potential mechanism linking regulation of fgf-21 to the switch of metabolism during starvation .

backgroundcar / rxr heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands . this constitutive activity of murine car can be inhibited by the inverse agonist ligand androstanol or increased by the agonist tcpobop . rxr agonists activate some rxr heterodimer complexes , which are termed permissive , while other non - permissive complexes are not responsive to such ligands.resultsdirect protein - protein interaction studies demonstrate that the rxr agonist 9-cis - ra increases interaction of car / rxr heterodimers with the coactivator src-3 , but also inhibits the ability of tcpobop to increase and androstanol to decrease coactivator binding . car transactivation of a response element with a five nucleotide spacer ( dr-5 ) is unaffected by 9-cis - ra or the synthetic rxr agonist lg1069 . in agreement with the inhibitory effect observed in vitro , these rexinoids block both the tcpobop mediated transactivation of this element and the androstanol dependent inhibition . in contrast , car transactivation of other response elements is increased by rexinoids . stable expression of car in a hepg2 derived cell line increases expression of the endogenous car target cyp2b6 . this expression is further increased by tcpobop but decreased by either androstanol or lg1069 , and lg1069 blocks the stimulatory effect of tcpobop but not the inhibitory effect of androstanol.conclusionwe conclude that car / rxr heterodimers are neither strictly permissive nor non - permissive for rxr signaling . instead , rexinoids have distinct effects in different contexts . these results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses .

at least one third of the 48 human members of the nuclear hormone receptor superfamily function as heterodimers with the retinoid x receptors , ( rxrs , nr2b1 - 3 ) . some , but not all of these heterodimers can be directly activated by rxr agonists , called rexinoids , which include the endogenous ligand 9-cis - retinoic acid ( 9-cis - ra ) and a number of synthetic compounds . the rexinoid responsive complexes are referred to as permissive and include the peroxisome proliferator activated receptor ( ppar ) and the farnesoid x receptor ( fxr ) heterodimers ; the retinoic acid receptor ( rar ) , and thyroid hormone receptor ( tr ) heterodimers are non - permissive [ 2 - 7 ] . the xenobiotic receptor car ( nr1i3 ) is one of several rxr heterodimers that has not been assigned to either category . initial results showed that car is an apparently constitutive transactivator able to activate gene expression in the absence of exogenously added ligands . the first car ligands identified , androstanol and androstenol , are inverse agonists that block this constitutive transactivation . more recently , it has been found that car mediates the characteristic response of a subset of drug metabolizing enzymes to a variety of structurally unrelated foreign compounds . these xenobiotic activators are referred to as " phenobarbital ( pb ) like , " based on the prototypic effects of the anti - epileptic agent on drug metabolism [ 12 - 14 ] . the most potent activator of murine car is the high affinity agonist ligand tcpobop ( 1,4-bis [ 2-(3,5-dichloropyridyloxy)]benzene ) . the effects of specific rxr ligands on the car / rxr heterodimer have not been characterized , although high concentrations of both 9-cis - ra and all - trans retinoic acid have been reported to block pb induction of the car targets cyp2b1 and cyp2b2 in primary rat hepatocytes . in addition , a recent report suggests that both compounds decrease tcpobop - dependent car transactivation . in both cases , these effects were primarily attributed to sequestration of limiting amounts of rxr into ligand - dependent homodimers or heterodimers with other receptors . we have used several approaches to specifically examine the responsiveness of the car / rxr heterodimer to rexinoids . direct biochemical measurements demonstrate that rexinoids can induce coactivator binding , but can also inhibit the effects of car agonist and inverse agonist ligands . in transient transfections , however , different car / rxr heterodimer binding sites show distinct responses to rxr activation . overall , these results significantly increase the potential complexity of the response of rxr heterodimers to rxr ligands and suggest a potential modulatory effect of rexinoids on xenobiotic responses . initial functional results suggested that transactivation by car / rxr heterodimers is not affected by rxr ligands , including 9-cis - ra or synthetic rexinoids , indicating that car is a non - permissive rxr partner . however , gel shift studies indicated that the mobility of the car / rxr complex bound to a dr-5 element is increased by the presence of 9-cis ra , suggesting that the rxr ligand can bind to the car / rxr heterodimer . an increase in the mobility of the car / rxr complex was also observed in the presence of tcpobop or androstanol , alone or in combination with 9-cis ra ( data not shown ) . to test the effects of rexinoids on coactivator binding in a defined biochemical system , surface plasmon resonance was used to characterize the effects of the various ligands on binding of car , rxr and car / rxr heterodimers to src-3 ( also known as actr , p / cip , rac3 , aib1 , tram-1 ) , an effective coactivator for car and many other members of the nuclear receptor superfamily . as expected , 9-cis - ra stimulated coactivator binding to rxr alone , and the basal interaction of car with src-3 was enhanced by tcpobop and decreased by androstanol ( fig . the interaction of src-3 with the preformed and characterized car / rxr heterodimer complex was increased by either tcpobop or 9-cis - ra ( fig . strikingly , however , coactivator binding to the heterodimer complex was decreased below the basal level in the presence of both ligands . in addition , src-3 binding to the heterodimer was decreased by androstanol and this effect was largely blocked by 9-cis - ra . these effects of 9-cis - ra on coactivator binding to the preformed heterodimer contrast with the strong stimulatory effect observed with rxr alone , confirming the characterization of this complex . these results demonstrate that the presence of the rxr ligand can significantly alter the response of the car / rxr heterodimer to car ligands . gst - src-3 fusion protein was immobilized on a flow cell and tested for interaction with receptors . a. overlaid results of injections of apo - rxr and rxr preincubated in the presence of 9-cis ra . b. overlaid results of injections of apo - car and car preincubated in the presence of tcpobop ( tc ) or androstanol ( an ) . c. overlaid results of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and tcpobop ( tc ) as indicated . d. overlaid results of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and androstanol ( an ) as indicated . to examine the effects of rxr ligands on car transactivation in a more functional context , the rexinoid lg1069 was combined with the car inverse agonist androstanol and the agonist tcpobop in a series of transient transfection studies using different response elements . initial studies were based on a well characterized binding site for car / rxr heterodimers , the dr-5 element from the promoter of the retinoic acid receptor 2 isoform . in agreement with the biochemical results , the rexinoid blocked both the stimulatory effect of the agonist and the inhibitory effect of the inverse agonist on this element ( fig . 100 nm lg1069 decreased the inhibitory effect of androstanol by as much as 70% and had an even stronger effect on tcpobop activation ( fig . consistent with previous results , deletion of the helix 12 af-2 motif of car eliminated both constitutive and ligand dependent transactivation . deletion of the same motif in rxr also strongly decreased transactivation , but did not completely eliminate ligand responses ( fig . a. hepg2 cells were cotransfected with vectors expressing wild type car ( car wt ) , a car af-2 deletion mutant ( car8 ) or an rxr af-2 deletion mutant ( rxr19 ) , together with a luciferase reporter containing the rare response element , in the presence or absence of androstanol , tcpobop and/or lg1069 . b. a car expression vector was cotransfected into hepg2 cells with the rare - tk - luc reporter construct in the presence of solvent ( open squares ) or 100 nm lg1069 ( solid diamonds ) and increasing amounts of either androstanol or tcpobop as indicated . luciferase activity is relative to that in the absence of ligand ( 100% ) . to test whether the apparent allosteric effects of lg1069 on car transactivation vary with different response elements , the effect of the rexinoid on transactivation directed by cytochrome p450 response elements in contrast to the results with the dr-5 site , but in agreement with the biochemical results , lg1069 alone had a stimulatory effect on the er-6 car response element from the promoter of the cyp3a4 gene ( fig . this effect was not due to rxr homodimer function , since no transactivation was observed with rxr alone . in contrast to both the dr-5 and biochemical results , lg1069 did not block the stimulatory effect of tcpobop . instead , the stimulatory effect of the two agonists was retained when they were combined . combining lg1069 with the inverse agonist androstanol resulted in a partial block in its inhibitory effect and an intermediate level of activity . similar results were observed with the tandem dr-4 sites from the cyp2b10 gene , except that lg1069 did not have a significant stimulatory effect on its own , but did augment the effect of tcpobop . it is interesting that these two cyp elements respond somewhat differently to rxr cotransfection , which increases responses directed by the cyp3a4 element but modestly decreases responses of the cyp2b10 element ( data not shown ) . on both elements , similar results were observed when 9-cis ra was substituted for lg1069 ( data not shown ) . these results show that the negative effects of rexinoids on tcpobop and androstanol signaling observed with the dr-5 site from the rar promoter are not observed with the dr-4 and er-6 sites from the cyp promoters . a. hepg2 cells were cotransfected with expression constructs for car and/or rxr together with a previously described luciferase construct containing a er-6 type element from the , human cyp3a4 promoter in the presence or absence of ligands . the luciferase activity presented above is relative to that directed by vector alone in the absence of ligands ( 100% ) . b. a similar transfection with a previously described reporter containing the tandem dr-4 elements from the mcyp2b10 gene . a previously described hepg2 derivative stably expressing mouse car was used to determine the effects of rxr agonists on car function in the context of native chromatin . expression of cyp2b6 , a human relative of cyp2b10 , is constitutively induced in this cell line . in contrast to the results with the transient transfections with the cyp response elements , cyp2b6 expression was decreased to a comparable extent by androstanol , lg1069 , and the combination of both ligands . cyp2b6 expression was increased by tcpobop , as expected , and this response was blocked by lg1069 treatment . initial functional results suggested that transactivation by car / rxr heterodimers is not affected by rxr ligands , including 9-cis - ra or synthetic rexinoids , indicating that car is a non - permissive rxr partner . however , gel shift studies indicated that the mobility of the car / rxr complex bound to a dr-5 element is increased by the presence of 9-cis ra , suggesting that the rxr ligand can bind to the car / rxr heterodimer . an increase in the mobility of the car / rxr complex was also observed in the presence of tcpobop or androstanol , alone or in combination with 9-cis ra ( data not shown ) . to test the effects of rexinoids on coactivator binding in a defined biochemical system , surface plasmon resonance was used to characterize the effects of the various ligands on binding of car , rxr and car / rxr heterodimers to src-3 ( also known as actr , p / cip , rac3 , aib1 , tram-1 ) , an effective coactivator for car and many other members of the nuclear receptor superfamily . as expected , 9-cis - ra stimulated coactivator binding to rxr alone , and the basal interaction of car with src-3 was enhanced by tcpobop and decreased by androstanol ( fig . the interaction of src-3 with the preformed and characterized car / rxr heterodimer complex was increased by either tcpobop or 9-cis - ra ( fig . strikingly , however , coactivator binding to the heterodimer complex was decreased below the basal level in the presence of both ligands . in addition , src-3 binding to the heterodimer was decreased by androstanol and this effect was largely blocked by 9-cis - ra . these effects of 9-cis - ra on coactivator binding to the preformed heterodimer contrast with the strong stimulatory effect observed with rxr alone , confirming the characterization of this complex . these results demonstrate that the presence of the rxr ligand can significantly alter the response of the car / rxr heterodimer to car ligands . gst - src-3 fusion protein was immobilized on a flow cell and tested for interaction with receptors . a. overlaid results of injections of apo - rxr and rxr preincubated in the presence of 9-cis ra . b. overlaid results of injections of apo - car and car preincubated in the presence of tcpobop ( tc ) or androstanol ( an ) . c. overlaid results of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and tcpobop ( tc ) as indicated . d. overlaid results of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and androstanol ( an ) as indicated . to examine the effects of rxr ligands on car transactivation in a more functional context , the rexinoid lg1069 was combined with the car inverse agonist androstanol and the agonist tcpobop in a series of transient transfection studies using different response elements . initial studies were based on a well characterized binding site for car / rxr heterodimers , the dr-5 element from the promoter of the retinoic acid receptor 2 isoform . in agreement with the biochemical results , the rexinoid blocked both the stimulatory effect of the agonist and the inhibitory effect of the inverse agonist on this element ( fig . 2a ) . in more detailed dose response studies , 100 nm lg1069 decreased the inhibitory effect of androstanol by as much as 70% and had an even stronger effect on tcpobop activation ( fig . consistent with previous results , deletion of the helix 12 af-2 motif of car eliminated both constitutive and ligand dependent transactivation . deletion of the same motif in rxr also strongly decreased transactivation , but did not completely eliminate ligand responses ( fig . a. hepg2 cells were cotransfected with vectors expressing wild type car ( car wt ) , a car af-2 deletion mutant ( car8 ) or an rxr af-2 deletion mutant ( rxr19 ) , together with a luciferase reporter containing the rare response element , in the presence or absence of androstanol , tcpobop and/or lg1069 . b. a car expression vector was cotransfected into hepg2 cells with the rare - tk - luc reporter construct in the presence of solvent ( open squares ) or 100 nm lg1069 ( solid diamonds ) and increasing amounts of either androstanol or tcpobop as indicated . luciferase activity is relative to that in the absence of ligand ( 100% ) . to test whether the apparent allosteric effects of lg1069 on car transactivation vary with different response elements , the effect of the rexinoid on transactivation directed by cytochrome p450 response elements was examined . in contrast to the results with the dr-5 site , but in agreement with the biochemical results , lg1069 alone had a stimulatory effect on the er-6 car response element from the promoter of the cyp3a4 gene ( fig . this effect was not due to rxr homodimer function , since no transactivation was observed with rxr alone . in contrast to both the dr-5 and biochemical results , lg1069 did not block the stimulatory effect of tcpobop . instead , the stimulatory effect of the two agonists was retained when they were combined . combining lg1069 with the inverse agonist androstanol resulted in a partial block in its inhibitory effect and an intermediate level of activity . similar results were observed with the tandem dr-4 sites from the cyp2b10 gene , except that lg1069 did not have a significant stimulatory effect on its own , but did augment the effect of tcpobop . it is interesting that these two cyp elements respond somewhat differently to rxr cotransfection , which increases responses directed by the cyp3a4 element but modestly decreases responses of the cyp2b10 element ( data not shown ) . on both elements , similar results were observed when 9-cis ra was substituted for lg1069 ( data not shown ) . these results show that the negative effects of rexinoids on tcpobop and androstanol signaling observed with the dr-5 site from the rar promoter are not observed with the dr-4 and er-6 sites from the cyp promoters . a. hepg2 cells were cotransfected with expression constructs for car and/or rxr together with a previously described luciferase construct containing a er-6 type element from the , human cyp3a4 promoter in the presence or absence of ligands . the luciferase activity presented above is relative to that directed by vector alone in the absence of ligands ( 100% ) . b. a similar transfection with a previously described reporter containing the tandem dr-4 elements from the mcyp2b10 gene . a previously described hepg2 derivative stably expressing mouse car was used to determine the effects of rxr agonists on car function in the context of native chromatin . expression of cyp2b6 , a human relative of cyp2b10 , is constitutively induced in this cell line . in contrast to the results with the transient transfections with the cyp response elements , cyp2b6 expression was decreased to a comparable extent by androstanol , lg1069 , and the combination of both ligands . cyp2b6 expression was increased by tcpobop , as expected , and this response was blocked by lg1069 treatment . a number of rxr heterodimer complexes have been categorized as either permissive or non - permissive based on their responses to rxr ligands . although there are some reported discrepancies , the ppar , fxr and ngf - ib / nurr1 complexes are generally considered permissive , while the non - permissive group includes the rar , tr and vdr complexes ( see [ 2 - 7 ] ) . in the permissive complexes , coactivators can presumably bind to the af-2 surface of both partners and binding to the rxr partner is stimulated by rexinoid agonists . this can result in distinct responses depending on which of the two partners is activated . in the case of the ppar/rxr complex , for example , activation with ppar or rxr specific ligands results in recruitment of quite different coactivators . among the non - permissive complexes , recent results suggest that the c - terminal helix 12 of rxr exerts allosteric effects on rar function . however , additional studies suggest that corepressor binding to the apo - rar component of the heterodimer blocks rexinoid induced binding of coactivators to rxr . these results suggest that the mechanisms that account for the lack of responsiveness of the non - permissive complexes are complex . as summarized in fig . 5 , the results described here demonstrate that the effects of rxr agonists on the car / rxr heterodimer can be quite different in different contexts . in vitro studies in the absence of dna demonstrate that car / rxr heterodimers can be activated by an agonist ligand for either receptor . this positive response to 9-cis - ra contrasts with the inhibition that would result from the loss of rxr to ligand - dependent homodimers , confirming that these studies reflect the activity of car / rxr heterodimers and demonstrating that such complexes can be permissive for rxr signaling . remarkably , however , coactivator binding is decreased in the presence of both of the activating ligands . similarly , the inhibitory effect of the inverse agonist ligand androstanol on coactivator recruitment is blocked by 9-cis - ra . total rna was prepared from a previously described hepg2 derivative stably expressing mcar and cultured in the presence of the indicated ligands ( an , androstanol ; lg , lg1069 ; tc , tcpobop ) for 9 hours . expression of cyp2b6 and the ef1 control was analyzed by northern blotting using appropriate cdna probes and quantitated by phosphorimager analysis . very similar results were obtained in 3 independent experiments ; a representative experiment is shown in transient transfections , the rexinoid lg1069 shows different effects on the ability of the xenobiotic receptor to transactivate various response elements . the rexinoid alone has no effect on transactivation of the rare , but strongly inhibits responses to both the agonist tcpobop and the inverse agonist androstanol . in contrast , lg1069 can stimulate transactivation of the response element from the cyp3a4 promoter . on that element it does not block the stimulatory effect of tcpobop , but decreases the inhibitory effect of the androstanol somewhat . on the tandem cyp2b10 elements , lg1069 does not have a stimulatory effect on its own and does not decrease the inhibitory effect of androstanol , but it does increase response to tcpobop . finally , expression of the endogenous cyp2b6 gene in car expressing hepg2 cells is modestly inhibited by lg1069 alone , androstanol alone and the combination of both . in this context , the rexinoid blocks activation by the agonist tcpobop . these rexinoid responses share the general theme of inhibition of some of the effects of car ligands by rxr ligands , but are clearly quite disparate . the differences may be based on variations in experimental approach or on more fundamental functional differences . for example , it is possible that the inhibitory effect of lg1069 in the stable car expressing cell line is based on the sequestration of limiting amounts of rxr into ligand - dependent homodimers , as has been suggested in previous studies using primary hepatocytes . however , similar effects were observed with the relatively large amounts of rxr used for the biochemical studies or with the transient transfections , which relied on cotransfected rxr . it is also possible that differences in the chromatin context could contribute to the differences between the effects observed with the endogenous and transiently transfected promoters . however , such differences can not account for the results with reporter plasmids that differ only in the identity of the response element . overall , we conclude that binding of both the dna and the rxr ligands can result in allosteric effects on the function of the car / rxr heterodimer . the disparities in these responses preclude a single , simple explanation for the effects of rexinoids on car / rxr heterodimers . nonetheless , one attractive possibility is that binding of the rxr agonist exerts direct inhibitory effects on either the binding or functional effects of the car ligands . some precedence for a significant allosteric effect of one heterodimer partner on another is provided by the inability of the insect ecdysone receptor to bind its steroid ligand in the absence of its heterodimer partner usp . the outcome of this inhibition of car ligand binding would be somewhat different depending on whether or not the rxr agonist was able to activate the basal transactivation of car / rxr heterodimer bound to a particular element . on the rare , for example , the rxr agonist might bind , but not increase transactivation beyond that observed in the absence of ligands . in this case , blocking tcpobop binding in the case of the cyp3a4 response element , however , activation by the rxr agonist is comparable to that observed with tcpobop alone . thus , blocking tcpobop binding would not result in the net inhibitory effect observed with the rare , but could account for the lack of additive effect of the two ligands . a variety of factors complicate this relatively simple model , including the specific cytoplasmic to nuclear translocation that is required for car transactivation in the liver . rexinoids by themselves are apparently unable to induce this translocation process in rat or mouse ( data not shown ) hepatocytes and thus can not directly activate car targets . more complex combinatorial effects of other coactivators or corepressors ( e.g. ) could also contribute to the observed responses . recent results suggest that car can interact with ncor or smrt in the presence of inverse agonists , although such interactions appear much weaker than those of other receptors . further characterization of potential car cofactors , as well as the development of sensitive and direct ligand binding assays will be required to define the molecular mechanisms that account for the diverse effects of rexinoids on car transactivation . overall , we conclude that the disparate effects of rexinoids on car / rxr heterodimers in different contexts add significantly to the potential complexity of the effects of rxr ligands on such nuclear receptor heterodimers , and demonstrate that classifying a particular complex as permissive or non - permissive can be an oversimplification . the results described here also suggest that rexinoids may have modulatory effects on xenobiotic responses , and that such effects may differ for different car / rxr target genes . a systematic examination of the effects of rxr ligands and retinoid status on a number of the growing set of car target genes the effect of ligands on coactivator binding was evaluated using the biacore instrument as described . for interaction of car , rxr and car / rxr heterodimer with src-3 , a gst - src-3 fusion protein containing the receptor interaction domain of the coactivator was expressed in bacteria and purified . lack of binding of free gst demonstrated that the surface was saturated with gst - src3 . purified car and rxr proteins were produced in bacteria and preincubated overnight at 4c alone or in combination , and in the presence or absence of 10 m androstanol , 10 m tcpobop , 10 m of 9 cis - ra , as appropriate . car alone , rxr alone , and car / rxr heterodimers bound to various ligands were injected individually into flow cells and binding was measured as described . the rare - tk - luc reporter plasmid with three copies of the dr-5 motif from the promoter of the mouse rar2 gene has been described previously , as has the cyp2b10-tk - luc reporter with two copies of the dr-4 type elements from the promoter of the mouse cyp2b10 gene and the cyp3a4-tk - luc reporter with three copies of the er-6 element from the human cyp3a4 promoter . the cdm8-mcar8 deletion construct containing a deletion of the last 8 amino acids and the mutant with 2 helix 3 mutations ( f171l , i174a ) that block ligand binding and response have been described previously , as has the cdm8-rxr19 construct with a deletion of the last 19 amino acids . hepg2 cells were maintained in dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal bovine serum ( hyclone ) . for transfections , 10hepg2 cells were plated in 24 well dishes with dmem supplemented with 10% charcoal stripped serum and transfected using the calcium phosphate precipitation method as described . the next morning typically , transfections included 100200 ng of luciferase reporter plasmids , 100 ng of -galactosidase ( -gal ) internal control plasmid ( cmv -gal ) , and 100 ng of cdm8 expression vectors . cells were assayed for luciferase ( promega ) activities 24 hours after addition of ligands and reporter expression was normalized to -galactosidase activity ( tropix ) according to the manufacturer 's directions . a previously described derivative of hepg2 cells stably expressing mcar was used for rna analysis . cells were subcultured and incubated in dmem supplemented with 10% charcoal stripped bovine serum overnight . various ligands were added and total rna was prepared 9 hours later and used for northern blotting using standard procedures . the effect of ligands on coactivator binding was evaluated using the biacore instrument as described . for interaction of car , rxr and car / rxr heterodimer with src-3 , a gst - src-3 fusion protein containing the receptor interaction domain of the coactivator was expressed in bacteria and purified . lack of binding of free gst demonstrated that the surface was saturated with gst - src3 . purified car and rxr proteins were produced in bacteria and preincubated overnight at 4c alone or in combination , and in the presence or absence of 10 m androstanol , 10 m tcpobop , 10 m of 9 cis - ra , as appropriate . car alone , rxr alone , and car / rxr heterodimers bound to various ligands were injected individually into flow cells and binding was measured as described . the rare - tk - luc reporter plasmid with three copies of the dr-5 motif from the promoter of the mouse rar2 gene has been described previously , as has the cyp2b10-tk - luc reporter with two copies of the dr-4 type elements from the promoter of the mouse cyp2b10 gene and the cyp3a4-tk - luc reporter with three copies of the er-6 element from the human cyp3a4 promoter . the cdm8-mcar8 deletion construct containing a deletion of the last 8 amino acids and the mutant with 2 helix 3 mutations ( f171l , i174a ) that block ligand binding and response have been described previously , as has the cdm8-rxr19 construct with a deletion of the last 19 amino acids . hepg2 cells were maintained in dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal bovine serum ( hyclone ) . for transfections , 10hepg2 cells were plated in 24 well dishes with dmem supplemented with 10% charcoal stripped serum and transfected using the calcium phosphate precipitation method as described . the next morning typically , transfections included 100200 ng of luciferase reporter plasmids , 100 ng of -galactosidase ( -gal ) internal control plasmid ( cmv -gal ) , and 100 ng of cdm8 expression vectors . cells were assayed for luciferase ( promega ) activities 24 hours after addition of ligands and reporter expression was normalized to -galactosidase activity ( tropix ) according to the manufacturer 's directions . a previously described derivative of hepg2 cells stably expressing mcar was used for rna analysis . cells were subcultured and incubated in dmem supplemented with 10% charcoal stripped bovine serum overnight . various ligands were added and total rna was prepared 9 hours later and used for northern blotting using standard procedures . i. t. and s. s. c. carried out the studies of car function in transiently transfected cells . s. s. c. carried out the studies of gene expression in stable car expressing cells . the rightward bar ( ----| ) symbolizes a blockage of the effect of the car ligand by rexinoids ( or decrease in the case of the repression of the cyp elements by androstanol ) ; ( - ) , no effect .

linear iga bullous dermatosis is a rare sulfone - responsive subepidermal blistering disorder of unknown etiology in which smooth linear deposits of iga are found in the basement membrane zone . chronic bullous dermatosis of childhood is equivalent to linear iga disease of adulthood and is characterized by an abrupt onset of large , widespread and tense bullae on a normal or erythematous base . in this case , we describe an unusual presentation of chronic bullous dermatosis in a 14-month - old saudi girl . histopathological examination revealed subepidermal cell poor blisters with linear deposition of iga , igg , igm , and c3 along the dermoepidermal junction . the unusual clinical , histopathological and immunofluorescence findings in this patient are discussed , with an account on the differential diagnosis in such cases along with a detailed review of the relevant literature .

chronic bullous dermatosis of childhood presents in prepubertal , often preschool , children and rarely in infancy . vesicles or bullae develop on an erythematous or normal base , occasionally giving rise to a so - called string of pearls , a characteristic lesion in which peripheral vesicles develop on a polycyclic plaque . they involve the buttocks , lower abdomen and genitalia and characteristically have a perioral distribution on the face . the disorder usually remits by the age of 6 - 8 years , but in one series , only 12% of the patients experienced persistent disease . in this case report , we present an unusual example of chronic bullous dermatosis of childhood , characterized by the presence of a subepidermal cell poor blister , fullhouse deposition of immunoglobulins including prominent deposition of iga at the dermo - epidermal junction in the absence of other autoimmune and connective tissue disorders . a 14-month - old girl from saudi was admitted to the dermatology ward because of a 2-month history of skin blistering with severe itching . on examination , multiple vesiculated and crusted skin lesions were found on the extremities , face , mouth , and genitalia [ figure 1 ] . no skin lesions on trunk or oral mucosa were noted . also , there was no history of drug intake , fever , or other systemic symptoms . a clinical differential diagnosis of chronic bullous disease of childhood and epidermolysis bullosa was made , and biopsies were taken from lesional and prelesional skin for routine histopathological and immunofluorescence studies , respectively . ( a ) chronic bullous disease of childhood . ( b ) chronic bullous disease of childhood . eroded and vesicular skin lesions on the external genitalia histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands . the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration . there was no evidence of an increase in polymorphous leukocytes or eosinophils [ figure 2a ] . direct immunofluorescence studies were performed and showed strong linear deposition of iga , igg , and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm ( 2 + ) in the same region [ figure 2b ] . the c3 deposits extended in a linear fashion to the skin appendages . according to the patient age , clinical distribution of the lesions along with the histological and immunofluorescence findings , a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita ( eba ) . direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone . ( direct if 200 ) based on this differential diagnosis , we performed a salt split test , which showed deposition of igg and c3 at the roof of blister . these findings led to a final diagnosis of chronic bullous dermatosis of childhood ( linear iga bullous dermatosis ) , with an unusual immunofluorescence pattern that is rarely described in the literature . histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands . the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration . there was no evidence of an increase in polymorphous leukocytes or eosinophils [ figure 2a ] . direct immunofluorescence studies were performed and showed strong linear deposition of iga , igg , and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm ( 2 + ) in the same region [ figure 2b ] . the c3 deposits extended in a linear fashion to the skin appendages . according to the patient age , clinical distribution of the lesions along with the histological and immunofluorescence findings , a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita ( eba ) . direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone . ( direct if 200 ) based on this differential diagnosis , we performed a salt split test , which showed deposition of igg and c3 at the roof of blister . these findings led to a final diagnosis of chronic bullous dermatosis of childhood ( linear iga bullous dermatosis ) , with an unusual immunofluorescence pattern that is rarely described in the literature . histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands . the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration . there was no evidence of an increase in polymorphous leukocytes or eosinophils [ figure 2a ] . direct immunofluorescence studies were performed and showed strong linear deposition of iga , igg , and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm ( 2 + ) in the same region [ figure 2b ] . the c3 deposits extended in a linear fashion to the skin appendages . according to the patient age , clinical distribution of the lesions along with the histological and immunofluorescence findings , a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita ( eba ) . direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone . ( direct if 200 ) based on this differential diagnosis , we performed a salt split test , which showed deposition of igg and c3 at the roof of blister . these findings led to a final diagnosis of chronic bullous dermatosis of childhood ( linear iga bullous dermatosis ) , with an unusual immunofluorescence pattern that is rarely described in the literature . chronic bullous dermatosis of childhood is the most common chronic bullous dermatosis in the first 10 years of life . the disease commonly involve the perioral skin , lower trunk , inner thighs , genitalia and perineum.[13 ] the mucous membranes may also be affected . bullae are the major clinical finding , and their appearance is characteristic with an annular arrangement of sausage - shaped bullae around resolving crusted lesions . the disorder is self - limiting and clears up within several months to three years . the clinical and histopathological differential diagnosis usually includes dermatitis herpetiformis , childhood form of eba and bullous pemphigoid . in our case , however , the presence of strong linear deposition of iga at the dermoepidermal junction and the results of the salt split test helped eliminate this possibility . furthermore , the presence of associated deposits of igg , igm , and c3 , which was confirmed by immunofluorescence studies in the absence of an associated autoimmune systemic disease is a rare event in this disorder and has only partially been reported previously in the literature . powell et al . had only deposits of iga with associated igg appearing in the early stages of the illness while our case has confirmed fullhouse deposition of ig 's and c3 . the lack of eosinophilic infiltration , besides the presence of iga deposits excludes an early onset of bullous pemphigoid . we also believe that the cell poor appearance of the blisters in this condition could be due to the age of the illness , as suggested by the 2-month history of the eruption . chronic bullous dermatosis of childhood is a common blistering disease , which can be seen in infants and preschool children . both dermatopathologists and clinical dermatologists should be aware of the unusual and rare histopathological and immunofluorescence features , which can be seen in such cases and may lead to an erroneous diagnosis of a more severe disorder like childhood eba .

advances in de novo sequencing technologies allow us to track deeper insights into microbial genomes for restructuring events during the course of their evolution inside and outside the host . bacterial species belonging to ochrobactrum genus are being reported as emerging , and opportunistic pathogens in this technology driven era probably due to insertion and deletion of genes . the ochrobactrumintermedium m86 was isolated in 2005 from a case of non - ulcer dyspeptic human stomach followed by its first draft genome sequence in 2009 . here we report re - sequencing of o. intermedium m86 laboratory adapted strain in terms of gain and loss of genes . we also attempted for finer scale genome sequence with 10 times more genome coverage than earlier one followed by comparative evaluation on ion pgm and illumina miseq . despite their similarities at genomic level , lab - adapted strain mainly lacked genes encoding for transposase protein , insertion elements family , phage tail - proteins that were not detected in original strain on both chromosomes . interestingly , a 5 kb indel was detected in chromosome 2 that was absent in original strain mapped with phage integrase gene of rhizobium spp . and may be acquired and integrated through horizontal gene transfer indicating the gene loss and gene gain phenomenon in this genus . majority of indel fragments did not match with known genes indicating more bioinformatic dissection of this fragment . additionally we report genes related to antibiotic resistance , heavy metal tolerance in earlier and re - sequenced strain . though snps detected , there did not span urease and flagellar genes . we also conclude that third generation sequencing technologies might be useful for understanding genomic architecture and re - arrangement of genes in the genome due to their ability of larger coverage that can be used to trace evolutionary aspects in microbial system .

human gastrointestinal ( gi ) tract is compartmentalized into different sections , which allows digestion and absorption of nutrients in the body . the distinct part of the gut has different physiological conditions that shape the gut microbiome ( or microbial diversity within the gut ) . the human intestinal microbiota is composed of 10 to 10 microorganisms whose collective genome ( microbiome ) contains at least 100 times as many genes as our own genome . as in most mammals , the gut microbiome is dominated by four bacterial phyla namely firmicutes , bacteroidetes , actinobacteria , and proteobacteria , representing more than 1000 different molecular species or phylotypes . stomach of the gi tract has very low ph ~ 2.5 , urea stress and microaerophilic environment making it extreme niche . to adopt up with this condition organism have to have some additional machinery to survive and perform its functions . there has been an estimate that human stomach comprises 32 phylotypes from proteobacteria , including 3 uncharacterized phylotypes . helicobacter pylori ( h. pylori ) , known causative agent of peptic ulcers and gastric cancer , colonize the stomach of approximately 50% of the world 's population . however now there are few recent reports indicating presence of bacteria other than h. pylori in the human stomach . our group had isolated a bacterium from non ulcer dyspeptic individual from north india in 2005 and was identified as ochrobactrum intermedium by 16s rrna sequencing and its first draft genome sequencing was reported by using ion torrent personal genome machine ( pgm ) , . to gain insights into genomic re - architecturing in terms of gain and loss during continuous passaging for several generations and also to evaluate sequencing platform for getting more refined data ( in terms of coverage ) , we re - sequenced the ochrobactrum intermedium m86 strain with illumina miseq and compared with 10 year earlier draft genome of the same strain . the 16s rrna based phylogenetic analysis showed ochrobactrum and brucella are closely related than helicobacter . urease gene ( urec ) based tree clearly indicated the presence of a distinct class of urease in ochrobactrum when compared to helicobacter and brucella ( data not shown ) . whole - genome re - sequencing of o. intermedium m86 was conducted with the illumina miseq . we obtained 5,384,581 paired - end reads of 300 bp using illumina miseq which was 4.1 fold higher than the reads obtained from ion torrent pgm machine . sequence yield per run is higher in illumina miseq than ion torrent pgm machine and reports also showed the error rate is higher in pgm 1.71% than illumina miseq , 0.4%.the number of error - free reads , without a single mismatch or indel , was 76.45% , 15.92% for , miseq , ion torrent respectively . this comparison revealed efficiency in terms of quantity and quality miseq is somewhat better than pgm in terms of output data . the genome of passaged / re - sequenced / laboratory adapted m86 comprised of two circular chromosomes with a total size of 4,683,452 bases with a 57% g + c content and no plasmids . of the 4415 ( cdss ) genes predicted , 38 trna and 5 rrna genes were found ( table 1 ) . the genes were distributed into 470 subsystems using rast . to check the quality of assemblies obtained from velvet with different k - mer values ; genome coverage , n50 values and maximum / median / average contig sizes were compared . it was observed that at k - mer = 25 , estimated coverage was 53% , 2,642,663 2(a ) , ( d ) , and ( e ) . in case of k - mer = 57 , n50 value was maximum but the coverage attained was 79% , moreover number of assembled nucleotides was less as compared to k - mer = 31 as mentioned in fig . 2(c ) , ( d ) and ( e ) . at k - mer = 31 , all the parameters were optimized and maximum coverage was obtained ( fig . the de novo assembly using the velvet and spades method generated 121 contigs , which were further assembled into 34 scaffolds . sequencing statistics of earlier genome ( did on ion pgm ) and resequenced strain ( on illumina ) was also compared . resequenced genome on illumina was better on ion pgm on all technical aspects like number of reads ( 5 times ) , genome coverage ( 10 times ) , n50 value ( 1.25 higher ) , number of scaffolds ( equal ) except run time ( 4 times more ) . this study was also aimed to enhance the quality of reads by re - sequencing for futuristic view of functional genomics . in our study , illumina miseq and pgm were performed to sequence genome of o. intermedium m86 and we could conclude that the illumina probably had higher coverage as compared to pgm with respect to total reads obtained and coverage , which could also be useful for larger genomes . for chromosome 1 and 2 , the is finder blast results showed unique insertion sequences / tandem repeats which were not reported in earlier draft genome but appeared in current analysis . unique insertion sequences that we detected only in re sequenced strain were is2 , is51 , is151 , is407 , isas1 , is30 , and is1595 . the rast annotation system showed absence of prophage encoding genes ( confirmed by phast ) in earlier strain , but were present in re sequenced strain , although they were not complete indicative of a possibility of gene exchange via transduction mechanism . reference based mapping of o. intermedium m86 draft genome and resequenced genome with o. intermedium lmg3301 revealed that resequenced genome appeared to have undergone insertion as well as deletion of some genes or islands ( fig . further investigation of the deleted portion belonged to phage proteins that were excised probably during passaging . anti - smash analysis revealed interesting findings with respect to absence of trps - t3pks system in the resequenced strain , with replacement of terpene and arylpolyene . such differences in biosynthetic gene cluster could assist the strains in developing and combating strategies . as was previously reported the strains of o. intermedium were resistant to all -lactam antibiotics except imipenem . this was confirmed in resequenced genome by rast annotation server and it showed the presence of -lactamase . in addition , metal - dependent hydrolases of the beta - lactamase superfamily i and beta - lactamase class c , other penicillin binding proteins , fluoroquinolones and streptothricin resistant genes were also found in resequenced as well as earlier genome but not reported so far . diverse ochrobactrum sps . have been reported from contaminated habitat which showed its role in bioremediation and tolerance to different heavy metals like arsenic , nicotine , and were found in earlier as well as resequenced strain . in addition , presence of genes coding for arsenic ( arsh , acr3 , arsenate reductase ) copper ( cute , ccmh , copc , copd , copper chaperone , copg , ccmm , copper - translocating p - type atpase , blue copper oxidase cueo precursor ) , cobalt - zinc - cadmium resistance ( co / zn / cd efflux system membrane fusion protein ) and mercuric reductase were also detected in earlier and resequenced strain indicating the necessity of these genes for growth under in - vitro as well as in - vivo environment . strain differences under selective or non - selective pressure may lead to formation of snps leading to inter and intra - strain diversity . around 8878 snps were detected among previous genome and resequenced o. intermedium m86 ( both chromosomes ) , wherein no snps detected in rpob , trpe , reca , dnak , aroc and gap . genes responsible for survival in acidic condition of stomach ( urec , ured , uree , uref and ureg of urease operon ) and flagellar assembly ( flic ) were devoid of snps indicating their stability under in - vitro conditions outside simulated niche . in chromosome i , we detected 2 gaps from which one fragment is absent in both the genomes , previous genome and resequenced genome . the other gap which is specific to resequenced genome includes phage related protein like phage tail protein , assembly protein . interestingly in chromosome 2 , a 5 kb fragment which was absent in previous genome and also in o. intermedium lmg 3301 strain was detected in a re - sequenced strain . further , tblastx analysis of this fragment showed the presence of integrase gene from rhizobium spp . we speculate that the insertion might be result of a horizontal gene transfer event in o. intermedium m86 . integrase gene product mainly helps in the site specific recombination through attb and attp site . construction of vector containing attachment site and integrase gene can be use as engineering tool for insertion of desired fragment of gene through site specific recombination . it is just because crossover of nucleic acid fragment and host integration site can occur at any 3 sublocation within a trna can occur , two with flanking symmetry ( anticodon - loop and t - loop tdna ) and the third at the asymmetric 3 end of the gene . apart from 5 kb indel , genes absent only in resequenced genome were phage encoded protein like phage tail coding protein , dna packaging protein , hk97 family phage genes which mainly play an important role in connecting the dna filled head to tail via connector . another gene holotricin-3 precursor which is a defense response gene and chloramphenicol acetyltransferase which provides resistant to chloramphenicol antibiotic were also absent . the stability and effect of indel events on the overall physiology , regulatory and colonization mechanisms in the passaged strain needs to be investigated for acid resistance , colonization , and persistence in hostile environment of acid and urea present in stomach . considering the opportunities and challenges of niche specialized o. intermedium strain ( among genus ochrobactrum ) that has a tendency of genome reduction in the technology driven era . our study is significant for this isolate of gastric origin in terms of genome architecture and genome rearrangement in terms of deletion as well as insertional events during routine maintenance under laboratory conditions . given the understanding of having such property of recombination and specialized process of adaptation in diverse environment , the more understanding of ochrobactrum genus at genomic level could be important to understand if specialized set of genes are assigned for environmental and clinical adaptations . strain o. intermedium m86 was weekly passaged for about 100 generations for this study and used as laboratory adapted strain in this study . then grown in macconkey broth ( himedia , india ) and after overnight incubation at 37 c purity of the culture was checked and pellet was collected by centrifugation . chromosomal dna isolation was done by using pure - link dna isolation kit ( invitrogen , carlsbad , usa ) and quality of the dna was checked by recording absorbance at a260/280 nm and agarose gel electrophoresis . phylogenetic analysis of 16s rrna genes for o. intermedium m86 , brucella melitensis biovar suis , helicobacter pylori atcc 43504 and o. intermedium lmg 3301 was done using mega v 6.0 with upgma method ( bootstrap = 1000 ) as described . similarly urease alpha subunit ( ure c ) of genera ochrobactrum , helicobacter and brucella was used to build upgma tree using mega . genome re - sequencing of m86 strain was done using illumina miseq m02845 sequencer was carried out using 2 300 bp chemistry . to check the quality of paired end poor quality reads ( phred quality below q30 ) and adapter sequences were removed using fastq trimmer and flexbar pre - processing tool respectively . to preserve mate integrity and to filter sequences by composition de novo assembly of prepossessed fastq reads was performed using velvet 1.2.10 ( k - mer = 25 , 31 , 57 ) and spades 3.0.0 ( k - mer 21 , 33 , 55 ) . further the quality of integrated contigs / scaffolds was evaluated using check bacterial contigs tool . contigs of o. intermedium m86 draft genome and resequenced genome were mapped against chromosome i and ii of o. intermedium lmg 3301 and visualized using brig ( fig . metabolic pathway prediction was performed on kaas to assign kegg orthology ( ko ) numbers to each predicted cds . comparative analysis was carried out between previous draft genome and resequenced genome for different features such as presence of insertion sequences , tandem repeats , phage genes , clustered regularly interspaced short palindromic repeats ( crispr ) , plasmid and secondary metabolite production genes using is finder , tandem repeats finder , phast , crisprfinder , blast and anti - smash 2.0 respectively . o. intermedium m86 draft genome and resequenced genome were compared with o. intermedium lmg 3301 for single nucleotide polymorphism ( snps ) detection using parsnp pipeline . strain o. intermedium m86 was weekly passaged for about 100 generations for this study and used as laboratory adapted strain in this study . then grown in macconkey broth ( himedia , india ) and after overnight incubation at 37 c purity of the culture was checked and pellet was collected by centrifugation . chromosomal dna isolation was done by using pure - link dna isolation kit ( invitrogen , carlsbad , usa ) and quality of the dna was checked by recording absorbance at a260/280 nm and agarose gel electrophoresis . phylogenetic analysis of 16s rrna genes for o. intermedium m86 , brucella melitensis biovar suis , helicobacter pylori atcc 43504 and o. intermedium lmg 3301 was done using mega v 6.0 with upgma method ( bootstrap = 1000 ) as described . similarly urease alpha subunit ( ure c ) of genera ochrobactrum , helicobacter and brucella was used to build upgma tree using mega . genome re - sequencing of m86 strain was done using illumina miseq m02845 sequencer was carried out using 2 300 bp chemistry . to check the quality of paired end poor quality reads ( phred quality below q30 ) and adapter sequences were removed using fastq trimmer and flexbar pre - processing tool respectively . to preserve mate integrity and to filter sequences by composition de novo assembly of prepossessed fastq reads was performed using velvet 1.2.10 ( k - mer = 25 , 31 , 57 ) and spades 3.0.0 ( k - mer 21 , 33 , 55 ) . further the quality of integrated contigs / scaffolds was evaluated using check bacterial contigs tool . contigs of o. intermedium m86 draft genome and resequenced genome were mapped against chromosome i and ii of o. intermedium lmg 3301 and visualized using brig ( fig . to predict gene coding regions ( cds ) prokka and rapid annotation subsystem technology ( rast ) were used . metabolic pathway prediction was performed on kaas to assign kegg orthology ( ko ) numbers to each predicted cds . comparative analysis was carried out between previous draft genome and resequenced genome for different features such as presence of insertion sequences , tandem repeats , phage genes , clustered regularly interspaced short palindromic repeats ( crispr ) , plasmid and secondary metabolite production genes using is finder , tandem repeats finder , phast , crisprfinder , blast and anti - smash 2.0 respectively . o. intermedium m86 draft genome and resequenced genome were compared with o. intermedium lmg 3301 for single nucleotide polymorphism ( snps ) detection using parsnp pipeline .

postprandial oxidative stress is characterized by an increased susceptibility of the organism towards oxidative damage after consumption of a meal rich in lipids and/or carbohydrates . micronutrients modulate immune system and exert a protective action by reducing low density lipoproteins ( ldl ) oxidation via induction of antioxidant enzymes . we evaluated the gene expression of oxidative stress ( hosp ) , inflammasome ( hip ) , and human drug metabolism pathways ( hdm ) and ox - ldl level at baseline and after the intake of red wine naturally enriched with resveratrol ( npvrw ) , in association with or without a mcdonald 's meal ( mcdm ) . the ox - ldl levels significantly increase comparing baseline ( b ) versus mcdm and decreased comparing mcdm versus mcdm + npvrw ( p 0.05 ) . percentages of significant genes expressed after each nutritional intervention were the following : ( 1 ) b versus mcdm , 2.88% hosp , 2.40% of hip , and 3.37% of hdmp ; ( 2 ) b versus mcdm + npvrw , 1.44% of hosp , 4.81% of hip , and 0.96% of hdmp ; ( 3 ) mcdm versus mcdm + npvrw , 2.40% of hosp , 2.40% of hip , and 5.77% of hdmp ; ( 4 ) b versus npvrw , 4.80% hosp , 3.85% hip , and 3.85% hdmp . npvrw intake reduced postprandial ox - ldl and the expression of inflammation and oxidative stress related genes . chronic studies on larger population are necessary before definitive conclusions .

epidemiological evidence has supported a protective role for diets low in saturated fat and rich in fruits and vegetables as well as a moderate wine consumption against the development and progression of cardiovascular ( cvd ) and chronic degenerative disease . the atherosclerotic processes underlying cardiovascular disease are intimately connected with a state of chronic inflammation , involving a variety of pathological changes such as endothelial cell activation , low density lipoprotein ( ldl ) modification , macrophage chemotaxis , and vessel smooth muscle cell migration . in industrialized societies , this excess in ldl particles promotes transport into the vessel wall where they undergo physicochemical modification , which facilitates their ingress into macrophages resulting in foam - cell formation . it is widely recognized that , together with oxidative stress , vascular inflammation , lipid deposition , and smooth muscle cell differentiation , oxidized low density lipoprotein ( ox - ldl ) level may play a major role in atherosclerosis and cardiovascular disease , and it is a commonly used marker for oxidative damage . in fact , oxidation of the lipids and apoproteins present in ldl leads to a change in the lipoprotein conformation by which ldl is better able to enter the monocyte - macrophage system of the arterial wall and promote the atherosclerotic process . the oxidative modification of ldl convert the native particles ox - ldl into pathogenic , immunogenic , and atherogenic particles . removal of ox - ldl from circulating blood is a promising therapeutic strategy against atherosclerosis and many other diseases . it is widely accepted that the consumption of fruits and vegetables prevents diseases related to the oxidative processes . for instance , individuals at high cardiovascular risk who improved their diet toward a mediterranean diet pattern , reached in antioxidants , showed significant reductions in cellular lipid levels and ldl oxidation . moreover , beneficial effects may partly help to explain the protective cvd effects achieved by foods and beverages containing polyphenols ( tea , vegetables , fruits , wine , etc . ) , that appears to be mediated via a plethora of biochemical pathways and signaling mechanisms , acting either independently or synergistically ( e.g. , decreasing ldl oxidation , improving endothelial function , increasing nitric oxide release , modulating inflammation and lipid metabolism , and improving antioxidant status ) . in particular , the reduction of oxidative stress and inflammation following red wine intake could possibly be due to its polyphenol , and resveratrol , content , as suggested by many studies . red wine ( rw ) consumption has shown to prevent the acute impairment of endothelial function that occurs following cigarette smoking or consumption of high fat meal and to modulate monocyte migration in healthy subjects . these effects must be considered independent of some of the protective effects of wine linked to ethanol , per se . it has been suggested that the phenolic content of rw may modulate leukocyte adhesion molecules , whereas both ethanol and polyphenols of rw may modulate soluble inflammatory mediators in high - risk patients . the phytoalexin trans-3,5,4-trihydroxystilbene ( trans - resveratrol ) is present in red grape skins and in wines . synthesis of resveratrol and its derivatives ( cis - resveratrol and resveratrol glucoside isomers ) , called all together stilbenes , is generally formed as natural defense ability of the vines against disease attack . the content of stilbenes in the final product depends mainly on the grape variety , but other factors like canopy management can stimulate the plant 's self - defense . focusing on those practices that can modulate both the qualitative parameters in the classical sense ( sugars , acidity , etc . ) , as well as the most truly innovative and related compounds useful to human health , growing methods can get high quality grapes and at the same time stimulate the stilbene synthesis . among these the unpruned vineyards can promote the natural presence of stilbenes too , whose positive role on human health is known . although different studies have reported positive data on gene expression after feeding animals with phenolic rich extracts or normal food , few human studies have shown the potential use of gene expression profiling in blood leukocytes to study the effects of meal consumption and the intake of red wine from nonpruned vineyard ( npvrw ) , on gene expression modulation of oxidative stress , inflammatory , and drug metabolism pathway , in association with ox - ldl . more understanding of the role of npvrw intake on oxidation of lipoproteins may allow rationally targeted dietotherapy that can beneficially alter postprandial biomarkers associated with cvd . to our knowledge , no research has compared the acute effects of npvrw in association with a high fat meal on postprandial biomarkers associated with cvd , including oxidative stress and anti - inflammatory and antioxidant activity . therefore , we set up a randomized , crossover , and controlled dietary intervention trial in healthy human volunteers to evaluate and compare the effects on ldl oxidative status after a mcdonald meal ( mcdm ) , alone or combined with the intake of 30 g alcohol from 250 ml npvrw . moreover , we investigated the gene expression of 208 gene related to oxidative stress , inflammation , and drug metabolism in the same conditions . we hypothesized that the magnitude of changes in postprandial responses would be less with the mcdm associated with npvrw , compared with the mcdm alone . this comparison aims to determine if resveratrol is implicated in the nutrigenomic effects of rw . the first aim of the present study was to assess the ox - ldl level in healthy subjects after the intake of 30 g alcohol from 250 ml npvrw , alone or combined with a mcdm . the second outcome was to examine changes of gene expression levels of 75 genes related to oxidative stress pathways ( hosp ) , 72 of human inflammasome pathways ( hip ) , and 61 genes of human drug metabolism ( hdm ) in the same conditions . as control , we repeated the same analysis after the intake of 30 g alcohol from 250 ml npvrw fasting , with respect to baseline . after the enrollment ( a ) , the study was conducted with four intervention arms : ( b ) baseline ; ( c ) mcdm ; ( d ) mcdm + 250 ml npvrw ( mcdm + npvrw ) ; ( e ) 250 ml npvrw . each arm was followed by a three - week washout period to avoid additive effects on treatments to follow . a total of 30 participants were consecutively recruited from those who participated in routine medical check - up at the section of clinical nutrition and nutrigenomic , at the university of rome tor vergata . all subjects were healthy and had no evidence of chronic disease . to be eligible for the study , participants had to meet the following inclusion criteria : age between 18 and 65 years , with a bmi 19 kg / m . exclusion criteria were as follows : pregnancy , active smoking , arterial hypertension ( 140/90 mm hg ) , body mass index ( bmi ) > 30 kg / m , acute or chronic diseases , autoimmune disease hiv / aids , neoplastic disease , intestinal disorders , vegetarianism , and use of antioxidants or vitamin supplements or any medication that could influence inflammation and oxidative stress . subjects daily consuming flavonoid - rich beverages , such as tea , herb tea , coffee , cocoa , and fruit juice , more than 500 ml ( as estimated from food frequency questionnaire ) , were also excluded . subjects were asked to exclude alcoholic beverages 15 days before the first intervention ( run - in period ) and during the study . natural foods rich in antioxidants intake were monitored so that individual diets had similar antioxidant contents throughout the study . at baseline , the clinical evaluation is based on anthropometric and body composition evaluation , quantification of ox - ldl , and a genomic evaluation of 208 genes belonging to the pathway of oxidative stress , inflammation , and drug metabolism . subjects were asked to maintain their usual lifestyle habits and to report any illness or abnormality presented during the study period . at the end of each arm of the study design , a clinician assessed any adverse effects from the interventions by going through a checklist of symptoms , including bloating , fullness , or indigestion , altered bowel habit , dizziness , and other symptoms that were possibly associated with the interventions . an informed consent was signed by all participants , in accordance with principles of the declaration of helsinki . nutritional status assessment , genomic analysis , settings and data collection were performed at the section of clinical nutrition and nutrigenomic , department of biomedicine and prevention of the university of rome tor vergata . after a 12-hour overnight fast , all subjects underwent anthropometric evaluation . all the individuals were instructed to take off their clothes and shoes before undergoing the measurements . waist and hip measures were taken using a flexible steel metric tape to the nearest 0.5 cm , with subjects standing with arms relaxed by their side and balanced on both feet . hip circumference was also measured according to international society for the advancement of kin anthropometry protocol taken at the greatest posterior protuberance of the buttocks . waist circumference was measured just above the iliac crest as recommended in the national institute of health guidelines . body weight ( kg ) was measured to the nearest 0.1 kg , using a balance scale ( invernizzi , rome , italy ) . height ( m ) was measured using a stadiometer to the nearest 0.1 cm ( invernizzi , rome , italy ) . body mass index ( bmi ) was calculated using the formula : bmi = body weight / height ( kg / m ) . body composition analysis was assessed by dxa ( i - dxa , ge medical systems , milwaukee , wi , usa ) , according to the previously described procedure , for evaluating soft tissues , that is , total body fat ( tbfat ) and total body lean ( tblean ) . the technique combined a total body scanner , an x - ray source , an internal wheel to calibrate the bone mineral compartment , and an external lucite / aluminium phantom to calibrate the fat compartment . individuals were asked to remove all clothing except for undergarments including shoes , socks , and metal items prior to being positioned on the dxa table . the entire body was scanned beginning from the top of the head and moving in a rectilinear pattern down the body to the feet . the coefficient of variation ( coefficient of variation = 100 sd / mean ) intra- and intersubjects ranged from 1% to 5% . the coefficient of variation for bone measurements is less than 1% ; coefficient of variation on this instrument for five subjects scanned six times over a nine - month period was 2.2% for tbfat and 1.1% for tblean . total body fat percentage ( pbf ) was calculated as tbfat mass divided by total mass of all tissues , considering also the total body bone ( tbbone ) , as follows : ( 1)pbf=(tbfat+tblean+tbbone)100 . blood samples ( 10 ml ) were collected into sterile tubes containing edta ( vacutainer ) . all materials were immediately placed on ice and plasma was separated by centrifugation at 1600 g for 10 min at 4c . laboratory tests included fasting glucose , hdl - cholesterol , ldl - cholesterol , triglycerides , aspartate aminotransferase ( ast ) , alanine transaminase ( alt ) , creatinine , fibrinogen , and c - reactive protein ( crp ) levels and were recorded at baseline . all clinical chemistry analyses , except plasma glucose , serum lipid , crp , and triglycerides analysis , were carried out with an advia 1800 chemistry system ( siemens healthcare ) , according to standard procedures . plasma glucose concentrations were measured using the glucose oxidase method with an automated glucose analyzer ( cobas integra 400 , roche diagnostics , indianapolis , in , usa ) ; serum lipid profile components were determined by standard enzymatic colorimetric techniques ( roche143 modular p800 , roche diagnostics , indianapolis , in ) . serum triglycerides were measured on the beckman synchron lx20 automated system by a coupled enzymatic method that produces a red - colored complex . all tests were performed using the same lot of reagents or assay plates in order to minimize variability due to differences in reagent lots . analyses were carried out at the accredited clinical chemical laboratories of the tor vergata hospital of rome , italy . blood sample was collected and stabilized in paxgene blood rna tubes ( pre analytix qiagen , hombrechtikon , switzerland ) and stored at 80c until rna extraction . the total rna of each collected sample was purified using the paxgene blood mirna kit according to the manufacturer 's instructions ( preanalytix qiagen , hombrechtikon , switzerland ) . aliquots of total rna were then quantified and assessed for quality by spectrophotometry ( nanodrop , wilmington , usa ) and agarose gel electrophoresis . we used specific rt2 profiler pcr arrays ( qiagen , netherlands ) : we focused on the human oxidative stress ( pahs-065za , qiagen , netherlands ) pathway , human inflammation ( pahs-097za , qiagen , netherlands ) pathway , and human drug metabolism ( pahs-002a , qiagen , netherlands ) pathway . each qrt - pcr experiment was performed in triplicate and repeated at least twice , according to manufacturer 's instruction ( qiagen , netherlands ) . the value used to plot relative gene expression was determined using the expression fold change ( fc ) = 2 . raw data were filtered for genes that were significantly changed above factor 1.0 within the 95% confidence interval ( p 0.05 ) for each experiment . finally , only genes with an absolute fc value of at least 1.5 and p value 0.05 ( indicating a statistical significance ) were considered as differentially expressed genes . a fasting blood samples were collected and stabilized in edta and stored at 80c until analysis . circulating ox - ldl level in plasma was measured by enzyme linked immunosorbent assay using the mab-4e6 antibody ( mercodia ab , uppsala , sweden ) , according to the customer protocol . the agricultural trial was started in 2012 on 12-year - old commercial vineyards , located in ponte di piave , treviso , ( north - east italy , 454302.93n , 12328:35 e , altitude 3 m asl ) . the variety was cabernet sauvignon , grafted on 1103 p , and the vineyard has a density of 3300 plants per hectare . the adopted training system was the free cordon ( a permanent spur cordon pruning with no wire above the cordon ) ; no pruning ( np ) treatment have been provided . for the np treatment a randomized block design was applied , with 3 plots , each one consisting of 2 rows ( one per treatment ) . fifteen vines per treatment were chosen and replicated 3 times ; the production of grapes was weighed , and the average weight of the bunch was made ; on a sample of berries the polyphenol content , total and extractable , according to the methodology proposed by di stefano and cravero , soluble solids content ( brix ) , total acidity ( ta ) , and ph were determined . the contents of some stilbenes , resveratrol , trans and cis , and trans - piceid were determined according to the methodology proposed by sun et al . . the selected dietary treatment was a high fat meal represented by a mcdonald 's meal ( mcdm ) consisting in number 1 big tasty bacon and number 1 small french fries ( 26.8% of total kcal from carbohydrates ; 18.2% of total kcal from protein , of which about 70% was comprised of animal proteins ; 55% of total kcal from total fat ) . the bromatological composition of mcdm was obtained using diet analyser software package dietosystem ( ds medica srl , milan , italy , version 12.00.13 . ) . atherogenic index ( ai ) , a parameter to determine the atherogenic risk of a diet , was calculated by the ratio between saturated ( c14 : 0 , c16 : 0 , c18 : 0 ) and unsaturated fat acids ( monounsaturated fatty acid ( mufa ) and polyunsaturated fatty acid ( pufa)):(2)ia=4c14 : 0+c16 : 0+c18 : 0mufa+pufa6+pufa3.thrombogenicity index ( ti ) was calculated by the ratio between saturated fat acids ( c14 : 0 , c16 : 0 , c18 : 0 ) and the sum of mufa and pufa : ( 3)it = c14 : 0+c16 : 0+c18 : 00.5mufa+0.5pufa6-fatty acids3-fatty acidspufa + 3pufa3 fatty acids+pufa 3-fatty acidspufa6-fatty acids1.cholesterol/saturated fatty acids index ( csi ) can be used as a fast and accurate way to assess the content of saturated fat and dietary cholesterol . the potential atherogenic risk of food referred to the cholesterol and saturated fat contained in the meal ; lower index suggests a low probability of incidence of cardiovascular disease : ( 4)csi=1.01g of saturated fat+0.05mg of cholesterol . a paired t - test or a nonparametric wilcoxon test was performed to evaluate differences before and after nutritional intervention . in all statistical tests performed , the null hypothesis ( no effect ) was rejected at the 0.05 level of probability . the differences between ox - ldl levels in the intervention arms were calculated as follows . % = ( ( z y)/y ) 100 , where % is the percentage variation of oxidation of ldl , calculated as ratio of absolute variation to the base value ; in particular z is the value of oxidation ldl after nutritional intervention ( npvrw , mcdm , and mcdm + npvrw ) and y is the value of oxidation if ldl at the nutritional intervention of reference ( b and mcdm ) . the value used to plot relative gene expression was determined using the expression fold change ( fc ) = 2 . data were normalized and we have considered an absolute fold change at least equal to 2.00 ( absolute fc 2.00 ) . transcripts were declared differently expressed only when p < 0.05 and fold changes were either < 0.8 ( downregulated ) or > 1.2 ( upregulated ) , as previously described . the npvrw treatment reported the following quality parameters : the soluble solids ( brix ) equal to 19.20 , a titratable acidity equal to 7.4 g / l , and a ph as 3.38 . regarding the content flavonoids and anthocyanins , the wine showed a content of total anthocyanins equivalent to 541 mg / kg of berry , a content of extractable anthocyanins equal to 317 mg / kg of berry . total flavonoids were detected equal to 1346 mg / kg and a content of extractable flavonoids equivalent to 575 mg / kg of berry . the macronutrient content of mcdm was 1144.04 kcal , 81.89 g of carbohydrates , 51.99 g of proteins ( 40.78 g of animal proteins , 10.60 g of vegetal proteins ) , 68.89 g of fats ( of which 21.71 g saturates , 30.71 g monounsaturates , and 13.96 g unsaturates ) , and 3.68 g of dietary fiber ( of which 1.51 g soluble , and 2.15 g insoluble fiber ) . the quality indices of mcdm were 1.97 of ai , 1.73 of ti , and 31.05 of csi . of the 30 initial participants initially enrolled , 24 subjects were eligible for the study . four subjects declined to participate during the first phase , and another two did not meet the inclusion criteria : one of them measured a bmi > 25 kg / m , one had a history of heart attack . in tables 1 and 2 the nutritional status characteristics and blood parameters of subjects are shown , before nutritional intervention . the value of obese subjects was obtained by dxa : total percentage of obese was estimated as a 58.33% of total subjects : 71.43% of female and 40.00% of men . according to bmi , 16.67% of total subjects were sarcopenic : 20.00% of male and 14.29% women were sarcopenic . the comparison of ox - ldl level in the nutritional intervention was shown in figure 2 . a significant increase ( p 0.05 ) of ox - ldl level was observed between b andmcdm , showing a % = 17.56% . the ox - ldl levels significantly decreased ( p 0.05 ) comparing mcdm versus mcdm + npvrw ( % = 20.94% ) . no significant value ( p > 0.05 ) was highlighted in b versus mcdm + npvrw ( % = 7.06% ) and in b versus npvrw ( % = 5.48% ) . in particular , we focused on different expression levels of 75 genes of oxidative stress , 72 genes of inflammasome and 61 genes of human drug metabolism pathways ( see supplement table s13 in supplementary material available online at http://dx.doi.org/10.1155/2015/317348 ) . the different fold change levels were analyzed for the condition : ( i ) b versus mcdm ; ( ii ) b versus npvrw ; ( iii ) b versus mcdm + npvrw ; ( iv ) mcdm versus mcdm + npvrw . after data normalization , considering an absolute fold change at least equal to 2.00 ( absolute fc 2.00 ) , we identified differential gene expression levels for each condition , as shown in figure 3 . in figures 4 , 5 , and 6 we showed the most representative genes of the studies pathways ; a fold change > 2.00 was considered significant ( p 0.05 ) . in figure 4 genes of inflammasome pathway are reported , comparing : ( i ) b versus mcdm ; ( ii ) b versus npvrw ; ( iii ) b versus mcdm + npvrw ; ( iv ) mcdm versus mcdm + npvrw : casp8 , caspase 1 apoptosis related cysteine peptidase ( nm_1228 ) ; ccl2 , chemokine ( c - c motif ) ligand 2 ( nm_2982 ) ; il6 , interleukin 6 ( interferon beta 2 ) ( nm_600 ) ; nfkb1 , nuclear factor of kappa light polypeptides gene enhancer in b - cells 1 ( nm_3998 ) . in figure 5 genes of oxidative stress pathway are reported , comparing ( i ) b versus mcdm ; ( ii ) b versus npvrw ; ( iii ) b versus mcdm + npvrw ; ( iv ) mcdm versus mcdm + npvrw : cat , catalase ( nm_1752 ) ; ccl5 , copper chaperone for superoxide dismutase ( nm_2985 ) ; gpx1 , glutathione peroxidase 1 ( nm_581 ) ; gpx2 , glutathione peroxidase 1 ( gastrointestinal ) ( nm_2083 ) ; sirt2 , sirtuin 2 ( nm_12237 ) ; sod1 , superoxide dismutase 1 soluble ( nm_454 ) ; txn , thioredoxin ( nm_3329 ) ; ucp2 , uncoupling protein 2 ( mitochondrial proton carrier ) ( nm_3355 ) . in figure 6 genes of human drug metabolism pathway are reported , comparing ( i ) b versus mcdm ; ( ii ) b versus npvrw ; ( iii ) b versus mcdm + npvrw ; ( iv ) mcdm versus mcdm + npvrw : adh4 , alcohol dehydrogenase 4-class 2 pi polypeptide ( nm_670 ) ; aldh1a1 , aldehyde dehydrogenase 1 family member a1 ( nm_689 ) ; alox5 , arachidonate 5-lipoxygenase ( nm_698 ) ; nat1 , n - acetyltransferase 1 ( arylamine n - acetyltransferase ) ( nm_662 ) ; nos3 , nitric oxide synthase 3 ( endothelial cell ) ( nm_603 ) . in the supplementary tables the fold change of all analyzed genes and related statistical significance were reported ( see tables s13 ) . dietary patterns have been associated with several cardiovascular risk factors such as blood pressure , obesity , serum lipids , and inflammatory markers . in postprandial state , cellular structures such as proteins , carbohydrates , nucleic acids , and lipids are damaged by oxidative processes . high fat diets are contributory atherosclerosis risk factors , since it can cause an acute modification in endothelial function . considering that after each meal , blood concentrations of glucose and lipids are raised , and this postprandial increase lasts for a rather long time ; these changes might be of importance in the process of atherosclerosis initiation and progression . moreover , after each meal the production of reactive oxygen species ( ros ) will lead to the activation of factor nuclear factor-b ( nf - kb ) . this factor is a transcription factor which is contributed in immunity , inflammation , and regulation of cell proliferation , growth , and apoptosis by controlling the expression of many genes . these are all among the mechanisms that might be contributed in the progression of atherosclerosis . although native ldl is exposed to all enzymatic and nonenzymatic oxidants , they are protected by a potent array of antioxidants in plasma . moreover , the ldl and other biomolecules are protected from free radical attack by the action of antioxidant capacity in the blood . the protective effects of enzymatic and nonenzymatic antioxidant activity are exerted in a network manner . therefore , plasma antioxidant status is the result of interaction and cooperation of various antioxidants . a decrease in ox - ldl was reported after consumption of beverage as source of polyphenols , such as grape juice , red wine , green tea , and cocoa drink and cranberries [ 36 , 37 ] . micronutrients modulate immune system and exert a protective action by reducing ldl - cholesterol oxidation via induction of antioxidant enzymes . according setorki et al . using high - dose vinegar with have demonstrated that grape seed proanthocyanidins with meal can lower the postprandial oxidative stress through decreasing oxidants concentration and increasing the level of serum antioxidants . moreover , according to observational studies , due to the synergism of different nutrients consumed in a daily diet , it could be difficult to separate out specific effects . however , it was of our interest investigating how polyphenols from rw intake , combined with mcdm , could be associated with circulating markers of oxidative stress and inflammation , then contributing to increase cardiometabolic risk . in our study , npvrw had a notably higher amount of trans - resveratrol and other stilbenes content compared to commercial wine . this might be an indication that less invasive pruning methods ( like the extreme case of no pruning ) can lead to a higher amount of resveratrol . according to our previous data , in the present study , after the consumption of mcdm with npvrw compared to mcdm , we observed a significant decrease of ox - ldl levels , with protective effect towards ldl oxidation . the opposite is observed for the mcdm compared to b. moreover , no significant effect on ox - ldl was highlighted after npvrw intake . the ox - ldl level , combined with transcriptomics analysis gives us further insights to understand the global effect of dietary patterns on health . in this study , mcdm was related to biological pathways associated with oxidative stress , inflammatory , and drug metabolism response . opposite to npvrw intake effect , after consumption of mcdm and mcdm + npvrw , we observed an upregulation of nf-b-1 gene expression , probably due to the consumption of a high fat meal . nf-b is the master regulator of the immune / inflammatory response ; by regulating nf-b , sirtuin-1 ( sirt-1 ) may inhibit the expression of genes involved in inflammation that is directly related with the nf-b signaling pathway . in addition , an increasing number of studies suggest that resveratrol plays an important role in protecting mitochondria and attenuating mitochondrial ros production , improving endothelium function , inhibiting inflammatory processes and decreasing the rate of endothelial apoptosis . activation of the redox - sensitive nf-b is believed to be an important component of the cascade of events triggered by psychosocial stress , leading to inflammation , thrombosis , and vascular damage . nf-b subunits are expressed ubiquitously and can be activated by a wide range of stimuli , such as ros , cytokines , infection , and dna damage ; however , their actions are regulated in a cell- and stimulus - specific manner , leading to a diverse spectrum of effects . sirt 1 , activated through both calorie restriction and resveratrol , mediates the beneficial impact of each on longevity and health , acting by deacetylating transcription factors , such as the tumor suppressor p53 , the forkhead box ( fox ) family of transcription factors foxo1 , foxo3 , and foxo4 and the transcription factors nf-b . it has therefore been suggested that resveratrol mimics the protective effects of dietary restriction , since it can activate the mammalian sirtuin in vitro assays . we observed different levels of sirt2 expression after consumption of the npvrw and mcdm + npvrw , compared to b , probably owed to the resveratrol present in red wine , as suggested by schirmer et al . . we have observed an upregulation of alcohol dehydrogenase ( adh4 ) in comparison between b and mcdm meal . probably , this is due to the presence of ethanol in bread of mcdm sandwich ; in fact in the other condition , adh4 is downregulated and aldehyde dehydrogenase ( aldh1a1 ) is upregulated , according to metabolic pathway of ethanol . monocyte chemoattractant protein-1 ( mcp-1 ) , also known as chemokine ( c - c motif ) ligand 2 ( ccl2 ) , is a proinflammatory chemokine and recruits and activates monocytes during the inflammatory response . our results , from the comparison between b and mcdm , suggest that ccl2 inhibits the transcription of il-6 , according to mandrekar et al . , who highlighted the role of ccl2 in the regulation of tnf- and il-6 however , the comparison between b versus npvrw , b versus mcdm + npvrw , and mcdm versus mcdm + npvrw shows a downregulation for each gene , ccl2 and il-6 , highlighting a control over inflammation genomic condition . as previously demonstrated , we have underlined the relation between catalase ( cat ) and ccl5 . in a proinflammatory situation , due to the consumption of high fat meal , cat downregulated the expression of ccl5 . on the other hand , comparing mcdm versus mcdm + npvrw we obtained a downregulation of cat and an upregulation of ccl5 . the positive effect of wine is highlighted in comparison between b and npvrw , such as in comparison between mcdm and mcdm + npvrw , in which txn is upregulated . in fact thioredoxin is critical for redox regulation of nf-b and it has general intracellular antioxidant activity and when upregulated or overexpressed protects against oxidative stress . according to poulsen et al . our results show an ucp2 upregulation in b versus npvrw , b versus mcdm + npvrw , and mcdm versus mcdm + npvrw comparison . we concluded that npvrw , in association with a high fat meal , could improve the upregulation of ucp2 preventing mitochondrial oxidative stress and hepatic fat accumulation . our results show a downregulation of thioredoxin ( txn ) gene in b compared to mcdm and a downregulation after mcdm + npvrw of the same gene . trx or txn is a 12 kda multifunctional protein with a redox - active site ( cys - gly - pro - cys ) , which is involved in dithiol / disulfide exchange reaction . furthermore , reduced trx prevents apoptosis via an inhibitory binding to apoptosis signal - regulating kinase 1 ( ask-1 ) , whereas this binding is lost when trx is oxidized . an increased trx expression could help to increase the reduction of intracellular proteins and other biomolecules as part of the antioxidant defense . our results suggest that downregulation of txn gene may be due to hazelnut supplementation but still it is still unclear what the interaction processes are . however , these data reflect gene expression profile and statistical predictions and need to be confirmed by further research . previous study reported that a simple change of 25% of energy load from fat to carbohydrate in a meal significantly improves postprandial proatherogenic factors . results observed in this exploratory study support the scientific evidence regarding the deleterious impacts of a high fat meal , such as the mcdm , which represents a usual meal of western dietary pattern . moreover , our results also indicate that expression of genes in pathways related to chronic disease are positively influenced by the enrichment of polyphenol during the meal , as demonstrated by the effects of npvrw intake . a limit of the study was due to the small number of participants , although for genomic studies a numerosity of 8 to 25 subjects is acceptable . in addition , it would be appropriate to evaluate the impact on gene expression of other factors associated with a healthy or unhealthy lifestyle , as the physical activity . moreover , another limit of the study was the short - term treatment , due to high level of quality indexes of mcdm , such as ai , csi , and ti that are referred to a higher amount of saturated fat and related to oxidized ldl and pathogenic factor . since the biomarkers used in our study are not commonly examined in the clinical setting , for example , gene expression of drug metabolism , inflammation , and oxidative stress , our results can only lead to limited conclusions . however , as previously reported , our data highlighted the positive effect of npvrw intake alone or in association with mcdm on ox - ldl , indicating that the antioxidant potential of the nutrients found in red wine may be an essential component to combatting chronic noncommunicable diseases linked to inflammation and oxidative stress . although more studies are recommended to investigate the role of antioxidant capacity on ldl oxidation and gene expression , our study confirms the need of a long term lifestyle intervention based on healthy diet , combined with a moderate intake of high quality red wine , to improve mechanisms of metabolic and cardiovascular diseases , such as oxidative stress and inflammation . in fact , whereas excessive alcohol consumption is among the leading contributors to morbidity and mortality worldwide , moderate alcohol consumption ( 2 drinks / day for men and 1 drink / day for women ) may have some health benefits . in conclusion , the idea that is possible to reduce the cardiovascular stress due to postprandial oxidative stress over the course of a meal may seem trivial ; however , these daily insults over time may lead to cvd . consuming rw may be a manner to reduce the magnitude of these daily insults , which potentially will reduce cvd risk . the results from this study demonstrate that consuming red wine , naturally enriched with resveratrol , results in lower postprandial ox - ldl concentration and lower inflammation , according to the vision of social medicine , for the prevention and control of cvd and chronic degenerative diseases . although the present data do not allow the conclusion that npvrw intake has an acute protective value for atherosclerosis , it seems reasonable to conclude that these results show the favorable acute effects on some risk factors of atherosclerosis , such as ox - ldl , and overexpression of inflammation and oxidative stress related genes . however , more research is needed on a larger population and on chronic effects of npvrw intake before definitive conclusions can be made .

this study analyzed the effect of diet enriched with 30% lipids on cytokines content in different tissues . swiss male mice were distributed into four groups treated for 8 weeks with control ( c , normolipidic diet ) ; soybean oil ( s ) ; lard ( l ) ; and hydrogenated vegetable fat ( h ) . we observed an increase in carcass fat in groups s and l , and the total amount of fatty deposits was only higher in group l compared with c group . the serum levels of free fatty acids were lower in the l group , and insulin , adiponectin , lipid profile , and glucose levels were similar among the groups . il-10 was lower in group l in mesenteric and retroperitoneal adipose tissues . h reduced il-10 only in retroperitoneal adipose tissue . there was an increase in il-6 in the gastrocnemius muscle of the l group , and a positive correlation between tnf- and il-10 was observed in the livers of groups c , l , and h and in the muscles of all groups studied . the results suggested relationships between the quantity and quality of lipids ingested with adiposity , the concentration of free fatty acids , and cytokine production in white adipose tissue , gastrocnemius muscle , and liver .

epidemiological studies reveal that approximately 2.3 billion adults will be overweight and 700 million will be obese in 2015 . obesity is a chronic subclinical inflammatory disease of multifactorial etiology , involving the ingestion of a high - calorie diet , a sedentary lifestyle , and genetic predisposition . the establishment of the disease can lead to the development of correlated morbidities such as diabetes mellitus type 2 , cardiovascular disease , and metabolic syndrome , among others , as a consequence of the imbalance of a complex system of energy balance [ 3 , 4 ] . the enlargement of adipocytes and the expansion of adipose tissue lead to local hypoxia and macrophage infiltration , causing an inflammatory response . in turn , the response changes the patterns of protein and gene expression of various bioactive molecules , called adipokines , produced by adipose tissue . adipokines are a group of proteins synthesized by white adipose tissue that act on the immune , cardiovascular , metabolic , and endocrine systems . examples include tnf- , leptin , adiponectin , resistin , il-6 , and il-10 [ 6 , 7 ] . in obese individuals , the expression of proinflammatory adipokines by adipose tissues tnf- and il-6 are involved in the development of insulin resistance and atherogenic processes . however , il-10 , a cytokine secreted primarily by monocytes / macrophages and lymphocytes , as well as adiponectin , secreted by adipocytes , possess anti - inflammatory and insulin - sensitizing properties by antagonizing il-6 and tnf-. low concentrations of il-10 and adiponectin have been associated with metabolic syndrome and diabetes mellitus type 2 [ 1012 ] . the il-10/tnf- ratio has been considered an important indicator of inflammatory status , and low values are associated with an increased risk of morbidity and mortality [ 13 , 14 ] . the lipid composition of the diet has a strong relationship with the development and persistence of obesity . acute ( 2 days ) or chronic ( 16 weeks ) treatment with a high - fat diet reduces the synthesis of adiponectin , suggesting that the serum lipid profile and lipid components of the diet are more related to the decrease of adiponectin than to obesity itself . in adult males , a positive correlation between body fat and the quantity or quality of lipids consumed has been described , and diets high in saturated and monounsaturated fatty acids induced greater adiposity compared with diets rich in polyunsaturated fatty acids . studies by our group have shown a link between a high - fat diet and different types of lipids in the diet with changes in gene expression , the synthesis and secretion of adipokines and obesity - related comorbidities [ 1821 ] . thus , the aim of this study was to investigate the effects of high - fat diets enriched with lard , soybean oil , or hydrogenated vegetable oil ( 30% lipids , w / w ) on body fat content , lipid profiles , serum glucose , insulin and adiponectin concentrations , and the tissue cytokines tnf- , il-10 , and il-6 . the experimental research committee of the so paulo federal university approved all procedures for the care of the animals used in this study ( protocol cep n1644/10 ) . thirty - day - old male swiss mice were used in this study and were kept under controlled conditions ( 12 h light : 12 h dark cycle at 22c 1c ) . during the experimental period , the animals were housed six per cage , receiving water and a specific diet ad libitum . the animals were assigned to four experimental groups , with 12 animals per group , according to their specific diet and treated for eight weeks as follows : ( 1 ) c ( control group ; the animals were treated for eight weeks with a standard ain diet ( american institute of nutrition ) ; ( 2 ) s ( soybean oil group ) ; ( 3 ) l ( lard group ) ; and ( 4 ) h ( hydrogenated vegetable oil group ) . the animals ' weights were evaluated weekly . all diets were prepared according to the recommendations of the ain and were normoproteic . all groups received a growth diet ( ain-93 g ) containing 20% protein and at least 7% essential fatty acids between the 30th and 60th day of life and a maintenance diet ( ain-93 m ) containing 14% protein and 2% soybean oil to provide the essential fatty acids between the 60th and 90th days of life . the compositions of the control , soybean , lard , and hydrogenated vegetable oil diets are described in table 1 . 500 mg of the prepared diets were used for lipid extraction , saponification , and fatty acid methylation as described by the american oil chemists - society , 2011 . the relative fatty acids content in the total lipids from the diet was quantified by gas - liquid chromatography using agilent technologies 7890a cg system , with an ionizable flame detector acoplado linked to the ezchrom elite cds software ( agilent technologies , inc . fatty acids were separated using an sp 2560 capillary column ( supelco , usa ) , with 100 m 0.25 mm 0.20 m measurements . the methylated fatty acids were identified by comparing their retention times to known standards ( nu - chek prep . results were expressed weight percentage ( g/100 g of total fatty acids table 2 ) . the animals were sacrificed by decapitation on the 90th day of life in the fasting state ( 8 h ) in the early morning to avoid chronobiological variations . trunk blood was collected and immediately centrifuged at 4c , and serum aliquots were taken and frozen at 80c to measure the concentrations of glucose , triacylglycerols , and total cholesterol using commercials kits from labtest diagnostic sa ( minas gerais , brazil ) and the concentrations of free fatty acids , insulin , and adiponectin using elisa ( linco research , inc . , usa ) . the retroperitoneal ( ret ) , epididymal ( epi ) , and mesenteric ( mes ) adipose tissues , gastrocnemius muscle ( gast ) , and liver were dissected and weighed , frozen in liquid nitrogen , and stored at 80c until the extraction of protein . was measured as described by stansbie et al . and standardized using the method described by oller do nascimento and williamson . briefly , an eviscerated carcass was autoclaved at 120c for 90 min and then homogenized with double its mass of water . triplicate aliquots of this homogenate were weighed and digested in 3 ml of 30% koh and 3 ml of ethanol for at least 2 h at 70c in capped tubes . after cooling , 2 ml of 12 n h2so4 was added , and the sample was washed three times with petroleum ether for lipid extraction . the results are expressed as grams of lipid/100 g of carcass . adipose tissue depots , gastrocnemius muscles , and livers ( 0.250.3 g ) were homogenized in ice - cold solubilization and total protein extraction buffer ( 100 mm tris , ph 7.5 , 10 mm ethylene acetic acid , 100 mm sodium fluoride , 10 mm sodium orthovanadate , 2 mm phenylmethylsulfonyl fluoride , 10 mm sodium pyrophosphate , and 0.1 mg / ml aprotinin ) . homogenates were centrifuged at 14000 g for 40 min at 4c , the supernatants were saved , and the protein concentrations were determined using a bradford assay ( bio - rad , hercules , california ) with bovine serum albumin as a reference . the results are expressed as the means sem . for multiple comparisons of means , one - way analysis of variance ( anova ) was performed with subsequent use of the tukey post hoc test . simple linear regression analysis was used to evaluate the correlation between cytokines ( il-10 and tnf- ) . body mass , white adipose tissue depots ( epi , ret , and mes ) , liver , and gast weights were similar among all groups . however , the s and l diets promoted a significant increase in carcass relative lipid content compared with group c. in addition , this parameter in group h was lower than in group s ( table 3 ) . exposure to the l diet after weaning caused an approximately 28% increase in the sum of the adipose tissue depots compared with the control group . the levels of serum glucose , triacylglycerols , total cholesterol , insulin , and adiponectin did not differ among the studied groups . nevertheless , the l diet caused a decrease in the serum concentrations of free fatty acids ( table 4 ) . tnf- levels did not change in any group or tissue studied compared with the control group ( figure 1(a ) ) . however , the l diet caused a decrease in il-10 levels in mes and ret white adipose tissue depots , and the h diet promoted a similar effect in the ret depot ( figure 1(b ) ) . furthermore , the il-6 content was elevated in mice that ingested the diet containing saturated fatty acids ( l ) ( figure 1(c ) ) . in group h , the il-10/tnf- ratio was higher in mes adipose tissue compared with groups s and l ( figure 1(d ) ) . we ran correlation tests for il-10 and tnf- and found the following to be positively correlated : group c , retroperitoneal fat depot , liver , and gastrocnemius muscle ; group s , epididymal and mesenteric fat depots and gastrocnemius muscle ; and group l , liver and gastrocnemius muscle . in group h , only mesenteric fat tissue did not correlate . the ingestion of high - fat diets for 8 weeks did not modify body or tissue weight . similar results were observed by others with eight or seven weeks of high - fat diet treatment [ 19 , 25 , 26 ] . however , other studies have reported an increase in body weight after 16 weeks of high - fat diet treatment [ 16 , 27 ] . as stated by noeman et al . , the high - fat diet effect is subtle but cumulative , needing at least 10 weeks to increase body weight . these results suggest that the difference in weight gain in the previous studies may be due to the period of treatment . despite the fact that the carcass lipid content in groups s and l was higher than in group c , this parameter was similar between groups c and h. the oxygen consumption after a meal was significantly lower in rats fed a lard diet than in rats fed a safflower oil or linseed oil diet . in this sense , dube et al . demonstrated that the ingestion of a hyperlipidic diet , enriched with lard , caused a decrease in the mrna expression of ucp-1 in brown adipose tissue . furthermore , the energy cost of lipid deposition from dietary fat is lower than from dietary carbohydrate . . demonstrated that enrichment of the diet with polyunsaturated fatty acids caused changes in adipose tissue metabolism , such as the increased uptake of diet - derived lipids favoring fat deposition . machado et al . reported that mice fed with a trans - fat - enriched diet for 16 weeks showed a reduction in subcutaneous and epididymal fat pads , suggesting an effect on the stimulation of lipolysis . taken together , these reports suggest that the effects on fat accumulation depend on the type and source of lipids present in the diet and could be related to the effects on energy expenditure and adipose tissue metabolism , such as lipoprotein lipase activity , lipogenesis , and lipolytic enzyme activities . the diet intervention did not alter the concentrations of serum glucose , triacylglycerol , total cholesterol , adiponectin , or insulin . previous studies have demonstrated no effect of high - fat diets on the serum adiponectin concentration [ 33 , 34 ] . however , a time - dependent effect of a high - fat diet on the adiponectin serum concentration has been shown ; 10 weeks of treatment increased and 18 weeks of treatment reduced the serum adiponectin concentration compared with the initial experimental period , suggesting a compensatory mechanism to maintain metabolic homeostasis , as this adipokine is associated with the maintenance of carbohydrate and lipid metabolism and also acts to improve insulin sensitivity [ 19 , 3640 ] . in fact , in our study , 8 weeks of a high - fat diet treatment did not modify serum glucose , triacylglycerol , total cholesterol , or adiponectin and insulin concentrations , in spite of the increasing adiposity in groups l and s. another possible explanation related to the similar serum lipid profile among the groups is the decrease in the carbohydrate content of the high - fat diets . the review from volek et al . reported that the serum total cholesterol level remained unchanged from baseline values , while both the hdl and ldl cholesterol levels increased and the tag levels dramatically decreased under a high - fat diet compared with high - carbohydrate diets . in the current study , the lard diet caused a decrease in the serum free fatty acid concentration in relation to the control diet . it has been stated that the plasma free fatty acids resulted from hydrolyzed triacylglycerol in chylomicrons and vldl by lipoprotein lipase activity and lipolysis in adipose tissue . one important mechanism for the removal of unesterified plasma fatty acids is through oxidation of these fatty acids in skeletal muscle and the heart . several studies have demonstrated that il-6 can increase fatty acid oxidation in myocytes [ 44 , 45 ] and in vivo . when we analyzed the il-6 content in gastrocnemius muscle , we verified an increase in group l compared with group c , which could partially explain the reduction in serum fatty acids in lard - treated animals . nutrients such as the fatty acids in special saturated fatty acids can activate intracellular pathways through tlr2 and tlr4 , leading to increased proinflammatory gene transcription [ 47 , 48 ] . in this sense , we analyzed the tnf- , il-6 , and il-10 concentrations in ret , epi , mes , gastrocnemius muscle , and liver . the literature is controversial about the effects of a high - fat diet on tnf- expression . a similar result was observed by flanagan et al . , who showed that tnf- gene expression in muscle was not significantly affected by the amount or type of dietary fat . hong et al . showed that 10-week - old mice treated with a high - fat diet ( 55% fat by calories ) for 3 weeks had lower il-10 levels in muscle than control mice , accompanied by a decrease in muscle insulin sensitivity . furthermore , il-10 treatment prevents muscle insulin resistance by decreasing obesity - associated macrophages and cytokines in muscle from high - fat diet - treated mice . in the present study , the il-10 gastrocnemius muscle content did not change in treated mice between 30 and 90 days of life with high - fat diets . however , the lard diet caused a decrease in il-10 in mes and ret , and the same result was observed in ret from group h without any change in glycemia or insulin plasma concentration , suggesting that the type of diet , age of the animal , and time of treatment could have different effects depending on the tissues evaluated . in addition , we can not dismiss the possibility that metabolic processes adapt to changes in dietary components during a specific period of life . the il-6 level in gastrocnemius muscle increased in group l compared with group c. the role of il-6 action in obesity - associated insulin resistance remains highly controversial ; wunderlich et al . showed that mice with a hepatic deficiency of il-6 receptor a ( il-6ra ) develop insulin resistance not only in the liver but also in skeletal muscle and wat . in addition , the il-6 level increases with exercise , a situation in which glucose uptake by muscles is high . it is interesting to note that , in the present study , the lard diet , which promoted a decrease in il-10 in adipose tissue depots , caused an increase in il-6 in gastrocnemius muscle . from these results , it is possible to suggest that the whole body of an adult animal tries to maintain carbohydrate and lipid metabolism homeostasis by modifying the expression and secretion of cytokines . in fact , by analyzing the ratio between il-10 and tnf- , which is used as an indicator of the inflammatory condition of the individual ( where lower values are associated with a poor prognosis ) [ 13 , 14 ] , we verified a similar result among groups in all tissues studied . in addition , a positive correlation between tnf- and il-10 was observed in the liver ( groups c , l and h ) and muscle ( all studied groups ) ( figure 2 ) , which could contribute to the maintenance of glucose , insulin , and lipid profiles at normal values . in summary , our results demonstrate that enrichment of the diet with soybean oil or lard elevated carcass lipid content , whereas enrichment with hydrogenated vegetable oil did not alter this parameter as compared to control diet ( 4% of soybean oil ) . only the lard diet modified the fatty acid serum concentration , decreased il-10 in adipose tissue depots and increased il-6 in gastrocnemius muscle . these suggested that the type of fatty acid present in the diet plays a part in determining the amount of carcass lipid content and serum fatty acid concentration and influences the cytokines content in the tissues .

in this article , we characterized tungsten oxide - decorated carbon - supported ptir nanoparticles and tested it for the electrooxidation reactions of ethylene glycol and ethanol . phase and morphological evaluation of the proposed electrocatalytic materials are investigated employing various characterization techniques including x - ray diffraction ( xrd ) and transmission electron microscopy ( tem ) . electrochemical diagnostic measurements such as cyclic voltammetry , chronoamperometry , and linear sweep voltammetry revealed that the tungsten oxide - modified ptir / vulcan nanoparticles have higher catalytic activity for ethylene glycol and ethanol electrooxidation than that of ptir / vulcan . a significant enhancement for electrooxidation of co - adsorbate monolayers occurred in the presence of a transition metal oxide relative to that of pure ptir / vulcan electrocatalyst . the likely reasons for this are modification on the pt center electronic structure and/or increasing the population of reactive oxo groups at the ptir / vulcan electrocatalytic interface in different potential regions .

an important task in the twenty - first century is to further develop fuel cells as alternative electrochemical devices for efficient generation of electricity . the low - temperature acid - type systems , such as hydrogen - oxygen polymer electrolyte membrane fuel cells and direct alcohol fuel cells , are , at present , the most commonly studied devices in many laboratories worldwide [ 1 , 2 ] . among organic compound fuels for anodic reactions in fuel cells , methanol has been historically most extensively studied , and more recently , other short chain liquid fuels such as ethanol and ethylene glycol have become important [ 411 ] . in the case of direct methanol fuel cells ( dmfc ) , slow electrode kinetics of methanol oxidation and methanol adsorption products ( which is mainly coads ) poisoning the surface of pt electrode at low temperature still hamper application [ 3 , 1215 ] . as a result , polyhydric and monohydric ( except methanol ) alcohols , such as ethylene glycol and ethanol , have been proposed as potential fc fuels which are much less volatile and less toxic than methanol . moreover , both alcohols have some of the largest volumetric energy densities , and they involve the transfer of a number of electrons that set a practical challenge for the effectiveness of catalysts . they also show lower permeability through membranes ( lower crossover effect ) [ 1618 ] . however , the electrochemical oxidation of short chain alcohol ( monohydric or polyhydric alcohols ) is much more complex than , for example , h2 oxidation . the main challenge generally for electrooxidation of polyhydric and monohydric alcohols to carbon dioxide is associated with the cleavage of the c c bond for complete conversion . due to incomplete oxidation , various intermediate species from electrooxidation in both alcohols it is well established that pt is rated as the most active material for oxidation of small organic molecules in acidic media , but poisoning by the intermediate by - product of co - adsorbed species of the binary or ternary platinum - based alloys with ru , sn , rh , pb , w , or mo [ 1927 ] was proposed to enhance the electrooxidation activity toward alcohols . alternatively , ir has also been employed as a co - metal for platinum - based catalysts in unitized polymer electrolyte fuel cells because of its high stability and resistance to corrosion [ 28 , 29 ] . the presence of ir , particularly iro2 , enhances the electrooxidation of methanol in direct methanol fuel cells due to providing a large number of oh groups that are adsorbed at relatively low potentials [ 3034 ] . moreover , cao et al . reported that the addition of ir into sn showed to be a promising alternative for pt - based catalysts for ethanol electrooxidation . demonstrated a positive effect for ethanol oxidation in which iro2 was incorporated to platinum - based catalysts . recently , adzic et al . revealed that the presence of a high content of ir atoms into ternary catalyst ptir / sno2/c enhances the complete electrooxidation of ethanol to co2 at a relatively low - onset potential . furthermore , ptir catalysts have been utilized for electrooxidation of ethylene glycol with positive results [ 37 , 38 ] . one of the numerous approaches to increase the electrocatalytic activity of platinum - based catalysts toward the oxidation of small organic compounds is the use of transition metal oxides as support systems for catalytic metal sites . the presence of transition metal oxides in the neighborhood of catalytic sites of noble metal catalysts results in an increasing population of oh groups at low potentials , thereby mitigating co poisoning of catalytically active platinum centers , possibly facilitating the cleavage of c this assumption is in accord with reports in which a significant improvement in oxidation of small organic molecules with metal oxides ( e.g. , wo3 , moo3 , tio2 , zro2 , v2o5 , and ceo2 ) modified by pt - based alloy catalysts has been observed [ 8 , 9 , 3943 ] . the present work will concentrate on the preliminary investigation of a carbon - supported ptir - based anodic catalyst with tungsten oxide as the additive prepared by the adsorption of tungsten acid . not only the peak current value during cyclic voltammetry ( cv ) tests in a broad potential region , but also the more specific performance in the low potential region will be evaluated from a more comprehensive point of view . thus , the electrocatalytic activity toward ethanol and ethylene glycol oxidation in comparison with that of ptir / c and wo3-modified ptir / c catalysts will be evaluated by cv , linear sweep voltammetry ( lsv ) , and chronoamperometry ( ca ) methods . all chemicals were commercial materials of analytical grade purity that were obtained from premetek ptir / c nanoparticles ( 20 % on vulcan xc-72 , pt : ir 1:1 ) . solutions were prepared using doubly distilled and subsequently deionized ( millipore milli - q ) water . argon was used to de - aerate the solutions and to keep an oxygen - free atmosphere over the solution during the measurements . some characteristics of catalytic particles were obtained using a libra 120 transmission electron microscope ( tem ) operating at 120 kv . samples for tem measurements were prepared by depositing drops of colloidal solutions of nanoparticles onto 400-mesh copper grids supporting a fromvar film ( agar scientific ) and , later , drying them in ambient laboratory conditions ( temperature , 20 2 c ) for 24 h prior to tem analysis . x - ray diffraction ( xrd ) patterns of the catalysts were obtained with a bruker d8 discover operating with a cu x - ray tube ( 1,5406 ) and vantec ( linear ) detector ( k = 1.5406 ) . the working electrode was glassy carbon , and the counter electrode was carbon rod . as a rule , all potentials in the present work were measured versus a k2so4-saturated hg2so4 reference electrode and were recalculated and reported versus the reversible hydrogen electrode ( rhe ) . the catalyst layer was fabricated through modification of the working electrode by immobilization of ptir / c nanoparticles . wo3 modification of the ptir / c catalyst was in accordance with the procedure described in our previous papers [ 8 , 9 ] . briefly , a solution of tungstic acid was prepared by passing an aqueous solution of 0.05 mol dm na2wo4 through a proton exchange resin . in a typical procedure , selected amount of ptir / c catalyst was added to 2 cm of 0.05 mol dm aqueous solution of tungstic acid . the resulting suspension was stirred for 24 h. during that process , the ptir / c nanoparticles interacted with tungstic acid to form tungsten oxide or hydrogen tungsten oxide bronzes . the supernatant solution was centrifuged and replaced with water in order to obtain a colloidal solution of tungsten oxide - modified ptir / c nanoparticles that was stable for months . the pt - to - tungsten ratio in the given catalyst system was determined with x - ray fluorescence ( xrf ) ; the targeted ratio was approximately 1:1 . to prepare a homogeneous catalyst layer on the glassy carbon working electrode surface , a 5-l aliquot of the catalyst dispersion was deposited using a micropipette ( the nominal loading of catalyst was approximately 160 g cm ) and allowed to dry under ambient conditions . prior to this step , when the catalyst layers had dried , 2 l of nafion ( 0.02 % alcoholic solution ) was dropped on top of the glassy carbon electrode surface covered with the catalyst and dried at room temperature . prior to the electrooxidation processes , the catalytic electrodes were scanned with 25 complete oxidation / reduction cycles between 0.0 and 0.8 v in 0.5 mol dm h2so4 at 50 mv s scan rate . the co - stripping experiments were carried out in 0.5 mol dm h2so4 electrolyte utilizing the glassy carbon electrode substrate onto which surface the appropriate catalyst was introduced . as a rule , a few cyclic voltammetric measurements ( at 50 mv s ) were recorded in the potential range from 0.0 to 0.8 v in the deoxygenated electrolyte . the co - saturated solution was prepared by flowing pure co ( from air liquide ) through the electrolyte for 10 min . the co adsorption process that was employed ( mainly on the surface of catalytic pt nanocenters ) was achieved by underpotential control at 0.1 v versus rhe for 5 min , after which the dissolved co was removed from the electrolyte by bubbling argon for 30 min maintaining the applied potential ( 0.1 v ) , in order to have a solution free of co. then , the adsorbed co monolayer was stripped by recording three cyclic voltammetric scans in the potential range from 0.0 to 0.9 v at a scan rate of 10 mv s. the measurements using these catalysts were repeated three or four times with freshly prepared electrodes , and the average results are presented here . the x - ray patterns of the ptir / vulcan nanoparticles in the presence and absence of the wo3 modifier are shown in fig . 1a , b. the broad diffraction peak centered at 20.025.0 in all the xrd pattern is attributed to the hexagonal carbon support . in the case of unmodified ptir 39.5 , 46.3 , and 67.7 correspond to the pt lattice planes ( pcpdf 04 - 0802 ) [ 45 , 46 ] . the diffractogram of the wo3-modified ptir / vulcan electrocatalyst shows peaks at 2 = 40.5 , 46.9 , and 66.2 , which are associated with pt reflections and the signals that originated from wo3 ( pcpdf 43 - 0679 ) . the results indicate that pt fcc is the main crystalline phase in the catalysts and that the presence of tungsten species resulted in the formation of crystalline aggregates . a peak shift is observed which could indicate the interaction between ptir / vulcan alloy and tungsten oxide . of the xrd patterns , none were metallic ir , or iridium oxide diffraction peaks have been observed because pt and ir metals have similar diffraction peak positions and crystalline structures [ 33 , 36 ] . to estimate the average particle sizes from scherrer s equation , the pt peak ( at 2 = 39.5 ) was used . the latter peak was chosen because it is located in a region where there are no interferences from the carbon support . the average pt particle sizes were obtained from the position and the full - width at half - maximum values of the pt peak ( at 2 = 39.5 ) . the values were in the ranges from 4 to 6 and 6 to 8 nm for unmodified and modified ptir / vulcan nanoparticles , respectively . higher values of the average particle sizes for the wo3-modified ptir / vulcan can be interpreted in terms of deposition of the wo3 crystalline monoclinic structure.fig . 1xrd diffractograms of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) xrd diffractograms of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) in order to get more information about the size , morphology , and distribution of nanoparticles on the carbon material , tem analysis was performed . figure 2 shows the tem images and distributions of the series of ptir / c and wo3-modified ptir / c nanoparticles . low magnification images show that the supported material was predominantly irregular spheres or spheroids of bimetallic nanoparticles that were homogeneously dispersed on the carbon ( vulcan xc-72 ) surfaces . the average particle sizes lie in the narrow range of 58 nm with a standard deviation of 1 nm , which is in agreement with the xrd results for wo3-modified ptir / vulcan and unmodified ptir / vulcan catalysts.fig . 2low magnification micrographs ( tem ) of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) low magnification micrographs ( tem ) of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) for initial electrochemical characterization , cyclic voltammetric curves of the ptir / vulcan and the wo3-modified ptir / vulcan nanoparticles deposited on glassy carbon electrode were obtained in 0.5 mol dm sulfuric acid - supporting electrolyte ( fig . 3 ) . in both cases , slight changes in the shape or current values of the cyclic voltammetric curves are observed . in the hydrogen adsorption / desorption region ( between 0.0 and 0.4 v versus rhe ) for the all proposed catalysts , some changes in the voltammetry are seen because of the dependence on the surface composition . . 3a ) is characterized by a single large peak in the hydrogen adsorption / desorption region , whereas the electrochemical behavior of electrodes made from the wo3-modified ptir / vulcan nanoparticles ( fig . . moreover , in the double layer region , which is between 0.4 and 0.7 v versus rhe , significant currents are recorded . this behavior is typical of electrocatalysts composed of transition metals dispersed on a carbon black support . it is apparent from fig . 3b that the oxidation peak appearing at about 0.2 v most likely reflects the intercalation of protons in tungsten oxide ( wo3 ) and tends to overlap with the hydrogen adsorption / desorption region of bare platinum ( at a potential lower than 0.4 v ) . this interpretation is difficult to prove because of the problem of unambiguously distinguishing contributions from the reversible reduction of tungsten oxide to hydrogen tungsten bronzes from the abovementioned hydrogen adsorption and desorption peaks originating from pt and ir.fig . 3cyclic voltammetric responses of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalytic systems in 0.5 mol dm h2so4 . scan rate = 10 mv s cyclic voltammetric responses of ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalytic systems in 0.5 mol dm h2so4 . scan rate = 10 mv s the behavior of the proposed catalytic layers was tested toward ethanol and ethylene glycol electrooxidation processes . representative cyclic voltammograms of these species at both ptir / vulcan and wo3-modified ptir / vulcan electrocatalysts deposited on glassy carbon electrode are shown in fig . 4b ) obtained at the ptir / vulcan and wo3-modified ptir / vulcan surface , respectively , show well - defined peaks for both forward and reverse scans in the investigated potential region . in the forward scan , the ethanol oxidation current at ptir / vulcan catalysts starts ( at 0.3 v ) and reaches the peak at 0.87 v , which is located at the same potential as compared to the wo3-modified ptir / vulcan , as illustrated in fig . , a single peak is developed at 0.7 v , which can be attributed to the oxidative decomposition of by - products .fig . 4cyclic voltammetric responses for oxidation of 0.5 mol dm ethanol ( a ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . scan rate = 10 mv s cyclic voltammetric responses for oxidation of 0.5 mol dm ethanol ( a ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . scan rate = 10 mv s similar measurements to those presented in fig . the cyclic voltammograms of wo3-modified ptir / vulcan and bare ptir / vulcan in the presence of ethylene glycol exhibited no significant oxidation current up to 0.27 and 0.35 v , respectively . two distinct peaks were observed in the potential range between 0.4 and 0.9 v in comparison to previous literature reports [ 3638 ] , and these processes can be attributed primarily to the oxidation of adsorbed organic species . in the case of electrooxidation of ethylene glycol , various intermediate oxidation products can be expected according to spectroscopic data [ 4752 ] including co , glycol aldehyde , glycolate , glyoxylate , oxalate , and formate . this is in contrast to the data obtained with ethanol , the oxidation of which produces only ch2cooh , co2 , and ch3cho . generally , in the presence of both fuels , the current density in the hydrogen region decreases in comparison to the voltammograms obtained in the supporting electrolyte alone due to their adsorption . additionally , fig . 4 displays that in both cases , the current density values are higher in the tested potential range confirming the enhancement of the electrooxidation of ethanol and ethylene glycol by the presence of the metal oxide wo3 . in addition , the onset potentials of ethanol and ethylene glycol oxidation shift toward more negative values which is especially pronounced in the case of ethylene glycol oxidation . this can be explained by the fact that transition metal oxides ( e.g. , wo3 and related compounds ) are known to activate interfacial water molecules ( from oh groups on wo3 ) at lower potentials which , in turn , promote the removal of poisoning species from the noble metal catalyst [ 8 , 9 , 14 , 5460 ] . figure 5 exhibits background - subtracted linear scan voltammograms ( lsvs ) for ethanol and ethylene glycol electrooxidation on ptir / vulcan and wo3-modified ptir / vulcan electrocatalysts deposited on glassy carbon substrate recorded in the potential range of 0.00.9 v. as can be seen for both fuels , the shape of the lsv curves is almost the same in the examined potential range . the only difference is that the current densities are higher for wo3-modified ptir / vulcan nanoparticles than those recorded on bare ptir / vulcan nanoparticles . the lsv experiments also confirm the shifting of the onset potential for ethanol and ethylene glycol electrooxidation on wo3-modified ptir / vulcan catalysts toward lower potential values . it is likely that the increases of the oxidation current densities observed in the lsv curves are associated with the addition of tungsten oxide . the decrease of the onset potential of ethylene glycol and ethanol oxidation is due to activation of interfacial water molecules forming oh species at lower anodic potential than the bare catalyst.fig . 5linear scan voltammetry responses for oxidation of 0.5 mol dm ethanol ( a ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalyst at 10 mv s scan rate . electrolyte = 0.5 mol dm h2so4 linear scan voltammetry responses for oxidation of 0.5 mol dm ethanol ( a ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalyst at 10 mv s scan rate . electrolyte = 0.5 mol dm h2so4 the effect of temperature for ethanol and ethylene glycol electrooxidation in bare and wo3-modified ptir / vulcan nanoparticles was also investigated . when the temperatures increase from 10 to 50 c , the oxidation current densities for both become higher . based on the arrhenius equation , the activation energy ( 45 and 49 kj mol , respectively ) can be determined from the slope calculated by linear regression by plotting ln(j ) as a function of reciprocal of temperature ( figure not shown ) . the value of activation energy is in good agreement with a previous report on alcohol electrooxidation . this observation suggests that the increase of temperature causes more facile oxidation kinetics and decreases poisoning by the intermediate species . in order to evaluate the electrocatalytic activity and also the long - term stability of tungsten oxide - modified ptir / vulcan nanoparticles for ethanol and ethylene glycol oxidation , chronoamperometric measurements were performed at low potentials ( fig . the polarization current density for the electrooxidation of both fuels on the investigated catalytic systems displays a rapid decrease in the first period of the experiment before reaching a stable value . the catalytic current developed for tungsten oxide - modified ptir / vulcan electrocatalysts for both ethanol and ethylene glycol always was significantly higher than those produced for bare ptir / vulcan nanoparticles in the tested time period . these results are consistent with those obtained by cyclic voltammetry . in the initial phase of the chronoamperometric experiments , it is likely that a higher number of free active sites are available for adsorbed ethanol or ethylene glycol molecules ( fast kinetic rate reaction ) , and during the next few minutes ( rate determining step ) , the amount of free catalyst sites is limited by poisoning by intermediate species , such as co , chx , ch3cho , ch3cooh ( for ethanol oxidation ) , glycol aldehyde , glycolate , glyoxylate , oxalate , and glycolate ( for ethylene glycol ) . in this regard , the improvement of catalytic properties observed by introduction of wo3 on pt - based nanoparticles surface can be associated with the oxophilic nature of tungsten oxide providing hydroxyl groups ( oh ) on the oxide surface at lower potential , which promotes electrooxidation of the surface co - poisoning intermediates species [ 8 , 9 , 5460].fig . 6chronoamperometric current - time responses ( recorded at 0.3 v ) for oxidation of 0.5 mol dm ethanol ( a ) with bare ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . electrolyte = 0.5 mol dm h2so4 chronoamperometric current - time responses ( recorded at 0.3 v ) for oxidation of 0.5 mol dm ethanol ( a ) with bare ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) and 0.5 mol dm ethylene glycol ( b ) with ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . electrolyte = 0.5 mol dm h2so4 regarding the stability of the electrocatalytic responses in the presence of ethylene glycol and ethanol , the long - term chronoamperometric and repetitive voltammetric measurements of wo3-modified ptir / c and bare ptir / c systems have been performed ( not shown here ) under the same conditions . in the potential range between 0.0 and 0.9 v , the catalytic peak currents decreased , remaining at 90 % than those of the first cycle after 100 cycles when the wo3-modified system has been used . during long - term chronoamperometric experiments ( 1 h ) , there was only 15 % decrease of catalytic currents in the case of tungsten oxides . in both experiments , no significant deactivation effect was observed that may imply dissolution of wo3 . in order to gather information of the ability of coads poisoning species to undergo oxidative desorption from the surface of the prepared pt - based catalysts , co - stripping voltammetry was performed . a typical coads - stripping curve on bare ptir / vulcan catalyst is presented in fig . it is characterized by a single sharp and prominent coads oxidation peak centered at 0.69 v with a coads oxidation onset potential of 0.590 v. in contrast , for the wo3-modified ptir / vulcan catalysts , two separated coads oxidation peaks with the position of the main peak potential at 0.62 v were found in the stripping voltammetric method ( fig . the onset potential for the main coads oxidation peak starts near 0.49 v. it becomes broadened and shifts to a more negative potential ( ca . 100 mv ) versus the main coads oxidation peak for the bare ptir / vulcan . for both catalysts , the hydrogen region was completely blocked by the full coverage with coads ; the main co - stripping ( oxidation ) peak appeared only during the first anodic cyclic , which indicates that all adsorbed co was oxidized and removed from the surface under this condition . this observation is in good agreement with the previous reports for metal oxides ( e.g. , wo3 , moo3 ) [ 8 , 9 , 62 , 63].fig . 7co - stripping voltammograms recorded at 10 mv s in 0.5 mol dm h2so4 for the ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . co adsorption was done at 0.1 v. solid curve shows the first cycles , while the dot curve shows the second cycles co - stripping voltammograms recorded at 10 mv s in 0.5 mol dm h2so4 for the ptir / vulcan ( a ) and wo3-modified ptir / vulcan ( b ) catalysts . co adsorption was done at 0.1 v. solid curve shows the first cycles , while the dot curve shows the second cycles the presence of two peaks on the co - stripping voltammetry has been described in the literature [ 6468 ] . two signals observed for oxidation of co - adsorbed curve on the wo3-modified ptir / vulcan catalyst suggest that two active sites for coads oxidation may exist , which are the likely case due to a variation in the interaction with the metal oxide . in a previous report , this effect for pt - based electrode has been explained by the presence of at least two different types of co adsorption products ( linearly or bridged bound ) which are characterized by different binding energies . moreover , the peak that appears at the lower potential may be ascribed the coads oxidation on the surface of wo3-modified ptir / vulcan catalyst corresponding to the interaction between the pt nanoparticles and tungsten oxide , whereas the peak at the higher potential may be attributed to adsorption on pt nanoparticles at a large distance from tungsten oxide . additionally , it can not be excluded that this peak originates from the redox reaction of a tungsten species . to estimate the electrochemically active surface area ( sa ) , the hydrogen adsorption / desorption system can not be used because of the overlap with the redox process of tungsten oxide . the co - stripping voltammetry method relies on forming a monolayer of strongly adsorbed co on the bare and modified ptir / vulcan catalysts . generally , co may form a linear , bridge bond to the surface leading to different numbers of electrons per site ( 2 and 1 , respectively ) . a linear adsorption may dominate if the co adsorption occurs at a potential close to 0 v. by analogy to these conditions , it was assumed that one monolayer of co adsorbed on pt ( linked linearly ) and that the coulombic charge required to oxidize adsorbed co to co2 is equal to 420 c cm [ 63 , 70 ] . the charge value required to estimate the electrochemically active surface area was determined by integrating the main co - stripping peaks . the obtained sa values for the catalysts were 57 and 54 m g for bare ptir / vulcan and wo3-modified ptir/ vulcan , respectively . it is reasonable to conclude that the electrochemical active surface area is not the major factor causing the difference of their catalytic activities for ethylene glycol and for ethanol electrooxidations . in other words , tungsten oxide species occupied slightly the electrochemically active surface area of modified ptir / vulcan nanoparticles . the same tendency has been noticed by others with different catalysts ( e.g. , pt , ptsn , and ptrh ) and with various transition metal oxides [ 57 , 62 , 63 , 71 ] . these effects may be explained by the ability of tungsten oxide in contact with aqueous solutions to increase the population of surface hydroxyl groups which likely play a major role in the coads removal . further work is needed to determine whether such catalysts are stable under test conditions for fuel cells to elucidate full reaction pathways . herein , we demonstrate the enhancement of the activity of catalysts composed of bimetallic ptir nanoparticles and tungsten oxide toward electrooxidation of ethylene glycol and ethanol . from both xrd and tem results , the average particle sizes were found to be in the range of 48 nm and uniformly dispersed on glassy carbon . the adsorbed layer of tungsten oxide on ptir / vulcan nanoparticles increases the catalytic currents and decreases the onset potentials for electrooxidation of ethylene glycol and ethanol . the above electrochemical measurements confirmed that the presence of tungsten oxide on the surface is beneficial in the electrooxidation of ethylene glycol and ethanol . the results also showed that the activity of the ptir / vulcan nanoparticles for oxidation of poisoning species ( coads ) is higher in the presence of tungsten oxide . the activation effect may involve direct specific interactions ( chemical or electronic ) between wo3 and both pt and ir metals .

noninvasive ventilation ( niv ) was introduced as an alternative to invasive mechanical ventilation for acute respiratory failure caused from exacerbations of chronic obstructive pulmonary disease in the 1980s , and its use gradually rose worldwide . seventy - eight patients ( 57 males , mean age 78.3 9.2 years ) undergoing niv were evaluated . of them , 48 ( 62.3% ) had acute hypercapnic respiratory failure because of a chronic obstructive pulmonary disease exacerbation , and the remaining 30 had acute hypercapnic respiratory failure from other causes , mainly cardiac failure . all patients were treated by niv using the bi - level positive airway pressure set up at high pressure / high backup rate . niv was successful in 67 subjects ( 85.9% ) and the patients were discharged , 57 of whom continued niv at home and ten had spontaneous breathing . niv was unsuccessful in eleven patients , ten of whom died and one was successfully treated by invasive mechanical ventilation . significant differences were detected for a higher basal glasgow coma scale score in successfully treated patients ( p = 0.007 ) , a higher basal acute physiology and chronic health evaluation score in unsuccessfully treated patients ( p = 0.004 ) , and a lower ph after 1 hour in unsuccessfully treated patients ( p = 0.015 ) . these findings show a very high rate of success of niv in patients with acute hypercapnic respiratory failure not only from chronic obstructive pulmonary disease but also from cardiac failure . this suggests that the use of invasive mechanical ventilation may be further reduced , with a decrease in its known complications as well .

the use of invasive mechanical ventilation by endotracheal intubation in acute respiratory failure is long known.1 noninvasive ventilation ( niv ) began to be considered as an alternative to invasive mechanical ventilation in acute respiratory failure caused from exacerbations of chronic obstructive pulmonary disease ( copd ) in the 1980s , and its use gradually rose worldwide . in 1998 , an international prospective survey reported that about one - third of patients initially treated with niv had to undergo endotracheal intubation,2 but 6 years later another large international prospective study reported a better outcome.3 also , niv effectiveness is supported by results of systematic reviews,4 and this has led to an increase in the application of this treatment , even in complicated situations such as a do - not - intubate order in elderly patients.5 however , in 2010 , manuel et al reasoned that there is little convincing evidence for the use of niv in severe , but stable copd and that what is less clear , however , is the quality of how niv is delivered to patients.6 indeed , a recent study focused its attention on the kind of niv setup , which can be based on high inflation pressure and high backup rate ( high - intensity niv ) or high pressure and low backup rate ( high - pressure niv ) . the authors reported that the pivotal role in managing by niv hypercapnic respiratory failure in copd patients is the high - pressure component.7 seventy - eight patients ( 57 males , mean age 78.3 9.2 years ) undergoing niv were evaluated . of them , 48 ( 62.3% ) had acute hypercapnic respiratory failure because of a copd exacerbation and the remaining 30 had acute hypercapnic respiratory failure from other causes , mainly cardiac failure . all patients were treated by niv using the bi - level positive airway pressure set up at high pressure / high backup rate . niv was successful in 67 subjects ( 85.9% ) and the patients were discharged , 57 of whom continued niv at home and ten were in a state of spontaneous breathing . niv was unsuccessful in eleven patients , ten of whom died and one was successfully treated by invasive mechanical ventilation . the mean age of successfully treated ( 77.7 9.4 years ) and unsuccessfully treated ( 82.1 8.1 years ) patients was not significantly different . significant differences were detected for a higher basal glasgow coma scale score in successfully treated patients ( p = 0.007 ) , a higher basal score of the acute physiology and chronic health evaluation score in unsuccessfully treated patients ( p = 0.004 ) , and a lower ph after 1 hour in unsuccessfully treated patients ( p = 0.015 ) . among the risk factors for failure of niv , the ph value seems particularly important,8 which corresponds with the current data . also , the acute physiology and chronic health evaluation confirmed the predictive value of a score higher than 29 , which was detected in patients with a negative outcome . overall , the current data , obtained in a pneumology unit , show that niv is able to manage the large majority of patients ( 86% ) with acute respiratory failure from copd exacerbations and also from other causes . a randomized study found that the use of niv as rescue therapy was associated with a lower number of patients meeting the endotracheal intubation criteria and with a lower mortality rate compared with endotracheal intubation.9 the factors underlying a negative outcome of niv warrant investigation in studies in large populations of patients . a recent study reported that patients with copd and obesity hypoventilation syndrome treated with niv because of acute hypercapnic respiratory failure had a response to treatment similar to patients with only copd.10 moreover , a low rate of endotracheal intubation need was recently reported in patients with acute respiratory failure from heart failure treated with niv,11 and this observation is pertinent for the patients in the current study . the findings show a very high rate of success of niv in patients with acute hypercapnic respiratory failure from copd as well as from other causes , including cardiac failure . this suggests that the use of invasive mechanical ventilation may be further reduced , with a decrease in its known complications as well .

background : neuromyelitis optica ( nmo ) and multiple sclerosis ( ms ) are autoimmune demyelinating diseases of the central nerve system . interleukin-7 ( il-7 ) and interleukin-7 receptor alpha ( il-7r ) were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes . the variants of both genes had been identified to be associated with ms susceptibility in caucasian , japanese and korean populations . however , the association of these variants with nmo and ms has not been well studied in chinese southeastern han population . here , we aimed to evaluate the association of six il-7 variants ( rs1520333 , rs1545298 , rs4739140 , rs6993386 , rs7816065 , and rs2887502 ) and one variant of il-7ra ( rs6897932 ) with nmo and ms among chinese han population in southeastern china.methods:matrix-assisted laser desorption / ionization time of flight mass spectrometry ( massarray system ) and sanger sequencing were used to determine the variants of il-7 and il-7ra in 167 nmo patients , 159 ms patients and 479 healthy controls among chinese han population in southeastern china . samples were excluded if the genotyping success rate < 90%.results : statistical differences were observed in the genotypes of il-7 rs1520333 in ms patients and il-7ra rs6897932 in nmo patients , compared with healthy controls ( p = 0.035 and 0.034 , respectively ) . there was a statistically significant difference in the genotypes of il-7 rs2887502 between ms and nmo patients ( p = 0.014 ) . and there were statistically significant differences in the rs6897932 genotypes ( p = 0.004 ) and alleles ( p = 0.042 ) between nmo - igg positive patients and healthy controls.conclusions:the study suggested that among chinese han population in southeastern china , the variant of il-7ra ( rs6897932 ) was associated with nmo especially nmo - igg positive patients while the variant of il-7 ( rs1520333 ) with ms patients . and the genotypic differences of il-7 rs2887502 between ms and nmo indicated the different genetic backgrounds of these two diseases .

neuromyelitis optica ( nmo ) and multiple sclerosis ( ms ) are autoimmune inflammatory demyelinating disorders of the central nervous system with unknown etiology , causing nontraumatic neurological disability in young adults . nmo is thought to be a subtype of ms but not an independent disease until the discovery of the anti - aquaporin 4 ( aqp4 ) antibody or nmo - igg . although the exact etiologies of both diseases are still unclear , it is sure that the t - helper cells ( th ) , directly or indirectly , participate in the pathogenesis and progress of ms and nmo by secreting various cytokines . over recent years , interleukin-7 ( il-7 ) had been widely considered to be a key cytokine controlling the differentiations and immune responses of several t - cells subsets . it is suggested that the function of il-7 was not only stimulating il-7 receptor alpha ( il-7r ) to promote the differentiation of th1 , but also involving in the survival and proliferation of pathogenic th17 cells in experimental autoimmune encephalomyelitis ( eae ) and ms patients . the serum il-7 can be seen as a potential marker monitoring the response to interferon- in ms patients . the genetic variants of il-7 and il-7ra had been identified to be associated with the susceptibility of ms and nmo in caucasian , japanese and some other populations in several studies . however , the results had not been replicated in chinese han population except our previous study on rs1520333 in il-7 and rs6897932 in il-7ra , in which no positive associations were observed . as the il-7/il-7r pathway might be a very attractive therapeutic target for inflammatory disorders , the genetic variants in this pathway were proposed to implicate in the pathogenesis of various autoimmune diseases . here , we enlarged the sample size to replicate the association of these 2 single nucleotide polymorphisms ( snps ) ( rs1520333 in il-7 and rs6897932 in il-7ra ) with ms and nmo . meanwhile , we assayed 5 more snps of il-7 ( rs1545298 , rs4739140 , rs6993386 , rs7816065 , and 2887502 ) in the enlarged samples of ms , nmo , and healthy controls . as described in our previous study , 110 unrelated nmo patients and 304 healthy controls were included . in this study , we recruited 57 more nmo patients and 178 more healthy controls . in total , 167 nmo patients ( 26 males and 141 females ) and 479 healthy controls ( 255 males and 224 females ) among chinese han population in southeastern china were included in this study . in addition , we recruited 159 ms patients ( 66 males and 93 females ) , and the diagnosis of which met the 2005 mcdonald criteria for ms . nmo - igg antibodies were detected with an indirect immunofluorescence assay using human embryonic kidney 293 cells transfected with recombinant human aqp4 gene ( each sample was measured at least twice , with the examiners unknowing the origin of the specimens . genomic dna was extracted from peripheral blood samples using a qiaamp dna blood minikit ( qiagen , hilden , germany ) . six variants in il-7 ( rs1520333 , rs1545298 , rs4739140 , rs6993386 , rs7816065 , and 2887502 ) and one variant in il-7ra ( rs6897932 ) were genotyped using sequenom massarray system ( sequenom , san diego , ca , usa ) in previous 106 nmo patients and 304 healthy controls , according to the manufacturer 's instructions at fudan - van andel research institute center , as we previously described . for quality control , sample duplicates whose genotypes had been identified by sequencing were included in each 96-well plate . then , the additional samples including 57 nmo patients , 159 ms patients , and 178 healthy controls were genotyped by sanger sequencing using an abi 3730 automated dna sequencer ( applied biosystems , usa ) . primer sequences for analysis of il7 and il7ra variants pcr : polymerase chain reaction . hardy the genotypes and allele frequencies were compared between cases and controls using the chi - square test or fisher 's exact test . continuous variables were shown as mean standard deviation ( sd ) . a p < 0.05 was considered as statistically significant . as described in our previous study , 110 unrelated nmo patients and 304 healthy controls were included . in this study , we recruited 57 more nmo patients and 178 more healthy controls . in total , 167 nmo patients ( 26 males and 141 females ) and 479 healthy controls ( 255 males and 224 females ) among chinese han population in southeastern china were included in this study . in addition , we recruited 159 ms patients ( 66 males and 93 females ) , and the diagnosis of which met the 2005 mcdonald criteria for ms . nmo - igg antibodies were detected with an indirect immunofluorescence assay using human embryonic kidney 293 cells transfected with recombinant human aqp4 gene ( euroimmun , lubeck , germany ) . each sample was measured at least twice , with the examiners unknowing the origin of the specimens . samples with twice positive results were reported to be nmo - igg positive . genomic dna was extracted from peripheral blood samples using a qiaamp dna blood minikit ( qiagen , hilden , germany ) . six variants in il-7 ( rs1520333 , rs1545298 , rs4739140 , rs6993386 , rs7816065 , and 2887502 ) and one variant in il-7ra ( rs6897932 ) were genotyped using sequenom massarray system ( sequenom , san diego , ca , usa ) in previous 106 nmo patients and 304 healthy controls , according to the manufacturer 's instructions at fudan - van andel research institute center , as we previously described . for quality control , sample duplicates whose genotypes had been identified by sequencing were included in each 96-well plate . then , the additional samples including 57 nmo patients , 159 ms patients , and 178 healthy controls were genotyped by sanger sequencing using an abi 3730 automated dna sequencer ( applied biosystems , usa ) . hardy weinberg equilibrium ( hwe ) was tested using the chi - square test . the genotypes and allele frequencies were compared between cases and controls using the chi - square test or fisher 's exact test . continuous variables were shown as mean standard deviation ( sd ) . a p < 0.05 was considered as statistically significant . all data were analyzed using spss 16.0 software for windows ( spss inc . , chicago , il , usa ) . after genotyping by sequenom massarray system , 4 nmo patients and 14 healthy controls were excluded for genotyping success rate < 90% . hence , there were 163 nmo patients , 159 ms patients , and 468 healthy controls analyzed . nmo - igg antibody was tested in the 57 newly recruited nmo patients , and 13 ( 29.5% ) were positive , added with previous 44 antibody positive nmo patients , there were 57 nmo - igg antibody positive patients in total . most of the snps in each group were under the hwe except il-7 rs2887502 in ms ( p = 0.033 ) and il-7ra rs6897932 in healthy controls ( p = 0.031 ) . for the c / c genotype of il-7 rs2887502 did not exist in ms patients and the frequency of c / c genotype was much higher in healthy control than that in the nmo - igg positive nmo patients ( 70.30% vs. 61.40% ) , the departure from dhw of both polymorphisms in ms and healthy controls were possibly due to the protective role of c / c genotype according to a previous study . characteristics of all chinese han participants in this study ms : multiple sclerosis ; nmo : neuromyelitis optica ; aqp4-ab : antiaquaporin-4 antibodies ; na : not applicable ; sd : standard deviation . the upper and the bottom panels indicate the homozygous genotypes , whereas the heterozygous genotype is shown in the middle one . hardy weinberg equilibrium tests for all chinese han participants in this study a p < 0.05 was considered statistically significant . ms : multiple sclerosis ; nmo : neuromyelitis optica ; il7 : interleukin-7 ; il7ra : interleukin-7 receptor alpha . as listed in table 4 , after enlarging the sample size for both previously studied snps , the frequency of the a / a genotype of rs1520333 in il-7 gene of ms patients was observed dramatically lower than healthy controls , which reached the statistical difference ( p = 0.035 ) . meanwhile , although statistical difference of the genotype was existed between total nmo and healthy controls in il-7ra rs6897932 ( p = 0.034 ) , we also found no statistical difference in the allele frequencies ( p > 0.05 ) . when comparisons were made between nmo - igg positive patients and ms patients or healthy control , we found that statistical significant differences of genotype and allele distributions were observed in il-7ra rs6897932 ( p = 0.004 and p = 0.042 ) between the nmo - igg positive patients and healthy controls . allele and genotype distributions of il7 and il7r variants among ms , nmo and healthy controls , n ( % ) * the results of fisher 's exact test . ms : multiple sclerosis ; nmo : neuromyelitis optica ; nmo : total neuromyelitis optica patients ; nmo : neuromyelitis optica - igg positive patients ; il7 : interleukin-7 ; il7ra : interleukin-7 receptor alpha . the results of five newly assayed il-7 snps are listed in table 5 , along with the results of corresponding chi - square test . the genotypes of il-7 rs2887502 were significantly different between nmo and ms patients ( p = 0.014 ) . and allele and genotype distributions of 5 newly studied il7 variants among ms , nmo and healthy controls , n ( % ) * the results of fisher 's exact test . ms : multiple sclerosis ; nmo : neuromyelitis optica ; nmo : total neuromyelitis optica patients . autoimmune diseases are complex trait that develop from intricate and poorly understood interactions between an individual 's genetics and the environmental exposures . the genetics of nmo susceptibility largely remain unknown . in this study , we totally investigated the association of 6 variants in il-7 and one variant in il-7ra with chinese nmo and ms patients , especially with the nmo - igg positive patients . we observed that the genotype of rs6897932 in il-7ra was statistically different between nmo patients and the healthy controls after including more nmo patients and healthy controls . while further comparing the nmo - igg positive patients with healthy controls , we found that the c / c genotype and c allele significantly decreased in nmo - igg positive nmo patients . in il-7 gene , the genotypes of il-7 rs2887502 were observed statistically different between nmo and ms patients in our present study . il-7 belongs to a superfamily of gamma - chain cytokine receptor , and its gene , il-7 , is located on chromosome 8q12 - 13 . functionally , il-7 binds to il-7r ( also termed as cd127 ) , which is encoded by the gene il-7ra , plays an essential role in the t cell survival and proliferation in human and animal model . a recent study revealed that the serum il-7 reflected the ms patients responsiveness to interferon- in th1-dirven ms , because of the important role of il-7 played in enhancing the th1 proliferation . in turn , numbers of previous studies pointed out the il-7/il-7ra variants were associated with the morbidity of nmo and ms in different populations and regions of the world . the rs6997932 was proved to be a causative variant that affected the expression of il-7ra . studies on different populations reached a consensus that this snp was strongly associated with ms , moreover , it was also associated with nmo in the studies performed in japanese and korean populations . some studies revealed that rs6993386 , rs1520333 , and rs7816065 of il-7 were associated with ms in caucasian populations , while rs1545298 , rs4739140 , and rs2887502 did not relate to this disease . previously , we performed a partial replication of the referred studies by assayed rs1520333 of il-7 and rs6897932 of il-7ra , however , we did not find any positive association of both snps with nmo . in consideration of the different genetic background among populations , we enlarged the sample size and investigated the associations of more snps with not only nmo patients , but also ms patients in the current study . interestingly , statistical significant differences were observed in the genotypes of il-7ra rs6897932 between the cohorts of total nmo patients and healthy controls . while further comparing the nmo - igg positive patients with healthy controls , significant differences existed in both distributions of genotypes and alleles in il-7ra rs6897932 . this result was coherent with the results of studies in japanese and korean populations , confirming that il-7ra rs6897932 might be a risky factor of nmo in asians . however , we did not observe the association of il-7ra rs6897932 with the pathogenesis of ms , which indicated the different genetic background among the east asians . in the comparisons between the ms patients and healthy controls , the genotypes of il-7 rs1520333 reached the statistical significant difference , which was first reported in asian . the significant difference of il-7 rs2887502 genotypes between nmo and ms patients was also firstly reported , which indicated the different genetic backgrounds of both diseases . although our study reported a potential association between il-7/il-7ra polymorphisms and ms / nmo , some limitations were present and should be addressed in the future . second , there was a disparity in the gender ratio , since the incidences of both diseases are higher in female than male . lastly , functional studies of the il-7 would be required to understand the actual effect of the il-7/il-7r complex in ms and nmo pathogenesis . in conclusion , the current study replicated some positive - associated snps , which were observed in other populations , in chinese nmo and ms patients . meanwhile , we investigated the associations of 5 more snps of il-7 with nmo and ms that were not investigated in asian previously . to our best knowledge , this study may provide a deeper understanding of the role of il-7/il-7r pathway playing in the pathogenesis of nmo and ms within different populations , which hints a new insight to the personal therapy for both diseases in the future . and this study suggested that it might be necessary to monitor the serum il-7 of nmo - igg positive patients during the treatment . this work was supported by grants from national key clinical specialty discipline construction program and key clinical specialty discipline construction program of fujian and the national natural science foundation of china ( no . 81125009 and no . this work was supported by grants from national key clinical specialty discipline construction program and key clinical specialty discipline construction program of fujian and the national natural science foundation of china ( no . 81125009 and no .

background : flavonoids comprise a large group of plant metabolites , 6,000 of which have been identified to date . some studies have shown the increased aerobic performance and maximal oxygen consumption ( vo2max ) and therefore fitness following quercetin intake as a result of elevated number of intracellular mitochondria caused by the flavonoid.methods:this double - blind clinical trial comprised 60 male students having an athletic history of at least 3 years . body composition , exercise performance , and some blood biomarkers were analyzed . the individuals were selected by convenient sampling , and then were assigned into four groups of equal number by using permuted block randomization . the first to fourth groups received a 500 mg supplemental quercetin capsule plus a 250 mg vitamin c pill , a 500 mg supplemental quercetin capsule plus a 250 mg placebo vitamin c pill , a 500 mg placebo quercetin capsule plus a 250 mg vitamin c pill , and a 500 mg placebo quercetin capsule plus a 250 mg placebo vitamin c pill , respectively , daily for 8 weeks . the participants were asked to continue their routine diet and physical activity during the study and they were monitored through phone calls or text messages.results:lean body mass , total body water , basal metabolic rate , and total energy expenditure increased significantly in the quercetin group after intervention . on the other hand , vo2max increased in the quercetin and quercetin + vitamin c groups following the intervention , non-significantly.conclusion:our findings suggest that supplementation with quercetin in athletes may improve some indices of performance .

flavonoids comprise a large group of plant metabolites , 6,000 of which have been identified to date . quercetin is a major natural flavonoid existing in the skin of many fruits and vegetables , as well as in leafy vegetables , berries , black tea , and various fruit juices . since it can be found in many plant - derived foods , it is present in most people 's diet . having many antioxidant , anticarcinogenic , anti - inflammatory , and heart - protecting effects , quercetin decreases the risk of cancers and various chronic diseases . thus , it is produced as a dietary supplement and is even added to some food and beverages . despite the large number of studies investigating the in vitro effects of quercetin , few have demonstrated its in vivo effects . there might be differences between the in vivo and in vitro effects of quercetin owing to its digestion , absorption , and metabolism effects . some animal studies have described the observed increased endurance performance and maximal oxygen consumption ( vo2max ) and therefore fitness following quercetin consumption as a result of elevated number of intracellular mitochondria caused by the flavonoid.[68 ] however , this association has not yet been proved in human studies . the improved physical performance caused by quercetin reported by some studies might be attributable to the decreased membranes in muscles , which in turn reduces the negative and exhaustive effects of excessive oxygen radicals during physical activity . proving this hypothesis to be right would mean quercetin to be able to reduce muscular damage and soreness , as well as neuromuscular dysfunction following exercise . however , quercetin has been reported to have contrasting antioxidant effects . although some studies found improved neuromuscular function and decreased soreness , others only mentioned improved muscular strength as a result of long - term quercetin consumption . however , they did not find quercetin to have significant effects on these indices . as mentioned previously , although in vitro and animal studies have suggested quercetin to have positive effects on athletic performance , inflammatory indices , and muscular damage , a definite judgment can not be made about its effects on athletes because of the fewer number of human studies and their contrasting findings . on the other hand , there is a growing interest in supplement use among athletes . therefore , this study evaluated the effects of supplementary quercetin on athletic performance , muscular damage , and body composition in male athlete students . this double - blind clinical trial involved 60 male students at isfahan university of medical sciences having an athletic history of at least 3 years . the subjects were excluded if they followed less than 70% of the study procedure or were unwilling to continue . the trial was approved by the research and ethics committee , faculty of nutrition , isfahan university of medical sciences , isfahan , iran , and registered in irct . the participants were first explained about the objectives and methods of the study . after obtaining written informed consents , height was measured to the nearest 0.5 cm by a tape meter in standing position without shoes . weight was measured in light clothing without shoes by a scale with an accuracy of 100 g , following which body mass index ( bmi ) was calculated as weight divided by squared height ( kg / m ) . a bioimpedence device was used to measure body water percentage and fat - free mass percentage . to evaluate body performance indices , exercise test was performed for all participants using the bruce protocol and hp cosmos treadmill ( mercury , germany ) . at the end of the test , vo2max and the individuals were then assigned into four groups of 15 using permuted block randomization . the first to fourth groups received a 500 mg supplemental quercetin capsule plus a 250 mg vitamin c pill , a 500 mg supplemental quercetin capsule plus a 250 mg placebo vitamin c pill , a 500 mg placebo quercetin capsule plus a 250 mg vitamin c pill , and a 500 mg placebo quercetin capsule plus a 250 mg placebo vitamin c pill , respectively , daily for 8 weeks . the participants were asked to continue their routine diet and physical activity during the study and they were monitored through phone calls or text messages . at the end of the intervention period , the remainder of the pills was evaluated to assess supplement intake . due to the double - blind nature of the study , neither the researchers nor the participants were aware of the content of the capsules . data were analyzed by paired t - test , analysis of covariance ( ancova ) , and repeated measure analysis of variance ( anova ) in spss15 . data were analyzed by paired t - test , analysis of covariance ( ancova ) , and repeated measure analysis of variance ( anova ) in spss15 . since four individuals were excluded due to unwillingness or other reasons , a total number of 56 participants were studied . mean ages of subjects in groups 1 to 4 were 20.93 1.53 , 21.50 2.17 , 21.21 1.52 , and 20.46 1.18 years , respectively ( p > 0.05 ) . table 1 shows the body composition indices among the four groups before and after the study . as seen , lean body mass ( lbm ) , total body water ( tbw ) , basal metabolic rate ( bmr ) , and total energy expenditure ( tee ) increased significantly in the first group . the comparison of body composition indices between four groups as presented in table 2 , body performance indices shows vo2max in group 1 significantly increased after the intervention . in addition , as the results of ancova after excluding the initial effects of vo2max suggested , vo2max increased in the first and third groups following the intervention . however , repeated measure anova did not reveal the differences to be significant . although the distance covered significantly increased in group 4 , ancova and repeated measure anova did not show significant differences . this randomized clinical trial comprised four groups who were evaluated to determine the effects of quercetin intake on physical and body performance and muscle injury . the results revealed significant differences in lactate dehydrogenase ( ldh ) , vo2max , tee , tbw , and lbm among the quercetin and vitamin c groups . however , after eliminating confounding effects of initial variables , only vo2max changes remained significant . our study is in line with the study of cureton et al . , who assessed the ergogenic effects of quercetin on 30 non - athletic men in a double - blind clinical trial . during 7 to 16 days , they administered 1 g of daily quercetin to the intervention group and the same amount of placebo to the control group . finally , they did not find significant changes either in ergogenic indices , such as phosphocreatine recovery time constant , vo2max , and perception of effort , during submaximal exercise test , or in the total work during a 10-min cycling with maximum power . similarly , ganio et al . performed a double - blind clinical trial on 11 non - athletic inactive participants ( five males and six females ) . although their intervention and control groups received , respectively , 1,000 mg of daily quercetin and placebo for 22 weeks , no significant differences were observed in terms of vo2max between the two groups . utter et al . also compared the effects of 250 mg of daily quercetin for 3 weeks with placebo among marathon runners and cyclists . they did not report any significant difference in the ratings of perceived exertion between the two groups . a crossover clinical trial by macrae et al . compared the effects of quercetin and a combination of quercetin and vitamins on 11 male cyclists during a 6 week period . although their results did not reveal significant differences in the total time of a 30 km ride and vo2max after the intervention , a significant increase was observed in peak power among the second group of cyclists . in a clinical trial , davis et al . evaluated the effects of quercetin on exercise performance among 12 volunteers . they divided the participants into two groups of intervention ( 500 mg daily quercetin ) and control ( 500 mg daily placebo ) . unlike in other studies , they found significant improvements in time to fatigue , vo2max , and endurance among non - athletic individuals during a 30 km bicycle ride . they performed a 12 week treatment with two doses of quercetin along with vitamin c and niacin on 941 male and female subjects aged 18 - 85 years . the participants were randomly divided into three groups of 500 mg daily quercetin , 1,000 mg daily quercetin , and placebo . their results did not show any significant differences between the intervention and placebo groups in terms of bmi or any other body composition indices . egert et al . investigated the effects of quercetin consumption on 93 obese subjects aged 25 - 65 in a crossover study . the participants received 6 weeks of 150 mg daily quercetin followed by a 5 week washout and a course of placebo . however , crp and body composition indices , including weight , waist circumference , body fat mass , and fat - free mass , did not significantly change after the intervention period . another study by egert et al . evaluated 35 healthy subjects in three groups of 50 , 100 , and 150 mg of daily quercetin . the study did not indicate any significant difference in resting energy expenditure or weight after a 2 week quercetin supplementation . similar to previous studies , the present study did not show any significant differences between the quercetin and placebo groups . therefore , it might be concluded that quercetin alone does not affect body composition and body performance indices . although different studies have used various doses of quercetin during various periods of time , we selected a dose within their range . however , higher doses might be able to make significant differences in the above - mentioned indices . since the findings of the present and previous studies are in contrast with in vitro and animal studies , a longitudinal research with long follow - up is suggested to evaluate the effects of quercetin . moreover , studies assessing the effects of quercetin on cardiovascular disease risk factors , endothelial dysfunction , and atherosclerosis incidence might reveal beneficial effects of this flavonoid . clinical trials with larger sample sizes of athletes and non - athletes are also recommended .

background : there are numerous reports of difficult laryngoscopy and intubation in patients with acromegaly . to date , no study has assessed the application of extended mallampati score ( ems ) for predicting difficult intubation in acromegalics . the primary aim of this study was to compare ems with modified mallampati classification ( mmp ) in predicting difficult laryngoscopy in acromegalic patients . we hypothesized that since ems has been reported to be more specific and better predictor than mmp , it may be superior to the mmp to predict difficult laryngoscopy in acromegalic patients.materials and methods : for this prospective cohort study with matched controls , acromegalic patients scheduled to undergo pituitary surgery over a period of 3 years ( january 2008-december 2010 ) were enrolled . preoperative airway assessment was performed by experienced anesthesiologists and involved a mmp and the ems . under anesthesia , laryngoscopic view was assessed using cormack - lehane ( cl ) grading . mmp and cl grades of i and ii were defined easy and iii and iv as difficult . ems grade of i and ii were defined easy and iii as difficult . data were used to determine the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of mmp and ems in predicting difficult laryngoscopy.results:seventy eight patients participated in the study ( 39 patients in each group ) . both mmp and ems failed to detect difficult laryngoscopy in seven patients . only one laryngoscopy was predicted to be difficult by both tests which was in fact , difficult.conclusion:we found that addition of neck extension did not improve the predictive value of mmp .

the modified mallampati classification ( mmp ) is widely used for pre - operative assessment of airway , and mallampati scores of iii and iv have been reported to be valuable in predicting difficult laryngoscopy in acromegalic patients as well . as it is known that mmp not only assesses pharyngeal structures but also head and neck mobility . it has been suggested that craniocervical extension relates to mouth opening and limited head and neck mobility can result in poor mmp . mashour and sandberg found that application of craniocervical extension improves the specificity and positive predictive value while retaining the sensitivity of the traditional mmp examination in a group of normal patients . the addition of craniocervical extension to the mmp was referred as the extended mallampati score ( ems ) . the incidence of difficult intubation in acromegalic patients is higher than the general surgical population . there are numerous reports of difficult laryngoscopy and intubation in patients with acromegaly.[913 ] to date , literature search did not reveal studies assessing the application of ems in for predicting difficult intubation in acromegalics . the primary aim of this study was to compare ems with mmp in predicting difficult laryngoscopy in acromegalic patients . we hypothesized that since ems has been reported to be more specific and better predictor than mmp , it may be superior to the mmp to predict the incidence of difficult laryngoscopy in acromegalic patients . our local ethics committee approved this prospective , observational , and controlled study and a written informed consent was obtained from all patients . over a period of 3 years ( january 2008-december 2010 ) 39 consecutive acromegalic patients presenting for for excision of pituitary tumor were enrolled . acromegalic features such as prognathism , macroglossia , and soft - tissue swelling were present variably in acromegalic group . for each acromegalic patient enrolled , the subsequent non - acromegalic patient with pituitary tumor was also enrolled to serve as a control . pre - operative airway assessment was performed by two experienced anesthesiologists and involved a mmp with patient sitting with the head in neutral position without phonation and then the ems with head in full extension , as described in literature . the view on mmp and ems was graded as follows : class i = soft palate , fauces , uvula , and pillars visible ; class ii = soft palate , fauces , and uvula visible ; class iii = soft palate and base of the uvula visible ; class iv = soft palate not visible at all . airway management plan for each patient was left to the discretion of the attending anesthesiologist who performed independent pre - operative assessment . standard monitoring prior to induction included electrocardiogram , non - invasive blood pressure , and pulse oximetry . after pre - oxygenation , general anesthesia was induced with fentanyl ( 2 mcg / kg ) and propofol ( 1.5 - 2 mg / kg ) . oropharyngeal airway and/or two - hand mask hold was used to facilitate mask ventilation , when required . after loss of all four twitches on the train - of - four obtained by ulnar nerve stimulation at the wrist , laryngoscopy was performed by one of the two attending anesthesiologists , using an appropriately sized macintosh laryngoscope blade and the laryngeal view was classified according to the method of cormack and lehane ( grade i = full view of the glottis , grade ii = glottis partly exposed , anterior commissure not seen , grade iii = only epiglottis seen , or grade iv = epiglottis not seen ) . however , it was allowed thereafter on instruction of the laryngoscopist in patients with initial laryngeal view grades iii and iv . both , mmp and ems grades iii and iv were considered predictors of difficult laryngoscopy , which was defined as cormack - lehane ( cl ) grades iii or iv ( before external laryngeal manipulation elm ) . intubation was defined as difficult if it required more than two attempts , change of blade , or use of a gum - elastic bougie / fiber optic bronchoscope / intubating laryngeal mask airway . the pre - operative assessment data were used to determine the accuracy of the two tests in predicting difficult intubation . several statistical measures were calculated that have been frequently used and are provided in appendix 1 . statistical analysis was done using software stata 9.1 ( college station , texas , usa ) . data are presented as mean ( standard deviation , sd ) , number or percentage . the demographic data were compared using two sample t - test with equal variance . the value of p < 0.05 was considered significant . a total of 78 patients participated in the study and one patient from control group was excluded from final analysis . in the control group , 39 patients were enrolled and one was excluded from final calculations because of inability to obtain cl grade . in the acromegalic group , a total of eight patients in acromegalic group and six patients in control group had a difficult airway manifesting as grade iii and iv on laryngoscopy without external laryngeal manipulations . demographic deatils of the study groups mean ( sd ) or n relationship between modified mallampati test and extended mallampati score with cormack - lehane grading of laryngoscopic view in the two study groups incidence of class iii , iv mmp assessment in normal and extended position was 18% and 31% in acromegalics . the sensitivity of both the positions was equal ( 13% ) whereas specificity for mmp and ems was 81% and 65% respectively in acromegalics . almost similar values of negative predictive value were obtained ( 78% for mmp and 74% for ems ) on contrary mmp was associated with higher positive predictive value ( 14% ) as compared to ems ( 1% ) [ table 3 ] . the incidence of class iii , iv mmp assessment in normal and extended position was 13% for both positions in control group . positive predictive value for mmp and ems was 60% and 0% respectively , whereas negative predictive value associated with these positions was 91% and 82% [ table 3 ] . statistical terms used for modified mallampati test and extended mallampati score as predicting tests for difficult laryngoscopy ( number or % ) the incidence of difficult intubation in general population has been described to be 5.8% in overall population , 6.2% for normal patients excluding obstetric and obese patients , 3.1% for obstetric patients and15.8% for obese patients by shiga et al . in meta - analysis of bedside screening test performance . mallampati test has been performed in with and without phonation and/or with different head or tongue positions in these studies , but despite theoretical arguments , this test has yielded poor to moderate sensitivity and moderate to fair specificity ( moderate discriminative power ) . pooled sensitivity of mmp is 49% and specificity being 86% , positive likelihood ratio 3.7 , and negative likelihood ratio 0.5 . diagnostic efficacy of these screening tests has been reported variously because of multiple factors like different patient characteristics , different test thresholds , inadequate statistical power and variable and low incidence of difficult intubation . incidence of class iii , iv mmp assessment in normal and extended position was 13% for both positions in control group . heterogeneity in mallampatti data has been reported with likely reasons being inconsistency and uncertainty in performing these tests . the incidence of difficult intubations in acromegalics in two retrospective studies has been described as 12 and 30/100 patients . in a study by schmidt et al . they defined difficult intubation as greater than two attempts at intubation , use of gum elastic bougie or change of laryngoscope blade . they reported 26% incidence of difficult laryngoscopy grade ( cl iii ) but with application of elm , it decreased to 10% . none of their patients had cl iv laryngoscopy grade . in our study , the incidence of class iii , incidence of difficult laryngoscopy as assessed by cl grade ( iii , iv ) was 20% in our study . the discrepancy may be because of the degree of acromegaly which may have led to more difficult airway . multiple bed side tests have been proposed for anticipation of difficult laryngoscopy and intubation , most common being the mmp . recently , the upper lip bite test has also been used and compared with mmp to predict difficult laryngoscopy in acromegalic patients . so far these tests have reported poor to moderate ( 20 - 62% ) sensitivity with moderate to fair specificity ( 82 - 97% ) as described by shiga et al . in their meta - analysis of 35 studies enrolling 50,760 patients . the diagnostic accuracy of these tests varies from trial to trial because of inter - observer differences , different test thresholds , inadequate statistical power and racial variation . the pooled sensitivity of mmp has been described 41 - 57% , pooled specificity is 81 - 90% . difficult intubation has been described variously as number of attempts at intubation , use of additional airway devices or difficult laryngoscopy grade ( cl iii , iv ) . it has been suggested that craniocervical extension relates to mouth opening and limited head and neck mobility can result in poor mmp as mouth opening is limited and submaximal in neutral position . found that application of craniocervical extension improves the specificity and predictive value of mallampati airway evaluation . the sensitivity of two positions was 83% whereas addition of craniocervical junction increased the specificity from 70% to 80% . we found a sensitivity of 50 and 0% in normal and extended position in control group while the values for acromegalics were 13% in each position . the specificity 94% and 84% was seen in normal and extended position in control group while values were lower in acromegalic group ( 81% in neutral position and 65% in extended position ) . addition of neck extension did not improve the predictive value of mmp , which may be ascribed to the racial variation in the studied population , small sample size , or inter observer variability and criteria for difficult intubation . despite theoretical arguments that craniocervical extension relates to mouth opening and limited head and neck movement may restrict mouth opening , we were not able to find any additional benefit of neck extension while doing mmp for pre - operative assessment of airway in either control or acromegalic group . currently available bed side screening tests for difficult airway have poor to moderate diagnostic value when used alone .

objectives . to compare the ability of endodontists to determine the size of apical pathological lesions and select the most appropriate choice of treatment based on lesions ' projected image characteristics using 2 d and 3 d images . study design . twenty - four subjects were selected . radiographic examination of symptomatic study teeth with an intraoral periapical radiograph revealed periapical lesions equal to or greater than 3 mm in the greatest diameter . cone - beam computed tomography ( cbct ) images were made of the involved teeth after the intraoral periapical radiograph confirmed the size of lesion to be equal to greater than 3 mm . six observers ( endodontists ) viewed both the periapical and cbct images . upon viewing each of the images from the two imaging modalities , observers ( 1 ) measured lesion size and ( 2 ) made decisions on treatment based on each radiograph . chi - square test was used to look for differences in the choice of treatment among observers . results . no significant difference was noted in the treatment plan selected by observers using the two modalities ( 2(3 ) = .036 , p > 0.05 ) . conclusion . lesion size and choice of treatment of periapical lesions based on cbct radiographs do not change significantly from those made on the basis of 2 d radiographs .

the clinical , radiographic , and histological diagnosis of periapical lesions has been a challenge [ 1 , 2 ] . current diagnostic methods help in fair assessment of accurate size and nature of a periapical lesion which determine the treatment and prognosis of the tooth in question [ 35 ] . making the correct pulpal and periapical diagnosis is helpful for treatment and prognosis of the tooth . even if biopsy is the gold standard in differentiating granulomas from cysts , clinicians are aware of the difficulty in obtaining biopsies in routine clinical practice [ 6 , 7 ] . therefore , there is an immediate need for a noninvasive method to diagnose lesions involving the periapical area . this could be due to fact that interpretation of images which influences success or failure also depends on several factors , including the clinician 's experience in interpreting images . however , due to inherent drawbacks such as superimposition and distortion , it is sometimes difficult to detect periapical ( pa ) lesions on two - dimensional images . two - dimensional radiographs can image pa lesions to be detected only when bone mineral loss reaches 30%50% due to structured noise of superimposed cortices . therefore , only 5055% of small and medium sized lesions can be diagnosed as periapical disease . reports indicate that cbct images provide clinically relevant information not found in periapical images [ 1013 ] . a recent study used cbct as the reference imaging modality ( gold standard ) to compare the accuracy of periapical and panoramic radiography in detecting periapical lesions . the question that is valid to ask now is , does evaluation of a periapical lesion with cbct change the estimation of size and choice of treatment among endodontists compared to periapical radiography ? selecting the most appropriate choice of treatment using the most accurate imaging modality would ultimately reduce cost and morbidity significantly in patients undergoing endodontic therapy . the purpose of this study was to compare lesion size and choice of treatment relative to the available radiographic information from periapical radiography and cbct . the null hypothesis is that there is no difference in the lesion size and choice of treatment of periapical lesions between conventional periapical radiographs and cbct images . the study group comprised twenty - four subjects , 11 women , and 13 men , with an average age of 53 years ( range 1888 years ) table 1 . approximately one hundred subjects were examined to achieve a study sample of twenty - four . all subjects reported to the endodontic division of the university of detroit mercy school of dentistry with symptoms suggestive of a periapical lesion . history of present illness and clinical examination ( including palpation , percussion , and cold test ) of the tooth involved was performed before any radiographic procedure was undertaken . subjects were recruited to the study only after the initial intraoral periapical radiograph showed a periapical lesion of size greater than 3 mm . both single rooted and multirooted teeth with periapical lesion size equal to or greater than 3 mm on intraoral periapical radiography if a patient had more than one tooth that qualified for the study , the tooth with the most severe symptoms and maximum size of the periapical lesion as determined by conventional radiography was selected . previously root - treated teeth and teeth with restorations were also included in the study . endodontically involved teeth that had history of trauma or radiographic evidence of fracture were excluded from the study . no specific control group was used for either of the radiographic modalities tested since teeth other than the study tooth in the same jaw were also imaged and served as internal controls . the study was reviewed and approved by the university of detroit mercy institutional review board ( udm irb protocol no . two - dimensional radiographs ( intraoral periapical ) were obtained with an intraoral dental x - ray machine ( planmeca intra , planmeca usa inc , il , usa ) using the paralleling axis technique and round collimator ( 2.86 diameter ) with variable kvp and mas and focal spot size 0.7 mm 0.7 mm . , long island city , ny , usa ) were used to record the images . schick cdr software was used to display images in real - time on a monitor . the images were stored as 8-bit tiff ( tagged image file format ) files at maximum quality ( 100% ) ( see figure 1 ) . adequate image quality essential for diagnosis was established in a subjective manner after comparing previous images obtained from this radiographic modality and standardizedwith the ones that would be used for this study . the size of the periapical lesions was measured with a measurement tool available in the schick software . teeth with periapical lesions equal to or greater than 3 mm as seen on the two - dimensional radiographs were further recommended for a cbct scan . dimensional calibration for size was applied to the two - dimensional images upon export to the study software . calibration was applied using the calibration tool in the software in which all the images were observed . a particular distance was measured on the periapical image obtained upon exposure , and the same distance was measured when this image was exported to the software through which the images were available for the observers to view . after explaining the cbct scan procedure and obtaining approval from the patient for the imaging procedure , a cbct scan of the study patient was acquired using the 1719 platinum next generation i - cat cbct scanner ( imaging sciences international , hatfield , pa , usa ) and standard scanning protocols ( pulsed exposure , 120 kvp , 3 to 7 ma , 14.7 second exposure time , 0.25 0.25 0.25 mm isotropic voxel size , 14 bit , maxilla or mandible ) ( see figure 2 ) . collimation was fully adjusted to include the maxilla or mandible only . a limited ( maxilla or mandible ) fov ( field of view ) the fov for the maxilla and mandible was 16 cm ( d ) 6 cm ( h ) . a cesium iodide flat panel detector was used to acquire images . only the arch ( maxillary / mandibular ) with the tooth and associated periapical lesion was scanned . all pertinent patient information on the scanned images was anonymized prior to being viewed by observers . scan data of subjects was exported from proprietary i - cat vision software ( i - cat vision q , imaging sciences international , hatfield , pa , usa ) to a previous version of i - cat software in.xstd format . images from both modalities were then imported into an interface viewing software , xv lite software ( apertyx inc . ( figure 1 : intraoral periapical radiograph , figure 2 : cbct image as viewed by observers ) the two - dimensional images were spatially calibrated according to known dimensions of the native images . periapical images were viewed in an office on a dell ultrasharp 2407wfp 24-inch widescreen flat panel lcd monitor ( dell inc . , round rock , texas , usa ) , running under microsoft windows xp professional sp-1 ( microsoft corp , redmond , wa , usa ) . the monitor specifications include a native resolution of 1920 1200 at 60 hz , pixel pitch of 0.27 mm , brightness of 450 cd / m , and contrast ratio of 1000 : 1 . the same monitor was not used for viewing both pa and cbct images for accession purposes . it was easier for observers to view cbct images at a monitor attached to the cbct scan acquisition hardware . the pa images were viewed by the observers at the principal investigator 's office as these images could be easily exported from the endodontic department upon capture to that office . three endodontists were senior faculty in the department of endodontics and three of them were junior part - time faculty . the observers were asked to perform the following with the two imaging modalities separately : ( a ) measure the extent of the periapical lesion ( greatest distance ( diameter ) in millimeters and ( b ) choose treatment for particular tooth ( root canal treatment , periapical surgery , root canal treatment and periapical surgery and no endodontic treatment ) . the observations were performed in two separate sessions : one for pa images and another for cbct images . this was done to enable observers could score all images between the two modalities in a timely fashion . all observers examined 24 images acquired from 24 different teeth ( 24 subjects ) from the two imaging modalities . overall , a total of 288 scores were that is , 6 observers x obtained ( 24 images for pa + 24 images for cbct ) each for lesion measurement and treatment choice . mann - whitney u test was used to assess for differences in the measurement of lesion sizes by all observers among the two modalities ( p > 0.05 ) . wilcoxon signed ranks test was used to look for differences in the measurement of lesion sizes among the two modalities by each observer . intraclass correlation coefficient was calculated among observers for each modality with respect to lesion size and choice of treatment . chi - square test was done to look for differences in the choice of treatment selected by all observers for the two modalities . the method of bland and altman was used to assess observer agreement of selected treatment between the two imaging modalities . the mean for lesion sizes among the two modalities for the study sample is shown in figure 3 . the 95% confidence interval for either the lower or upper limit of agreement was less than two standard deviations ( 2sd = 22.86 = 5.72 ) . table 2 shows no statistically significant difference between the two modalities in terms of the lesion size measured by individual observers ( mann - whitney u = 9720.500 , z = .916 , p > 0.05 ) . the intraclass correlation coefficient for the observers was 0.465 for treatment decision , and the high agreement ( 0.947 ) for lesion size . chi - square test revealed no significant difference in the treatment plan selected by observers between the two modalities ( (3 ) = .036 , p > 0.05 ) . for some treatment decisions , there was equal or almost equal selection between the two modalities . figure 5 shows trend among observers in selecting treatments between the two modalities . the 95% confidence interval for either the lower or upper limit of agreement was less than two standard deviations ( 2sd = 22.86 = 5.72 ) . this implies that the chance that observers chose different treatments from the two imaging modalities is minimal . the intraclass correlation among observers for each modality and both the imaging modalities together with reference to treatment selection and lesion size was calculated . ( tables 4 and 5 ) . though there was no statistical significance between the two modalities in terms of lesion size measured by individual observers , there was appreciable variability when the lesion size measurements of all the six observers were averaged ( figure 6 ) . with the introduction of cone beam ct in dentistry , several applications of this advanced imaging modality , including endodontics , are being investigated . in endodontics , the use of cbct involves diagnosis of pathosis , treatment planning , and diagnosis of trauma to dentoalveolar structures and evaluation of previously root - treated teeth , especially for missed canals [ 1619 ] . even though the benefits of cbct in endodontics are well understood , this particular study was undertaken to assess if the visualization of the third dimension as seen in cbct images would alter the choice of treatment made by endodontists . it is also interesting that the number of various treatment decisions ( root canal treatment , periapical surgery , root canal treatment plus periapical surgery , and no endodontic treatment ) was equal between the two imaging modalities . this led investigators of this study to believe that although cbct images show more information , this additional information would not necessarily translate into selection of a treatment different from the decisions made with the periapical images . in our study , six endodontists viewed images from both modalities ( pa and cbct ) and made their treatment decisions . the intraclass correlation with regards to the treatment decision was only moderate ( 0.465 ) . this could be attributed to the varying clinical experience levels ( novice to highly skilled ) . previous studies indicate that the long - term stability of observers in detecting periapical radiolucencies on conventional radiographs was satisfactory . two observers were well versed in interpreting cbct images and were using this technology on a regular basis while others had only knowledge of the cbct technology but were not well experienced in interpreting images . with reference to lesion size , there was , however , a high level of agreement ( 0.947 ) among observers between the two imaging techniques . this finding is in accordance with similar studies which confirm that periapical lesion measurements ( when adequately calibrated ) are the same between cbct images and traditional two - dimensional radiographic images . it is important to analyze the difference in radiation doses from the two imaging modalities under investigation in this study . several studies have assessed the effective radiation dose from different cbct scanners available in the market . comparisons in radiation dose between medical ct and cbct have also been made through scientific studies [ 23 , 24 ] . the mean organ dose to organs such as the brain , thyroid , and parotid gland becomes extremely important while performing a cbct scan . cbct doses also differ with reference to the particular scanning protocol that is being selected for a specific study . short scan times and standard resolution ( not high resolution ) scan protocols would reduce the radiation doses from these procedures . the potential risk to the patient due to this increase in radiation dose can be minimized by using a limited field of view ( fov ) . newer cbct scanners customized specially for endodontic purposes have the ability to scan only the particular tooth in question . however , in our study , the cbct scanner utilized did not have the ability to scan only the tooth and the entire jaw ( maxilla / mandible ) had to be imaged . the effective dose using the 2007 icrp ( international council on radiation protection ) calculation for the scanner and the protocols used in this study varies from 40 to 69 sv . the effective dose from the next generation cbct scanner used in this study is comparable to the dose from newer cbct machines currently being marketed only for endodontic purposes , for example kodak 9000 , with an effective dose of 21 sv for a 4 5 cm jaw sextant . also , we used a scan protocol with an option to obtain higher resolution images since there was a need to display the best quality images for the observers to view . the scan resolution used in this study is similar to other studies of this nature . also lesion size differences between the two imaging modalities can be compared only when highly resolvable images are available . this study is not without its limitations : ( i ) the study sample ( n = 24 ) is relatively small and it is not known if a larger sample would have produced a different outcome , ( ii ) history and clinical information ( signs and symptoms ) of study subjects were not provided to the observers at the time of the viewing sessions , so it is not certain if this information would have produced a significant difference in treatment selection between the two imaging modalities , ( iii ) the clinical experience of observers varied from novice to most skilled , ( iv ) the radiation dose from the cbct imaging procedure in the presence of an already existing radiographic procedure ( periapical radiographs ) should be validated , and a separate study comparing the effective dose of radiation used in cbct imaging and conventional radiography for endodontic purposes is required , and ( v ) it is not known if a different resolution of cbct images would have affected the results since native resolution of cbct images was used by observers with no image enhancements ( other than brightness and contrast ) being performed for either modalities . it is now well recognized that prospects for the use of cbct in endodontics in the future appear promising . however , there are several factors such as diagnostic yield , radiation dose and selection criteria to be considered before this technology becomes incorporated into the diagnostic armamentarium in endodontics . since interpretation of cbct images requires additional training , care must be taken to avoid misinterpretation by clinicians who have not had the required training . for example , with an untrained eye , artifacts such as beam hardening due to metallic restorations on cbct images could be misdiagnosed as a carious lesion . endodontists aspiring to use this technology should either train themselves or seek the help of a maxillofacial radiologist who can interpret the images for them . also , when cbct images do not offer additional clues to diagnosis , periapical radiographs should be referred for more information . therefore , the notion that cbct would replace conventional periapical radiographs is far - fetched . another factor to be considered is the viability and cost - effectiveness of using cbct in a clinical setting . not all endodontic offices currently include cbct in their diagnostic work up . at the present time , the request for cbct scans for endodontic purposes is not uniform between specialists for the same reason . the cost of a cbct scan is several fold compared to a conventional periapical radiograph and the increase in cost needs to be justified . in conclusion , it was determined from our study that the choice of treatment in endodontics does not change significantly even with the inclusion of an advanced imaging modality such as cone beam ct . the results of this study , however , should be interpreted with caution . although cbct performed almost the same as periapical radiography , the potential advantages of cbct in other areas of endodontics including assessing proximity of teeth to anatomic structures , such as mandibular canal and maxillary sinus , can not be underestimated . this study also proves the robust diagnostic potentials of traditional two - dimensional imaging modalities . further studies with a larger sample size are underway to further confirm the results obtained from this study and identify appropriate areas in endodontics where cbct imaging would play a vital role .

we examined the association between sex , age , insulin regimens and glycemic control in 133 japanese children and adolescents , 42 males and 61 females aged 16.8 7.0 yr , with type 1 diabetes mellitus ( t1 dm ) . the patients were divided into 5 age groups and were also classified according to the insulin regimen . the annual median hba1c level in males ( 7.3 0.2% ) was similar to that in females ( 7.2 0.2% ) . in regard to the age of the patients , the median hba1c levels in patients aged 1519 yr ( 7.9 0.4% ) was significantly higher than those aged 59 yr ( 7.2 0.1% ) and those aged 20 yr ( 6.6 0.4% , p<0.05 , respectively ) . on the other hand , there were no significant relationships between the hba1c values and the insulin regimens . in conclusion , difficulty in management of diabetes due to emotional issues and endocrinological factors during adolescence may play a possible role in the deterioration of diabetes control . on the other hand , the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm .

in recent years , a variety of insulin regimens have been used for the treatment of type 1 diabetes mellitus ( t1 dm ) in children and adolescents ( 1 ) . multiple daily injections ( mdi ) of rapid- ( ra ) and long - acting ( l ) insulin analogues have been used widely in these patients . moreover , use of continuous subcutaneous insulin infusion ( csii ) has been extended to those in whom mdi is either impractical or ineffective . these measures have been reported to improve hyperglycemia with reducing the occurrence of severe hypoglycemia in the young patients ( 1,2,3,4 ) . on the other hand , the international society for pediatric and adolescent diabetes ( ispad ) recommends a target range for hba1c of less than 7.5% in pediatric patients of all ages with t1 dm in its clinical practice consensus guidelines ( 5 ) . several studies have demonstrated that the recent hba1c levels of young patients with t1 dm on an intensive insulin treatment using newer insulins are still higher than the goal ( 6,7,8 ) . in addition , it is known that optimization of metabolic outcome in females and adolescent patients is more difficult despite intensification of insulin therapy ( 6 , 7 ) . we examined the annual mean hba1c levels during 2008 in japanese children and adolescents with t1 dm and compared them in relation to the sex and age of the patients and the insulin regimens employed to identify the factors associated with glycemic control . one hundred and three patients , 42 males and 61 females aged 16.8 7.0 yr , with a duration of diabetes of at least one year were enrolled in this study . the patients were divided into 5 age groups : group a , 14 yr ( n=6 ) ; group b , 59 yr ( n=17 ) ; group c , 1014 yr ( n=18 ) ; group d , 1519 yr ( n=26 ) ; and group e , 20 yr ( n=36 ) . they were also classified according to the insulin regimens used as follows : twice daily insulin injections , n=10 ; thrice daily insulin injections , n=14 ; four times daily insulin injections , n=49 ; five times daily insulin injections , n=15 ; and csii , n=15 . in regard to the insulin preparations used , 8 patients used premixed insulin analogue ( aspart+nph or lispro+nph ) , and 2 used twice - daily injections of a mixture of regular ( r ) and neutral protamine hagedorn ( nph ) insulins . six patients used varying combinations of premixed insulin analogue , ra ( aspart or lispro ) and l ( detemir or glargine ) , 5 used premixed insulin analogue , ra or l , and one used thrice - daily injections of premixed insulin analogue and ra . in the 49 patients taking 4 injections daily , 42 ( 85.7% ) used ra as bolus insulin and l as basal insulin , 3 used ra as bolus insulin and nph as basal insulin and 4 used ra and r as bolus insulin and l as basal insulin . in the 15 patients taking 5 injections daily , 12 ( 80.0% ) used ra as bolus insulin and twice - daily injections of l as basal insulin , 2 used ra as bolus insulin and nph and l as basal insulin and one used ra and/or r as bolus insulin depending on their daily schedule and twice - daily injections of l as basal insulin . all the patients were instructed to self - monitor their blood glucose levels at home . determination of the insulin regimens and adjustments of the insulin dose were performed by one pediatrician , who was mainly involved in treatment of the patients at an outpatient clinic on the basis of the blood glucose profiles determined by self - monitoring and the lifestyles and daily activity schedules of the patients . the patients visited the outpatient clinic each month or at least every two or three months . we compared the annual mean hba1c levels from january to december 2008 in relation to the sex and age of the patients and the insulin regimens employed . the hba1c level was determined by an hplc method ( normal reference range , 3.35.8% ) . the statistical significance of differences between groups was analyzed by the mann - whitney u test and kruskal - wallis test ( dr . spssii ) . the statistical significance of differences between groups was analyzed by the mann - whitney u test and kruskal - wallis test ( dr . spssii ) . the overall mean annual hba1c level in 2008 was 7.2 1.1% ( 4.59.7% ) . in the 103 patients , the mean hba1c level was < 7.5% , which is defined as optimal in the ispad clinical practice consensus guidelines ( 5 ) , in 59.2% of the patients , while it was > 9.0% , which is considered to be predictor of poor outcome in the same guidelines , in 6.8% of the patients . there was no significant difference in the median hba1c level between the male and female patients ( 7.3 0.2% vs. 7.2 0.2% ) . in regard to the relation with age , the median hba1c level in group d was significantly higher than the levels in groups b and e ( 7.9 0.4% vs. 7.2 0.1% and 6.6 0.4% , p<0.05 , respectively ; table 1 ) . there were no significant differences in median hba1c level among groups a , b , c and e. in group d , hba1c levels of < 7.5% were achieved in only 8 ( 29.6% ) patients ; conversely , 6 ( 22.2% ) patients showed poor glycemic control with hba1c levels of > 9.0% . table 1 differences in the hba1c level in relation to age at the time of the studyage groupnhba1c ( % ) group a ( 14 yr)67.6 0.2group b ( 59 yr)177.2 0.1group c ( 1014 yr)187.3 0.3group d ( 1519 yr)267.9 0.4*group e ( 20 yr)366.6 0.4*vs . the results were expressed as medians se . * vs. groups b and e , the results were expressed as medians se . in regard to the frequency of insulin regimens , of the 103 patients , 70 ( 72.8% ) received three or more injections of insulin , and 15 ( 14.6% ) received csii . among the 26 patients in group d , 22 ( 84.6% ) used multiple injections of insulin or csii . in addition , all the young children in group a received either multiple injections or csii . table 2 shows the distribution of the median hba1c levels among the various insulin regimens . we could not find any statistically significant differences in relation to the insulin regimens employed . table 2 differences in the hba1c level in relation to the insulin regimens employedinsulin regimensnhba1c ( % ) twice - daily injections107.1 0.2thrice - daily injections147.6 0.3injections 4 times daily497.2 0.1injections 5 times daily157.0 0.2csii157.3 0.5there were no significant relationships between the hba1c values and the insulin regimens ( kruskal - wallis test ) . the results were expressed as medians se . there were no significant relationships between the hba1c values and the insulin regimens ( kruskal - wallis test ) . there was no significant difference in the median hba1c level between the male and female patients ( 7.3 0.2% vs. 7.2 0.2% ) . in regard to the relation with age , the median hba1c level in group d was significantly higher than the levels in groups b and e ( 7.9 0.4% vs. 7.2 0.1% and 6.6 0.4% , p<0.05 , respectively ; table 1 ) . there were no significant differences in median hba1c level among groups a , b , c and e. in group d , hba1c levels of < 7.5% were achieved in only 8 ( 29.6% ) patients ; conversely , 6 ( 22.2% ) patients showed poor glycemic control with hba1c levels of > 9.0% . table 1 differences in the hba1c level in relation to age at the time of the studyage groupnhba1c ( % ) group a ( 14 yr)67.6 0.2group b ( 59 yr)177.2 0.1group c ( 1014 yr)187.3 0.3group d ( 1519 yr)267.9 0.4*group e ( 20 yr)366.6 0.4*vs . * vs. groups b and e , p<0.05 , respectively ( kruskal - wallis test ) . in regard to the frequency of insulin regimens , of the 103 patients , 70 ( 72.8% ) received three or more injections of insulin , and 15 ( 14.6% ) received csii . among the 26 patients in group d , 22 ( 84.6% ) used multiple injections of insulin or csii . in addition , all the young children in group a received either multiple injections or csii . table 2 shows the distribution of the median hba1c levels among the various insulin regimens . we could not find any statistically significant differences in relation to the insulin regimens employed . table 2 differences in the hba1c level in relation to the insulin regimens employedinsulin regimensnhba1c ( % ) twice - daily injections107.1 0.2thrice - daily injections147.6 0.3injections 4 times daily497.2 0.1injections 5 times daily157.0 0.2csii157.3 0.5there were no significant relationships between the hba1c values and the insulin regimens ( kruskal - wallis test ) . the results were expressed as medians se . there were no significant relationships between the hba1c values and the insulin regimens ( kruskal - wallis test ) . good glycemic control is critical in patients with t1 dm . a target range of < 7.5% is recommended in the clinical practice consensus guidelines of the ispad for all pediatric age groups ( 5 ) . the long - term microvascular and macrovascular complications of diabetes mellitus , the sequelae of acute hypoglycemia and the central nervous system alterations associated with hyper- and hypoglycemia are considered to be avoidable if this glycemic goal can be achieved . nevertheless , it still appears to be difficult to achieve this glycemic goal in young people with t1 dm , even when intensive insulin therapy using the newer insulins is adopted ( 6,7,8,9 ) . danne et al . of the hvidoere study group on childhood diabetes ( 7 ) reported that the mean hba1c level in 1998 of 2,780 children and adolescents with t1 dm aged 018 yr from 21 international pediatric diabetes centers in 17 countries was 8.62 0.03% , which was not different from that in 1995 of 2,101 patients aged 1118 yr from the same centers , despite adoption of intensive insulin regimens . de beaufort et al . of the same study group ( 8) reported that the mean hba1c level in 2005 among 2,062 patients with t1 dm aged 1118 yr was 8.2 1.4% . they concluded that even intensive insulin regimens with increased use of csii were not effective for improving the hba1c levels in this large cohort of adolescents with t1 dm . these results suggest that it might be difficult to obtain significant improvement of the glycemic outcome in young patients with t1 dm despite substantial changes in the therapeutic approaches . some studies have shown an association of poor glycemic control with the female gender and adolescent age ( 6,7,8 ) . endocrinological factors play a possible role in aggravating glycemic control in females as compared to males , especially in the pubertal age group . in addition , females at this age suffer more frequently from behavioral problems , including eating disorders and subthreshold disorders , as compared with males ( 10 , 11 ) . moreover , the practice of insulin omission to control body weight is frequently used by females of mid - teen age , which induces poor glycemic control ( 12 ) . nonetheless , we did not find any significant differences in the median hba1c levels in relation to gender , even during adolescence , in the present study . on the other hand , we found the highest median hba1c levels among the patients in group e. most of the patients with an hba1c > 9.0% were adolescents . some studies have suggested that decreased peripheral insulin sensitivity during adolescence , perhaps caused by hypersecretion of growth hormone , is the major reason for deterioration of glycemic control at this age ( 13 , 14 ) . moreover , the majority of adolescent patients with poor glycemic control had psychological and/or familiar problems in the present study . adolescence is a period in which young people try to gain independence from the family , but the demand for greater independence may not be equaled by the need to take greater responsibility for diabetes management ( 15 ) . it may be difficult to motivate proper diabetes management during this period , but it is important for diabetes teams to elicit it by providing continual mental support and diabetes education ( table 3 ) . table 3 factors in deterioration of glycemic control during adolescence1)decreased peripheral insulin sensitivity , perhaps caused by hypersecretion of growth hormone2)corrupted lifestyles including irregular daily diets and activity patterns3)spread of psychological and/or familiar problems4)inequality between demand for greater independence and responsibility for diabetes management5)difficulties in motivation for diabetes management there was no significant relationship between the hba1c levels and the insulin regimens used in the present study ; thus , the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm . nevertheless , we consider this to be one of the reasons why our patients were treated with optimal insulin regimens taking into consideration the patients lifestyles , customs and daily activity schedules , as well as the glucose profiles for the individual patients . patients who achieved satisfactory glycemic control even though they received twice - daily injections of insulin did not need either mdi or csii . for some reason , such as their lifestyles and daily activity schedules , they could not be treated with mdi ; however , the twice - daily injections seemed to be more optimal for their treatment . patients who were treated with thrice - daily injections sometimes missed their bolus injections at lunchtime during school hours while on mdi with injections a times daily . this was the major reason why they chose the regimen of thrice - daily injections of insulin . on the other hand , patients on mdi or csii eventually required further intensification of the insulin regimens to improve glycemic control when the optimal glycemic outcome could not be accomplished by conventional insulin therapy . some patients who were treated with injections 5 times daily subsequently required twice - daily injections of basal insulin to attain stable glucose levels throughout the day . mdi and csii should be introduced in motivated patients of any age , including very young children , if they fail to achieve optimal glycemic control with twice- or thrice - daily injections of insulin ( 16 , 17 ) . consequently , the insulin regimen should be individualized for each patient , and an optimal insulin regimen should yield satisfactory metabolic outcomes . in addition , diabetic education and a team approach by the diabetes care team is indispensable to achieve good glycemic control . adolescents with t1 dm showed significant higher annual serum hba1c levels than patients from the other age groups . difficulty of diabetes management due to emotional issues and endocrinological factors during puberty may play a possible role in deterioration of diabetes control in this age group . on the other hand , the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm .

with the development of rapid freezing of human oocytes , many programs have reported ivf success rates comparable to those achieved with fresh eggs and thawed frozen embryos . egg freezing is now gaining professional and regulatory acceptance as a safe and effective technique for women who wish to avoid discarding excess embryos , who face fertility - threatening medical treatments , or who want to preserve their eggs for use when they are better situated to have a family . this article focuses on the uses of and justification for egg freezing , the path to professional acceptance , the variability in success rates , and the controversy over freezing eggs for social rather than medical reasons . it also addresses the emergence of egg banking as a separate sector in the infertility industry , the regulatory issues that it poses , and its effect on egg donation . key here is the legal control of stored eggs by banking women and their options when they wish to dispose of those eggs . the analysis is framed around empowerment and alienation . egg freezing is generally empowering for women , but the donation or sale of unused eggs to infertile women , egg bankers , and researchers also raises issues of alienation .

mastery of the human reproductive process took another step forward in january 2013 when the american society of reproductive medicine ( asrm ) announced that oocyte cryopreservation was no longer experimental . several clinics in europe and the united states had reported births since 2000 , and many programs were offering it without reliable evidence of efficacy . until january 2013 the asrm had called the practice experimental because the birth rate after thawing and insemination of frozen eggs was not as high as the birth rate from fresh or frozen embryos . professional recognition of the non - experimental status of oocyte cryopreservation is a noteworthy step in the history of assisted reproduction . with an already existing matrix of laws and practices for assisted reproductive technologies ( art ) the technology will also expand reproductive freedom in several important ways , particularly for women facing cancer treatment , wanting an egg donor , or seeking to preserve fertility against the ravages of time . those who argue that the field is under regulated will find a counterexample of sorts . while it empowers women in some respects , it creates unwanted pressure and alienation in others . those who gain the most may be the egg sellers and entrepreneurs who have emerged to fill and create the market demand for egg freezing . to illustrate these points , this article maps the developments in egg freezing , and shows how this evolving field is affected by and in turn may affect reproductive practices . as such , it may guide practice and policy for egg freezing and future reproductive innovations . part ii discusses the moral , medical , and social options that egg freezing offers to women . part iii examines how a market in frozen eggs might develop , though originally intended for personal use only . part iv looks at the effect of egg freezing on egg donation for infertility and research . part v comments on the problem of moving innovative therapies into a regulatory setting , usually through professional society guidelines . part vi concludes by showing how new reproductive technologies are shaping the meaning of reproduction even as existing attitudes and sex roles influence those uses . to understand the importance of egg freezing , one must first recall the problems with getting eggs , the difficulty in freezing and thawing them , and the variable success rates of clinics engaged in the practice . the role that the asrm has played in this story is deferred to the end . getting eggs , unlike sperm , has always posed special problem because of their relative scarcity and location in the body . although a woman is born with the maximum number of eggs that she will ever have , they are lost as she ages . the ability to hyperstimulate ovaries and then surgically retrieve multiple mature oocytes has been a driving force in the take - off of ivf and assisted reproduction . it has also made egg donation to infertile women and gestational surrogacy viable , as well as donation to researchers , and eventually for clinical use in regenerative medicine . sperm and embryos have long been frozen , often for lengthy periods , and then after thawing used successfully to initiate pregnancy . because of their larger size and volume of fluid , oocytes were not easily frozen and thawed . a slow freezing technique was tried first in the laboratory , and then with women who lacked other healthy embryos , but success was mixed compared to use of fresh or frozen - thawed embryos . crystals would form and interfere with meiotic spindle segregation in metaphase 2 of the oocyte development . a few art programs in italy ( where legal constraints on discarding or freezing embryos gave incentives for investigation of egg freezing ) eventually developed vitrification , a method of flash freezing to 270 f with a glycol cryoprotectant , which prevented the crystallization and meiotic disruption that had made slow freezing so unreliable . when the eggs were warmed and then a single sperm injected into cytoplasm ( intracytoplasmic sperm injection ( icsi ) ) , higher success rates occurred . the technique soon spread to the united states and europe . with mounting data on live - births after vitrification , the asrm announced that egg freezing and thawing are no longer experimental in younger , highly selected populations for particular purposes . despite the asrm 's pronouncement that oocyte cryopreservation is no longer experimental for several uses , programs vary in their skill and success rate in the art of freezing . the larger and more experienced programs will have success rates comparable to the use of fresh or frozen embryos , said to be 3050% per frozen - thawed egg , but few programs without considerable experience score that high . art programs that report offering oocyte cryopreservation , over 50% of them have never thawed and inseminated frozen eggs and had live births thereafter . as with other medical technologies , it is easy for practitioners to claim comparable expertise without showing comparable results . the careful consumer will closely parse the numbers and pick only programs with well - established success rates . the asrm 's pronouncement of non - experimental status for egg freezing empowers women in various ways , but it also risks selling them an ineffective service , particularly if they are in their mid - thirties or older , when their eggs will have already aged considerably . it may also distance them from the meaning of producing the female germ cells so necessary for reproduction by breaking the bond that exists between women as producers and consumers of their body 's reproductive inputs , thus raising the commodification issues that weave though the ethics of assisted reproduction . in addition the asrm 's approval of egg freezing empowers fertility programs and independent egg freezing firms with a new profit center and incentives to market that service . this section and the one following addresses these uses and comments on how egg freezing links empowerment and alienation in women producing eggs for freezing . egg freezing is a clear advantage for those couples undergoing ivf who have qualms about freezing embryos but few about freezing eggs . the qualms might be specifically moral , or they may reflect a more general concern about not having too many embryos left in the freezer . for either group egg freezing is empowering . they will be able to avoid the risk of reduced efficiency from an ivf cycle due to their reluctance to discard embryos or to have too many leftover . instead of limiting the number of eggs inseminated to those that they would transfer to the uterus ( discarding the extra eggs or not retrieving them at all ) , now they may retrieve all eggs , inseminate only the number that can be safely transferred , and freeze surplus eggs for later thawing and use . if the stored eggs were no longer needed , they could be discarded without the ethical frisson of discarding frozen embryos . the catch in this approach is that there is no guarantee that the eggs chosen to be thawed and inseminated will yield the desired number for safe transfer and pregnancy , thus not providing the safety net they sought . the attraction to egg freezing is that if the first transfer is unsuccessful , they will not have to undergo another stimulation and retrieval cycle to obtain more eggs . there would be the additional cost in freezing and thawing eggs , but if a comparable success rate will be achieved , the price to the patient would be worth it . while empowering ivf patients with strong moral compunctions about embryo discard , egg freezing also opens the door to state disempowerment of patients with no such compunction . as right to life groups become more aware of art practices with fertilized eggs and embryos , which under their ethos are new persons who must be protected , they may support laws similar to those passed in italy that limit the number of embryos that can be created at one time and require that all embryos be transferred to the uterus . with extra eggs frozen and available for later thawing and insemination , such laws would limit the number of discarded embryos while , if the technique is effective , they would present no substantial interference with the efforts of an infertile couple to have a family . without the vatican in the vicinity , the political clout or inspiration to pass such disempowering laws in the united states may be absent . many people will view ivf as too far removed from abortion to spur legislation , particularly legislation that limits the ability of infertile couples to have families . nor would such limitations automatically follow from the state personhood amendments which periodically threaten but have not yet been passed . despite popular belief , merely defining the fertilized egg as a person under state law would not directly say what must be done with embryos outside of the body . to carry the discussion of constitutional issues a bit further , assume , however , that the state legislated directly to limit the number of eggs fertilized to the two or three that could be safely transferred to the uterus at one time , and required that all fertilized eggs be placed in the uterus . the purpose of such a law would be to reduce the chance that more embryos would be created and frozen , thus leading to their eventual death by discard or removal from storage . this justification would draw on the recognition in roe v. wade ( and casey and gonzalez thereafter ) that the state may protect prenatal life and potential personhood prior to viability as long as it did not substantially burden the core right to abortion . since the embryos are not in the body , embryo protection laws would not fall under the abortion right recognized in roe . but such laws could limit the safety , efficacy , and cost of ivf to such an extent that it interfered with a woman 's right to use ivf to have a family . while not yet recognized as such by the supreme court , many scholars would argue that that right is founded in a more general privacy or liberty right to have a family , and not a right to terminate a pregnancy as such . on this view a law limiting the number of eggs fertilized , requiring that all be transferred to the uterus , and prohibiting freezing embryos would appear to be a substantial burden on that right . yet if egg freezing is no longer experimental , the state could argue that eggs not fertilized could be frozen and inseminated at a later time , without having to go through another hyperstimulation and retrieval cycle does not burden or infringe the right at all . as we will see in part iv , the non - experimental label is a misnomer of sorts . yes , some programs have very good success rates after thawing and insemination , while others have had limited or no experience with egg freezing even though they offer it . favorable success rates have usually occurred with younger , healthier women and not older women or those whose fertility problems are related to egg quality . given the uncertainties of whether the two or three eggs inseminated will cleave and reach the stage appropriate for transfer , limiting the number of eggs inseminated with no option to inseminate more and freeze the extra embryos might mean that a woman ends up without enough viable embryos from that cycle to achieve pregnancy . she would then have to go through another costly and burdensome stimulation and retrieval cycle . until egg freezing were a comparable effective substitute for embryo freezing , such a policy would arguably so burden the woman 's freedom to reproduce as to be unconstitutional . this argument assumes that close or substantial comparability of efficacy would be required for a law limiting the number of eggs inseminated to be constitutional . recent circuit court cases on abortion restrictions , such as the constitutionality of hospital privileges requirements and following the fda label for medical abortions , indicate otherwise . the 5th circuit in a recent texas case refused to uphold the preliminary injunction issued by the district court simply because the law at issue made abortion more expensive or harder to access , on them ground that the situations before and after the new restrictive law . if the supreme court applies this more lenient standard to abortion laws , it is likely to do so for laws that interfere with infertile couples having children by restricting the number of eggs inseminated or embryos transferred . egg freezing also enhances freedom for women undergoing cancer treatment who want to preserve their fertility . if they had a spouse or partner , they could undergo a stimulation and retrieval cycle , create embryos , and freeze them for later use . they could do the same without an available partner , if they were willing to use donor sperm to create embryos . if they lack a partner and willingness to use a sperm donor , egg freezing would be empowering because it both protects their fertility and gives them choice over the genetic father of their post - treatment children . a similar need might arise with women with genetic diseases or other conditions , such as premature ovarian failure , who had not yet found a partner but wanted to ensure they had healthy eggs at a later point in their life for reproduction . offering egg freezing to cancer patients involves the disadvantage of needing a controlled stimulation cycle to harvest eggs , which delays chemotherapy and may lead to high patient estrogen levels . while exposure to more estrogen is generally not a good idea for estrogen sensitive breast cancers , the amount of exposure during one retrieval cycle might not be so great as to counterindicate it . with the time interval between diagnosis and the start of treatment varying among malignancies the controversy among clinicians , especially oncologists , about the risk of high estrogen levels might lead some clinicians and patients to attempt single egg retrieval in a natural cycle or immature oocyte retrieval with in vitro maturation of early stage oocytes . information is sparse about whether women who have used this option have been able to successfully preserve their fertility , mainly because so few have attempted pregnancy with thawed eggs after treatment . but a few babies have been born , and as long as treatment is not unduly delayed and ovarian stimulation is not contraindicated , few would argue against this option . the risk that it might lead to children whose mother dies from a recurrence of the cancer is not a sufficient ground not to proceed . egg freezing has also been touted as a way to provide women who are still fertile with insurance against their biological clock . women in their twenties or early thirties will be able to devote themselves to career without losing reproductive potency . by freezing eggs they will still have healthy eggs available for when they are ready to start a family . rescheduling motherhood in this way does sound empowering , but again nothing is as simple as it sounds . imogen goold and julian savelescu and others have articulated the general case for non - medical or social egg freezing . egg insurance against future infertility will enable equal participation in employment , allow women time to find a compatible partner , and enable them to postpone pregnancy until they are emotionally and psychologically ready to have a child . assuming safety and efficacy their arguments are generally persuasive but need to be broken down into the differing perspectives that different age groups of women will bring to egg freezing . the equal participation in employment argument emphasizes the importance of enabling women to obtain an education and invest in career without losing the opportunity to have children . a more desirable approach to this dilemma , of course , would be to change traditional employment models so that women do not face restrictions from time off for child - bearing and rearing . egg freezing is no substitute for those efforts , but it may provide some women with reassurance that they can commit themselves to education and work without losing their fertility . with marriage and children such a dominant cultural narrative for women , they may be more concerned about their relational status , their ability to find a suitable mate , and whether they are emotionally and psychologically ready for children . this pressure weighs even heavier if they are at risk for premature ovarian failure or other medical conditions that will limit their supply of healthy eggs . age group differences will arise with both careerist and relational concerns . to ensure the greatest fertility , it would be best for women to freeze their eggs in their early or late 20s . at this point many women but not all would usually have completed their education and started on a career . if they have not yet had children , and have no partner or immediate plans to do so , banking eggs would appear to enable them to live their lives without the biologic clock ticking so loudly in their ears . also , since health insurance is not likely to cover egg freezing until its efficacy is much better established , some of them might have the reources to cover the costs of thawing , insemination , and caring for a child . yet the optimism bias of the relatively young may make the risk of future infertility seem quite distant . surely , they say to themselves , they will find a man and settle down in the next few years , so why undergo the intrusion and cost of egg freezing ? only the most risk averse or those with a yen for the latest technological fix , may be willing to take the hormones and pay out cash for the egg insurance that they may never need to cash in . the early twenties perspective on women in this cohort may become acutely aware of the loss that they will experience if they postpone conception too long . if they are not in a relationship conducive to having children , a cold look at the facts may lead them to stock the egg freezer . as they slip into their late 30s , the internal pressure to freeze eggs will grow . at this point egg freezing may still their anxiety and allow them to get their workplace , relational , or psychological states in order . yet freezing post-35 may not give them the fertility they hope for , if only because the viability of their eggs will also have diminished . preferences here will vary with the importance of having children with their own eggs versus that of a donor and the varying degrees of reliability of actually producing children with different techniques . egg freezing for teenagers and those in their 20s is the most likely to be successful but the least likely to be needed ( stimulation and retrieval , not to mention cost , is hardly trivial ) . for women in their late 20s or early 30s one alternative would be to do a retrieval cycle , freeze some of their eggs , and inseminate some with donor sperm and then freeze those embryos . when they are ready to reproduce with a partner , they can thaw the frozen eggs and try with those . if that does n't work and donor sperm is acceptable to them , they can use the previously frozen donor sperm inseminated embryos . or they could use a donor egg and their partner sperm , and if no partner , an egg and sperm donor ( or already created embryo ) . the same would go for women who freeze after 35freeze some eggs and create embryos with donor or partner sperm , and then when ready for children , use the frozen eggs and then frozen embryos . if none of those work , then they still have the option of using donor eggs with partner or donor sperm ( or buying an already created frozen embryo ) . sarah elizabeth richards , a strong proponent of freezing eggs , ignores in her fervency the lower success rate with freezing eggs after 35 or these other options for motherhood . her 2013 book motherhood rescheduled : the new frontier of egg freezing and other writings present egg freezing as a culture narrative shift that allows women to one is struck , however , by how she oversells the technology as relief from the angst of finding the right mate and having children . richards begins one op ed ( why i froze my eggs ( and you should , too ) ) recounting how between the ages of 3638 she spent $ 50,000 of her savings to freeze 70 eggs in the hope that they would help her have a family in her mid-40s when she would be ready with a mate . for richards it was the best investment she ever made , because it stopped the deep sadness that without a reliable partner she was losing her dream of being able to have children. egg freezing snapped away the punishing pressure to seek a new mate before she was ready . the most powerful gender equalizer of all the ability to control when to have children. just as men in their 40s ( or even later ) could have children , she could too . here 's the rub . $ 50,000 for egg freezing at 37 is an expensive and probably ineffective way to quiet the ticking fertility clock . if a woman is listening carefully , this move will only lessen the thrum , not quiet it altogether because the viability of late-30s eggs is much less than that of eggs frozen in one 's 20s or even early 30s . none should think this form of insurance will cash out with the child that they want . with or without egg freezing they will be able to bear and rear children , though perhaps not with the genetic tie that egg freezing provides . the rescheduling motherhood narrative , so empowering for some , might be disempowering for others . rather than you may choose to freeze, some might read the message sent as this aspect leapt to the fore when a report gurgled up from the internet about a top law firm that offered egg freezing to new associates . some saw here exploitive law firms wanting to feast on the energy of lower paid lawyers by reassuring them that devotion to the firm would not wreck their family plans . no worries , they could simply put them off with egg freezing . whatever this hypothetical firm 's agenda , it will be important that egg freezing be structured as an opportunity not a requirement ( though the two may be difficult to separate ) . young women wanting to start families earlier rather than later should be protected in that choice , and not pressured to think they must defer children because of the demands of launching a career . indeed , if their employer or medical insurance is paying , it may create even more pressure to do so , even if the firm is simply trying to stay competitive . and entrepreneurial art programs or egg banks will market this service to the relevant age groups . expect broadcast , print , or internet advertisements that play on infertility fears and offer freezer discounts . egg freezing and other reproductive technologies are only empowering if they allow women ( and men ) the choice to use them . egg freezing fails that test if it is read as a prescription that all women entering the workforce or reaching a certain age should store their eggs . indeed , the chance to use it will also require the $ 10,000 to go through a stimulation and retrieval cycle to freeze eggs and pay ongoing egg storage costs as well as a tolerance for the medical intrusion involved . and a perception that egg or embryo donation , which lacks the genetic connection of using one 's own eggs , will not fill the gap . as more women freeze eggs for social reasons , the question of what they will do with their eggs when no longer wanted will arise . will they discard them ? donate or sell them to other women , to egg banks , or to researchers ? similar dispositional questions will arise with eggs frozen by women not wanting to create more embryos than can be transferred , or by women facing cancer or other health issues . here egg freezing empowers women to assert ownership and control over the eggs they have produced . women banking eggs are the owners of them , and will have ultimate dispositional control over them until they choose to transfer that control to another . how they will or should exercise that control is the question . egg freezing will thus be a legal as much as a medical or psychosocial transaction . the women involved will sign a contract with the bank about their rights to remove their eggs at any time and how they should be disposed of in the case of death , failure to pay storage fees , or other contingencies . presumably they may write a check on their egg account , designating themselves or another as payee . egg banking thus raises the possibility that egg storers will sell their eggs to other women or researchers who are in the market for egg donors and find those eggs cheaper or desirable for other reasons . the national organ transplantation act allows buying and selling eggs and sperm , though a few states have a ban . unlike eggs stored at a later age when women are going through infertility or stored for cancer treatment , eggs stored by women in their 20s or earlier 30s may be as good as healthy donor eggs and now available at a better price . if they are of an entrepreneurial bent or simply want to recover some of the costs they have invested in egg freezing , one can expect some women who freeze their eggs to participate in the market for donor eggs . of course , with so many eggs available from storage , the price of eggs may drop or rise depending on the fertility , health , and genetic desirability of the woman offering the eggs . laboratory and biobank regulations to ensure health and safety of storage facilities beyond existing fda regulations are likely to follow , as well as liquidity and other banking controls . as women come to view storing eggs as a banking transaction , with the possibility of accruing interest ( in the form of rising prices ) or independent sale , a sense of alienation or commodification might accompany egg freezing . although their goal at time of freezing is to reserve their own fertility , they will also have to think of their eggs as possible future commodities that are transferred to others , either on grounds of altruism or profit . the forms they will be asked to sign for future disposition of stored eggs will remind them of this contingency as well as the infectious disease and other fda screening that will then have to be done to prepare for or done at a later time for those contingencies . at some point women freezing their eggs may come to see them as another commodity on the reproductive market / that arose from their non - market efforts to preserve their fertility . only later are do they become marketeers , either as entrepreneurs or sellers in their own right , but that possibility hovers from the inception . freezing eggs solely for infertility purposes and then selling them later may be less alienating than if sale is intended from the start ( eg going through a retrieval cycle for the purpose of selling eggs ) . a greater degree of alienation may also enter the already partially alienated practice of egg donation . as the next section shows , the gift frame that largely surrounds paid egg donation for both donors and recipients will shift toward a more commercial frame , akin to the frame that now surrounds sperm donation . this is not harmful in itself , but frame shifting will necessarily require adjustment in the social and psychological valence that eggs now carry . the work she has undergone is no longer for herself alone , once she decides to sell them . as eggs become a commodity on the market , gender may be less important than it previously was . fungibility will also be a challenge . because of the genetic tie and its meaning for women banking their eggs , egg are not fungible . they will also vary in other genetic endowments , eg , mutational load for common and rarer disease and the age and viability of the eggs . except for the family tie , eggs from women of comparable ages and health may be fungible but of little interest to others because they are older and not that much less expensive than egg donors recruited for that purpose . egg freezing empowers women by giving them a way to preserve oocytes against the threat of age or disease . it also empowers the doctors and firms that provide those services , opening additional ways to sell art services and egg banking itself . a particular aspect of that empowerment is the creation of a novel kind of biobank , with a set of issues different from those that arise in tissue , dna , and sperm banks . to store eggs to use those eggs for reproduction , she will need a specialist in thawing and inseminating those eggs , and then placing them in her uterus . egg freezing will thus increase demand for art services , some of which would not otherwise have been requested . the specialists providing art may add egg banking to their portfolio of services , contract it out , or refer to established banks . since eggs will need to be frozen after retrieval , the art programs are likely to do that themselves . they will also prefer to thaw , inseminate , and implant embryos because of the additional charges generated . actual storage of the eggs might be at the clinic site or contracted or referred off - premises , as increasingly occurs with frozen embryos . as egg freezing proliferates , independent banks might emerge to handle administration of deposits , storage fees , and sending frozen eggs per depositor instruction to a site or program where thawing and insemination or other disposition will occur . regulations to ensure proper storage , accounting , and response to depositor instructions may be needed . legal protection for loss of stored eggs should also be provided , perhaps based on the cost of production . in the early stages of egg freezing there will be few depositors and few non - clinic egg banks . as the practice grows and art clinics get out of the longterm storage business , the number of off - premise egg banks will grow . the use of frozen eggs from paid egg donors will also give a boost to expansion of this sector . at some point full service reproductive banks might develop , with one entity banking eggs , sperm , and embryos , or eggs and embryos . when thawing and use is desired , stored gametes or embryos may then be transferred to the art programs that specialize in thawing , insemination , and implantation . reproductive biobanks serve different purposes than the many biobanks that now exist for tissue , dna , and other samples , but common issues arise . all biobanks have to face issues of who may deposit , the conditions of deposit , who may withdraw samples , legal protection for the privacy and quality of the sample , and how rights or claims in products that arise from the banking ( less important for reproductive banks ) are allocated . egg banking focuses attention on the choice of the depositor because she must invest money and body to obtain the banked entity . her physical , monetary , and emotional investment in the deposit may be greater than someone giving a sample of dna or tissue from an operation for research . her ability to have her genetic child may ride on the deposit , which itself may have an alienating aspect from her embodiment that other biobanking does not . other banks may focus more on research into prevention or treatment of health and disease , potentially affecting thousands , if not millions of persons . dna biobanks to uncover gene associations with common or rare diseases , such as the 1000 genome project in the united states or the uk biobank , have great research significance . other biobanks may amass tissue that could be used in treatment , such as the embryonic stem cell bank proposed for racial and ethnic variety . some banks may be more concerned than are gamete or embryo banks about intellectual property rights in resulting discoveries or the need to contact depositors about later annotations or calls on variations of unknown significance . each instance of banking will have its own set of challenges that depend on the precise use at issue . only reproductive banks will enable an infertile woman or couple to have a child , and need to be regulated accordingly . egg freezing will also empower the participants in the current system of paid egg donation , though less the egg donors themselves than the entrepreneurs and commercial providers . these effects , however , may make finding egg donors easier or more efficient , thus empowering recipients who lack eggs to have families . an additional benefit is that it will increase the supply of donor eggs sufficiently so that professional guidelines calling for informing or not using egg donors who have been through six donation cycles might not so drive up the price of eggs that they become out of reach for some women or lead to harmful effects on repeat donors . the asrm 's 2013 statement cited better coordination between egg donor and recipient as a reason for finding egg freezing non - experimental . no longer would it be necessary to have the donor and recipient 's cycle synchronized so that retrieval , fertilization , and embryo transfer take place at the same time and place . now the donor eggs could be retrieved and then frozen , and thawed and inseminated at the most convenient time and place for the recipient without fretting about donor compliance for synchrony with the recipient 's uterus . egg freezing , however , may facilitate more far - reaching changes in the egg donation system . that system increasingly relies on brokers to identify donors , match them up with recipients , and then have the stimulation , retrieval , insemination , and transfer done by the recipient 's physician . with the increasing reliability of egg freezing , especially from younger women , egg bank entrepreneurs may recruit several donors of differing ethnicity for egg production , have their eggs retrieved , genetically screened , and then freeze them for later sale as demand for eggs of women with that donor profile arise . the egg bank ( which might be run by a fertility doctor with experience in egg freezing and egg donation ) will pay the donor 's fee and retrieval costs up front for an initial supply of eggs . since one donor could produce 1025 eggs , it would be possible to sell frozen eggs in batches of 46 to women seeking them at a cheaper price than if recipients had hired the egg donor and her entire output themselves . the thawing and insemination could occur at the frozen egg bank ( and its associated doctors ) or could be shipped to affiliated programs which are trained in the bank 's thawing techniques . the upfront investment in egg production might lead to greater number of sales of eggs per donor , thus increasing the egg entrepreneur 's profits . an atlanta ivf program had tried for many years to develop safe and effective egg freezing . the program owners then decided to start a commercial egg bank that would recruit egg donors and sell batches of six eggs to couples / women for half the price of a recruited egg donor . its costs of recruitment , retrieval , and donor fees , which would roughly be the cost for a donation to a single donor , could be recovered by selling the now frozen donated eggs to several donors . depending on the number of eggs retrieved and the demand for a donor with those characteristics ( age , education , looks , proven fertility , etc ) a single donation via the frozen egg bank route could make egg donation cheaper for recipients and more profitable for the egg agency / art program . it would also lead to fewer women acting as donors , since one donor could provide eggs to several recipients . such a change in the egg donation system would entail some reframing of how egg donation is currently perceived . as professor rene almeling shows in her insightful study of egg and sperm donation , egg donation is now framed as a gift to infertile women , albeit for a fee . while the money is the key to women choosing to be egg donors , the gift nature of the situation is also important , and very different than how sperm donation is framed by sperm banks and experienced by sperm donors . would donors be as easily recruited for contributions to a frozen egg bank , which then sells their eggs in batches to recipients ? although this change would inject more commodification into egg exchanges , the donors would still be helping infertile women , indeed , more than the one that their donation would traditionally have aided . their need to meet or know about the recipient could also be met by providing that information prior to the donation and informed of it when other women wish to buy their eggs from the bank . key here , of course , is whether this system would maintain or increase demand for frozen relative to fresh donor eggs . would a woman or couple seeking an egg donation be willing to forego the notion that the donor donated just for them ? if the success of egg freezing is comparable to initiating successful pregnancies , information is available about the donor , and the price is lower , the use of donor eggs from egg banks might well meet the needs of recipients , and thus reinforce this practice . it may also make it easier to keep track of half - siblings born as a result and enable contact with the donor , if she has chosen identity release on the eighteenth birthday of offspring . indeed , it may lead to a much greater use of donor eggs obtained from egg banks , which will cost less and may give a greater range of choice , though it will lack the chance to meet or talk with the donor , as now sometimes occurs . a further alienation from the body and the person may result . the expansion of egg donation in this way depends on the legal infrastructure for ownership of eggs . it assumes that the egg donor is transferring all of her ownership interests in her eggs to the egg donor entrepreneur who recruits her and pays her fee and the costs of stimulation and retrieval , and then sells or transfers her eggs to others as he or she finds fit . the donor would retain no rights , nor would the recipient who previously would have gained title to all of them , since the recipient would be purchasing only a portion of the donor 's output . there is a gain in efficiency here at a cost in greater alienation or commodification of gametes , though it may also be easier for resulting offspring to contact each other and their genetic mother . in the transition period before such arrangements proliferate , couples or women who arrange for an egg donor will ordinarily be paying for and acquiring her entire output . but they may not need all of them , and instead of inseminating all , might choose to share ( for a part of the expenses ) with other women looking for an egg donor . this might be feasible in a fresh cycle for both , but logistics might be easiest if the recipient freezes eggs that she does not use to create embryos . as purchaser of the donor 's output in that cycle , she would be free to freeze some of them and sell or share with others . this would turn the recipient into an entrepreneur who then sells some of the eggs she purchased from the donor to another . to facilitate such a market , egg banks specializing in donor eggs may buy her surplus and sell accordingly . no doubt other problems will arise as the field grows , beyond the issues already noted . a shortage of eggs has plagued some embryonic stem cell researchers , in part because of bans on paying egg donors ( even though egg donors for infertility can be paid ) . the successful production of embryonic stem cell lines through somatic cell nuclear transfer will spur demand for research eggs as will the uk 's recent approval of experimental treatment of mitochondrial dna defects using defect - free partial egg donors . with a growing number of women freezing and banking their eggs , eventually some of them will donate unwanted eggs to researchers , to banks that will provide them to researchers , or paid egg donors will be recruited specifically to donate to research , with unused eggs at the time frozen for later research . a main barrier to the shortage of research eggs has been ethical concerns about the propriety of paying egg donors for research eggs , even though it is now well - accepted that paying women who donate eggs to infertile women is acceptable . the legal prohibition on paying for organs never extended to blood and gametes , and only a few states ban payment for eggs . in the united states , however , the controversy over federal funding of embryonic stem cell research led to a funding policy of not funding research with cell lines derived from embryos created from paid gamete donors . several states followed suit by banning any use of paid gametes in stem cell research , even though they allowed fertility donors to be paid . new york has now removed that ban but governer jerry brown vetoed such a bill in california . in any case , the federal limit on paying gamete or embryo donors in federally funded stem cell research still remains . with a growing number of women freezing and banking their eggs , eventually some of them will donate unwanted eggs to researchers , to banks that will provide them to researchers , or paid egg donors will be recruited specifically to donate to research , with unused eggs at the time frozen for later research . freezing technology will not dissolve moral concerns about paying egg and sperm donors , but it should make eggs so widely available at a reasonable price that many researchers will be able to go forward with non - federally funded research . as scientific interest in using oocytes in research takes off , the availability of eggs from women who have banked eggs initially for themselves and from donors recruited for that purpose should meet most scientific needs . the movement of egg freezing from research to innovative therapy to accepted practice illustrates the trajectory of much medical development and the pitfalls along the way . the united states does not have a national fertility regulatory agency to announce when success rates with egg freezing are acceptable , nor are such judgments within the jurisdiction of the fda . as a result , it fell to a professional society of doctors offering ivf ( and egg freezing ) to play that role . indeed , it took that responsibility quite seriously because of earlier criticism from a presidential bioethics council that too many new reproductive technologies were being used without adequate testing . basic ivf itself was highly innovative and full of risks to resulting children , but when it succeeded in 1978 , doctors inside and outside the u.k . the ability to hyperstimulate the ovaries and remove several eggs , fertilizing and transferring some embryos to the uterus , and freezing surplus embryos for later use , gave a great boost to the field . the use of icsiwas also introduced without controlled trials , first for problems of male infertility , and then for a wide array of other situations , so that it is now used in 50% of basic ivf . many other aspects of art have also been adopted without controlled trials or systematic investigation , including different embryo cultures , the use of assisted hatching , when to transfer embryos to the uterus , preimplantation genetic diagnosis , egg freezing , and the like . art is not exceptional here this pattern is typical of many areas of medicine . to deal with criticisms that too many assisted reproductive innovations were being used without adequate study the asrm issued a report in 2009 on the definition of experimental procedures. it proclaimed that procedures , tests , treatments or other interventions for the diagnosis or treatment of infertility are considered experimental or investigational until the published medical evidence regarding their risks , benefits , and overall safety and efficacy is sufficient to regard them as established medical practice. medical evidence relevant to determining whether something is established medical practice must be derived from appropriately designed , peer - reviewed , published studies performed by multiple independent investigators. i d . it went on to say that the asrm will state specifically in official publications that a procedure is considered experimental or investigational and will remove a procedure from that status when evidence warrants it . the asrm also warned that procedures classified as experimental or investigational should not be represented or marketed to patients as established or routine medical practice. advertisements and other materials describing or relating to that procedure should state specifically that the procedure is not established medical practice and is classified by the asrm as experimental or investigational . patients choosing that procedure should be counseled about the experimental nature of the procedure and have that counseling documented . the asrm report , however , does not require that a physician offering an investigational procedure do so only in the context of a research study . an experimental treatment must be labeled as such , regardless of whether it is offered as part of a research project blessed by an irb . the asrm 's handling of the problem of when an experimental or innovative practice moves to established status illustrates some of the issues that arise with use of a professional association to make those decisions . issuance by a self - interested and resource - limited professional association risks establishing minimum rather than optimal standards of care , with no effective means of enforcement . despite best efforts , those standards may be internally inconsistent , are time - dependent , and may be quickly outmoded . unsurprisingly , they may also reflect the interests of those issuing the guidelines or their sponsors . several of these problems are evident in the asrm guideline on egg freezing , a practice initially offered or marketed to women without good evidence that it was safe and effective . as noted , the asrm practice committee had taken the position that egg freezing is experimental and not ready for prime time use because the live birth rate was significantly less than the success with fresh or frozen - thawed embryos . when it announced in 2013 that egg freezing was no longer experimental in younger , highly selected populations for particular purposes , it based its conclusion both on preliminary data on safety and four random controlled trials of fresh versus vitrified / warmed oocytes , finding that implantation and clinical pregnancy rates are similar in young , highly selected populations to success rates with fresh eggs or frozen - thawed embryos . it noted , however , that these results may not be generalizable , and clinic specific success rates should be used to counsel patients whenever possible . also , large observational studies of clinical practice suggest that success rates overall may be lower . while finding that egg freezing was no longer experimental for patients receiving gonadotoxic therapies for cancer or other diseases , by woman with genetic disease , and couples with ethical or legal constraints on freezing embryos , it took a different position with regard to egg freezing to circumvent reproductive aging in healthy women . it found no data to support the safety , efficacy , ethics , emotional risks , and cost - effectiveness of oocyte crypreservation for this indication. one may question why the same data that supports recognition for approved uses would not also support recognition for this use , other than the population of older women who been the main customers for egg freezing . so how to explain the asrm 's inconsistency in 2013 to certify some uses of egg freezing as non - experimental and others , such as social uses of egg freezing , as still experimental . in its view there was no data to support the safety , efficacy , ethics , emotional risks , and cost - effectiveness of oocyte crypreservation for the sole purpose of circumventing reproductive aging in healthy women. presumably the asrm would like to see studies of women who have banked eggs for this purpose , especially over age 35 , who were its main customers so far in the the data was not overwhelming for the non - experimental uses that had been approved , simply because there had been so few studies . the data for social uses ( the health effects of doing so , the live - birth rate of pregnancy after freezing and thawing , psychosocial reactions , and more ) will not be immediately forthcoming because those uses have been relatively few , offspring born even fewer , and studies of them non - existent . initially the asrm 's hedge on freezing as social insurance smacked some women as less consumer protection than old - fashioned , male - dominated medical paternalism . women were quick to argue that they should be informed of that option so that they could make their own choice . in fact , behind its labeling rhetoric this was all that the asrm was urging : fully informed choice . the asrm feared that some clinics or agencies would tout social uses as a way to sell ivf cycles and egg banking services , when there are too many uncertainties to justify the costs and physical burdens of doing so , particularly for women over 35 who appeared at this time to be the most likely users . some uk doctors agree with this position because of lack of benefit to justify the harm imposed . in addition , if programs were not fully informing patients of egg freezing 's non - established status , they would be violating the principle of informed consent . the asrm 's position was actually less protective than it purported to be , which is one of the problems with professional self - regulation . art programs could not sell egg freezing as standard , established therapy , but they could advertise and sell it as long as they labeled it truthfully as experimental. nor did it require that a program bring egg freezing to an irb as a research study . in the end , informed consent would be the watch word here , as seemingly everywhere else in bioethics . a similar strategy is seen here in the asrm 's practice committee 's discussion of limits on the number of donor egg cycles . in the abstract , the practice committee does not say that more than six cycles should not be done , but only that donors be informed of the possibility of undocumented but possible health risks . in the conclusions it states that it is prudent to limit the number of stimulated cycles to approximately six. the slushy language here shows another limitation in professional society regulation , in this case to try to avoid mandatory statements that are based on insufficient data or that could lead to charges of antitrust collusion . the map provided here of how egg freezing fits into the ethical , legal , and medical matrix of existing art practice has mentioned several kinds of regulation that might apply or be needed . there already are fda guidelines on handling donor gametes in the laboratory , which will apply if women who freeze eggs for their own use wish to sell or donate them to others . if egg producers had not been tested for various infectious diseases at the time of freezing , they will have to be tested before their eggs can be used by others . existing state laws such as those in california and in new york will be a model for ensuring safe operation , as will the federal clinical laboratory improvement act . but egg banking raises special issues concerning the depositor 's need to be sure that deposited eggs are safely preserved and returned to the depositor or to a designated recipient . there will be many similarities and differences from traditional cash banking , which may yield some insights about how egg freezing and other forms of biobanking should be addressed . an issue of general regulatory concern with biobanking is what damages if one 's banked sperm , embryos , cord blood , or other tissue has been lost , stolen , or damaged . because of the family importance of the banked materials or their need in specific medical procedures , money damages may be hard to calculate and will have an alienating effect on those persons who have lost banked material . beyond health , safety , and banking issues , there is a need for clear recognition of the woman 's ownership of and hence dispositional control of her eggs . while such a result would seem to follow from previous cases and practices , any regulatory regime should start with recognition of her legal rights in her eggs . until she has validly transferred full or partial control to a spouse , partner , physician , bank , or researcher , it is her right to decide whether to produce eggs , store them , have them inseminated , or transfer full or partial control to another . by the same token , once she does so , she loses that control and may have no further say in whether they are used to produce research embryos or embryos for infertile persons . a full regulatory regime is lacking for many aspects of art and egg freezing is no exception . women , however , play a special role both as providers of gametes and as gestators . no man can reproduce without an egg source and/or gestator , while women can gestate without using their own egg . traditionally , they have also played a much larger role in rearing , though more involvement by fathers is occurring in the united states and in many european countries . art expands both women 's career , education , and fertility options , and makes special demands of them . sometimes they will resort to art to help the man overcome his infertility , for example , by going through ivf when he has sperm problems , or making sure that he will have progeny when his wife lacks ovaries or uterine function by employing a surrogate gestator or an egg donor . women 's resort to art , however , is largely driven by their own desire for children , reflecting personal and cultural narratives of the importance of reproduction for female identity . science not sex becomes the primal scene , with the glare of laboratory lights replacing the darkness of the fallopian tubes . new narratives fit ivf and embryo freezing into old ones of husband and wife having children . surrogate motherhood challenged that narrative because it was so directly an alienation of gestation from rearing and so explicitly for a price ( though a diminished gift rationale remained ) . it devalued the female genetic tie but kept the gestating mother as rearing mother . for women who valued genes over gestation , it was a greater challenge . both areas came to privilege preconception / implantation intent over the later wishes of the gestational surrogate . egg donors also lost out on rearing rights if the recipient 's gestation and rearing had been agreed to in advance . with egg donation and surrogacy now widely accepted , egg freezing seems empowering . by shifting or rescheduling genetic motherhood to a later time , it enables women to enter the workforce , establish career , and undergo cancer treatment with less of a chance of missing out on genetic motherhood . on this view , the empowering aspect , though firmly rooted in cultural narratives of the importance of female reproduction , is more important than any accompanying alienation or shift in art business and profit models . the main task is to make freezing as safe and effective as other forms of art , bring the cost down , and ensure that women know what they are getting into . another perspective , while crediting the empowering narrative , will remind women that there are costs here as well , such as the likely low success rate of freezing after 35 and the need to find an experienced program . this translates to storing eggs at the earliest age possible , which alters the passage of women to early adulthood . ? it also means resisting a de rigueur attitude to freezing and pro - life efforts to diminish the right to freeze and discard embryos . the trick is to find the sweet spot between these goals . in striking that balance within the prevailing market framework , women may come to view their eggs not only as products of their body essential for their genetic motherhood , but also as products that can be streamed into commerce . this can arise from women who no longer need to store eggs and want to recoup their investment or make a profit . it will also arise in the egg donor market , which despite payment has residues of gift economy . now egg donors will be simply paid for their ovarian bounty , with less of a connection to the recipients they are helping . it may also lead them to negotiate prices pegged to the number of eggs produced . once everyone adjusts , the rough edges may smooth , increasing welfare and flourishing for most participants . as usual , how it is done rather than that it is done will probably turn out to be the key issue . the onslaught of genetic screening now waiting in the wings will be a far more important development for all concerned . mapping egg freezing on to these issues and showing how regulation largely by professional societies emerge with all its flaws will help in devising practices and policies for egg freezing and other innovations in assisted reproduction .

summarythe slx1-slx4 endonuclease required for homologous recombination and dna repair in eukaryotic cells cleaves a variety of branched dna structures . the nuclease subunit slx1 is activated by association with a scaffolding protein slx4 . at the present time , little is known about the structure of slx1-slx4 or its mechanism of action . here , we report the structural insights into slx1-slx4 by detailing the crystal structure of candida glabrata ( cg ) slx1 alone and in combination with the c - terminal region of slx4 . the structure of slx1 reveals a compact arrangement of the giy - yig nuclease and ring domains , which is reinforced by a long helix . slx1 forms a stable homodimer that blocks its active site . slx1-slx4 interaction is mutually exclusive with slx1 homodimerization , suggesting a mechanism for slx1 activation by slx4 .

the human slx1-slx4 structure - selective endonuclease plays a key role in dna repair , homologous recombination , replication fork restart , and telomere maintenance ( svendsen and harper , 2010 ) . the genes encoding saccharomyces cerevisiae slx1 and slx4 were discovered in a genetic screen for mutations that are synthetic lethal in the absence of the sgs1 helicase , a protein that is important for genome stability ( mullen et al . , 2001 ) . homology searches subsequently identified the slx1 and slx4 genes in higher eukaryotes ( andersen et al . , 2009 ; fekairi et al . , 2009 ; muoz et al . , slx1 is an evolutionarily conserved protein that contains an n - terminal giy - yig nuclease domain ( also called uri domain ) and a c - terminal zinc - finger domain . giy - yig domains also are present in homing nucleases , the bacterial nucleotide excision - repair nuclease uvrc , and several type ii restriction enzymes ( dunin - horkawicz et al . , 2006 ) . the mechanism of substrate binding and cleavage for giy - yig family members has been elucidated by crystallographic studies of protein - dna complexes obtained for two restrictases , namely r.eco29kl ( mak et al . , 2010 ) and hpy188i ( sokolowska et al . , 2011 ) . the slx4 subunit of the slx1-slx4 nuclease is thought to provide a scaffold that coordinates the actions of a number of proteins involved in dna processing ( cybulski and howlett , 2011 ) . for example , vertebrate slx4 is a large , multi - domain protein that interacts with several dna repair proteins ( andersen et al . , 2009 ; fekairi et al . , 2009 ; muoz et al . , 2009 ; salewsky et al . , 2012 ; svendsen et al . , 2009 ) : ( 1 ) the n - terminal region of human slx4 binds the msh2-msh3 mismatch - repair complex and xpf - ercc1 nucleotide excision - repair enzyme ; and ( 2 ) the c - terminal portion of slx4 binds the telomeric proteins trf2 and rap1 , the plk1 kinase , and the mus81-eme1 endonuclease . in all organisms studied to date , slx1 binds to the extreme c - terminal region of slx4 , which contains an evolutionarily conserved helix - turn - helix motif . interestingly , in vitro studies have shown that slx4 stimulates the endonuclease activities of slx1 , mus81-eme1 , and xpf - ercc1 ( hodskinson et al . , 2014 ; muoz et al . , 2009 ; wyatt et al . , the importance of slx4 is demonstrated by the observation that biallelic mutations in slx4 ( also known as fancp ) are associated with the cancer - prone disorder fanconi anemia ( bogliolo et al . , 2013 ; kim et al . , 2011 ; stoepker et al . , although the amino acid sequence of slx4 is evolutionarily diverse , the c - terminal region of all slx4 proteins contains a conserved c - terminal domain ( ccd ) that underpins the interaction with slx1 and a dna - binding sap domain found in many dna repair proteins ( andersen et al . , 2009 ; aravind and koonin , 2000 ; fekairi et al . , 2009 ; muoz et al . , 2009 ; svendsen et al . , , there are few other discernible domains , whereas slx4 proteins from higher eukaryotes ( e.g. , worms , flies , and humans ) contain one or two copies of a ubz family zinc - finger domain known as ubz4 ; the mei9 interaction like region ( mlr ) ; and a broad - complex , tramtrack , and bric - a - brac ( btb ) domain ( stogios et al . , 2005 ) . the role of slx1-slx4 in dna repair has been studied extensively ( cybulski and howlett , 2011 ; sarbajna and west , 2014 ; wyatt and west , 2014 ) . although deletion of slx1 in yeast does not affect the response to dna damage , it has been shown that slx1-slx4 plays a role in maintaining the integrity of ribosomal loci , which contain tandem repeats that frequently lead to replication fork arrest ( coulon et al . , 2004 ) . it is possible that slx1-slx4 is involved in the collapse of stalled forks and the resolution of recombination intermediates , such as holliday junctions ( hjs ) , after fork recapture ( gritenaite et al . , 2014 ) . in human cells , transient depletion of slx4 leads to an increased sensitivity to alkylating and crosslinking agents , indicating the importance of slx4 for the repair of dna inter - strand crosslinks ( icls ) and protein - dna adducts . depletion of slx4 also reduces the efficiency of double - strand break repair and leads to genome instability ( garner et al . , 2013 ; muoz et al . , 2009 ; sarbajna et al . , 2014 ; svendsen et al . , 2009 ; wechsler et al . , collectively , these observations indicate that slx1 and/or slx4 have relatively well - conserved roles in processing dna intermediates that arise at stalled or collapsed replication forks , particularly when cells are treated with dna - damaging agents that interfere with normal replication fork progression . purified s. cerevisiae slx1-slx4 cleaves various dna substrates in vitro , including splayed - arm structures , model replication forks , 5-flaps , and hjs ( fricke and brill , 2003 ) . for the 5-flap substrates , the major cleavage site lies in the 5 single - stranded arm at the junction between single- and double - stranded dna . whereas slx1 alone possesses weak nuclease activity , purified human slx1-slx4 exhibits related activities and cleaves 5-flaps , 3-flaps , replication forks , and hjs . of particular interest , slx1-slx4 and mus81-eme1 cooperate during hj resolution , with slx1-slx4 performing the initial nick such that mus81-eme1 can resolve the nicked hj without substrate dissociation ( wyatt et al . , 2013 ) . although there is a significant amount of functional information available for slx1-slx4 and its associated proteins , structural and mechanistic insights for this enzyme are lacking . here we report the crystal structure of the slx1 nuclease , obtained using candida glabrata slx1 ( cg - slx1 ) . the protein forms a compact structure with the giy - yig nuclease and ring - finger domains interacting with each other , and the structural arrangement is reinforced by a long helix . we find that cg - slx1 forms a stable homodimer in the absence of cg - slx4 . importantly , the crystal structure of cg - slx1 in complex with cg - slx4 , together with biochemical analyses , demonstrate that cg - slx1 homodimerization is mutually exclusive with the formation of a cg - slx1-slx4 heterodimer , revealing a likely regulatory mechanism for slx1 endonuclease activity . to obtain structural information for slx1 , we purified c. glabrata slx1 protein ( cg - slx1 ) alone and in complex with the conserved c - terminal domain of slx4 ( cg - slx1-slx4 ) . when the enzymatic activities of cg - slx1 and cg - slx1-slx4 were verified using synthetic dna substrates ( for sequences , see figures s1a and s1b ) , we found that cg - slx1 exhibited little or no activity , whereas the cg - slx1-slx4 complex cleaved a diverse set of branched dna structures ( figure 1 ) . the observed nuclease activity was inherent to slx1 , as substitution of an invariant residue in the active site ( slx1 ) abolished the nuclease activity of cg - slx1-slx4 ( figure 1b , bottom ) . we observed that the hj was the preferred substrate for cg - slx1-slx4 , followed by 5-flap , splayed arm , and 3-flap dna structures ( figures 1b [ top ] and 1c ) . in contrast , the enzyme failed to cleave gapped , nicked , and double- or single - stranded dna substrates . a similar substrate preference was reported for full - length yeast ( fricke and brill , 2003 ) and human ( wyatt et al . , 2013 ) enzymes , although cg - slx1-slx4 was comparatively more active on 3-flaps . enzyme titration experiments with the 5-flap substrate revealed that the primary cleavage product , a nicked or gapped dna duplex , was processed further to generate a shorter dna duplex ( figure 1a ) . however , time - course experiments carried out with limiting amounts of protein revealed that the major reaction was a single nick that converted the 5-flap into a linear duplex ( figure 1b ) . this is consistent with the inability of cg - slx1-slx4 to cleave nicked or gapped dna duplexes when protein is limiting . to gain more insight into the mechanism by which cg - slx1-slx4 cleaves 5-flaps , we prepared two different substrates , each p - labeled on a different oligonucleotide ( figures s1c and s1d ) . time - course experiments revealed two independent cleavage sites : a primary incision that removed the 5-flap , and a less efficient incision reaction that removed the duplex arm . collectively , our results provide additional evidence for the promiscuous nature of slx1-slx4 , suggesting that this biochemical property has been conserved throughout evolution . we next obtained crystals of cg - slx1 , which belonged to the p43212 space group , and diffracted x - rays to 2.34 resolution . the protein was predicted to contain a zinc finger , so diffraction data were collected at the zinc absorbance peak wavelength ( 1.280 ) , and the structure was solved by zinc single - wavelength anomalous diffraction ( sad ) ( table s1 ) . the asymmetric unit of the crystal contained one slx1 molecule and the structure was refined to an rfree of 25.1% ( table s1 ) . fragments of simulated annealing composite omit electron density maps are presented in figure s2a . we noted that the loop regions of the zinc - finger domain had higher b factors and less - well - defined electron density maps , indicating the flexibility of this part of the structure . the cg - slx1 monomer consists of two distinct domains : ( 1 ) an n - terminal giy - yig or uri nuclease domain , and ( 2 ) a c - terminal zinc - finger domain . in the cg - slx1 structure , these two domains interact with each other and form an oblong molecule approximately 70 long and 3040 wide ( figure 2 ) . the overall structure is reinforced by a long helix ( helix 6 , located between the two domains and comprising residues 176214 ) that spans the entire molecule and provides a scaffold for the two domains . the central element of the nuclease domain is a sheet consisting of five strands arranged 2-1-3-6-7 ( numbered in the amino acid sequence , figure s2c ) , with strands 2 and 3 oriented antiparallel to 1 , 6 , and 7 ( figure 2 ) . the sheet is flanked by six helices ; short helix 1 and long helix 6 are located on one side of the sheet and four helices ( 25 ) are positioned on the other side . the 25 region also contains a hairpin formed by strands 4 and 5 ( figure 2 ) . comparisons of cg - slx1 structure with related giy - yig nucleases can be found in the supplemental results and in figures s2d the active site of cg - slx1 is highly conserved with other giy - yig nucleases ( figure s2h ) , and we therefore propose that the catalytic mechanism of slx1 is identical to that described for hpy188i ( sokolowska et al . , 2011 ) . details of the organization of the active site and the catalytic mechanism proposed for cg - slx1 can be found in the supplemental results and in figures s2h and s2i . the c - terminal part of the cg - slx1 structure comprises a zinc - finger domain . our structure shows that it is a ring finger closely related to domains found in ubiquitin ligases ( supplemental results ; figures s2j inspection of the cg - slx1 crystal packing revealed that a 2-fold crystallographic axis generated a very tight protein dimer , with a total buried surface area of 4,729 ( figure 3a ) . at the dimer interface , helix 2 of one monomer was positioned snugly in a groove located between the nuclease and ring domains of the other subunit . notably , the side chain of arg72 , found at the n terminus of helix 2 , was inserted into a pocket on the surface of the other subunit , where it interacted with the backbone carbonyls of glu3 and gln5 . another prominent interaction that stabilized the homodimer involved tyr86 from the slx1-specific 4-5 hairpin of one subunit and helix 7 from the ring domain of the other subunit . contacts were also made between glu121 , tyr122 , and tyr86 from one subunit and thr271 , ile272 , and ile273 of the other . together , these residues formed a network of charged and van der waals interactions through their side chains . two symmetrical copies of this part of the interface were located close to each other and formed a continuous zipper of interdigitated amino acid side chains ( figure 3b ) . the extensive dimerization contacts and large buried surface area prompted us to investigate whether cg - slx1 dimerization occurs in solution . to do this , we used gel filtration coupled to multi - angle light scattering ( gf - mals ) ( figure 3c ) and analytical ultracentrifugation ( auc ) ( figure s3a ) . the molecular weight ( mw ) of cg - slx1 measured by gf - mals was 80.3 kda and 72.0 kda by auc , suggesting that cg - slx1 exists as a homodimer in solution ( calculated mw of 72.0 kda ) . first , we used gf - mals to determine that the mw of cg - slx4 alone was 19.0 kda . this is in agreement with the theoretical mw of 19.7 kda and indicates that cg - slx4 is monomeric in solution . for cg - slx1-slx4 , the measured mw was 57.0 kda ( gf - mals ) and 47.0 kda ( auc ) . importantly , these data indicate that the cg - slx1-slx4 complex contains one molecule of slx1 and one molecule of slx4 ( calculated mw of 57.0 kda ) . our results therefore demonstrate that cg - slx1 alone exists as a stable homodimer , but , in the presence of cg - slx4 , interacts with this subunit to form a stable heterodimeric complex . close inspection of the cg - slx1 homodimeric structure showed that the active site of each subunit was partially blocked by the ring domain of the other subunit ( figures 3a and s4 ) . indeed , cg - slx1 ( figure 1a ) as well as the s. cerevisiae ( fricke and brill , 2003 ) and human ( muoz et al . , 2009 ) enzymes are inactive in the absence of slx4 . to gain further insight into the cg - slx1 homodimerization , we prepared a series of cg - slx1 variants with substitutions in dimer interface residues . additional gel filtration experiments demonstrated that cg - slx1 homodimerization and cg - slx1-slx4 complex formation are mutually exclusive , and the exchange between the two forms can be promoted by high salt concentration ( supplemental results ; figures s3d and s3e ) . in summary , furthermore , they indicate that cg - slx1 dimerization provides a physical block to the active site . thus , these findings provide a potential mechanism by which slx1 nuclease activity is inhibited . nonetheless , cg - slx1-dna interactions can be modeled based on the conservation of the active site between slx1 and giy - yig restrictases , for which high - resolution protein - dna structures are available . to this end , we superimposed the eco29ki structures ( protein data bank [ pdb ] i d 3nic [ mak et al . , 2010 ] ) on our cg - slx1 structure , which resulted in a good fit between dna from the eco29ki structures and the surface of slx1 . the modeling revealed that cg - slx1 contains two groups of potential dna - binding residues , all but one of which are located in the nuclease domain ( figure 4a ) . the first group ( arg35 , arg38 , gln39 , and gln191 ) could interact with the non - cleaved dna strand toward its 5 end from the active site . based on the polarity of the dna at the active site , these residues are predicted to contact regions of flap and splayed arm substrates that contain only double - stranded dna ( figure 4b ) . arg35 , arg38 , and gln39 were all located in the vicinity of the phosphate groups of the modeled dna . sequence alignment of fungal slx1 proteins revealed that arg35 and gln39 are strictly conserved and arg38 is partially conserved ( figure s2c ) . non - conserved gln191 was also located in this region but was positioned farther from the substrate in our cg - slx1-dna model . the second group of predicted dna - binding residues ( arg72 , gln77 , his80 , and his84 ) was located on the opposite face of the active site relative to the first group . these residues are predicted to contact the 5 region of the cleaved strand that exists as single - stranded dna in flap and splayed arm substrates . in the eco29kl - dna structure , the straight dna duplex extended farther away from these residues , suggesting that the single - stranded portion of the splayed arm or 5-flap substrates must be bent to interact with the protein . gln77 , his80 , and his84 were all located in helix 2 very close to the metal - coordinating active site residue glu79 . among fungal slx1 proteins , although his80 is substituted by a tryptophan in most species , his84 is always replaced with a charged residue . interestingly , both arg72 and the region around his80 are buried in the cg - slx1 homodimer , which likely contributes to its inhibition ( figure s4a ) . to verify the importance of the potential dna - binding residues identified in our model , we prepared several cg - slx1 variants in which these residues were individually substituted with alanine . the cg - slx1 mutants were purified in the context of the cg - slx1-slx4 complex and enzyme activity was determined using the 5-flap substrate ( figure 4c ) . these mutants showed either a complete loss of nuclease activity ( r35a and q39a ) or substantially impaired activity ( r38a , r72a , q77a , q191a , h80 , and h84 ) . we also tested the ability of these slx1 variants to bind the 5-flap substrate using electrophoretic mobility shift assays ( emsas ) ( figure 4d ) . cg - slx1 alone did not exhibit dna - binding properties , while cg - slx1-slx4 formed a protein - dna complex with reduced mobility . at high protein concentrations , two protein - dna complexes were observed , possibly indicating the binding of two slx1 molecules to dna . the r38a and q39a mutants showed slightly stronger substrate binding compared to the wild - type protein , despite exhibiting severe catalytic defects . importantly , arg38 and gln39 are located close to the active site and gln39 forms a hydrogen bond with active site residue arg36 . its greatly reduced activity suggests that arg72 , which is located in the vicinity of the scissile phosphate , has a more important role in aligning the substrate for cleavage than enhancing substrate affinity . the remaining mutants , namely r35a , q77a , h80a , h84a , and q191a , all displayed defects in dna binding . his84 is located in helix 2 , which we postulate to participate in binding the single - stranded portion of the 5-flap dna . this result further underscores the importance of helix 2 in the dna - binding interface . we have therefore identified two regions of cg - slx1 that are important for protein - dna interactions : ( 1 ) one predicted to bind double - stranded portion of the dna , and ( 2 ) the other potentially interacting with the single - stranded flap . our initial crystallization trials with cg - slx1-slx4 did not yield any crystals , so to gain further insight into cg - slx1-slx4 interaction , we performed additional deletion studies . based on bioinformatic predictions and limited proteolysis experiments , we designed four truncated forms of slx4 , comprising residues 557685 ( cg - slx4 ) , 608685 ( cg - slx4 ) , 557698 ( cg - slx4 ) , or 647726 ( cg - slx4 ) ( figure all of these fragments were soluble when expressed in escherichia coli ( figure s5b ) , but only the ccd3 variant co - purified with cg - slx1 ( figures s5c and s5d ) . when we analyzed the activity of cg - slx1-slx4 on the 5-flap dna , we found that it displayed slightly less activity than cg - slx1-slx4 and that the cleavage sites for both ccd variants were similar ( figure s5e ) . they belonged to the p63 space group and diffracted x - rays to 1.8 ( table s1 ) . the structure was solved by molecular replacement using the cg - slx1 model and the structure of cg - slx4 was traced manually ( figures 5 and s2b ) . the overall conformation of cg - slx1 domains did not change significantly upon slx4 binding , although the location of the ring domain changed slightly ( figures s5f and s5 g ) . when cg - slx1 and cg - slx1-slx4 were superimposed using the giy - yig domains , the position of the ring domain residues differed by up to 3.5 . the 4-5 hairpin was not formed in the cg - slx1-slx4 complex and this part of the structure adopted a different conformation ( figure 5a ) . its closest structural homologs are ff domains mediating protein - protein interactions ( bedford and leder , 1999 ) . for example , prp40 ff1 domain ( pdb i d 2b7e [ gasch et al . , 2006 ] ) can be superimposed on cg - slx4 with an rmsd of 1.6 over 37 c atoms . notably , however , the characteristic phenylalanine residues of the ff domain are not conserved in cg - slx4 . cg - slx4 was positioned in a cleft between the nuclease and ring domains , and most of the interactions between the cg - slx1 and cg - slx4 occurred through hydrophobic contacts ( figure 5b ) . in particular , the second short helix of cg - slx4 was placed snugly in a hydrophobic groove on the cg - slx1 surface . the groove lined up with the side chains of cg - slx1 phe4 ( nuclease domain ) and tyr257 , ile292 , and ile273 ( ring domain ) . the side chain of cg - slx4 phe681 , which is conserved in most fungal slx4 sequences ( figure s5a ) and is located in the second short helix , was inserted into a pocket on the cg - slx1 surface . the hydrophobic interactions were reinforced further by stacking of cg - slx1 tyr257 with cg - slx4 tyr679 . in addition , the backbone carbonyl of this tyrosine formed a hydrogen bond with the n1 of the conserved trp288 in cg - slx1 . additional residues at the cg - slx1-slx4 interface included cg - slx1 ile73 , tyr122 , and tyr125 , all of which interacted with the c terminus of the last helix of cg - slx4 and the following loop ( residues 714720 ) . the importance of the latter interaction was confirmed by the lack of binding between cg - slx1 and slx4 , in which the c - terminal helix of the ccd is absent . in murine slx4 , the c1536r mutation abolished its interaction with slx1 ( castor et al . , 2013 ) . this cysteine corresponds to cg - slx4 gly716 located at the c terminus of the last helix of cg - slx4 . insertion in this position of an arginine residue with a large side chain would lead to steric clashes with cg - slx1 , explaining the effect of c1536r mutation . importantly , the position of cg - slx4 in the slx1-slx4 heterodimer overlapped with the position of one of the slx1 monomers in the cg - slx1 homodimer ( figures s5f and s5 g ) . in fact , several residues , including cg - slx1 tyr122 and ile273 , participated in both cg - slx1 homodimerization and cg - slx1-slx4 heterodimerization , thus explaining why the formation of cg - slx1 homodimeric and cg - slx1-slx4 heterodimeric complexes are mutually exclusive events . slx4 was located away from the predicted dna - binding interface of cg - slx1 and is not expected to form contacts with the substrate ( figure s4b ) . in conclusion , the studies presented here provide the first structural information for slx1 and its interaction with slx4 . structure - selective endonucleases pose an inherent threat to genome integrity because broken dna ends can facilitate chromosome rearrangements and genome alterations that are potentially tumorigenic . it is therefore crucial to keep these endonucleases tightly regulated to ensure cleavage of the correct dna substrate at an appropriate time in the cell cycle . recent studies have revealed that these regulatory mechanisms operate at various levels : protein expression ( courcelle et al . , 2001 ) , post - translation modification ( mus81-eme1/mms4 , yen1 ) ( blanco et al . , 2014 ; matos et al . , 2011 , 2013 ) , nuclear localization ( gen1 ) ( chan and west , 2014 ) , conformational changes ( fen1 ) ( kim et al . , 2001 ; storici et al . , 2002 ) , and protein - protein interactions ( e.g. , xpg , xpf - ercc1 , and slx - mus ) ( arajo et al . , 2001 ; li et al . , 1994 ; our data suggest a mechanism of slx1 regulation through inhibitory homodimerization that would keep slx1 , a promiscuous and potentially dangerous endonuclease , latent and ensure that its activity is tightly regulated in cells . purified cg - slx1 , at a concentration of 10 mg / ml , was subjected to crystallization screens at 18c using the sitting - drop vapor diffusion method . prior to crystallization , the protein was dialyzed against buffer containing 20 mm hepes - naoh ( ph 7.5 ) , 350 mm nacl , and 1 mm dtt . crystals were obtained with 1.4 m sodium citrate tribasic dihydrate and 0.1 m hepes - naoh ( ph 7.5 ) . purified cg - slx1-slx4 , at a concentration of 10 mg / ml , was subjected to crystallization screens at room temperature using the sitting - drop vapor diffusion method . prior to crystallization , the protein was dialyzed against buffer containing 20 mm hepes - naoh ( ph 7.5 ) , 150 mm nacl , and 1 mm dtt . crystals were obtained with 0.2 m magnesium chloride hexahydrate , 0.1 m bis tris ( ph 6.5 ) , and 25% ( v / v ) peg 3350 in the presence of a splayed arm dna substrate with 15-bp double - stranded portion and 5-nt single - stranded overhangs . x - ray diffraction data for cg - slx1 were collected at beamline 14.1 at berliner elektronenspeicherring - gesellschaft fr synchrotronstrahlung ( bessy ) ( mueller et al . , 2012 ) , and data for cg - slx1-slx4 were collected at beamline id29 at european synchrotron radiation facility ( esrf ) . data for the phasing and refinement of cg - slx1 structure were collected at 1.280 wavelength . diffraction data used for refinement of the cg - slx1-slx4 structure were collected at 0.97626 . phases for cg - slx1 were determined using single - wavelength anomalous diffraction in autosol module in phenix ( adams et al . , 2010 ) , using data collected at zn peak wavelength ( 1.280 ) . phases for cg - slx1-slx4 were determined by molecular replacement using phaser - mr module in phenix ( adams et al . , 2010 ) . the structure of cg - slx1 was used as the starting model for molecular replacement . interactive model building was performed in coot ( emsley et al . , 2010 ) and refinement with phenix with r - free , calculated with 5% of unique reflections . the structures of cg - slx1 and cg - slx1-slx4 were refined with 95.7% and 99.7% residues in the favored region of the ramachandran plot , respectively . structure validation was carried out using molprobity analysis ( chen et al . , 2010 ) . structural analyses , including superpositions , and structural figures were prepared in pymol ( http://www.pymol.org ) . simulated annealing composite omit maps were calculated in cns 1.3 ( brnger et al . , 1998 ) . description of protein purification , oligomeric state analysis , nuclease assays , and emsa can be found in the supplemental experimental procedures .

backgroundpediatric pelvic fractures are associated with high - energy trauma and injury to other systems , leading to an increased incidence of complication and mortality . previous studies analyzed the pediatric population as a whole , including both children and adolescents . the purpose of this study was to examine whether adolescents with pelvic fracture have different complication and mortality rates compared to younger children and adults.methodsusing the national trauma data bank , 37,784 patients below the age of 55 years with pelvic fractures were identified and divided into children ( age < 13 years ) , adolescents ( age 1317 years ) , and adults ( age > 17 years ) . descriptive statistics and bivariate and multivariate analyses were performed.resultschildren had an increased odds of death [ odds ratio ( or ) 2.29 , 95 % confidence interval ( ci ) 1.962.67 ] and complications ( or 1.36 , 95 % ci 1.201.55 ) , whereas adolescents had a decrease in odds of death ( or 0.89 , 95 % ci 0.741.06 ) and complications ( or 0.70 , 95 % ci 0.610.81 ) compared to the adult population.conclusionsadolescents with pelvic fractures exhibit a different physiologic response to the children and adult populations . this emphasizes the need to distinguish these subpopulations in future epidemiological research and treatment planning .

pelvic fractures comprise < 0.2 % of all pediatric fractures but constitute up to 5 % of admissions to level i pediatric trauma centers . like adults , pediatric pelvic fractures are associated with high - energy trauma and injury to other systems , leading to an increased incidence of complication and mortality [ 24 ] . due to the pliable nature of the child s skeleton , severe soft tissue trauma may occur without producing skeletal injury [ 4 , 5 ] . thus , a child presenting with a pelvic fracture should be suspected of having a multiorgan injury with an increased potential for injury , complications , and death [ 6 , 7 ] . during the transition period between childhood and adulthood , known as adolescence , children gain size and strength , making their body increasingly resistant to extrinsic injury . however , previous studies on pediatric pelvic fractures analyzed the pediatric population as a single group of both children and adolescents [ 2 , 3 , 711 ] . biological , psychological , social , and environmental changes influence the onset and termination of adolescence . physiologic changes that occur during adolescence , such as increase in muscle size and bone mass , doubling in heart size and lung vital capacity , and rise in blood pressure , blood volume , and hematocrit ( particularly in boys ) , may lead to a theoretical protective effect against trauma . similar to growing children , adolescents have an osteoblast : osteoclast activity ratio > 1 , which increases their capacity to heal fractures . these physiological advantages , together with the increase in distance between internal organs , weight to surface area ratio , and cardiovascular reserve , may make adolescents particularly resilient to traumatic injury . the purpose of this study is to examine whether adolescents with pelvic fracture have different complication and mortality rates compared to younger children and adults . we identified the study population by means of the national trauma data bank ( ntdb version 7.1 ) . this ntdb version contained over 2.7 million cases from over 900 us trauma centers between the years 2002 and 2006 . the data were imported and merged into a single dataset from the 13 ntdb files using sas version 9.2 ( sas institute , cary , nc ) . for the purpose of our study , all burn or penetrating injuries were excluded , which reduced the group to 1.7 million cases . if a patient s multiple icd-9 diagnosis codes contained at least one of the following codes , the patient was considered to have a pelvic fracture : 808.2 , 808.3 , 808.4 , 808.41 , 808.42 , 808.43 , 808.49 , 808.5 , 808.51 , 808.52 , 808.53 , 808.59 , 808.8 , 808.9 . acetabular fractures were excluded from the study . those entries without a pelvic fracture were removed , giving a total of 54,459 cases of pelvic fractures . finally , all adults aged 55 years and older were removed from the study , yielding a final study population of 37,784 . this final study population was subdivided into our three groups of interest : children ( younger than 13 years old ) , adolescents ( aged 1317 years ) , and adults ( aged 1854 years ) . severe complication was defined as having at least one of the following complications recorded : renal failure , pneumonia , bacteremia , acute respiratory distress syndrome ( ards ) , deep vein thrombosis ( dvt ) , or a pulmonary embolism . prehospital risk factors such as sex , race , age , arrival in shock ( systolic blood pressure < 90 mmhg ) , injury severity score ( iss ) , head injury , and mechanism of injury were also analyzed to determine their association with the main outcomes . descriptive statistics were performed on the entire study population ( n = 37,784 ) . to determine associations between risk factors and the main outcomes of interest , bivariate analysis was conducted between each prehospital risk factor and two main outcomes ( mortality and severe complication ) . for this and subsequent assessment , open fractures ( only 4.2 % of pelvic fractures and highly variable in presentation and complication profile ) and unknown mechanisms of injury were excluded , yielding a study group of 24,684 . a subpopulation of severe pelvic injuries was also created to determine if any pediatric subgroup has better outcomes . for this population , an abbreviated injury scale ( ais ) score of <3 , signifying minor and moderate injuries , and all patients with lower extremity fractures besides pelvic fractures were excluded . this created a study subgroup of 2,487 patients with isolated severe pelvic fractures . for both the main study population and the subgroup of severe pelvic injury , prehospital risk factors were determined to be significant using the mantel this method produced crude odds ratios ( ors ) and 95 % confidence intervals ( cis ) , and those variables of significance were included for the multivariate analysis . logistic regression analyses including those variables of significance were performed to determine the association between the prehospital risk factors and the two main outcomes . each model included a specific age group ( children , adolescent , and adult ) as compared to all other ages in order to assess the importance of age and outcome after sustaining a pelvic fracture . the hosmer lemeshow goodness - of - fit test was performed on each model to determine whether or not the observed event rates matched the expected event rates . descriptive statistics were performed on the entire study population ( n = 37,784 ) . to determine associations between risk factors and the main outcomes of interest , bivariate analysis was conducted between each prehospital risk factor and two main outcomes ( mortality and severe complication ) . for this and subsequent assessment , open fractures ( only 4.2 % of pelvic fractures and highly variable in presentation and complication profile ) and unknown mechanisms of injury were excluded , yielding a study group of 24,684 . a subpopulation of severe pelvic injuries was also created to determine if any pediatric subgroup has better outcomes . for this population , an abbreviated injury scale ( ais ) score of <3 , signifying minor and moderate injuries , and all patients with lower extremity fractures besides pelvic fractures were excluded . this created a study subgroup of 2,487 patients with isolated severe pelvic fractures . for both the main study population and the subgroup of severe pelvic injury , prehospital risk factors were determined to be significant using the mantel this method produced crude odds ratios ( ors ) and 95 % confidence intervals ( cis ) , and those variables of significance were included for the multivariate analysis . logistic regression analyses including those variables of significance were performed to determine the association between the prehospital risk factors and the two main outcomes . each model included a specific age group ( children , adolescent , and adult ) as compared to all other ages in order to assess the importance of age and outcome after sustaining a pelvic fracture . the hosmer lemeshow goodness - of - fit test was performed on each model to determine whether or not the observed event rates matched the expected event rates . our study s overall incidence rate of pelvic fractures was 26 cases per 10,000 trauma admissions per year for adults , 4 cases per 10,000 trauma admissions per year for adolescents , and 5 cases per 10,000 trauma admissions per year for children . severe pelvic fractures had an incidence rate of 3.5 cases per 10,000 per year for adults , 0.47 cases per 10,000 per year for adolescents , and 0.63 cases per 10,000 per year for children . descriptive statistics of demographics , injury mechanism , injury severity , treatment , and complications are presented in table 1 . after sustaining a pelvic fracture , survival was 91.4 % for the total population . when separated by age group , adolescents appeared to have the best survival statistics ( 93.2 % ) , while children had the worst ( 89.8 % ) . adolescents had the lowest percentage of severe complication ( 11.4 % ) , children had a slightly higher percentage ( 13.0 % ) , and adults had the highest percentage ( 15.7 % ) .table 1frequency distribution of characteristics for the entire population of pelvic fractures , n = 37,784variableage grouptotal , n ( % ) children ( < 13 years ) , n ( % ) , n = 5,325adolescents ( 1317 years ) , n ( % ) , n = 4,052adults ( 1854 years ) , n ( % ) , n = 28,407gender male2,349 ( 44.8)1,888 ( 46.7)17,393 ( 61.3)21,630 ( 57.4 ) female2,897 ( 55.2)2,157 ( 53.3)10,983 ( 38.7)16,037 ( 42.6)race caucasian3,570 ( 72.3)2,745 ( 73.4)18,301 ( 69.9)24,616 ( 70.7 ) african american474 ( 9.6)341 ( 9.1)2,717 ( 10.4)3,532 ( 10.1 ) asian / pacific islander73 ( 1.5)54 ( 1.4)504 ( 1.9)631 ( 1.8 ) hispanic490 ( 9.9)394 ( 10.6)3002 ( 11.5)3,886 ( 11.2 ) native american31 ( 0.6)31 ( 0.9)201 ( 0.8)263 ( 0.8 ) other299 ( 6.1)173 ( 4.6)1,440 ( 5.5)1,912 ( 5.4)survival survived4,766 ( 89.8)3,753 ( 93.2)25,853 ( 91.5)34,372 ( 91.4 ) died542 ( 10.2)273 ( 6.8)2,404 ( 8.5)3,219 ( 8.6)severe complication yes693 ( 13.0)462 ( 11.4)4,465 ( 15.7)5,620 ( 14.9 ) no4,632 ( 87.0)3,590 ( 88.6)23,942 ( 84.3)32,164 ( 85.1)fracture type open183 ( 3.4)96 ( 2.4)1,316 ( 4.6)1,595 ( 4.2 ) closed5,142 ( 96.6)3,956 ( 97.6)27,078 ( 95.4)36,176 ( 95.8)fracture location ilium703 ( 13.7)622 ( 15.7)3,797 ( 13.9)5,122 ( 14.1 ) ischium162 ( 3.1)153 ( 3.9)1,151 ( 4.2)1,466 ( 4.0 ) pubis2,615 ( 42.0)1,632 ( 41.2)11,281 ( 41.4)15,078 ( 41.5 ) multiple330 ( 6.4)220 ( 5.5)1,799 ( 6.6)2,349 ( 6.5 ) unspecified673 ( 13.1)342 ( 8.6)2,328 ( 8.6)3,343 ( 9.2 ) other1,120 ( 21.7)996 ( 25.1)6,878 ( 25.3)8,994 ( 24.7)systolic blood pressure ( mmhg ) < 90878 ( 16.5)524 ( 12.9)4,615 ( 16.3)6,017 ( 15.9 ) 901392,869 ( 53.9)2,680 ( 66.2)16,704 ( 58.8)22,253 ( 58.9 ) 140 + 1,578 ( 29.6)848 ( 20.9)7,088 ( 24.9)9,514 ( 25.2)moi motor vehicle1,329 ( 24.9)2,224 ( 54.9)11,520 ( 40.6)15,073 ( 39.9 ) motorcycle119 ( 2.2)91 ( 2.3)2,674 ( 9.4)2,884 ( 7.6 ) pedestrian170 ( 3.2)102 ( 2.5)566 ( 1.9)838 ( 2.2 ) crush93 ( 1.8)31 ( 0.7)732 ( 2.6)856 ( 2.3 ) high fall592 ( 11.1)108 ( 2.6)2,680 ( 9.5)3,380 ( 9.0 ) low fall1,216 ( 22.8)63 ( 1.6)1,393 ( 4.9)2,672 ( 7.1 ) unknown1,806 ( 34.0)1,433 ( 35.4)8,842 ( 31.1)12,081 ( 31.9)ais of pelvis none3,323 ( 62.4)2,660 ( 65.6)19,331 ( 68.1)25,314 ( 67.0 ) 19 ( 0.2)11 ( 0.3)96 ( 0.3)116 ( 0.3 ) 21,313 ( 24.6)869 ( 21.5)5,153 ( 18.2)7,335 ( 19.4 ) 3610 ( 11.5)450 ( 11.1)3,129 ( 11.0)4,189 ( 11.1 ) 453 ( 1.0)45 ( 1.1)522 ( 1.8)620 ( 1.6 ) 517 ( 0.3)17 ( 0.4)176 ( 0.6)210 ( 0.6 ) 60 ( 0.0)0 ( 0.0)0 ( 0.0)0 ( 0.0)iss < 254,338 ( 81.5)2,816 ( 69.5)19,721 ( 69.4)26,875 ( 71.1 ) 25987 ( 18.5)1,236 ( 30.5)8,686 ( 30.6)10,909 ( 28.9)pneumonia yes102 ( 1.9)125 ( 3.1)1,232 ( 4.3)1,459 ( 3.9 ) no5,223 ( 98.1)3,927 ( 96.9)27,175 ( 95.7)36,325 ( 96.1)acute respiratory stress syndrome yes61 ( 1.1)55 ( 1.4)573 ( 2.0)689 ( 1.8 ) no5,264 ( 98.9)3,997 ( 98.6)27,834 ( 98.0)37,095 ( 98.2)deep vein thrombosis yes27 ( 0.5)21 ( 0.5)503 ( 1.8)551 ( 1.5 ) no5,298 ( 99.5)4,031 ( 99.5)27,904 ( 98.2)37,233 ( 98.5)bacteremia yes10 ( 0.2)26 ( 0.6)196 ( 0.7)232 ( 0.6 ) no5,315 ( 99.8)4,026 ( 99.4)28,211 ( 99.3)37,501 ( 99.4)renal failure yes32 ( 0.6)15 ( 0.4)225 ( 0.8)272 ( 0.7 ) no5,293 ( 99.4)4,037 ( 99.6)28,182 ( 99.2)37,512 ( 99.3)pulmonary embolism yes15 ( 0.3)5 ( 0.1)160 ( 0.6)180 ( 0.4 ) no5,310 ( 99.7)4,047 ( 99.9)28,247 ( 99.4)37,604 ( 99.6)icu stay < 1 week4,890 ( 91.8)3,545 ( 87.5)24,086 ( 84.8)32,521 ( 86.1 ) 1 week435 ( 8.2)507 ( 12.5)4,321 ( 15.2)5,263 ( 13.9)hospital stay < 1 week3,727 ( 70.0)2,406 ( 59.4)13,778 ( 48.5)19,911 ( 52.7 ) 1 week1,598 ( 30.0)1,646 ( 40.6)14,629 ( 51.5)17,873 ( 47.3)major procedure crif1 ( 0.02)2 ( 0.05)20 ( 0.07)23 ( 0.08 ) orif245 ( 4.62)395 ( 9.71)4,691 ( 16.47)5,331 ( 14.1 ) closed dislocation reduction7 ( 0.13)19 ( 0.47)117 ( 0.41)143 ( 0.39 ) open dislocation reduction12 ( 0.23)15 ( 0.37)127 ( 0.45)154 ( 0.43 ) no major procedure5,060 ( 95.0)3,621 ( 89.4)23,452 ( 82.6)32,133 ( 85.0)moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation frequency distribution of characteristics for the entire population of pelvic fractures , n = 37,784 moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation most of the pelvic fractures were closed ( 95.8 % ) . pubic rami fractures consistently had the highest prevalence across all age groups ( 41.5 % ) . the most common known mechanism of injury was motor vehicle accident ( 39.9 % ) , followed by high - energy fall ( 9.0 % ) and motorcycle accident ( 7.6 % ) . most patients had an iss < 25 ( 71.1 % ) . when divided by age , more children had an iss < 25 ( 81.5 % ) than adolescents ( 69.5 % ) or adults ( 69.4 % ) . adolescents also had less reports of hypovolemic shock on arrival ( systolic pressures below 90 mmhg ) ( 12.9 % ) than children ( 16.5 % ) and adults ( 16.3 % ) who had comparable numbers . for this assessment , open fractures and unknown mechanisms of injury were excluded , yielding a study group of 24,684 . the results of the bivariate analysis ( reporting crude ors and 95 % cis ) for this population are depicted in table 2 . as compared to the rest of the population , children had a small increase in odds of death ( or 1.27 , 95 % ci 1.111.44 ) , but a slight decrease in odds of severe complication ( or 0.83 , 95 % ci 0.750.93 ) . adolescents had decreases in both odds of death ( or 0.85 , 95 % ci 0.721.00 ) and severe complication ( or 0.78 , 95 % ci 0.690.89 ) . adults had a slight decrease in odds of death ( or 0.92 , 95 % ci 0.821.02 ) , but a small increase in odds of severe complication ( or 1.27 , 95 % ci 1.161.39 ) . individuals suffering from hypovolemic shock had substantially increased odds of death ( or 5.89 , 95 % ci 5.326.51 ) and severe complication ( or 3.62 , 95 % ci 3.333.93 ) . sustaining a head injury also increased the odds of death ( or 2.96 , 95 % ci 2.593.39 ) and complication ( or 3.23 , 95 % ci 2.903.61 ) . an iss 25 greatly increased the odds of death ( or 11.1 , 95 % ci 9.9212.5 ) and complication ( or 9.19 , 95 % ci 8.489.97 ) . motor vehicle accidents caused the largest increase in odds of death ( or 1.46 , 95 % ci 1.321.62 ) and severe complication ( or 1.53 , 95 % ci 1.421.65).table 2bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) , n = 24,684predictorsoutcomedeathsevere complicationmale1.24 ( 1.131.37)1.41 ( 1.311.52)not caucasian1.14 ( 1.041.26)1.00 ( 0.921.08)pediatric ( < 18 years)1.09 ( 0.981.21)0.79 ( 0.720.86)children ( < 13 years)1.27 ( 1.111.44)0.83 ( 0.750.93)adolescent ( 1317 years)0.85 ( 0.721.00)0.78 ( 0.690.89)adults ( 1854 years)0.92 ( 0.821.02)1.27 ( 1.161.39)hypovolemic shock5.89 ( 5.326.51)3.62 ( 3.333.93)head injury2.96 ( 2.593.39)3.23 ( 2.903.61)iss 2511.1 ( 9.9212.5)9.19 ( 8.489.97)motor vehicle accident1.46 ( 1.321.62)1.53 ( 1.421.65)motorcycle accident1.34 ( 1.161.54)1.34 ( 1.201.49)fall from height0.61 ( 0.520.72)0.61 ( 0.540.69)low - energy fall0.41 ( 0.330.51)0.36 ( 0.300.43)crush0.43 ( 0.290.63)0.66 ( 0.520.84)pedestrian versus auto1.40 ( 1.111.77)1.13 ( 0.931.38)iss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) , n = 24,684 iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes . adjusted ors and 95 % cis are presented in table 3 . when controlling for mechanism of injury and other prehospital conditions , children had more than twice the odds of death ( or 2.29 , 95 % ci 1.962.67 ) and increased odds of severe complication ( or 1.36 , 95 % ci 1.201.55 ) relative to the adult group after sustaining a pelvic fracture . adolescents , on the other hand , had a slight decrease in odds of death ( or 0.89 , 95 % ci 0.741.06 ) and lower odds of severe complication ( or 0.70 , 95 % ci 0.610.81 ) relative to the adult group after sustaining a pelvic fracture.table 3multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)model predictorsoutcomedeath , n = 24,684severe complication , n = 22,846children ( < 13 years)2.29 ( 1.962.67)1.36 ( 1.201.55)adolescents ( 1317 years)0.89 ( 0.741.06)0.70 ( 0.610.81)adults ( 1854 years)referentreferentmale1.19 ( 1.071.33)1.38 ( 1.261.50)not caucasian1.00 ( 0.901.12)hypovolemic shock4.52 ( 4.055.05)2.81 ( 2.563.08)head injury1.60 ( 1.371.86)1.70 ( 1.501.92)iss 259.15 ( 8.0710.4)7.41 ( 6.798.09)motor vehicle accident3.98 ( 0.3940.2)1.13 ( 0.921.41)motorcycle accident4.61 ( 0.4646.8)1.18 ( 0.921.51)fall from height3.27 ( 0.3233.1)0.80 ( 0.631.03)low - energy fall3.18 ( 0.3231.7)0.77 ( 0.581.02)pedestrian versus auto4.50 ( 0.4544.8)crush2.42 ( 0.2325.3)0.95 ( 0.691.34)hosmer lemeshow0.00690.17bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or deathhosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death hosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval a subgroup analysis was performed for patients determined to have a severe , isolated , closed pelvic injury ( ais 3 ) . table 4 demonstrates the descriptive statistics for this subpopulation ( n = 2,487 ) . adolescents ( n = 255 ) still appeared to have a lower incidence of mortality ( 3.1 % ) and lower incidence of a severe complication ( 8.2 % ) compared to adults ( n = 1,886 ) and children ( n = 346 ) . adolescents contributed the most to motor vehicle accident ( 89.4 % ) , which was the most common mechanism of injury in the severe pelvic fracture group . similar to the whole population , the severe pelvic injury group demonstrated more children with an iss < 25 ( 82.1 % ) . for cases of hypovolemic shock on arrival , adolescents had the fewest reports of systolic pressures below 90 mmhg ( 6.7 % ) , while children ( 15.9 % ) had the largest contribution . children had the lowest incidence of hospital stay and intensive care unit ( icu ) admission , while adolescents and adults were relatively comparable.table 4frequency distribution of characteristics for individuals with isolated , closed , severe pelvic fractures ( ais3 ) , n = 2,487variableage grouptotal , n ( % ) children ( < 13 years ) , n ( % ) , n = 346adolescents ( 1317 years ) , n ( % ) , n = 255adults ( 1854 years ) , n ( % ) , n = 1,886gender male127 ( 38.7)92 ( 36.2)1,109 ( 58.9)1,328 ( 53.8 ) female201 ( 61.3)162 ( 63.8)775 ( 41.1)1,138 ( 46.2)race caucasian235 ( 77.1)168 ( 74.3)1,208 ( 73.0)1,611 ( 73.7 ) african american31 ( 10.2)30 ( 13.3)170 ( 10.3)231 ( 10.6 ) asian / pacific islander4 ( 1.3)1 ( 0.4)24 ( 1.5)29 ( 1.3 ) hispanic29 ( 9.5)20 ( 8.9)200 ( 12.1)249 ( 11.4 ) native american1 ( 0.3)2 ( 0.9)7 ( 0.4)10 ( 0.5 ) other5 ( 1.6)5 ( 2.2)46 ( 2.7)56 ( 2.5)survival survived321 ( 92.8)247 ( 96.9)1,774 ( 94.1)2,342 ( 94.2 ) died25 ( 7.2)8 ( 3.1)112 ( 5.9)145 ( 5.8)severe complication yes32 ( 9.3)21 ( 8.2)209 ( 11.1)262 ( 10.5 ) no314 ( 90.8)234 ( 91.8)1,677 ( 88.9)2,225 ( 89.5)fracture location ilium41 ( 11.9)47 ( 18.4)228 ( 12.1)316 ( 12.7 ) ischium7 ( 2.0)7 ( 2.8)53 ( 2.8)67 ( 2.7 ) pubis222 ( 64.2)143 ( 56.1)1,059 ( 56.2)1,424 ( 57.3 ) multiple31 ( 9.0)30 ( 11.8)277 ( 14.7)338 ( 13.6 ) unspecified10 ( 2.9)4 ( 1.6)70 ( 3.7)84 ( 3.4 ) other35 ( 10.0)24 ( 9.3)199 ( 10.5)258 ( 10.3)systolic blood pressure ( mmhg ) < 9055 ( 15.9)17 ( 6.7)176 ( 9.3)248 ( 10.0 ) 90139173 ( 50.0)178 ( 69.8)1,197 ( 63.5)1,548 ( 62.2 ) 140 + 118 ( 34.1)60 ( 23.5)513 ( 27.2)691 ( 27.8)moi motor vehicle127 ( 36.7)228 ( 89.4)1,094 ( 58.0)1,449 ( 58.3 ) motorcycle17 ( 4.9)6 ( 2.4)196 ( 10.4)219 ( 8.8 ) pedestrian17 ( 4.9)5 ( 2.0)70 ( 3.7)92 ( 3.7 ) crush13 ( 3.8)5 ( 2.0)101 ( 5.4)119 ( 4.8 ) high fall52 ( 15.0)4 ( 1.5)248 ( 13.2)304 ( 12.2 ) low fall120 ( 34.7)7 ( 2.7)177 ( 9.3)304 ( 12.2)ais of pelvis 3321 ( 92.7)231 ( 90.6)1,606 ( 85.2)2,158 ( 86.8 ) 420 ( 5.8)17 ( 6.6)225 ( 11.9)262 ( 10.5 ) 55 ( 1.5)7 ( 2.8)55 ( 2.9)67 ( 2.7)iss < 25284 ( 82.1)169 ( 66.3)1,308 ( 69.4)1,761 ( 70.8 ) 2562 ( 17.9)86 ( 33.7)578 ( 30.6)726 ( 29.2)pneumonia yes3 ( 0.9)12 ( 4.7)53 ( 2.8)68 ( 2.7 ) no343 ( 99.1)243 ( 95.3)1,833 ( 97.2)2,419 ( 97.3)acute respiratory stress syndrome yes1 ( 0.3)1 ( 0.4)28 ( 1.5)30 ( 1.2 ) no345 ( 99.7)254 ( 99.6)1,858 ( 98.5)2,457 ( 98.8)deep vein thrombosis yes0 ( 0.0)1 ( 0.4)19 ( 1.0)20 ( 0.8 ) no346 ( 100.0)254 ( 99.6)1,867 ( 99.0)2,467 ( 99.2)bacteremia yes1 ( 0.3)1 ( 0.4)6 ( 0.3)8 ( 0.3 ) no345 ( 99.7)254 ( 99.6)1,880 ( 99.7)2,479 ( 99.7)renal failure yes2 ( 0.6)1 ( 0.4)7 ( 0.4)10 ( 0.4 ) no344 ( 99.4)254 ( 99.6)1,879 ( 99.6)2,477 ( 99.6)pulmonary embolism yes1 ( 0.3)0 ( 0.0)7 ( 0.4)8 ( 0.3 ) no345 ( 99.7)255 ( 100.0)1,879 ( 99.6)2,479 ( 99.7)icu stay < 1 week326 ( 94.2)224 ( 87.8)1,676 ( 88.9)2,226 ( 89.5 ) 1 week20 ( 5.8)31 ( 12.2)210 ( 11.1)261 ( 10.5)hospital stay < 1 week256 ( 74.0)151 ( 59.2)1,011 ( 53.6)1,418 ( 57.0 ) 1 week90 ( 26.0)104 ( 40.8)875 ( 46.4)1,069 ( 43.0)major procedure crif0 ( 0.0)0 ( 0.0)1 ( 0.1)1 ( 0.1 ) orif31 ( 9.0)45 ( 17.6)501 ( 26.5)577 ( 23,1 ) closed dislocation reduction0 ( 0.0)2 ( 0.8)7 ( 0.4)9 ( 0.4 ) open dislocation reduction0 ( 0.0)0 ( 0.0)13 ( 0.7)13 ( 0.5 ) no major procedure315 ( 91.0)208 ( 81.6)1,364 ( 72.3)1,887 ( 75.9)moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation frequency distribution of characteristics for individuals with isolated , closed , severe pelvic fractures ( ais3 ) , n = 2,487 moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation similar to what was done with the whole population , bivariate analysis was also performed on the severe pelvic fracture subpopulation ( table 5 ) . as compared to the adults , children had higher odds of death but lower odds of severe complication . however , adolescents had a crude decrease in odds of death and severe complication after sustaining a pelvic fracture compared to the adults . individuals suffering from hypovolemic shock , head injury , and iss 25 had an increase in odds of death and severe complications . motor vehicle accidents caused the largest increase in odds of death ( or 1.58 , 95 % ci 1.102.26 ) , while motorcycle accidents accounted for the largest increase in severe complication ( or 1.58 , 95 % ci 1.062.34).table 5bivariate analysis for isolated closed pelvic fractures with an associated ais 3 , comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)predictorsoutcomedeath , n = 2,487severe complication , n = 2,487male1.11 ( 0.791.56)1.60 ( 1.222.08)not caucasian1.53 ( 1.092.15)1.26 ( 0.971.64)pediatric ( < 18 years)0.92 ( 0.621.37)0.78 ( 0.571.06)children ( < 13 years)1.31 ( 0.842.05)0.85 ( 0.571.25)adolescent ( 1317 years)0.50 ( 0.241.02)0.74 ( 0.471.18)adults ( 1854 years)1.09 ( 0.731.62)1.29 ( 0.941.77)hypovolemic shock13.0 ( 9.0618.6)7.35 ( 5.449.93)head injury4.41 ( 2.946.60)5.91 ( 4.288.16)iss 2527.5 ( 9.9212.5)14.9 ( 10.720.7)motor vehicle accident1.58 ( 1.102.26)1.32 ( 1.011.72)motorcycle accident1.62 ( 0.982.67)1.58 ( 1.062.34)fall from height0.63 ( 0.341.16)0.74 ( 0.481.13)low - energy fall0.095 ( 0.0230.38)0.35 ( 0.200.61)crush0.85 ( 0.371.97)0.95 ( 0.521.75)pedestrian versus auto1.33 ( 0.602.93)1.33 ( 0.602.93)bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death bivariate analysis for isolated closed pelvic fractures with an associated ais 3 , comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes ( table 6 ) . when controlling for mechanism of injury and other prehospital conditions and using the adult group as a referent , children had nearly double the odds of death after sustaining a severe pelvic fracture , whereas adolescents had a large decrease in odds of death ( or 0.40 , 95 % ci 0.180.91 ) . compared to adults , children had potentially increased odds of severe complication after sustaining a severe pelvic fracture ( or 1.07 , 95 % ci 0.651.75 ) , whereas adolescents had potentially decreased odds of severe complication ( or 0.66 , 95 % ci 0.391.14 ) following a severe pelvic fracture.table 6multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3 , using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)model predictorsoutcomedeath , n = 2,487severe complication , n = 2,487children ( < 13 years)1.90 ( 1.063.41)1.07 ( 0.651.75)adolescent ( 1317 years)0.40 ( 0.180.91)0.66 ( 0.391.14)adults ( 1854 years)referentreferentmale1.31 ( 0.951.80)not caucasian1.34 ( 0.902.01)hypovolemic shock9.93 ( 6.5215.1)6.56 ( 4.579.42)head injury1.70 ( 1.052.77)2.51 ( 1.723.66)iss 2519.3 ( 10.535.5)10.5 ( 7.2315.2)motor vehicle accident0.96 ( 0.621.50)0.98 ( 0.651.49)motorcycle accident1.27 ( 0.722.23)low - energy fall0.25 ( 0.061.12)0.96 ( 0.471.99)hosmer lemeshow0.290.14bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or deathhosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3 , using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death hosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval in this model , an iss > 25 , hypovolemic shock , and head injury yielded a substantial increase in odds of death ( table 6 ) . descriptive statistics of demographics , injury mechanism , injury severity , treatment , and complications are presented in table 1 . after sustaining a pelvic fracture when separated by age group , adolescents appeared to have the best survival statistics ( 93.2 % ) , while children had the worst ( 89.8 % ) . adolescents had the lowest percentage of severe complication ( 11.4 % ) , children had a slightly higher percentage ( 13.0 % ) , and adults had the highest percentage ( 15.7 % ) .table 1frequency distribution of characteristics for the entire population of pelvic fractures , n = 37,784variableage grouptotal , n ( % ) children ( < 13 years ) , n ( % ) , n = 5,325adolescents ( 1317 years ) , n ( % ) , n = 4,052adults ( 1854 years ) , n ( % ) , n = 28,407gender male2,349 ( 44.8)1,888 ( 46.7)17,393 ( 61.3)21,630 ( 57.4 ) female2,897 ( 55.2)2,157 ( 53.3)10,983 ( 38.7)16,037 ( 42.6)race caucasian3,570 ( 72.3)2,745 ( 73.4)18,301 ( 69.9)24,616 ( 70.7 ) african american474 ( 9.6)341 ( 9.1)2,717 ( 10.4)3,532 ( 10.1 ) asian / pacific islander73 ( 1.5)54 ( 1.4)504 ( 1.9)631 ( 1.8 ) hispanic490 ( 9.9)394 ( 10.6)3002 ( 11.5)3,886 ( 11.2 ) native american31 ( 0.6)31 ( 0.9)201 ( 0.8)263 ( 0.8 ) other299 ( 6.1)173 ( 4.6)1,440 ( 5.5)1,912 ( 5.4)survival survived4,766 ( 89.8)3,753 ( 93.2)25,853 ( 91.5)34,372 ( 91.4 ) died542 ( 10.2)273 ( 6.8)2,404 ( 8.5)3,219 ( 8.6)severe complication yes693 ( 13.0)462 ( 11.4)4,465 ( 15.7)5,620 ( 14.9 ) no4,632 ( 87.0)3,590 ( 88.6)23,942 ( 84.3)32,164 ( 85.1)fracture type open183 ( 3.4)96 ( 2.4)1,316 ( 4.6)1,595 ( 4.2 ) closed5,142 ( 96.6)3,956 ( 97.6)27,078 ( 95.4)36,176 ( 95.8)fracture location ilium703 ( 13.7)622 ( 15.7)3,797 ( 13.9)5,122 ( 14.1 ) ischium162 ( 3.1)153 ( 3.9)1,151 ( 4.2)1,466 ( 4.0 ) pubis2,615 ( 42.0)1,632 ( 41.2)11,281 ( 41.4)15,078 ( 41.5 ) multiple330 ( 6.4)220 ( 5.5)1,799 ( 6.6)2,349 ( 6.5 ) unspecified673 ( 13.1)342 ( 8.6)2,328 ( 8.6)3,343 ( 9.2 ) other1,120 ( 21.7)996 ( 25.1)6,878 ( 25.3)8,994 ( 24.7)systolic blood pressure ( mmhg ) < 90878 ( 16.5)524 ( 12.9)4,615 ( 16.3)6,017 ( 15.9 ) 901392,869 ( 53.9)2,680 ( 66.2)16,704 ( 58.8)22,253 ( 58.9 ) 140 + 1,578 ( 29.6)848 ( 20.9)7,088 ( 24.9)9,514 ( 25.2)moi motor vehicle1,329 ( 24.9)2,224 ( 54.9)11,520 ( 40.6)15,073 ( 39.9 ) motorcycle119 ( 2.2)91 ( 2.3)2,674 ( 9.4)2,884 ( 7.6 ) pedestrian170 ( 3.2)102 ( 2.5)566 ( 1.9)838 ( 2.2 ) crush93 ( 1.8)31 ( 0.7)732 ( 2.6)856 ( 2.3 ) high fall592 ( 11.1)108 ( 2.6)2,680 ( 9.5)3,380 ( 9.0 ) low fall1,216 ( 22.8)63 ( 1.6)1,393 ( 4.9)2,672 ( 7.1 ) unknown1,806 ( 34.0)1,433 ( 35.4)8,842 ( 31.1)12,081 ( 31.9)ais of pelvis none3,323 ( 62.4)2,660 ( 65.6)19,331 ( 68.1)25,314 ( 67.0 ) 19 ( 0.2)11 ( 0.3)96 ( 0.3)116 ( 0.3 ) 21,313 ( 24.6)869 ( 21.5)5,153 ( 18.2)7,335 ( 19.4 ) 3610 ( 11.5)450 ( 11.1)3,129 ( 11.0)4,189 ( 11.1 ) 453 ( 1.0)45 ( 1.1)522 ( 1.8)620 ( 1.6 ) 517 ( 0.3)17 ( 0.4)176 ( 0.6)210 ( 0.6 ) 60 ( 0.0)0 ( 0.0)0 ( 0.0)0 ( 0.0)iss < 254,338 ( 81.5)2,816 ( 69.5)19,721 ( 69.4)26,875 ( 71.1 ) 25987 ( 18.5)1,236 ( 30.5)8,686 ( 30.6)10,909 ( 28.9)pneumonia yes102 ( 1.9)125 ( 3.1)1,232 ( 4.3)1,459 ( 3.9 ) no5,223 ( 98.1)3,927 ( 96.9)27,175 ( 95.7)36,325 ( 96.1)acute respiratory stress syndrome yes61 ( 1.1)55 ( 1.4)573 ( 2.0)689 ( 1.8 ) no5,264 ( 98.9)3,997 ( 98.6)27,834 ( 98.0)37,095 ( 98.2)deep vein thrombosis yes27 ( 0.5)21 ( 0.5)503 ( 1.8)551 ( 1.5 ) no5,298 ( 99.5)4,031 ( 99.5)27,904 ( 98.2)37,233 ( 98.5)bacteremia yes10 ( 0.2)26 ( 0.6)196 ( 0.7)232 ( 0.6 ) no5,315 ( 99.8)4,026 ( 99.4)28,211 ( 99.3)37,501 ( 99.4)renal failure yes32 ( 0.6)15 ( 0.4)225 ( 0.8)272 ( 0.7 ) no5,293 ( 99.4)4,037 ( 99.6)28,182 ( 99.2)37,512 ( 99.3)pulmonary embolism yes15 ( 0.3)5 ( 0.1)160 ( 0.6)180 ( 0.4 ) no5,310 ( 99.7)4,047 ( 99.9)28,247 ( 99.4)37,604 ( 99.6)icu stay < 1 week4,890 ( 91.8)3,545 ( 87.5)24,086 ( 84.8)32,521 ( 86.1 ) 1 week435 ( 8.2)507 ( 12.5)4,321 ( 15.2)5,263 ( 13.9)hospital stay < 1 week3,727 ( 70.0)2,406 ( 59.4)13,778 ( 48.5)19,911 ( 52.7 ) 1 week1,598 ( 30.0)1,646 ( 40.6)14,629 ( 51.5)17,873 ( 47.3)major procedure crif1 ( 0.02)2 ( 0.05)20 ( 0.07)23 ( 0.08 ) orif245 ( 4.62)395 ( 9.71)4,691 ( 16.47)5,331 ( 14.1 ) closed dislocation reduction7 ( 0.13)19 ( 0.47)117 ( 0.41)143 ( 0.39 ) open dislocation reduction12 ( 0.23)15 ( 0.37)127 ( 0.45)154 ( 0.43 ) no major procedure5,060 ( 95.0)3,621 ( 89.4)23,452 ( 82.6)32,133 ( 85.0)moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation frequency distribution of characteristics for the entire population of pelvic fractures , n = 37,784 moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation most of the pelvic fractures were closed ( 95.8 % ) . pubic rami fractures consistently had the highest prevalence across all age groups ( 41.5 % ) . the most common known mechanism of injury was motor vehicle accident ( 39.9 % ) , followed by high - energy fall ( 9.0 % ) and motorcycle accident ( 7.6 % ) . most patients had an iss < 25 ( 71.1 % ) . when divided by age , more children had an iss < 25 ( 81.5 % ) than adolescents ( 69.5 % ) or adults ( 69.4 % ) . adolescents also had less reports of hypovolemic shock on arrival ( systolic pressures below 90 mmhg ) ( 12.9 % ) than children ( 16.5 % ) and adults ( 16.3 % ) who had comparable numbers . for this assessment , open fractures and unknown mechanisms of injury were excluded , yielding a study group of 24,684 . the results of the bivariate analysis ( reporting crude ors and 95 % cis ) for this population are depicted in table 2 . as compared to the rest of the population , children had a small increase in odds of death ( or 1.27 , 95 % ci 1.111.44 ) , but a slight decrease in odds of severe complication ( or 0.83 , 95 % ci 0.750.93 ) . adolescents had decreases in both odds of death ( or 0.85 , 95 % ci 0.721.00 ) and severe complication ( or 0.78 , 95 % ci 0.690.89 ) . adults had a slight decrease in odds of death ( or 0.92 , 95 % ci 0.821.02 ) , but a small increase in odds of severe complication ( or 1.27 , 95 % ci 1.161.39 ) . individuals suffering from hypovolemic shock had substantially increased odds of death ( or 5.89 , 95 % ci 5.326.51 ) and severe complication ( or 3.62 , 95 % ci 3.333.93 ) . sustaining a head injury also increased the odds of death ( or 2.96 , 95 % ci 2.593.39 ) and complication ( or 3.23 , 95 % ci 2.903.61 ) . an iss 25 greatly increased the odds of death ( or 11.1 , 95 % ci 9.9212.5 ) and complication ( or 9.19 , 95 % ci 8.489.97 ) . motor vehicle accidents caused the largest increase in odds of death ( or 1.46 , 95 % ci 1.321.62 ) and severe complication ( or 1.53 , 95 % ci 1.421.65).table 2bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) , n = 24,684predictorsoutcomedeathsevere complicationmale1.24 ( 1.131.37)1.41 ( 1.311.52)not caucasian1.14 ( 1.041.26)1.00 ( 0.921.08)pediatric ( < 18 years)1.09 ( 0.981.21)0.79 ( 0.720.86)children ( < 13 years)1.27 ( 1.111.44)0.83 ( 0.750.93)adolescent ( 1317 years)0.85 ( 0.721.00)0.78 ( 0.690.89)adults ( 1854 years)0.92 ( 0.821.02)1.27 ( 1.161.39)hypovolemic shock5.89 ( 5.326.51)3.62 ( 3.333.93)head injury2.96 ( 2.593.39)3.23 ( 2.903.61)iss 2511.1 ( 9.9212.5)9.19 ( 8.489.97)motor vehicle accident1.46 ( 1.321.62)1.53 ( 1.421.65)motorcycle accident1.34 ( 1.161.54)1.34 ( 1.201.49)fall from height0.61 ( 0.520.72)0.61 ( 0.540.69)low - energy fall0.41 ( 0.330.51)0.36 ( 0.300.43)crush0.43 ( 0.290.63)0.66 ( 0.520.84)pedestrian versus auto1.40 ( 1.111.77)1.13 ( 0.931.38)iss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) , n = 24,684 iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes . adjusted ors and 95 % cis are presented in table 3 . when controlling for mechanism of injury and other prehospital conditions , children had more than twice the odds of death ( or 2.29 , 95 % ci 1.962.67 ) and increased odds of severe complication ( or 1.36 , 95 % ci 1.201.55 ) relative to the adult group after sustaining a pelvic fracture . adolescents , on the other hand , had a slight decrease in odds of death ( or 0.89 , 95 % ci 0.741.06 ) and lower odds of severe complication ( or 0.70 , 95 % ci 0.610.81 ) relative to the adult group after sustaining a pelvic fracture.table 3multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)model predictorsoutcomedeath , n = 24,684severe complication , n = 22,846children ( < 13 years)2.29 ( 1.962.67)1.36 ( 1.201.55)adolescents ( 1317 years)0.89 ( 0.741.06)0.70 ( 0.610.81)adults ( 1854 years)referentreferentmale1.19 ( 1.071.33)1.38 ( 1.261.50)not caucasian1.00 ( 0.901.12)hypovolemic shock4.52 ( 4.055.05)2.81 ( 2.563.08)head injury1.60 ( 1.371.86)1.70 ( 1.501.92)iss 259.15 ( 8.0710.4)7.41 ( 6.798.09)motor vehicle accident3.98 ( 0.3940.2)1.13 ( 0.921.41)motorcycle accident4.61 ( 0.4646.8)1.18 ( 0.921.51)fall from height3.27 ( 0.3233.1)0.80 ( 0.631.03)low - energy fall3.18 ( 0.3231.7)0.77 ( 0.581.02)pedestrian versus auto4.50 ( 0.4544.8)crush2.42 ( 0.2325.3)0.95 ( 0.691.34)hosmer lemeshow0.00690.17bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or deathhosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death hosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval a subgroup analysis was performed for patients determined to have a severe , isolated , closed pelvic injury ( ais 3 ) . table 4 demonstrates the descriptive statistics for this subpopulation ( n = 2,487 ) . adolescents ( n = 255 ) still appeared to have a lower incidence of mortality ( 3.1 % ) and lower incidence of a severe complication ( 8.2 % ) compared to adults ( n = 1,886 ) and children ( n = 346 ) . adolescents contributed the most to motor vehicle accident ( 89.4 % ) , which was the most common mechanism of injury in the severe pelvic fracture group . similar to the whole population , the severe pelvic injury group demonstrated more children with an iss < 25 ( 82.1 % ) . for cases of hypovolemic shock on arrival , adolescents had the fewest reports of systolic pressures below 90 mmhg ( 6.7 % ) , while children ( 15.9 % ) had the largest contribution . children had the lowest incidence of hospital stay and intensive care unit ( icu ) admission , while adolescents and adults were relatively comparable.table 4frequency distribution of characteristics for individuals with isolated , closed , severe pelvic fractures ( ais3 ) , n = 2,487variableage grouptotal , n ( % ) children ( < 13 years ) , n ( % ) , n = 346adolescents ( 1317 years ) , n ( % ) , n = 255adults ( 1854 years ) , n ( % ) , n = 1,886gender male127 ( 38.7)92 ( 36.2)1,109 ( 58.9)1,328 ( 53.8 ) female201 ( 61.3)162 ( 63.8)775 ( 41.1)1,138 ( 46.2)race caucasian235 ( 77.1)168 ( 74.3)1,208 ( 73.0)1,611 ( 73.7 ) african american31 ( 10.2)30 ( 13.3)170 ( 10.3)231 ( 10.6 ) asian / pacific islander4 ( 1.3)1 ( 0.4)24 ( 1.5)29 ( 1.3 ) hispanic29 ( 9.5)20 ( 8.9)200 ( 12.1)249 ( 11.4 ) native american1 ( 0.3)2 ( 0.9)7 ( 0.4)10 ( 0.5 ) other5 ( 1.6)5 ( 2.2)46 ( 2.7)56 ( 2.5)survival survived321 ( 92.8)247 ( 96.9)1,774 ( 94.1)2,342 ( 94.2 ) died25 ( 7.2)8 ( 3.1)112 ( 5.9)145 ( 5.8)severe complication yes32 ( 9.3)21 ( 8.2)209 ( 11.1)262 ( 10.5 ) no314 ( 90.8)234 ( 91.8)1,677 ( 88.9)2,225 ( 89.5)fracture location ilium41 ( 11.9)47 ( 18.4)228 ( 12.1)316 ( 12.7 ) ischium7 ( 2.0)7 ( 2.8)53 ( 2.8)67 ( 2.7 ) pubis222 ( 64.2)143 ( 56.1)1,059 ( 56.2)1,424 ( 57.3 ) multiple31 ( 9.0)30 ( 11.8)277 ( 14.7)338 ( 13.6 ) unspecified10 ( 2.9)4 ( 1.6)70 ( 3.7)84 ( 3.4 ) other35 ( 10.0)24 ( 9.3)199 ( 10.5)258 ( 10.3)systolic blood pressure ( mmhg ) < 9055 ( 15.9)17 ( 6.7)176 ( 9.3)248 ( 10.0 ) 90139173 ( 50.0)178 ( 69.8)1,197 ( 63.5)1,548 ( 62.2 ) 140 + 118 ( 34.1)60 ( 23.5)513 ( 27.2)691 ( 27.8)moi motor vehicle127 ( 36.7)228 ( 89.4)1,094 ( 58.0)1,449 ( 58.3 ) motorcycle17 ( 4.9)6 ( 2.4)196 ( 10.4)219 ( 8.8 ) pedestrian17 ( 4.9)5 ( 2.0)70 ( 3.7)92 ( 3.7 ) crush13 ( 3.8)5 ( 2.0)101 ( 5.4)119 ( 4.8 ) high fall52 ( 15.0)4 ( 1.5)248 ( 13.2)304 ( 12.2 ) low fall120 ( 34.7)7 ( 2.7)177 ( 9.3)304 ( 12.2)ais of pelvis 3321 ( 92.7)231 ( 90.6)1,606 ( 85.2)2,158 ( 86.8 ) 420 ( 5.8)17 ( 6.6)225 ( 11.9)262 ( 10.5 ) 55 ( 1.5)7 ( 2.8)55 ( 2.9)67 ( 2.7)iss < 25284 ( 82.1)169 ( 66.3)1,308 ( 69.4)1,761 ( 70.8 ) 2562 ( 17.9)86 ( 33.7)578 ( 30.6)726 ( 29.2)pneumonia yes3 ( 0.9)12 ( 4.7)53 ( 2.8)68 ( 2.7 ) no343 ( 99.1)243 ( 95.3)1,833 ( 97.2)2,419 ( 97.3)acute respiratory stress syndrome yes1 ( 0.3)1 ( 0.4)28 ( 1.5)30 ( 1.2 ) no345 ( 99.7)254 ( 99.6)1,858 ( 98.5)2,457 ( 98.8)deep vein thrombosis yes0 ( 0.0)1 ( 0.4)19 ( 1.0)20 ( 0.8 ) no346 ( 100.0)254 ( 99.6)1,867 ( 99.0)2,467 ( 99.2)bacteremia yes1 ( 0.3)1 ( 0.4)6 ( 0.3)8 ( 0.3 ) no345 ( 99.7)254 ( 99.6)1,880 ( 99.7)2,479 ( 99.7)renal failure yes2 ( 0.6)1 ( 0.4)7 ( 0.4)10 ( 0.4 ) no344 ( 99.4)254 ( 99.6)1,879 ( 99.6)2,477 ( 99.6)pulmonary embolism yes1 ( 0.3)0 ( 0.0)7 ( 0.4)8 ( 0.3 ) no345 ( 99.7)255 ( 100.0)1,879 ( 99.6)2,479 ( 99.7)icu stay < 1 week326 ( 94.2)224 ( 87.8)1,676 ( 88.9)2,226 ( 89.5 ) 1 week20 ( 5.8)31 ( 12.2)210 ( 11.1)261 ( 10.5)hospital stay < 1 week256 ( 74.0)151 ( 59.2)1,011 ( 53.6)1,418 ( 57.0 ) 1 week90 ( 26.0)104 ( 40.8)875 ( 46.4)1,069 ( 43.0)major procedure crif0 ( 0.0)0 ( 0.0)1 ( 0.1)1 ( 0.1 ) orif31 ( 9.0)45 ( 17.6)501 ( 26.5)577 ( 23,1 ) closed dislocation reduction0 ( 0.0)2 ( 0.8)7 ( 0.4)9 ( 0.4 ) open dislocation reduction0 ( 0.0)0 ( 0.0)13 ( 0.7)13 ( 0.5 ) no major procedure315 ( 91.0)208 ( 81.6)1,364 ( 72.3)1,887 ( 75.9)moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation frequency distribution of characteristics for individuals with isolated , closed , severe pelvic fractures ( ais3 ) , n = 2,487 moi mechanism of injury , ais abbreviated injury scale , iss injury severity score , icu intensive care unit , crif closed reduction internal fixation , orif open reduction internal fixation similar to what was done with the whole population , bivariate analysis was also performed on the severe pelvic fracture subpopulation ( table 5 ) . as compared to the adults , children had higher odds of death but lower odds of severe complication . however , adolescents had a crude decrease in odds of death and severe complication after sustaining a pelvic fracture compared to the adults . individuals suffering from hypovolemic shock , head injury , and iss 25 had an increase in odds of death and severe complications . motor vehicle accidents caused the largest increase in odds of death ( or 1.58 , 95 % ci 1.102.26 ) , while motorcycle accidents accounted for the largest increase in severe complication ( or 1.58 , 95 % ci 1.062.34).table 5bivariate analysis for isolated closed pelvic fractures with an associated ais 3 , comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)predictorsoutcomedeath , n = 2,487severe complication , n = 2,487male1.11 ( 0.791.56)1.60 ( 1.222.08)not caucasian1.53 ( 1.092.15)1.26 ( 0.971.64)pediatric ( < 18 years)0.92 ( 0.621.37)0.78 ( 0.571.06)children ( < 13 years)1.31 ( 0.842.05)0.85 ( 0.571.25)adolescent ( 1317 years)0.50 ( 0.241.02)0.74 ( 0.471.18)adults ( 1854 years)1.09 ( 0.731.62)1.29 ( 0.941.77)hypovolemic shock13.0 ( 9.0618.6)7.35 ( 5.449.93)head injury4.41 ( 2.946.60)5.91 ( 4.288.16)iss 2527.5 ( 9.9212.5)14.9 ( 10.720.7)motor vehicle accident1.58 ( 1.102.26)1.32 ( 1.011.72)motorcycle accident1.62 ( 0.982.67)1.58 ( 1.062.34)fall from height0.63 ( 0.341.16)0.74 ( 0.481.13)low - energy fall0.095 ( 0.0230.38)0.35 ( 0.200.61)crush0.85 ( 0.371.97)0.95 ( 0.521.75)pedestrian versus auto1.33 ( 0.602.93)1.33 ( 0.602.93)bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death bivariate analysis for isolated closed pelvic fractures with an associated ais 3 , comparing predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes ( table 6 ) . when controlling for mechanism of injury and other prehospital conditions and using the adult group as a referent , children had nearly double the odds of death after sustaining a severe pelvic fracture , whereas adolescents had a large decrease in odds of death ( or 0.40 , 95 % ci 0.180.91 ) . compared to adults , children had potentially increased odds of severe complication after sustaining a severe pelvic fracture ( or 1.07 , 95 % ci 0.651.75 ) , whereas adolescents had potentially decreased odds of severe complication ( or 0.66 , 95 % ci 0.391.14 ) following a severe pelvic fracture.table 6multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3 , using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals)model predictorsoutcomedeath , n = 2,487severe complication , n = 2,487children ( < 13 years)1.90 ( 1.063.41)1.07 ( 0.651.75)adolescent ( 1317 years)0.40 ( 0.180.91)0.66 ( 0.391.14)adults ( 1854 years)referentreferentmale1.31 ( 0.951.80)not caucasian1.34 ( 0.902.01)hypovolemic shock9.93 ( 6.5215.1)6.56 ( 4.579.42)head injury1.70 ( 1.052.77)2.51 ( 1.723.66)iss 2519.3 ( 10.535.5)10.5 ( 7.2315.2)motor vehicle accident0.96 ( 0.621.50)0.98 ( 0.651.49)motorcycle accident1.27 ( 0.722.23)low - energy fall0.25 ( 0.061.12)0.96 ( 0.471.99)hosmer lemeshow0.290.14bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or deathhosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3 , using predictors upon arrival to the emergency department with the outcomes death and severe complication ( odds ratios and 95 % confidence intervals ) bold values signify 95 % confidence intervals that are completely greater than 1 , indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course : pneumonia , bacteremia , deep vein thrombosis , pulmonary embolism , renal failure , acute respiratory distress syndrome , or death hosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval in this model , an iss > 25 , hypovolemic shock , and head injury yielded a substantial increase in odds of death ( table 6 ) . in this study , we analyzed data gathered from the ntdb to characterize the risks for mortality and complications associated with pelvic fractures in adolescents as compared to younger children and adults . compared to adults , adolescents were shown to have decreased mortality and severe complication rates , while children have an increased mortality rate but decreased severe complication rate . in the subanalysis of severe pelvic fractures , the same pattern emerged , although the results for complication rates were not statistically significant . mortality rates as low as 1.4 % to as high as 25 % have been reported [ 5 , 10 , 11 , 14 , 15 ] . banerjee et al . reported a 16 % mortality rate in 44 patients related mainly to the associated injuries and not to the pelvic fractures themselves . another study reported 5 % overall mortality for 722 pediatric pelvic fractures versus 17 % among similar injuries in the adult population . comparable to previously reported studies , the current study reported 10.2 % survival for children and only 6.8 % for adolescents , compared to 8.5 % in adults with pelvic fractures . the pattern of lower survival in children and higher survival in adolescents compared to adults was also seen in the analysis of severe pelvic injuries ( table 4 ) . this study illustrates that simply comparing pediatric mortality to that of adults may be an inadequate evaluation due to the fact that adolescents and children sustaining pelvic injuries may have different outcomes . central nervous system head injury was cited as the most common cause of death in children with pelvic fractures in two recent studies [ 2 , 11 ] . other causes of death in children with pelvic fractures , reported in these studies , include multiorgan failure and visceral injuries [ 2 , 11 ] . in a study that examined the national pediatric trauma registry ( nptr ) , hospital type ( children s or general ) and iss were the only significant parameters in the multivariate analysis . hemorrhage from a pelvis fracture - related vascular injury was reported as the cause of death in only 0.3 % of children , compared with 3.4 % of adults . multivariate analysis showed hypovolemic shock , head injury , and iss 25 to correlate with an increased mortality rate in a patient with any type of pelvic fracture ( severe or not ) . the multivariate model for severe pelvic injuries also demonstrated that , despite their lower iss , children had a significantly higher risk of mortality ( or 1.90 , 95 % ci 1.063.41 ) and adolescents had a significantly lower risk of mortality ( or 0.40 , 95 % ci 0.180.91 ) as compared to adults . whereas many studies report the rate of associated injuries , very few studies report the rate of complications after pediatric pelvic fractures . according to a previous study of 120 children ( age < 16 years ) with pelvic fractures , complications included 13.3 % wound complications , 8.3 % urinary tract infections , 4.2 % septicemias , 1.7 % ards , and one case ( 0.8 % ) of pulmonary embolus ( pe ) . in the current study , there were 346 children ( age < 13 years ) with severe pelvic injuries , with a lower overall complication rate of 9.3 % . there was one case ( 0.3 % ) of ards and one case of pe ( 0.3 % ) , two cases of renal failure ( 0.6 % ) , and no cases of septicemia . the 255 adolescents with severe pelvic injury had an even lower complications rate of 8.2 % , with one ards ( 0.4 % ) , one septicemia ( 0.4 % ) , one renal failure ( 0.4 % ) , one dvt , but no pe . urinary tract infection ( uti ) was not considered to be a significant complication in our study . however , in the severe pelvic injury group , there were three children ( 0.87 % ) , three adolescents ( 1.2 % ) , and 30 adults ( 1.59 % ) with uti . the most common severe complication was pneumonia : three children ( 0.87 % ) , 12 adolescents ( 4.7 % ) , and 53 adults ( 2.8 % ) . previous studies have reported a high percentage of associated injuries in the pediatric pelvic fracture population . in one study surveying 166 cases , there were 38.6 % head traumas , 19.9 % significant chest traumas , and 19.3 % abdominal / visceral trauma . additionally , an incidence of 1127 % of associated abdominal injuries [ 2 , 10 , 11 , 1517 ] , 27 % associated chest injuries , and up to 50 % of associated head injuries have been previously reported [ 2 , 10 , 18 ] . the proportion of associated injuries ( head , thorax , and abdomen , respectively ) in the current study was 7.5 , 4.1 , and 4.6 % for children ; 18 , 7.1 , and 8.6 % for adolescents . the proportion of a depressed consciousness level [ glasgow coma scale ( gcs ) score < 15 ] in the population of severe pelvic fractures was 19.4 % in children and 31.3 % in adolescents ( 25 % if adolescents and children are grouped together ) . other pertinent findings in this study included a 57 times higher incidence of pelvic fractures in adults versus children and adolescents , a higher incidence of pelvic fractures in white non - hispanic males , the vast majority of the injury mechanism of pelvic fractures being blunt trauma from motor vehicle accidents , and a predominance of non - operative treatment for these fractures . these findings are largely in accordance with previous reports [ 2 , 3 , 8 , 11 ] . the main strengths of the current study is that this is the largest cohort used for an investigation of this sort , and the national representation of academic and non - academic centers of various levels provide far - reaching generalizability . while the ntdb includes a very large sample of trauma centers from all over the united states , it is a convenience sample that consists solely of data submitted by participating hospitals , leading to a disproportionate number of larger hospitals with younger and more severely injured patients . the quality of data entered into the ntdb is solely dependent on the participants entering the data ; however , the ntdb is continuously working to screen , clean , and standardize the quality of the data entered into the database . the larger population provided by the ntdb may be the reason for a higher mortality seen in our pediatric population , unlike the previous studies with smaller sample sizes . the ntdb also reported more pelvic fractures in children than in adolescents , which contributed to our pediatric population s higher mortality rate . we also used pelvis ais 3 to indicate a severe pelvic injury , while other studies have used a cutoff of ais 4 . additionally , since the ntdb does not code separately for pelvic ais , we derived our pelvic ais score by using the lower extremity ais score and excluding all patients with other lower extremity injuries . assuming an associated lower extremity fracture rate of about 25 % , this may have influenced our descriptive statistics . however , this allowed us to compare similar injuries and better isolate the effect of age . the ntdb also does not provide blood gas values such as base deficit and lactate , and , therefore , our analysis was limited to the use of hypovolemic shock on arrival . this suggests that there could be interactions between variables that were not included in the model . these variables might not have been collected by the ntdb or may be latent factors that are not identified . such discrepancies may have altered our final parameter estimates by either inflating or deflating their values . finally , we chose the age group of 1317 years to represent adolescence . biological , as well as social and environmental , changes influence the beginning of adolescence , making the precise beginning age questionable . because of this , our age group was chosen based on previous publications [ 1 , 12 ] . in conclusion , the results of this study suggest that the adolescents with pelvic fractures behave as a separate group from the children and adult populations . these results emphasize the need for further epidemiological research on disparate injury response between pediatric subpopulations defined by level of maturity . there should be further research aimed towards designating physiological criteria besides age by which to place pediatric patients in their respective subpopulations . this study highlights the fact that the mortality rate seen in a given pediatric population may not be an accurate measurement , as our lower mortality rate largely stems from the adolescents increased survival , while the children may actually have a higher mortality rate than the adults . meir marmor md was involved in the study design , data analysis , and writing .

introductionserratia marcescens is a gram - negative bacillus which belongs to the family enterobacteriaceae . it is a facultative anaerobe and produces red pigment at room temperature . it naturally occurs in soil and water as well as the intestines , and it is responsible for nosocomial infections . there have been few reports about community acquired pneumonia of serratia.case presentationthis report presents a 37-year - old man with hemoptysis , fever , and shortness of breath . the clinical and laboratory examinations revealed that the patient had pseudohemoptysis due to s. marcescens pneumonia , on an immunocompromised pattern , because of the coexistence of sarcoidosis ( stage 1).conclusionappropriate antibiotic therapy for serratia was administered , and the patient s symptoms regressed . the patient is healthy and asymptomatic after 1-year follow - up . to the best of the authors knowledge , this is the first reported case of a pseudohemoptysis in a patient with pulmonary sarcoidosis .

it naturally occurs in soil and water as well as the intestines , and it is responsible for nosocomial infections . there have been few reports about community acquired pneumonia of serratia . this report presents a 37-year - old man with hemoptysis , fever , and shortness of breath . the clinical and laboratory examinations revealed that the patient had pseudohemoptysis due to s. marcescens pneumonia , on an immunocompromised pattern , because of the coexistence of sarcoidosis ( stage 1 ) . the patient is healthy and asymptomatic after 1-year follow - up . to the best of the authors knowledge , this is the first reported case of a pseudohemoptysis in a patient with pulmonary sarcoidosis . serratia , is an opportunistic pathogen , mainly responsible for infecting the urinary system , respiratory tract , central nervous system , and bloodstream.1 infections of colonized individuals are associated with invasive devices , surgery , and immunocompromised states . serratia ubiquitously thrives in moist environments and frequently contaminates liquids and equipment . in the outpatient setting , it has been associated with community - acquired pneumonias , but only limited case reports exist , and usually are associated with some degree of immunocompromise . this paper reports a case where the poor living environment and coexistence of sarcoidosis were likely the predisposing factors that developed pneumonia from serratia . a 37-year - old man presented in the emergency department complaining of productive cough with blood - tinged sputum , intermittent fever , and shortness of breath for the last 20 days . his past medical history was remarkable only for arterial hypertension treated with atenolol . on examination his axillary temperature was 37.8c , blood pressure was 140/80 mm hg with a normal heart rate of 90 bpm , and oxygen saturation spo2 : 94% . , the patient had a mildly elevated white blood cell count of 10,900 cells/l , with normal differential . the hemoglobin level was 10.6 g / dl , with a hematocrit of 32.5% ; the platelet count was 455,000/l . a basic metabolic panel was notable for an elevated serum creatinine of 2.52 mg / dl , with urea of 57 mg / dl , and normal electrolytes . serum angiotensin converting enzyme was slightly elevated at 57 iu / ml ( normal range 852 iu / ml ) . the acute renal failure was attributed to the serratia infection , since the patient did not previously have this disorder and serratia is known to induce renal failure.1 there was no lymphadenopathy observed in general examination , but chest x - radiography demonstrated bilateral hilar prominence , lower - right lung lobe opacification , and right hemidiaphragm elevation ( figure 1 ) . chest high - resolution computed tomography scan was performed according to the department s protocol for investigating the thorax . the scan revealed consolidation of the anterior segment of the right lower lobe , and bilateral paratracheal , subcarinal , and hilar lymphadenopathy ( figure 2 ) . pulmonary function testing after remission of the productive cough ( 2 days ) showed a restrictive pattern with a forced vital capacity ( fvc ) of 49% predicted , a forced expiratory volume in one second ( fev1 ) of 47% predicted , and an fev1/fvc ratio of 71% . the total lung capacity was 66% of predicted , and the residual volume was 79% of predicted . there was reduction of the diffusion lung capacity for carbon monoxide to 68% of predicted . serologic studies for human immunodef iciency , hepatitis b virus , and a tuberculin skin test were negative . urine antigen for streptococcus pneumoniae and legionella were given upon admission , but they were also negative . no evidence of inflammation , granulomas , malignancy , vasculitis , or alveolar wall necrosis were found on biopsy . cell / l , with 78% alveolar macrophages , 12% lymphocytes , and 10% neutrophils . balf flow cytometry showed that 69% of the lymphocytes were t - lymphocytes , 7% were b - lymphocytes , and the t4/t8 ratio was 1.9 , while the blood t4/t8 ratio was 1.4 . microbiology cultures of the balf grew serratia marcescens , which was sensitive to ciprofloxacin , tobramycin , and amikacin . the smear for acid - fast bacilli was negative , and cultures for mycobacteria remained negative after 6 weeks . the patient was treated with ciprofloxacin 500 mg twice daily for 3 weeks . after the third day of treatment , the cough improved , and the reddish appearance of the sputum completely resolved . on follow - up after 1 month , chest x - radiography presented complete remission on right lower lobe infiltrate , while the perihilar lymphadenopathy persisted . after 1-year follow - up , the patient remains asymptomatic , and on chest x - radiography the infiltrate has completely resolved , while enlargement of the perihilar lymph nodes persists ( figures 3 and 4 ) . this is a case of pseudohemoptysis due to pulmonary infection with s. marcescens and concomitant sarcoidosis unexpectedly found . s. marcescens is a gram - negative , rod - shaped bacterium in the enterobacteriaceae family , found in soil , water , plants , and human gut flora . at room temperature pseudohemoptysis , which is the appearance of bright reddish or pinkish colored sputum without the evidence of any red blood cells , has been well described in colonization or infection with serratia and is attributed to this pigment . in this patient , similarly to previously published cases , no red blood corpuscles were seen on direct microscopic examination of his sputum.2 lymph node hyperplasia occurs in serratia infections and may persist after treatment . this is due to the marked follicular , primary and secondary germinal center and medullar hyperplasia.3 in this case , the lymph node biopsy did not show any of these features but only nonnecrotizing granulomas without evidence of infection . the coexistence of sarcoidosis and opportunistic infection in the absence of any immunosuppressive therapy has previously been documented.4 an infection can be the presenting problem subsequently leading to the diagnosis of sarcoidosis , as in this case . in sarcoidosis immunopathogenesis , the immune system undergoes a reactivity change . primary features are a reduction in circulating cd4 + lymphocytes ( t - helper cells ) , while their numbers in tissue increase , which is associated with a marked increase in tissue cytokine production , particularly interferon- , granulocyte - macrophage colony - stimulating factor , and interleukin-2.5,6 this t - helper 1 ( th-1 ) cytokine profile recruits macrophages , eventually forming a granulomatous reaction.7 in contrast to these highly active tissue - based responses , the immunity related to the circulatory cells expression is depressed by cutaneous anergy and poor responses to antigen recall testing . in addition , the production of inhibitory cytokines by macrophages appears to interfere with the normal immune response . girard et al reported five cases and reviewed 65 additional cases of opportunistic infections associated with sarcoidosis.8 while the majority of these occurred in patients receiving corticosteroids and were accompanied by cd4 lymphocytopenia , they described opportunistic infections in four untreated individuals . observations have suggested a pathogenetic role of gram - negative infections in sarcoidosis by driving an interleukin-18 response as another feature of the th-1 pathway.9 this has been postulated for infections with hemophilus influenzae and moraxella catarrhalis.10 in summary , this report describes a rare case of an opportunistic infection in an outpatient setting . in this case , the sarcoidosis was the underlying condition that induced an infection by gram - negative bacilli . written informed consent was obtained from the patient upon discharge for publication of this case report and all accompanying images .

merkel cell carcinoma ( mcc ) , a highly aggressive skin tumour with increasing incidence , is associated with the newly discovered merkel cell polyomavirus ( mcpyv ) . studies on mcc and mcpyv as well as other risk factors have significantly increased our knowledge of mcc pathogenesis , but the cells of origin , which could be important targets in future therapies , are still unknown . merkel cells ( mcs ) , the neuroendocrine cells of the skin , were believed to be at the origin of mcc due to their phenotypic similarities . however , for several reasons , for example , heterogeneous differentiation of mccs and postmitotic character of mcs , it is not very likely that mcc develops from differentiated mcs . skin stem cells , probably from the epidermal lineage , are more likely to be cells of origin in mcc . future studies will have to address these questions more directly in order to identify the physiological cells which are transformed to mcc cells .

merkel cell carcinoma ( mcc ) is a highly aggressive skin malignancy mainly affecting elderly and immunosuppressed people . mcc is a rare tumour , but its incidence , especially in men , has risen during the last two decades in several countries . for instance , an annual increase of 8% between 1986 and 2001 has been reported for the united states , and in the netherlands , mcc incidence rates have doubled between 1993 and 2007 . in scandinavia , however , incidence of mcc did not rise between 1995 and 2005 . mcc owes its name to characteristics it shares with merkel cells ( mcs ) , the neuroendocrine cells of the skin [ 5 , 6 ] . interest in mcc has significantly increased during the last years due to the discovery of merkel cell polyomavirus ( mcpyv ) and its implication in mcc pathogenesis [ 79 ] . studies on mcpyv and other molecular risk factors like mutations in the atoh1 gene have greatly advanced our understanding of mcc pathogenesis . the monoclonal integration of viral dna in a large proportion of mccs suggests that viral infection precedes tumourigenesis [ 7 , 11 ] . furthermore , mcpyv t antigens are required for maintenance of virus - carrying mcc cell lines . binding of large t antigen to cell cycle regulators or of small t antigen to the translation regulator 4e - bp1 are potential mechanisms by which integrated mcpyv could transform cells [ 11 , 13 ] . mcpyv may thus at least contribute to mcc formation , being the first molecular risk factor identified in mcc . additionally , mutations in several genes , including the tumour suppressor atoh1 , encoding a transcription factor involved in merkel cell differentiation , and pik3ca , which codes for phosphatidylinositol 4,5-bisphosphate 3-kinase , catalytic subunit , alpha isoform , have been found in subsets of mcc [ 10 , 14 ] . such mutations might either act in concert with mcpyv during tumourigenesis , or cause mcc formation in the absence of the virus . if it was known from which cell type mcc originates , one could develop more specific therapies which target these particular cells . in the present paper , we start with a brief discussion of cells of origin in cancer and subsequently elaborate on different cell types which have been hypothesized to be at the origin of mcc . last but not least , we briefly discuss how mcpyv might affect the potential mcc precursors . cells of origin in cancer have been defined as cells which acquire the first genetic hit or hits that culminate in the initiation of cancer . given the extended lifetime and self - renewal of physiological stem cells , these cells which assure homeostasis of rapidly self - renewing tissues therefore , it is not surprising that various studies have identified stem cells as cells of origin in cancer , for example , hematopoietic stem cells in chronic myeloid leukemia ( cml ) or crypt stem cells in intestinal cancer . in the skin , youssef et al . identified long - term resident progenitor cells of the interfollicular epidermis and the upper infundibulum as cells of origin in basal cell carcinoma ( bcc ) , using clonal analysis in mice . these long - lived progenitor cells could , however , also be called epidermal stem cells according to sellheyer . it is noteworthy that this stem cell population is also the cell of origin of merkel cells in mice [ 2224 ] . furthermore , squamous cell carcinoma ( scc ) could be induced in hair follicle stem cells as well as in cells immediately exiting the bulge , but not in transit amplifying cells , which are more developmentally restricted [ 25 , 26 ] . generally , it should be noted that the terms stem cells and progenitor cells are often used interchangeably , as a sharp distinction is not always easy . the second group of cells of origin in cancer are committed progenitor cells which differ from stem cells by their much more restricted differentiation potential . such cells play an important role in the acute phase of cml , but have also been identified as cells of origin in solid tumours . examples include medulloblastoma arising from granule neuron progenitors [ 28 , 29 ] and breast cancer developing from luminal epithelial progenitors . third , even differentiated cells could give rise to cancer , as any cell which is able to proliferate could become a cell of origin in cancer , provided it acquires mutations which restore the ability to self - renew and prevent differentiation to a postmitotic state . for instance , a study in mice strongly suggests that malignant peripheral nerve sheath tumours arise from differentiated glia . moreover , differentiated endocrine cells in the pancreas appear to be a target for oncogenic transformation . however , it should be noted that in both cases , less differentiated progenitor cells can not be definitively excluded as cells of origin . before turning to the origin of mcc , it is important to keep in mind that the terms cell - of - origin in cancer and cancer stem cell are different [ 15 , 33 ] . the cell - of - origin is a physiological cell which becomes tumourigenic due to genetic alterations . are a cell population within a tumour which constantly self - renews and is able to generate all types of cells present in this tumour , thus preserving the tumour . briefly , the cell - of - origin acquires tumourigenic properties , whereas the cancer stem cell sustains tumourigenic properties . regarding the cancer stem cell concept , early histological and ultrastructural analyses of the so - called trabecular carcinoma of the skin [ 36 , 37 ] revealed similarities to merkel cells [ 5 , 6 , 36 ] , leading to the currently used designation merkel cell carcinoma moreover , the reported morphological observations led to the conclusion that mcc may most probably originate from merkel cells ( mcs ) [ 5 , 6 , 36 ] . this traditional view of mcc origin ( figure 1 ) was further corroborated by the discovery that mcc and mcs share a similar immunophenotype [ 1 , 38 ] . shared features include presence of the merkel cell marker cytokeratin 20 ( ck20 ) in mcc [ 39 , 40 ] as well as biosynthesis of synaptophysin [ 4144 ] , ncam / cd56 [ 41 , 45 , 46 ] , and numerous endocrine markers . although at first glance , these observations strongly support the hypothesis that mcc emerges from transformed mcs , there are quite a few data which question this view . for instance , the neural cell adhesion molecule l1 ( cd171 ) , a relative of ncam , is produced by mcc cells , but not by mcs . moreover , the arrangement of intermediate filaments , including ck20 and neurofilaments , differs between mcc and mcs : in mccs , whirl - or plaque - like aggregates are observed , whereas in mcs , the intermediate filament cytoskeleton is loosely and diffusely arranged . last but not least , the tyrosine kinase receptor c - kit , which has been detected in the majority of mccs , is mostly absent from human mcs . second , in a study on fetal and human skin , no proliferative merkel cells could be detected , strongly suggesting that human merkel cells are postmitotic . in line with these findings , more recent lineage tracing analyses revealed that in mice , adult merkel cell homeostasis is ensured by differentiation of epidermal progenitors , not through the proliferation of differentiated mcs . given that the possibility to restore proliferative potential is a prerequisite for a cell to become cell - of - origin in cancer , it is highly unlikely that postmitotic mcs would be the source of a malignancy . third , differences in tissue localization argue against mcs as cells - of - origin for mcc . whereas the majority of mcs is located in the basal layer of the epidermis , mccs are mostly found in the dermis and subcutis . however , it should be noted that a minority of 3.2% to 9.1% of reported cases are partly or even fully localized in the epidermis . finally , the observed heterogeneity in mcc rather favors less differentiated cells as cells of origin . in detail , mccs associated with diverse differentiation patterns have been described : squamous , squamous and sarcomatous , melanocytic , eccrine , leiomyosarcomatous , rhabdomyoblastic , and fibrosarcomatous differentiation . although there is a certain immunophenotypical diversity in mcs themselves , the multitude of differentiation patterns in mcc rather points to stem or early progenitor cells as cells - of - origin . it is , of course , conceivable that distinct stem or progenitor populations account for mccs with distinct differentiation patterns . in the following sections , we will therefore discuss stem cell populations in skin which might serve as cell of origin in mcc . as mentioned before , in mice , therefore , epidermal stem cells or long - lived epidermal progenitor cells appear to be good candidates for a mcc cell - of origin ( figure 1 ) . along this line , lemasson et al . as ck14 is a marker of the basal epidermal layer which comprises the epidermal stem cells , and as mcs in mice have ck14-positive progenitors [ 2224 ] , the authors argued that a malignant transformation of epidermal stem cells could underlie mcc , and that the transformed cells could then serve as cancer stem cells for mcc . in the same study , however , contradictory to the findings of lemasson et al . , abbas and bhawan reported the absence of nestin in all 11 mcc cases investigated . it can not be excluded that these discrepancies were caused by different immunostaining protocols , as high variability of nestin immunoreactivity in skin due to procedural differences has been described . nevertheless , both studies showed the presence of ck19-positive cells in mcc [ 59 , 62 ] . ck19 has been used as an epidermal stem cell marker , but is also present on mature merkel cells . this might be an argument against hair follicle bulge stem cells as cells of origin in mcc , as ck15 is found in these cells . summarizing , there are several hints pointing to an epidermal stem / progenitor cell origin of mcc , but no experimental proof so far . based on their histomorphological pattern as well as results of immunohistochemical stainings , dermal neuroendocrine cells have been suggested as a potential source for mcc [ 51 , 67 ] . this suggestion points to a second stem cell population present in skin , dermal stem cells ( dscs ) derived from the neural crest lineage [ 68 , 69 ] ( figure 1 ) . interestingly , the transcription factor sox2 , which is expressed by neural crest - derived stem cells from human skin , has been detected in mcc by immunohistochemistry . nine mcc samples were all sox2-positive , with more than 50% of cells exhibiting nuclear staining . on the other hand , sox2 is expressed by epidermal progenitors in the mouse tongue during their differentiation to sensory cells , demonstrating that it can not be regarded as an exclusive neural crest - derived stem cell marker . therefore , although mcc cells express several proteins abundant in neural cells , strong hints to a neural crest origin of mcc are currently missing . in addition to the neural crest - derived stem cells mentioned before , a further stem cell population has been isolated from murine and human dermis and termed skin - derived precursors ( skps ) [ 73 , 74 ] . these cells are able to differentiate into both neural and mesodermal cell types . in mice , facial skps are generated from the neural crest , whereas dorsal trunk skps derive from the somites ( figure 1 ) . although there are a lot of similarities between skps and the dscs mentioned before , both cell types differ in that skps lack the robust expression of the neurotrophin receptor p75 which is characteristic for dscs . given their dermal localization and their broad differentiation potential , skps should be regarded as a further potential cell of origin for mcc . if the mcpyv plays an important role in mcc genesis , as suggested by several recent studies ( reviewed in [ 8 , 9 ] ) , it should target the putative cells of origin ( figure 1 ) . it is noteworthy that jc polyomaviruses have been shown to preferentially infect stem cells or progenitor cells with a low degree of differentiation . if mcpyv exhibits a similar preference , this would argue for a stem rather than a merkel cell at the origin of mcc . along the same line , unpublished findings from our laboratory suggest that human merkel cells are normally devoid of mcpyv large t antigen ( ltag ) . among 733 mcs detected by ck20 immunostaining in human skin areas prone to mcc development , none exhibited ltag immunopositivity in the adjacent section . although this does not prove the absence of mcpyv from differentiated mcs , it makes them less likely as a target for mcpyv infection . despite intense research on mcc during the last years , the cells of origin of this cutaneous malignancy remain elusive . merkel cells ( mcs ) , originally the favourite candidate for such a role , appear less likely to give rise to mcc now , mainly because of mcc heterogeneity and the postmitotic character of mcs . stem cells located in the skin , most probably of the epidermal lineage , appear to be more probable cells of origin for mcc . however , as experimental evidence for a stem cell origin is missing , too , more direct approaches to tackle the origin - of - mcc - question are needed .

three mechanisms have been proposed for the role of glutathione ( gsh ) in regulating cisplatin ( cddp ) sensitivities that affects its ultimate cell - killing ability : ( i ) gsh may serve as a cofactor in facilitating multidrug resistance protein 2- ( mrp2- ) mediated cddp efflux in mammalian cells , since mrp2-transfected cells were shown to confer cddp resistance ; ( ii ) gsh may serve as a redox - regulating cytoprotector based on the observations that many cddp - resistant cells overexpress gsh and -glutamylcysteine synthesis ( -gcs ) , the rate - limiting enzyme for gsh biosynthesis ; ( iii ) gsh may function as a copper ( cu ) chelator . elevated gsh expression depletes the cellular bioavailable cu pool , resulting in upregulation of the high - affinity cu transporter ( hctr1 ) which is also a cddp transporter . this has been demonstrated that overexpression of gsh by transfection with -gcs conferred sensitization to cddp toxicity . this review describes how these three models were developed and critically reviews their importance to overall cddp cytotoxicity in cancer cell treatments .

cisplatin ( cddp ) has been the mainstay for the treatment of a broad spectrum of human malignancies since it was approved by the fda about 30 years ago . it has been used in the first - line treatment modalities of human malignancies , including testicular , ovarian [ 2 , 3 ] , cervical , bladder , head and neck , and small cell lung cancers ( sclcs ) [ 3 , 7 , 8 ] . once patients develop resistance to platinum ( pt ) drugs , other effective treatment options become limited . it is well known that cddp acts on multiple cellular targets representing diverse signal transduction pathways . it is therefore conceivable that multiple mechanisms are involved in cddp resistance , including reduction of drug transport and increased dna adduct tolerance and repair [ 9 , 10 ] . in this review , we focus on the role of glutathione ( gsh ) system in cddp resistance . the first mechanism involves the effects of gsh on the atp - binding cassette ( abc ) transporter - mediated cddp transport ; the second mechanism involves the redox - regulating capacity of gsh in detoxifying cddp toxicity ; the third mechanism involves regulation of the intracellular copper pool that affects cddp uptake . the abc transporters are a family of cytoplasmic membrane - spanned proteins that function as efflux pumps for eliminating antitumor agents , xenobiotics , and anionic lipophilic endogenous constituents [ 1113 ] . therefore , many of these transporters are also known as multidrug resistance proteins ( mrps ) . gsh may serve as substrate for conjugation reaction with cddp prior to mrp - mediated transport . ishikawa and ali - osman first reported the formation of a pt(gs)2 conjugate in l1210 leukemia cells . these investigators also reported that elimination of pt(gs)2 across the membrane requires atp , suggesting the involvement of an energy - dependent transporter of gsh - conjugate ( termed gs - x pump ) in the elimination of pt(gs)2 complex . later , gene encoding mrp2 , member abcc2 in the abc transporter family , was cloned ; it was found that transfection of mrp2 into hek-293 cells conferred cddp resistance ( 10-fold ) in the transfected cells . these results demonstrated that mrp2 is the gs - x pump for the elimination of cddp . mrp2 , like mrp1 , can also transport gssg itself , an oxidized form of gsh , with relatively higher affinity than does gsh [ 18 , 19 ] . thus , these abc transporters can be considered as regulators of intracellular gssg - gsh homeostasis and the associated redox maintenance ( see below ) . many studies have demonstrated direct interactions between gsh and abc transporters [ 20 , 21 ] , suggesting that gsh may induce conformational changes that facilitate mrp - mediated substrate transport . mrp2 is also known as a canalicular multispecific organic anion transporter ( cmoat ) because of its high level of expression in the hepatic canalicular compartment and because it mediates the transport of a broad spectrum of nonbile salt organic anions from the liver into bile . cmoat - deficient ( tr- ) wistar rats are mutated in the gene encoding mrp2 , leading to defective hepatobiliary transport of a whole range of substrates , including bilirubin glucuronide . mrp2 mrna and protein levels can be markedly induced by treatments with metalloid salts including sodium arsenite [ as(iii ) ] and potassium antimonyl tartrate in primary rat and human hepatocytes . expression of mrp2 in primary rat hepatocytes is also induced by cddp . in one study , a single subcutaneous injection of cddp ( 5 mg / kg ) into male sprague - dawley rats resulted in > 10-fold induction of mrp2 in renal brush - border membranes within one day of treatment whereas nonsignificant induction of mrp2 levels was found in the livers . in normal rats , ~47% of the initial cddp dose the finding that increased expression of mrp2 in renal bbm upon injection of cddp suggests that this transporter may be involved in the excretion of cddp by the kidney . since levels of mrp2 are already high in the hepatocytes , this may explain why only marginal increases of mrp2 was seen in the livers of cddp - treated animals . moreover , a recent report showed that elevated mrp2 levels seemed to affect the efficacy of cddp - based chemotherapy in hepatocellular carcinoma hcc . while ishikawa and ali - osman initially reported that formation of pt(gs)2 complex reached a maximal level after 12 hrs in l1210 cells treated with 20 m cddp , corresponding to ~60% of the intracellular pt content , however , berners - price and kuchel studied the reaction of cddp with gsh inside intact red blood cells using h spin - echo nuclear magnetic resonance ( nmr ) and detected no formation of pt - gsh bonds within 4 hrs of incubation . recently , kasherman et al . studied the interactions of cddp with cell extracts prepared from ovarian cancer cells and found very little pt(gs)2 by the [ h , n]hsqc approach . since cddp can bind to many thiol - containing proteins and its primary cytotoxic target dna , once inside the cells , these cellular constituents will compete against gsh for cddp binding . these investigators found that the majority of glutathionated complexes were in fact in high molecular mass fraction ( whereas the molecular mass of pt(gs)2 is 809 ) . therefore , the significance of gsh - cddp binding as an important step for cddp elimination remains somewhat controversial . gsh is an abundant thiol - containing tri - peptide ( glu - cys - gly ) , constituting 110 mm in mammalian cells . de novo biosynthesis of gsh is controlled by the rate - limiting enzyme , glutamate - cysteine ligase ( gcl , also known as -glutamylcysteine synthetase , -gcs ) which consists of a catalytic ( heavy ) ( -gcsh ) and a regulatory ( light ) subunit ( -gcsl ) . -gcs carries out the initial ligating reaction of glutamine ( glu ) and cysteine ( cys ) . production of gsh is accomplished by the subsequent reaction involving glycine ( gly ) by gsh synthetase ( figure 1 ) . gsh can be oxidized into gssg by gsh peroxidase using h2o2 as a substrate whereas gssg can be reduced back to gsh by gssg reductase using nadph as a cofactor . therefore , gsh - gssg system provides an important redox buffer in living organisms . as alluded to earlier , the transport activity of mrp2 ( and other members of the mrp family ) is regulated by gsh availability . since -gcs is the rate - limiting enzyme for the biosynthesis of gsh , coregulation of -gcs and mrps would facilitate the efflux activity ( figure 1 ) . indeed , we found that a number of cytotoxic agents can simultaneously induce the expression of both -gcsh and mrp , including cddp , carcinogens , and prooxidants [ 30 , 31 ] . furthermore , enhanced expression of -gcsh / mrp1 was found in colorectal cancers , which are associated with inflammation - associated oxidative stress [ 32 , 33 ] . these observations , taken together , strongly suggest that gsh/-gcs system is a molecular sensor of oxidative stress conditions . previous studies have reported that exposure of cultured cells to cddp led to the development of cddp resistance which was correlated with increased cellular gsh levels [ 3439 ] . moreover , gsh depletion by buthionine - sulfoximine ( bso ) has been associated with increased sensitivity to cddp [ 8 , 1417 ] . in many cases , when -gcs mrna contents were measured , elevated levels of -gcsh mrna were also correlated with cddp resistance . these studies have been widely taken as that intracellular gsh levels play an important role in regulating cddp resistance [ 34 , 4043 ] , perhaps through a gsh - mediated cytoprotective mechanism . however , molecular mechanisms as how gsh functions as a cytoprotector for cddp resistance have not been elucidated . one , these studies frequently used cddp - treated cells and the observations were mostly correlative in nature . whether elevated expression of gsh indeed is causally responsible for the cddp resistance needs to be critically evaluated . two , as alluded to above , induction of -gcs , and thus gsh , in cddp - treated cells may be an oxidative stress - induced phenomenon because -gcs / gsh is a sensor / regulator of reactive oxygen species ( ross ) imbalance , and its expression can be induced by a wide array of cytotoxic insults . in many cases , elevated expression of -gcs / gsh under cytotoxic insult three , also as alluded above , normal cells usually contain millimolar levels of gsh . stoichiometrically , these abundant amounts of thiol compound may already be sufficient to neutralize the cytotoxic effects of cddp which are usually in micromolar ranges . thus , increased gsh content found in most of cddp - treated cells may not necessarily have a major impact in the detoxification mechanism of cddp . as will be described below , we found that elevated expression gsh per se induces cellular sensitization to cddp treatment . both transcriptional regulation and posttranscriptional regulation have been reported for the upregulation of -gcsh by various cytotoxic assaults . transcriptional regulation is mediated by an antioxidant response element ( are , 5-tgagtca ) located at the promoter of the -gcsh allele , which interacts with the nf - e2-related transcription factor ( nrf2 ) . under unstressed conditions , majority of nrf2 is in the cytosol and bound to kelch - like ech - associated protein ( keap1 ) which functions as a substrate adaptor for a cullin - dependent e2 ubiquitin ligase complex and targets nrf2 for ubiquitination and proteasomal degradation . because keap1 is a redox - sensitive e3 ligase , oxidative stress conditions induce keap1 sulfhydryl group modification and conformational changes , resulting in nrf2 release from proteasomal degradation and allowing it to translocate to the nucleus . by heterodimerizing with the small maf protein as coactivator , together , they bind to the are and transactivate -gcsh expression . expression of mrp2 is also apparently regulated by nrf2 signal . livers from hepatocyte - specific gclc/-gcsh - knockout mice showed elevated expression of nrf2 and mrp2 due to elevated oxidative stress . we demonstrated that oxidative stress induces -gcsh mrna stabilization through the p38 map kinase pathways . under oxidative stress conditions , p38 map kinase activates mapkapk2 , which promotes translocation of mrna - stabilizing factor hur from the nucleus to the cytoplasmic compartment , where it stabilizes -gcsh mrna by binding to the au - rich motif located at the 3 untranslated region . the accumulated -gcsh mrna produces elevated levels of gsh that feed back to suppress -gcsh mrna stabilization and the nrf2 signal - mediated -gcsh transcription as mentioned above . in 1998 , while we were studying the coregulation of -gcsh and mrp1 by cddp , we found that transfection of recombinant dna encoding the -gcsh subunit alone was sufficient to enhance gsh levels in the transfected cells . surprisingly , we also found that these stable -gcsh - transfected cells exhibited hypersensitivity instead of resistance to the toxicity of cddp . hypersensitivity to the toxic effect of cddp was associated with enhanced uptake of cddp in these transfected cells . however , transporter for the uptake of cddp was not available at the time . cddp transporter was later identified as the high - affinity cu transporter ( hctr1 ) and the mechanism of this hypersensitization was due to the upregulation of hctr1 in these transfected cells ( see below ) . mutagenized yeast cells with a transpose library after selecting mutants that were able to grow in the presence of a toxic dose of cddp . one of the mutants was defective in the mac1 gene which encodes a copper concentration - dependent transcription factor for the expression of several genes involved in the uptake of iron and copper . these investigators subsequently determined that yctr1 is the target gene that could recapitulate the cddp - resistance phenotype observed in the mac1 mutant . likewise , murine embryonic fibroblasts derived from mctr1(/ ) animals exhibited reduced cddp accumulation as compared with their respective wild - type counterparts . the discovery that ctr1 is involved in pt drug transport underscores the important role of cu metabolism in the efficacy of pt drug chemotherapy . copper is an essential micronutrient for cell survival , yet cu overload is toxic . to meet their need for cu while avoiding toxicity , all living organisms from yeast to humans have developed an evolutionarily conserved system to regulate cu homeostasis . this system consists of a transporter ( ctr1 ) for cu acquisition , chaperones ( hah1 , cox17 , and ccs ) for cu delivery to various intracellular compartments , and exporters ( atp7a and atp7b ) for cu elimination ( figure 2 ) . although cu entry can also be carried out by divalent metal transporter 1 ( dmt1 ) , which transports a broad range of divalent metal ions including cu(ii ) , the majority of cu acquisition is accomplished by ctr1 , which transports cu(i ) . the belgrade rat , which has a mutation in dmt1 , has no cu - deficient phenotype . extracellular cu exists in the oxidized form [ cu(ii ) ] which is converted into cu(i ) by membrane - bound cupric reductases , relevant to the yeast fre1 and fre2 reductases [ 51 , 52 ] , for hctr1-mediated transport . expression of yeast ctr ( yctr1 and yctr3 ) is transcriptionally upregulated under cu - deplete conditions and is downregulated under cu - replete conditions . during cu - depleted conditions , the transcription factor mac1p binds to the metal binding sequence [ 5-tttgc(t / g)c(a / g ) ] located at the promoters of yctr1 and yctr3 [ 5355 ] and turns on the expression of these genes . under cu - replete conditions , mac1 dissociates from the promoters , resulting in shut - down of the expression of yctr1 and yctr3 . in the meantime , the transcription factor ace1 is activated to induce the expression of genes encoding cu - chelating proteins ( cup1 and crs5 ) and the antioxidant superoxide dismutase ( sod1 ) [ 5658 ] to protect cells from cu overload . both mac1 and ace1 contain zinc finger ( zf ) motifs that function as metallosensors . a transcriptional regulation mechanism is also involved in cu(i)-dependent regulation of the drosophila ctrb gene , a homologue of yctr1 and yctr3 . in addition to transcriptional regulation , posttranslational regulation has been reported for yctr1 and yctr3 proteins in response to cu stress . available information in the literature regarding the mechanisms of regulation of human copper transporter ( hctr1 ) is controversial . it has been reported that regulation of mammalian ctr1 is controlled at the posttranslational levels . elevated cu levels induce the trafficking of hctr1 from the plasma membrane to endosomal / lysosomal compartments where ctr1 is degraded [ 61 , 62 ] . we investigated the regulation of hctr1 expression in response to cu concentration stress and found that levels of hctr1 mrna were decreased in cultured small cell lung cancer ( sclc ) cells treated with cuso4 but were increased in cells treated with the cu - depleting agent bathocuproine disulfonic acid . we further demonstrated that the zf transcription factor sp1 plays an important role in the transcriptional regulation of hctr1 . these results , taken together , indicate that expression of hctr1 is upregulated under copper deplete condition but is downregulated under copper replete conditions . the findings that hctr1 expression is regulated by intracellular cu bioavailability prompted us to investigate whether sensitization of gsh - overproducing cells in the -gcsh - transfected cells to cddp was due to reduced intracellular bioavailable cu contents , resulting in upregulation of hctr1 expression . it has been well established that cu can form a cu - gsh complex by directly interacting with its internal cysteine - sh residue of gsh . however , unlike formation of pt(gs)2 , formation of cu(gs ) is almost a spontaneous reaction and requires no enzymatic involvement [ 6365 ] , resulting in reduced intracellular bioavailability of cu levels . we found that the -gcsh - transfected cells indeed showed cu deficiency as evidenced by the reduced enzymatic activities of cu , zn superoxide dismutase ( sod1 ) and mitochondrial cytochrome c oxidase ( cco ) as compared with those in the nontransfected cells . moreover , reduction of cu content in ceruloplasmin , a copper - containing ferroxidase that plays an important role in mammalian iron homeostasis , was also found in the transfected cells . levels of hctr1 expression in the three independently established -gcsh - transfected cells were 3- to 4-fold increases as compared with that in the parental cells . no substantial decreases in atp7a or atp7b levels were observed . moreover , increased hctr1 expression in these -gcsh - transfected cells was associated with enhanced cddp uptake . sensitization of -gcsh - transfected cells to cddp could be reversed when these cells were treated with gsh - depleting agent buthionine - sulfoximine ( bso ) . this reversal was associated with the reduced expression of hctr1 due to the reinstated cu bioavailability in the transfected cells as analyzed by activities of the cu signature enzymes . taken together , our finding demonstrated that elevated levels of gsh per se can sensitize cddp toxicity . the elucidation that gsh functions as a cu chelator in upregulating its transporter hctr1 has important implications in cancer chemotherapy using platinum - based antitumor agents . we note that recent report has shown that another copper chelator , tetrathiomolybdate , can enhance cddp sensitivity in ovarian cancer animal model . the three major mechanisms that control cddp sensitivity by gsh described in the paper reflect the complexity of a small peptide that can regulate the efficacy of cddp toxicity . the significance of each of these mechanisms may depend upon various cell types and/or different cell physiologic conditions . as alluded to above , cddp can interact with many cellular targets and affect many signal transduction pathways to elicit its cytotoxicity . likewise , gsh is an important redox regulator and redox signaling can affect many important cellular processes . future investigations are needed to address the roles of gsh in the global effects of cddp sensitivity .

backgroundinconsistent results were reported in recent literature regarding the association between methylenetetrahydrofolate reductase ( mthfr ) c677t / a1298c polymorphisms and the susceptibility of congenital heart disease ( chd ) . in this study , we performed a meta - analysis to investigate the associations by employing multiple analytical methods.methodsliterature search was performed and published articles were obtained from pubmed , embase and cnki databases based on the exclusion and inclusion criteria . data were extracted from eligible studies and the crude odds ratios and their corresponding 95% confidence intervals ( cis ) were calculated using random or fix effects model to evaluate the associations between the mthfr c677t / a1298c polymorphisms and chd development . subgroup based analysis was performed by hardy weinberg equilibrium , ethnicity , types of chd , source of control and sample size.resultstwenty-four eligible studies were included in this meta - analysis . significant association was found between fetal mthfr c677 t polymorphism and chd development in all genetic models . the pooled ors and 95% cis in all genetic models indicated that mthfr c677 t polymorphism was significantly associated with chd in asian , but not caucasian in subgroup analysis . the maternal mthfr c677 t polymorphism was not associated with chd except for recessive model . moreover , neither maternal nor fetal mthfr a1298c polymorphism was associated with chd.conclusionthe fetal mthfr c677 t polymorphism may increase the susceptibility to chd . fetal mthfr c677 t polymorphism was more likely to affect asian fetus than caucasian . the mthfr a1298c polymorphism may not be a risk of congenital heart disease .

approximately , chd accounts for 28% of the major congenital anomalies ( van der linde et al . , 2011 ) . the generally accepted prevalence of chd was about 8 per 1000 live births , which poses a serious challenge to healthcare ( bernier et al . , 2010 ) . remarkable progresses have been achieved in chd diagnosis and cardiac surgery during the past decades , resulting in an increased survival rate of neonates with chd ( greutmann and tobler , 2012 ) . however , more patients with chd have grown up who comprised of a special population : patients with grown - up congenital heart disease ( guch ) ( khairy et al . it was reported that the prevalence of patients with guch was estimated to be 4 per 1000 adults . long - term medical care and related resource cost are needed for patients with guch , and rapidly increase healthcare burden . thus , it is very important to characterize the etiology of congenital heart disease , which has not been well understood yet . washington infant study have indicated that the cause of chd was multifactorial , and both genetic background and environmental factors may play important roles in the development of chd ( richards and garg , 2010 ; shieh et al . , importantly , due to the advances in molecular techniques , accumulating evidences have suggested that genetic factors were dominant ( bruneau , 2008 ) . it was known that a large proportion of chds were characterized with aneuploidy or abnormal chromosomal number ( blue et al . , 2012 ; pierpont et al . , 2007 ) . about 50% of children who were born with trisomy 21 have atrial and ventricular septal defects or atrioventricular canal lesion . with completion of the human genome project it has been reported that the mutations in single genes including tbx5 , jag1 , nkx2.5 and gata4 have been associated with the development of chd ( basson et al . , 1997 ; the association between folic acid metabolism and the development of chd has been explored recently . maternal supplement of folic acid has been proved to reduce the incidence of chd as well as other congenital heart disease ( van beynum et al . , 2010 ) . single nucleotide polymorphisms of many genes involved in the folate pathway have been identified to affect the function of the genes or folic acid metabolism and thus increase the risk of chd ( locke et al . , 2010 ; shaw et al . , 2009 ) . the flavin adenine dinucleotide - dependent enzyme 5,10-methylenetetrahydrofolate reductase ( mthfr ) catalyzes the reduction of methylenetetrahydrofolate to 5-methyltetrahydrofolate , which is required for the remethylation of homocysteine to methionine ( ueland et al . , 2001 ) hyperhomocysteinemia was believed to be a high risk for the development of heart defects ( verkleij - hagoort et al . , 2006 , 2007 ) . elevating the level of 5-methyltetrahydrofolate , a major circulating folic acid , prevented chd by reducing maternal homocysteine plasma level ( lamers et al . , 2004 ) . therefore , the polymorphisms of mthfr may be closely related to the risk of chd . it was reported that two mthfr snps including mthfr c677 t ( p.ala222val , i d : rs1801133 ) and mthfr a1298c ( p.glu429ala , rs1801131 ) were potentially associated with chd ( van driel et al . , 2008 ) . the amino acid transition in mthfr c677 t ( ala - val ) has resulted in a thermolabile protein associated with reduced enzyme activity in vivo , which may increase plasma homocysteine level ( huhta and hernandez - robles , 2005 ) . the mthfr a1298 c has also been reported to moderately reduce mthfr activity in vivo ( weisberg et al . , 1998 ) . to date , a large number of studies regarding the associations between mthfr gene polymorphisms and risk of chd have been published . herein , we performed a meta - analysis of all published studies until january 2013 to investigate the association between the two snps ( mthfr 677ct and mthfr 1298ac ) and chd patients and their mothers . the pubmed , embase , web of knowledge and cnki ( china national knowledge infrastructure ) database searches were performed to identify all the eligible papers . the search terms were used as the following : ( mthfr or methylenetetrahydrofolate reductase or folic acid ) and ( variant or polymorphism or snp ) and ( congenital heart disease or heart defect or chd or congenital anomalies ) . moreover , potentially relevant studies were evaluated by reviewing the titles and abstracts , and studies matching the criteria were carefully retrieved . if more than one study was published using the same data , only the study with a larger population was included . the eligible studies should meet the following inclusion criteria : ( 1 ) investigation of association between the mthfr polymorphisms ( including c677 t or a1298c or both ) and congenital heart disease ; ( 2 ) a case control study ; ( 3 ) providing sufficient data on genotype frequencies of the mthfr c677 t and/or a1298c polymorphisms and sufficient data for calculation of an odd ratio ( or ) with 95% confidence interval ( ci ) . the exclusion criteria were as follows : ( 1 ) reviews , case report , editorial or comment ; ( 2 ) a duplicated study ; ( 3 ) studies providing insufficient data or data in poor quality ; and ( 4 ) studies without control . based on the inclusion and exclusion criteria , data extraction from each study was performed by two authors ( wang , hou ) independently to ensure that the data extraction were accurate . the following information was extracted from each study : ( 1 ) name of the first author ; ( 2 ) year of publication ; ( 3 ) country of origin ; ( 4 ) ethnicity of the study population ; ( 5 ) source of controls ( population based or hospital based ) ; ( 6 ) sample size of case and controls ; ( 7 ) types of congenital heart disease ; ( 8) genotype distributions in cases and controls ; and ( 9 ) whether population involved in the study was in hardy weinberg equilibrium ( hwe ) . meta - analysis was performed to evaluate the association between mthfr polymorphisms and risk of developing chd . firstly , crude ors with 95% cis were calculated to assess the strength of the correlation between the mthfr c677t / a1298c polymorphisms ( including maternal and fetal ) and risk of chd . pooled ors and 95% cis were calculated for the multiplicative , co - dominant , dominant , and recessive genetic models respectively . the significances of pooled ors were analyzed by z tests , and the criteria for statistically significant were p < 0.05 . a q test was conducted to determine the possible heterogeneity , and p < 0.10 or i > 50% indicated an obvious heterogeneity . pooled ors ( 95% ci ) were calculated by random effects model ( dersimonian laird method ) or fix effects model ( mantel haenszel method ) . subgroup analysis was performed by ethnicity , types of chd , source of controls and sample size ( n < 100 vs. n > 100 ) . sensitivity analysis were performed to evaluate the stability of the results by removing one case control study each time to assess the influence of the individual data on pooled ors . moreover , the egger quantitative tests were also performed , and p < 0.05 was considered statistically significant . to obtain reliable data , two authors ( wang , hou ) have performed the statistical analysis independently by using the same data and the programs . data analyses were performed using stata version 12 ( stata corporation , college station , texas , usa ) . the pubmed , embase , web of knowledge and cnki ( china national knowledge infrastructure ) database searches were performed to identify all the eligible papers . the search terms were used as the following : ( mthfr or methylenetetrahydrofolate reductase or folic acid ) and ( variant or polymorphism or snp ) and ( congenital heart disease or heart defect or chd or congenital anomalies ) . moreover , potentially relevant studies were evaluated by reviewing the titles and abstracts , and studies matching the criteria were carefully retrieved . if more than one study was published using the same data , only the study with a larger population was included . the eligible studies should meet the following inclusion criteria : ( 1 ) investigation of association between the mthfr polymorphisms ( including c677 t or a1298c or both ) and congenital heart disease ; ( 2 ) a case control study ; ( 3 ) providing sufficient data on genotype frequencies of the mthfr c677 t and/or a1298c polymorphisms and sufficient data for calculation of an odd ratio ( or ) with 95% confidence interval ( ci ) . the exclusion criteria were as follows : ( 1 ) reviews , case report , editorial or comment ; ( 2 ) a duplicated study ; ( 3 ) studies providing insufficient data or data in poor quality ; and ( 4 ) studies without control . based on the inclusion and exclusion criteria , data extraction from each study was performed by two authors ( wang , hou ) independently to ensure that the data extraction were accurate . the following information was extracted from each study : ( 1 ) name of the first author ; ( 2 ) year of publication ; ( 3 ) country of origin ; ( 4 ) ethnicity of the study population ; ( 5 ) source of controls ( population based or hospital based ) ; ( 6 ) sample size of case and controls ; ( 7 ) types of congenital heart disease ; ( 8) genotype distributions in cases and controls ; and ( 9 ) whether population involved in the study was in hardy weinberg equilibrium ( hwe ) . meta - analysis was performed to evaluate the association between mthfr polymorphisms and risk of developing chd . firstly , crude ors with 95% cis were calculated to assess the strength of the correlation between the mthfr c677t / a1298c polymorphisms ( including maternal and fetal ) and risk of chd . pooled ors and 95% cis were calculated for the multiplicative , co - dominant , dominant , and recessive genetic models respectively . the significances of pooled ors were analyzed by z tests , and the criteria for statistically significant were p < 0.05 . a q test was conducted to determine the possible heterogeneity , and p < 0.10 or i > 50% indicated an obvious heterogeneity . pooled ors ( 95% ci ) were calculated by random effects model ( dersimonian laird method ) or fix effects model ( mantel haenszel method ) . subgroup analysis was performed by ethnicity , types of chd , source of controls and sample size ( n < 100 vs. n > 100 ) . sensitivity analysis were performed to evaluate the stability of the results by removing one case control study each time to assess the influence of the individual data on pooled ors . moreover , the egger quantitative tests were also performed , and p < 0.05 was considered statistically significant . to obtain reliable data , two authors ( wang , hou ) data analyses were performed using stata version 12 ( stata corporation , college station , texas , usa ) . totally , we have identified 288 potentially relevant studies by employing the search strategy described above . based on obvious irrelevance to mthfr and chd in titles , 248 papers from the 288 potentially relevant papers were excluded . after reading the abstracts of the remaining 40 studies , 7 studies were further excluded , as 6 studies were reviews and one study was a duplicated study . to further polish target studies , of these , 9 studies were excluded , due to insufficient data , data with poor quality or papers without control . after careful screening , 24 eligible studies were finally included in this meta - analysis ( balderrabano - saucedo et al . , 2013 ; bozovic et al . , 2011 ; galdieri et al . , 2007 ; garcia - fragoso et al . , 2010 ; gong et al . , 2009 , 2012 ; hobbs et al . , 2010 ; junker et al . , 2001 , 2005 , 2009a , 2009b ; liu et al . , 2005 ; peng et al . , 2009 ; sanchez - urbina et al . , 2012 ; shaw et al . , 2005 ; storti et al . van driel et al . , 2008 ; wang , 2006 ; wintner et al . , 2007 ; xu et al . , 2010 ; yan and li , 2003 ; zhu et al . , 2006 ) . the search strategy and inclusion / exclusion of studies were shown in a flow chart ( fig . 1 ) . among these studies , fourteen studies investigated the maternal mthfr c677 t polymorphism with 1745 cases and 2044 controls and nineteen studies investigated the fetal mthfr c677 t polymorphism with 2697 cases and 3434 controls . in addition , there were 4 studies investigating maternal mthfr a1298c polymorphism with 432 cases and 532 controls and 5 studies investigating fetal mthfr a1298c polymorphism with 945 cases and 1074 controls . 9 studies included both maternal and fetal mthfr polymorphisms and 5 studies included both mthfr c677 t and a1298c polymorphisms . moreover , 11 studies were performed in caucasian and 12 studies were performed in asian . the genotype and allele distributions of maternal c677 t and a1298c polymorphisms in all the studies included were shown in table 2 . for the fetal polymorphisms , the genotype and allele frequencies of c677 t and a1298c were shown in table 3 . for the maternal mthfr c677 t polymorphism , the results indicated no statistically significant association between the polymorphism and the susceptibility to chd in all genetic models except for recessive model and co - dominant model ( t vs. c : or = 1.103 , 95% ci 0.9991.218 ; tt vs. cc : or = 1.254 , 95% ci 1.0121.553 ; tt + ct vs. cc : 1.105 , 95% ci 0.9581.275 ; tt vs. tc + cc : or = 1.235 , 95% ci 1.0241.489 ) ( table 4 fig . 2 ) . in the subgroup analysis by ethnicity , no significant association was observed in asian population in all genetic models except for recessive model ( t vs. c : or = 1.204 , 95% ci 0.9731.490 ; tt vs. cc : or = 1.454 , 95% ci 0.9212.295 ; tt + ct vs. cc : or = 1.170 , 95% ci 0.8021.707 ; tt vs. tc + cc : or = 1.431 , 95% ci 1.0112.024 ) ( table 4 ) . no association was detected in caucasians in all genetic models . in the stratified analysis by types of chd , there was a significant association between c667 t and asd / pda , however , the results were not reliable because only one study was performed in asd / pda patients ( table 4 ) . in the subgroup of source of control , association was only observed in the recessive model of hospital based control subgroup ( tt vs. tc + cc : or = 1.550 , 95% ci 1.1062.170 ) ( table 4 ) . in the sample size subgroup analysis , there was no significant association between chd and maternal c677 t in all genetic models of large sample studies . however , we have observed a significant association in co - dominant model and recessive model with small sample studies ( table 4 ) . for the fetal mthfr c667 t polymorphism , the overall results suggested a significant association of polymorphism with chd susceptibility ( t vs. c : or = 1.271 , 95% ci 1.1781.372 ; tt vs. cc : or = 1.610 , 95% ci 1.3741.885 ; tt + ct vs. cc : or = 1.258 , 95% ci 1.1201.414 ; tt vs. tc + cc : or = 1.565 , 95% ci 1.3701.788 ) ( table 5 , fig . fetal mthfr c677 t was associated with chd in asian populations for all genetic models , however , no significant association was found in caucasian ( table 5 ) . in the stratified analysis by types of chd , significant associations were detected between fetal mthfr c677 t and all types of chd for all genetic models ( table 5 ) . similar significant association was also observed in cd and asd / pda , however , the positive result in cd was not reliable because it was derived from one study . interestingly , a significant association was observed in hospital based control subgroup rather than in population based control subgroup . by considering sample size , significant results were also found in all genetic models in both small and large sample subgroups ( table 5 ) . for mthfr a1298c polymorphism , the results showed no significant association between this polymorphism and chd risk in either maternal or fetal groups ( maternal : t vs. a : or = 1.043 , 95% ci 0.8551.271 ; cc vs. aa or = 1.109 , 95% ci 0.6921.775 ; cc + ac vs. aa : or = 1.108 , 95% ci 0.8561.435 ; cc vs. ac + aa : or = 0.735 , 95% ci 0.4671.157 ; fetal : c vs. a or = 0.938 , 95% ci 0.8121.083 ; cc vs. aa : or = 1.058 , 95% ci 0.7191.558 ; cc + ac vs. aa : or = 0.871 , 95% ci 0.7281.042 ; cc vs. ac + aa : or = 1.184 , 95% ci 0.8151.721 ) . in the subgroup analysis of either maternal or fetal polymorphisms , there was no statistically significant association in each subgroup by ethnicity , types of chd , source of controls and sample size under all genetic models ( table 6 ) . as shown in table 4 , heterogeneity between studies was significant ( p < 0.10 ) under additive , and recessive genetic models for maternal mthfr c677 t . moreover , evidence for heterogeneity between studies was also found in all genetic models for fetal mthfr c677 t . for the mthfr a1298c polymorphism no evidence for heterogeneity between studies was detected for maternal mthfr c677 t in the co - dominant and dominant models , for maternal and fetal mthfr a1298c under all genetic models except for maternal recessive model . in the sensitivity analysis , the overall association between the maternal mthfr c677 t genotype and chd was not substantially changed by excluding one study at a time ( data not shown ) . similar results were also found in fetal mthfr c677 t , maternal and fetal mthfr a1298c . except for dominant model of fetal mthfr c677 t and co - dominant model of maternal a1298c polymorphisms , no publication bias could be detected by employing egger 's test for studies on maternal mthfr c677 t polymorphism ( t vs. c : p = 0.647 ; tt vs. cc : p = 0.324 ; tt + ct vs. cc p = 0.533 ; tt vs. tc + cc : p = 0.269 ) ; fetal mthfr c677 t polymorphism ( t vs. c : p = 0.077 ; tt vs. cc : p = 0.110 ; tt vs. tc + cc : p = 0.057 ) ; maternal mthfr a1298c polymorphism ( c vs. a : p = 0.882 ; cc + ac vs. aa : p = 0.330 ; cc vs. ac + aa : p = 0.107 ) ; and fetal mthfr a1298c polymorphism ( c vs. a : p = 0.493 ; cc vs. aa : p = 0.576 ; cc + ac vs. aa : p = 0.576 ; cc vs. ac + aa : p = 0.576 ) . the results of egger 's test suggested publication bias in dominant model of fetal mthfr c677 t polymorphism ( tt + ct vs. cc : p = 0.006 ) and co - dominant model of maternal a1298c polymorphism ( cc vs. aa : p = 0.049 ) . the begg 's tests of corresponding genetic models in forest plots were shown in figs . 2 and 3 . totally , we have identified 288 potentially relevant studies by employing the search strategy described above . based on obvious irrelevance to mthfr and chd in titles , 248 papers from the 288 potentially relevant papers were excluded . after reading the abstracts of the remaining 40 studies , 7 studies were further excluded , as 6 studies were reviews and one study was a duplicated study . to further polish target studies , of these , 9 studies were excluded , due to insufficient data , data with poor quality or papers without control . after careful screening , 24 eligible studies were finally included in this meta - analysis ( balderrabano - saucedo et al . , 2013 ; bozovic et al . , 2011 ; galdieri et al . , 2007 ; garcia - fragoso et al . , 2010 ; gong et al . , 2009 , 2012 ; hobbs et al . , 2010 ; junker et al . , 2001 , 2005 , 2009a , 2009b ; liu et al . , 2005 ; peng et al . , 2009 ; sanchez - urbina et al . , 2012 ; shaw et al . , 2005 ; storti et al . van driel et al . , 2008 ; wang , 2006 ; wintner et al . , 2007 ; xu et al . , 2010 ; yan and li , 2003 ; zhu et al . , 2006 ) . the search strategy and inclusion / exclusion of studies were shown in a flow chart ( fig . 1 ) . among these studies , fourteen studies investigated the maternal mthfr c677 t polymorphism with 1745 cases and 2044 controls and nineteen studies investigated the fetal mthfr c677 t polymorphism with 2697 cases and 3434 controls . in addition , there were 4 studies investigating maternal mthfr a1298c polymorphism with 432 cases and 532 controls and 5 studies investigating fetal mthfr a1298c polymorphism with 945 cases and 1074 controls . 9 studies included both maternal and fetal mthfr polymorphisms and 5 studies included both mthfr c677 t and a1298c polymorphisms . moreover , 11 studies were performed in caucasian and 12 studies were performed in asian . the genotype and allele distributions of maternal c677 t and a1298c polymorphisms in all the studies included were shown in table 2 . for the fetal polymorphisms , the genotype and allele frequencies of c677 t and a1298c were shown in table 3 . for the maternal mthfr c677 t polymorphism , the results indicated no statistically significant association between the polymorphism and the susceptibility to chd in all genetic models except for recessive model and co - dominant model ( t vs. c : or = 1.103 , 95% ci 0.9991.218 ; tt vs. cc : or = 1.254 , 95% ci 1.0121.553 ; tt + ct vs. cc : 1.105 , 95% ci 0.9581.275 ; tt vs. tc + cc : or = 1.235 , 95% ci 1.0241.489 ) ( table 4 fig . 2 ) . in the subgroup analysis by ethnicity , no significant association was observed in asian population in all genetic models except for recessive model ( t vs. c : or = 1.204 , 95% ci 0.9731.490 ; tt vs. cc : or = 1.454 , 95% ci 0.9212.295 ; tt + ct vs. cc : or = 1.170 , 95% ci 0.8021.707 ; tt vs. tc + cc : or = 1.431 , 95% ci 1.0112.024 ) ( table 4 ) . no association was detected in caucasians in all genetic models . in the stratified analysis by types of chd , there was a significant association between c667 t and asd / pda , however , the results were not reliable because only one study was performed in asd / pda patients ( table 4 ) . in the subgroup of source of control , association was only observed in the recessive model of hospital based control subgroup ( tt vs. tc + cc : or = 1.550 , 95% ci 1.1062.170 ) ( table 4 ) . in the sample size subgroup analysis , there was no significant association between chd and maternal c677 t in all genetic models of large sample studies . however , we have observed a significant association in co - dominant model and recessive model with small sample studies ( table 4 ) . for the fetal mthfr c667 t polymorphism , the overall results suggested a significant association of polymorphism with chd susceptibility ( t vs. c : or = 1.271 , 95% ci 1.1781.372 ; tt vs. cc : or = 1.610 , 95% ci 1.3741.885 ; tt + ct vs. cc : or = 1.258 , 95% ci 1.1201.414 ; tt vs. tc + cc : or = 1.565 , 95% ci 1.3701.788 ) ( table 5 , fig . fetal mthfr c677 t was associated with chd in asian populations for all genetic models , however , no significant association was found in caucasian ( table 5 ) . in the stratified analysis by types of chd , significant associations were detected between fetal mthfr c677 t and all types of chd for all genetic models ( table 5 ) . similar significant association was also observed in cd and asd / pda , however , the positive result in cd was not reliable because it was derived from one study . interestingly , a significant association was observed in hospital based control subgroup rather than in population based control subgroup . by considering sample size , significant results were also found in all genetic models in both small and large sample subgroups ( table 5 ) . for mthfr a1298c polymorphism , the results showed no significant association between this polymorphism and chd risk in either maternal or fetal groups ( maternal : t vs. a : or = 1.043 , 95% ci 0.8551.271 ; cc vs. aa or = 1.109 , 95% ci 0.6921.775 ; cc + ac vs. aa : or = 1.108 , 95% ci 0.8561.435 ; cc vs. ac + aa : or = 0.735 , 95% ci 0.4671.157 ; fetal : c vs. a or = 0.938 , 95% ci 0.8121.083 ; cc vs. aa : or = 1.058 , 95% ci 0.7191.558 ; cc + ac vs. aa : or = 0.871 , 95% ci 0.7281.042 ; cc vs. ac + aa : or = 1.184 , 95% ci 0.8151.721 ) . in the subgroup analysis of either maternal or fetal polymorphisms , there was no statistically significant association in each subgroup by ethnicity , types of chd , source of controls and sample size under all genetic models ( table 6 ) . as shown in table 4 , heterogeneity between studies was significant ( p < 0.10 ) under additive , and recessive genetic models for maternal mthfr c677 t . moreover , evidence for heterogeneity between studies was also found in all genetic models for fetal mthfr c677 t . for the mthfr a1298c polymorphism , significant heterogeneity was only found in recessive model of maternal polymorphism . no evidence for heterogeneity between studies was detected for maternal mthfr c677 t in the co - dominant and dominant models , for maternal and fetal mthfr a1298c under all genetic models except for maternal recessive model . in the sensitivity analysis , the overall association between the maternal mthfr c677 t genotype and chd was not substantially changed by excluding one study at a time ( data not shown ) . similar results were also found in fetal mthfr c677 t , maternal and fetal mthfr a1298c . except for dominant model of fetal mthfr c677 t and co - dominant model of maternal a1298c polymorphisms , no publication bias could be detected by employing egger 's test for studies on maternal mthfr c677 t polymorphism ( t vs. c : p = 0.647 ; tt vs. cc : p = 0.324 ; tt + ct vs. cc p = 0.533 ; tt vs. tc + cc : p = 0.269 ) ; fetal mthfr c677 t polymorphism ( t vs. c : p = 0.077 ; tt vs. cc : p = 0.110 ; tt vs. tc + cc : p = 0.057 ) ; maternal mthfr a1298c polymorphism ( c vs. a : p = 0.882 ; cc + ac vs. aa : p = 0.330 ; cc vs. ac + aa : p = 0.107 ) ; and fetal mthfr a1298c polymorphism ( c vs. a : p = 0.493 ; cc vs. aa : p = 0.576 ; cc + ac vs. aa : p = 0.576 ; cc vs. ac + aa : p = 0.576 ) . the results of egger 's test suggested publication bias in dominant model of fetal mthfr c677 t polymorphism ( tt + ct vs. cc : p = 0.006 ) and co - dominant model of maternal a1298c polymorphism ( cc vs. aa : p = 0.049 ) . the begg 's tests of corresponding genetic models in forest plots were shown in figs . 2 and 3 . to date , it is known that genetic and environmental risks may be the causes of congenital heart diseases . importantly , numerous studies have suggested the role of folic acid metabolism in the chd development ( ueland et al . , 2001 ) . mthfr is a key enzyme in folic acid conversion process , and its activity may be related with a variety of diseases including chd ( li et al . , 2013 ; long et al . , 2012 ) . it was reported that the c677 t mutation of mthfr could render the enzyme thermolabile with approximately 50% reduced activity and increased plasma homocysteine concentrations ( huhta and hernandez - robles , 2005 ) . therefore , the variants of the mthfr gene may modulate the activity of mthfr and may be an important determinant of chd development . several studies have reported the potential association between mthfr polymorphisms ( c677 t and a1298c ) and chd , however , the results were not consistent ( balderrabano - saucedo et al . , 2013 ; two studies have performed meta - analysis in association between mthfr c677 t polymorphism and chd two years ago ( nie et al . , 2011 ; yin et al . , 2012 ) . however , the mthfr a1298c polymorphism was not analyzed in either of the two studies . we have analyzed the association between mthfr polymorphisms and chd by multiple methods in all genetic models and included more recent studies . to our knowledge , this is the first meta - analysis on association between mthfr polymorphisms and chd including both c677 t and a1298c . for the mthfr c677 t polymorphism , most studies have indicated that maternal c677 t was not a strong risk of chd , however , some reports have suggested its potential role in chd development . in our finding , no statistically significant difference was detected in genotype or allele frequencies of mthfr c677 t polymorphism in the mothers of chd patients compared with controls . only marginal association between maternal c677 t polymorphism and chd was found in recessive model . the finding was consistent with the previous studies involving both maternal and fetal c677 t polymorphism . particularly , we found a significant association between maternal c677 t and chd in recessive genetic models of asian subgroup , however , similar result was not observed in caucasian . this discrepancy of association between asian and caucasian groups may be attributed to the different genetic background and environmental factors . our results have indicated that fetal mthfr c677 t polymorphism was significantly associated with chd in all genetic models . it was evident that fetal mthfr c677 t polymorphism was an important risk in the development of chd . to explain the results , we speculated that decreased fetal mthfr enzyme activity may result in a local hyperhomocystein environment , in which the heart could not develop normally ( lu et al . , 2011 ) . these evidences have supported the viewpoint that fetal mthfr c677 t polymorphism was more important than maternal mthfr c677 t polymorphism , and concentration of homocystein in fetus may influence heart development rather than maternal homocystein concentration . in addition , we found that fetal mthfr c677 t was significantly associated with chd in asian , while no statistically significant association was found in caucasian population . consistent with the result from recessive model of maternal analysis , the fetal mthfr c677 t was more likely to be associated with chd in asian than caucasian . the results have validated the notion that mthfr c667 t may be in combination with other genetic background and environment factors to affect the fetal heart development . by considering the source of controls , the association between mthfr c667 t polymorphism and chd was significant in hospital based control group , though , not significant in population based control . the confounding results from two subgroups categorized by source of control have indicated that hospital based and population based control was not homogenous in this study . we believe that the comparability between cases and controls contributes to the disagreement of these two subgroups . for the mthfr a1298c polymorphism , we found no statistically significant association between this polymorphism and chd either in maternal or fetal analysis . our finding has demonstrated that mthfr a1298c may not be a risk of congenital heart disease development . however , some studies indicated that the interaction between mthfr 1298 c allele and folic acid supplement increased the risk of having a child with chd ( van driel et al . considering that minimal eligible studies included in our meta - analysis , this result should be validated with more studied and large pooled samples in future . in subgroup analysis by ethnicity , only one study was performed in asian population to investigate fetal a1298c polymorphism and four studies were performed in caucasian population . because of the importance of mthfr polymorphisms in asian chd development , we suggest that more studies investigating association between a1298c polymorphism and chd be performed in asian population . although we made these findings in this meta - analysis , there were several limitations . first , our study was mainly based on unadjusted odd ratios , and the potential covariates including gender , age , vitamin supplement , smoking or other environmental factors , which might influence the final results , were unable to control . we have investigated the study heterogeneity including geographic region , ethnicity , and source of control . however , none of them was identified as the potential source of heterogeneity between studies by meta - regression ( data not shown ) . we estimated that other unknown confounding factors may help explain the between - study heterogeneity . however , only a few studies included in our meta - analysis have classified their cases by types of chd . to analyze this issue despite the limitations mentioned above , the present study has demonstrated that the fetal mthfr c677 t polymorphism is an important risk of developing congenital heart diseases . our findings also suggest that mthfr a1298c polymorphism does not increase the susceptibility to chd . interestingly , we found that fetal mthfr c677 t polymorphism more likely affects asian fetus than caucasian fetus in the development of chd .

tracheobronchial damage is very rare in clinical practice and represents no more than 1% of all injuries caused by blunt trauma . nearly 80% of patients die before reaching the hospital . most ruptures are observed in the right main bronchus and are located within 2 cm of the carina trachea . the highest mortality rate applies to patients with bilateral bronchial injuries . nonspecific symptoms , additionally masked by complaints regarding other damaged organs , delay the diagnosis and surgical treatment . the aim of this article is to present one particular clinical case and to discuss it in conjunction with a literature review .

chest injuries constitute barely 34% of the consequences of blunt trauma , whereas nearly 25% of post - traumatic deaths are caused by injuries to the chest . such injuries are present in over half the patients after multifocal trauma . statistically , airway trauma is most commonly observed among men under 40 years of age , and the male - female ratio is 3 : 1 [ 1 , 2 ] . 47% ; nearly 80% of injuries are located less than 2 cm from the carina . left - sided bronchial injuries constitute 32% of cases , while only 2% involve trauma to both main bronchi . the rate of post - traumatic tracheal ruptures is around 19% ; they are most often located in the cervical trachea ( 1015% ) . the nonspecificity of the clinical symptoms , which often overlap with symptoms arising from other injured organs , results in diagnostic difficulties and delays the start of treatment . the symptom most often mentioned in clinical practice is subcutaneous emphysema ; other frequently observed symptoms include dyspnea , changes of vocal timbre , hemoptysis , and bleeding ( if the injury involves the bronchial arteries ) . imaging examinations reveal pneumothorax , sometimes bilateral . increased air leakage in the drainage system after the connection of a suction device to the pleural drain is an important symptom that suggests the possibility of airway disruption . in the case of injuries located in the thoracic part of the respiratory tract , mediastinal emphysema with the accumulation of air in the subcutaneous tissue may be the only symptom present . when airway injury is suspected , the primary diagnostic examination is bronchofiberoscopy , which also enables the assessment of the extent of the injury . in the case of intensified bleeding , classic rigid bronchoscopy is recommended . the patient was a 25-year - old male mine worker , who suffered a multiorgan injury when he was buried by falling rocks . on admission to the intensive care unit , mechanical ventilation was introduced along with pharmacological therapy with catecholamines and antibiotics . after a chest radiogram revealed right - sided pneumothorax and costal fractures ( ribs ii v ) the patient s crushed right foot was treated surgically ( amputation at chopart s joint ) , while the fractured medial condyle of the femur was treated conservatively . due to the observed incidents of reduced respiratory fitness parameters , computed tomography of the chest the examination visualized irregularities in the outline of the left main bronchus and a consolidation of gas bubbles in this area , which prompted the suspicion of bronchial injury ( figures 1 a , b , 2 a , b present a tomographic reconstruction of the respiratory tract ) . tomographic image reconstructions : deformation of the wall of the left main bronchus tomographic image reconstructions : vascular system and lung parenchyma diagnostic investigation was supplemented with bronchofiberoscopic examination , revealing hyperemia and massive edema of the mucosa in the proximal segment of the left main bronchus , immediately next to the tracheal bifurcation . consequently , the attempt to reach the left main bronchus with the endoscope was unsuccessful . the patient was referred for further treatment at the thoracic surgery center in wrocaw with a suspicion of airway disruption . 500 ml in volume ) and extensive hematomas in the tissues of the thoracic integuments . prepare of the mediastinal tissues revealed the completely amputated left main bronchus ; the whole length of the membranous part of the bronchus was ruptured . subsequently the detached bronchus was anastomosed with the trachea , and the anastomosis was wrapped with an intercostal muscle flap . the patient s hospitalization continued at the intensive care unit of the lower silesian lung diseases center . , the drain from the left pleura was removed ; 3 days later , the right pleural drain was removed as well . on the 6 day after the thoracotomy , the airway was reevaluated with an endoscope , revealing stenosis at the tracheobronchial anastomosis ; the endoscope could not pass through this location . additionally , a large amount of mucous secretion , which filled the whole bronchial tree , was evacuated . consequently , the secretion accumulated again , resulting in a repeated bronchoaspiration after less than 20 h. it was deemed necessary to perform tracheotomy . after the patient s cardiopulmonary parameters stabilized on the 14 postoperative day , the patient was referred for further treatment on the thoracic surgery department . the patient was discharged from the center on the 18 postoperative day . after less than 6 weeks , the patient was readmitted with symptoms of left main bronchus occlusion . for the past several days , the patient had manifested symptoms of airway infection : he was fever and he had dyspnea . an out - patient computed tomography ( ct ) scan demonstrated anastomotic stenosis ( fig . obturation of the left main bronchus at the anastomosis site obstruction of the left bronchial tree with preserved lung aeration a chest radiogram ( fig . 5 ) taken on the day of readmission ( 7 days after the ct exam ) revealed complete atelectasis of the left lung . radiological image of left lung atelectasis bronchofiberoscopic examination showed that the lumen of the left main bronchus was very narrow : its diameter did not exceed 3 mm . an attempt was made to perform mechanical dilatation , but without success . the diagnostic investigation was supplemented with chest ct , revealing obturation of the left bronchial tree ( fig . computed tomography image : obstruction of the left bronchial tree computed tomography image : left lung atelectasis chest computed tomography : bronchographic reconstruction scans first postoperative day a decision was made to perform surgical intervention . thoracotomy was repeated with sleeve resection of the stenosed left main bronchus ( length : 11.5 cm ) , and the bronchus was anastomosed with the trachea ( the posterior wall with single sutures , and the anterior wall with a continuous suture ) . figure 9 shows the chest radiogram obtained on the 1 postoperative day , while figure 10 presents a radiogram from the 6 postoperative day . chest x - ray from the day preceding the patient s discharge from the day of readmission , the patient was administered broad - spectrum antibiotic treatment . on the third postoperative day , the drain placed in the left pleura was removed . he continued to be monitored as an outpatient he attended follow - up visits , during which the site of anastomosis was evaluated with an endoscope . the first such examination took place 2 weeks after discharge , demonstrating permeability of the anastomotic site and no signs of inflammation . subsequent follow - up examinations took place every three months until the end of february , 2015 . the final examination showed a stable endoscopic image with satisfactory width of the left main bronchial lumen . post - traumatic airway injuries are very rare in clinical practice . due to the character and multifocality of the traumatizing force , symptoms of other organs are often much more pronounced and mask the symptoms related to the respiratory system . the mortality rate is 60% for bilateral bronchial damage , 26% for tracheal injuries , and , respectively , 16% and 8% for isolated injuries of the right and the left bronchial tree . statistically , the median time until diagnosis is estimated at approximately 9 days , and until the start of surgical treatment at 25 days . the median time until diagnosis and treatment is , respectively , 30 and 42 days for left - sided bronchial injuries , 1 and 3 days for right - sided injuries , and 3 and 23 days for tracheal injuries . the type , extent , and possible consequences of a blunt trauma are determined by 3 factors : compression , truncation , and shock wave . , the energy is dispersed as the tissues buckle under compression ; if the deformation is too quick , the tissues are ruptured . the first one assumes that the action of a violent compression force in the anteroposterior dimension results in its transverse dispersion . consequently , the pulmonary parenchyma is pressed against the chest wall , and the lateral forces cause the lung to press on the tracheal bifurcation . this is presumably the dominant mechanism for crushing injuries . according to the second theory , confirmed in experimental canine studies , the rising compression of the chest and trachea with a closed glottis leads to a rapid increase of airway pressure ( especially in the trachea and large bronchi ) . when the respiratory tract s elasticity is overcome , a rupture occurs ( usually of the membranous part ) . the third theory assumes that during quick deceleration the force disperses , resulting in traumatization of the trachea and the bronchial tree . this mechanism best explains the injuries occurring in traffic accident victims [ 8 , 9 ] . injuries of the left bronchial tree are observed less frequently ; this is due to the close proximity of the aortic arch , which stabilizes the bronchus . the force injuring the bronchus simultaneously causes a rupture of the aortic wall , which most often results death on the scene . in the present case , the traumatic mechanism was probably somewhat different , as evidenced by the ct image and the intraoperative situation . the dynamism of the traumatic process was surprising in the context of the relatively inconspicuous clinical manifestation . the lack of sleeve resection of the injured bronchus during the first procedure is worth discussing . the decision was made in view of the patient s severe condition including cardiopulmonary failure , the impossibility of making technical provide for the anesthesiological and surgical team , and the intraoperative surprise factor . the literature features recommendations stating that reconstruction should be attempted in the case of bronchial injuries located proximally to the subsegmental branches , while the distal segments should be resected . the survival rates of patients treated with surgery are better than those of patients treated conservatively . the mortality rate is lower in patients in whom bronchial reconstruction was performed than in patients in whom the injured bronchial segment was resected with the peripheral fragment of pulmonary parenchyma ( 3% vs. 13% ) . in the case of tracheal injuries , the mortality rate was estimated at 6% in patients treated with surgery and 66% in patients treated conservatively . the present case raises the question whether covering the anastomotic site with a pedicled intercostal muscle contributed to the occurrence of stenosis . recommendations that can be found in the medical literature include the use of reversed parietal pleural flaps , pericardial fat , or fragments of the serratus anterior , pectoralis major , or latissimus dorsi muscle . in the long term , intercostal muscles show a tendency to calcify [ 7 , 11 ] , as was the case in the present patient 9 weeks passed between the first and the second procedure . another question open for discussion is the possibility of dilatation the lumen of the stenosed bronchus with a laser . however , in view of complete lung atelectasis , the symptoms of infection , and the young age of the patient , a decision was made to perform surgical treatment . this case study was presented at the extended conference of the polish thoracic surgeon club ( march 2021 , 2015 in karpacz ) .

it was recently demonstrated that tlr4 activation via dietary lipids triggers inflammatory pathway and alters insulin responsiveness in the fat tissue during obesity . here , we question whether other tlr family members could participate in the tlr - mediated inflammatory processes occurring in the obese adipose tissue . we thus studied the expression of tlr1 , tlr2 , tlr4 , and tlr6 in adipose tissue . these receptors are expressed in omental and subcutaneous human fat tissue , the expression being higher in the omental tissue , independently of the metabolic status of the subject . we demonstrated a correlation of tlrs expression within and between each depot suggesting a coregulation . murine 3t3-l1 preadipocyte cells stimulated with pam3csk4 induced the expression of some proinflammatory markers . therefore , beside tlr4 , other toll - like receptors are differentially expressed in human fat tissue , and functional in an adipocyte cell line , suggesting that they might participate omental adipose tissue - related inflammation that occurs in obesity .

toll - like receptors ( tlrs ) are transmembrane receptors initiating a range of host defense mechanisms in response to microbial products [ 1 , 2 ] . activation of the tlrs leads to activation of intracellular signaling pathways which results in the production of inflammatory cytokines , or chemokines , inducing the development of antigen - specific adaptive immunity . these receptors recognize pathogen - associated molecular patterns as well as host - derived ligands released by various cell types during immune responses . this leads to signaling events resulting in acute host responses necessary to kill the pathogens . this activation is beneficial for the host but can become deleterious if resulting in chronic inflammation . beside their role in innate and adaptive immune responses , tlrs have been recently described to regulate energy metabolism , mostly through acting on adipose tissue . in particular , it was demonstrated that tlr4 , the receptor for recognition of gram - negative bacterial cell wall components , was able to sense free fatty acids and to induce insulin resistance in adipose tissue . tlr2 , as tlr4 , has been shown to be activated by saturated free fatty acids and is implied in bacterial lipoprotein recognition . unlike other tlrs , which are functionally active as homodimers , tlr2 can form heterodimers with tlr1 or tlr6 that will recognize distinct molecular patterns of lipopeptides and can discriminate between tri- and diacylated lipopeptides [ 8 , 9 ] . our purpose is to demonstrate that beside tlr4 , tlr2 and its partners tlr1 and tlr6 are potentially susceptible to play also a role in adipose tissue inflammation . epidemiological studies have clearly demonstrated a relationship between intraabdominal fat depots and metabolic abnormalities related to obesity [ 10 , 11 ] . in this regard , subcutaneous and omental adipose tissues display different metabolic features [ 12 , 13 ] such as differences in lipolysis or adipokine secretion . since omental tissue is related to a higher degree of inflammation , a high expression of tlrs in this tissue may correspond to an implication of these receptors in obesity - related inflammation . we analyzed , tlr1 , tlr2 , tlr6 , and tlr4 expression in paired human adipose omental and subcutaneous samples from subjects with different glycaemic status . we previously demonstrated that inflammation correlates with a decrease of lipogenesis and that tlr4 stimulation interferes with adipocyte differentiation . in order to determine whether tlr2 activation can be also implied in proinflammatory stimulation or interact with adipogenesis , we studied the impact of pam3csk4 ( a tlr1/tlr2 agonist ) exposure on 3t3-l1 preadipocyte cell line . insulin , dexamethasone and , isobutyl-1-methylxanthine were purchased from sigma chemical co. ( st louis , mo , usa ) . the synthetic bacterial lipoprotein n - palmitoyl - s-[2,3-bis(palmitoyloxy)-(2rs)-propyl]-[r]-cysteinyl-[s]-seryl-[s]-lysyl-[s]-lysyl-[s]-lysyl-[s]-lysine ( pam3csk4 ) was from invivogen ( san diego , ca , usa ) . tetramethylbenzidine ( tmb ) was from bd biosciences ( franklin lakes , nj , usa ) . forty - one caucasian female subjects ( seven lean volunteers and thirty - four obese patients ) attending either the dpartement de chirurgie gnrale et endocrinienne , chru de lille or the department of endocrinology of the clnica universitaria de navarra were enrolled in the study . subjects were further classified into 3 groups according to the recently established diagnostic thresholds ( based on an oral glucose tolerance test , ogtt ) for diabetes and lesser degrees of impaired glucose regulation ( normoglycaemia : fasting plasma glucose concentration ( fpg ) < 100 mg / dl and 2-h pg < 140 mg / dl after ogtt ; glucose intolerant : fpg > 100 mg / dl and < 125 mg / dl or 2-h pg between 140 and 199 mg / dl after ogtt ; type 2 diabetes mellitus : fpg 126 mg / dl or 2-h pg 200 mg / dl after ogtt ; table 1 ) . the lean group included patients undergoing surgery due to benign diseases , such as cholecystectomy , while the 34 obese patients strictly met the criteria for bariatric surgery . in both groups of patients surgery informed consent was obtained from all subjects and the experimental design was approved by the hospitals ' ethical committees responsible for research . biopsies were obtained from both subcutaneous and omental adipose tissues of lean and obese volunteers . 3t3-l1 preadipocytes were maintained and cultured in dmem ( gibco , paisley , scotland , uk ) containing 10% ( vol / vol ) fetal calf serum ( gibco ) . 3t3-l1 cells were differentiated into adipocytes as previously described . briefly , 2-day postconfluent 3t3-l1 preadipocytes ( designated day 0 ) were fed dmem containing 10% fcs , 10 g / ml insulin , 1 m dexamethasone , and 0.5 mm 3-isobutyl-1-methylxanthine for 2 days . cells were then fed dmem supplemented with 10% fcs and 5 g / ml insulin until day 10 . human samples were homogenized using an ultra - turrax t25 basic equipment ( ika werke gmbh , staufen , germany ) . total rna was extracted from 3t3-l1 cells or from human adipose samples using rneasy lipid kit ( qiagen , courtaboeuf , france ) . one microgram of total rna was transcribed into cdna using cdna archive kit ( applied biosystems , foster city , ca , usa ) . each cdna sample was analyzed for gene expression by quantitative real - time pcr ( qpcr ) using the fluorescent taqman 5-nuclease assay on an applied biosystems 7900ht sequence detection system . the taqman real - time pcr was performed using 2 taqman master mix and 20 premade taqman gene expression assays ( applied biosystems ) . the mrnas levels were normalized to that of acidic ribosomal phosphoprotein ( 36b4 ) , a gene whose expression is unaffected by adipogenesis . for human samples , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was used as reference gene , since it was previously described to exhibit a low coefficient of variation and no significant differences in mrna levels between samples of the different phenotypical groups . the data are given as the ratio of the target gene mrna to that of gapdh or 36b4 mrna level . concentrations of cytokines and chemokines were measured by duoset elisa development system according to manufacturer instructions ( r&d systems , abingdon , uk ) . to further confirm the specific tlr2 activation and to exclude potential endotoxin contamination of pam3csk4 agonist leading to tlr4 activation , we used a monoclonal anti - tlr2 antibody to inhibit its biological activity ( clone t2.5 from hycult biotechnologies , uden , the netherlands , igg1 isotype ) [ 18 , 19 ] . the negative control was performed using another igg1 monoclonal antibody ( 15h6 , interchim , montluon , france ) . statistical analysis was performed with the spss software package ( 14.0.2 , chicago , il ) . according to sample size , the test on ranks was performed and two - tailed exact p - values are given . the mrna levels between lean and obese patients or in cell culture studies were analyzed by u mann - whitney 's test . comparisons of mrna levels between subcutaneous and omental adipose tissues were performed using the paired wilcoxon test . correlations between continuous variables were determined using the nonparametric spearman 's rank correlation . the threshold of significance was set at p < .05 . we first globally analyzed tlr1 and tlr2 mrna expression in omental and subcutaneous adipose tissue of 34 morbidly obese ( bmi = 46.4 5.6 kg / m ) and 7 lean subjects ( bmi = 21.4 2.4 kg / m ) by qpcr . we observed an increased expression of tlrs in omental fat compared to subcutaneous adipose tissue ( 1.42- , 1.35- , 1.35- , and 1.40-fold increase for tlr1 , tlr2 , tlr4 , and tlr6 , resp . ; the nonparametric paired wilcoxon test was performed between subcutaneous and omental values for each gene and demonstrated a significant difference . p - values are , respectively , tlr1 : 1,03 10 ; tlr2 : 3.84 10 ; tlr4 : 1,29 10 ; tlr6 : 2.30 10 . this difference was significant in obese normoglycemic and obese glucose intolerant groups , never in lean group and significant for tlr4 and tlr6 in diabetic subjects . we then performed spearman 's correlation analysis ( nonparametric analysis based on rank ) between each tlr expression and each depot ( data not shown ) . for a given tlr , expressions was strongly correlated in omental and subcutaneous adipose tissues ( e.g. , individuals with high tlr1 expression in the omental depot also showed a high subcutaneous tlr1 level ) . in each given depot , a correlation between the four tlrs expression was also observed ( e.g. , individuals with a high tlr1 expression in subcutaneous depot also display a high expression of all other tlrs in this depot ) . mann - whitney analysis was performed to detect an association between tlrs expression and glycaemic status . no correlation between expression levels of tlrs and glycaemic status was observed among obese subjects . the only significant value obtained was between lean and obese normoglycemic subjects in omental tissue for tlr1 and tlr2 ( p = 3.73 10 and 3.58 10 , resp . ) to determine whether toll - like receptors are expressed and functional in an adipocyte cell line model , total mrna was isolated from 3t3-l1 preadipocytes as well as fully differentiated adipocytes , and qpcr was performed . considering differentiated adipocytes , the expression of tlr4 mrna was 76 times higher than that of tlr1 , 4.6 times higher than that of tlr2 and 30 times higher than that of tlr6 . this relative expression difference was also observed in preadipocyte but with a lower range ( figure 3 ) . differentiation into adipocyte has no significant impact on tlr2 and tlr6 expression levels while tlr1 expression is lowered ( 5-fold ) and tlr4 is enhanced ( 4-fold ) . we wanted to demonstrate that , besides tlr4 as already published , other tlrs can be functional in 3t3-l1 cell line . we therefore stimulated 3t3-l1 preadipocytes and fully differentiated adipocytes with pam3csk4 ( a tlr1/tlr2 agonist ) and measured the expression level of inflammation markers by qpcr after 4 hours of induction . mrna coding for il6 , ccl2 , ccl11 , nos2 , ccl5 , and ptgs2 was highly induced by pam3csk4 stimulation in preadipocytes as well as in fully differentiated adipocytes ( figure 4 ) . to further demonstrate the specificity of this stimulation and exclude the eventuality of tlr4 stimulation via endotoxin contamination a monoclonal antibody of the same isotype was used as a control and demonstrated no effect on the pam3csk4 induction . therefore , the stimulation of proinflammatory markers by pam3csk4 can specifically be attributed to tlr2 ( figure 5 ) . cell supernatants were collected and cytokine and chemokine secretions were measured by elisa at different time points after pam3csk4 stimulation . preadipocytes were mainly responsible for release of il6 , whereas the ccl2 , ccl5 , and ccl11 concentrations observed were in the same range for both preadipocytes and adipocytes ( figure 5 ) , demonstrating that adipocytes are able to secrete proinflammatory products via tlr2/tlr1 activation in both states of differentiation . we previously demonstrated that tlr4 stimulation by lps was able to impair adipocyte differentiation of 3t3-l1 cells . to demonstrate that tlr2/tlr1 is also able to interfere with this process , we added 1 or 10 ng / ml pam3csk4 to the differentiating medium throughout the differentiation process . microscopic observation of lipid - laden cells stained with oil - red - o showed a slight reduction of lipid droplets in 3t3-l1 cells cultured with pam3csk4 at day four . since ppar is a key regulator of adipocyte differentiation , we monitored its expression level during the differentiation process . ppar mrna was detected 2 days after onset of differentiation and was further elevated at days 6 and 10 . treatment with pam3csk4 led to a 50% reduction of ppar expression ( figure 6 ) , demonstrating that tlr2/tlr1 activation impairs adipogenesis . obesity is defined as a low - grade chronic inflammatory disease associated with a moderate increase of circulating inflammatory factors . this inflammation causes or worsens insulin resistance in insulin - responsive tissues such as adipose tissue , muscle , or liver . one of the causative factors of this inflammation process is the adipose tissue itself via its early infiltration with immune cells ( mainly macrophages ) and via its autocrine and paracrine secretion of pro - and anti - inflammatory cytokines . importantly , it was shown that macrophage infiltration is more prominent in visceral fat than in subcutaneous fat [ 13 , 24 ] , thereby reinforcing the notion that intra - abdominal fat amount and metabolic abnormalities are correlated , as clearly shown by several epidemiological studies [ 10 , 11 ] . one member of the tlr family , namely tlr4 , was reported to participate to the development of inflammation and insulinresistance at the adipose level [ 5 , 25 ] . we thus hypothesized that other functional members of the tlr family of innate immune receptors might also participate in these processes . since , like tlr4 , tlr2 was demonstrated to sense fatty acids when dimerized either with tlr1 or tlr6 we first focused our search on this receptor and on its dimerization partners tlr1 and tlr6 . we found that tlr4 , tlr1 , tlr2 , and tlr6 are significantly overexpressed in omental adipose tissue . since a preferential macrophage infiltration into obese omental versus subcutaneous fat was demonstrated , we can not exclude that toll - like receptors expression in adipose tissue is mainly due to macrophages as suggested by others , but has to be demonstrated since many other adipose tissue cell types can express toll - like receptors . we observed a strong correlation between each tlr expression within a given adipose depot and that at the individual level , each tlr expression is correlated positively in subcutaneous and omental depots . , a cross - regulation between tlr4 and tlr2 expression was demonstrated after activation of 3t3-l1 cells with lps , a tlr4 agonist . whether lps is the effector signal triggering overexpression of tlrs in human omental tissue could be hypothesized but other tlr ligands could also be involved . endotoxemia was shown to participate in the initiation of obesity and insulin resistance , therefore serum lps concentration as well as free fatty acid concentration would be interesting to be evaluated in relation to tlrs expression . recently , resistin was demonstrated to be able to bind to tlr4 , and other endogenous ligands such as hmgb1 or hyaluronan fragments were also reported as tlr4 or tlr2 activators . whether different tlr activators in obesity are of endogenous or exogenous origins or even of both , remained to be investigated and will be crucial for a better comprehension of this chronic inflammation . we previously demonstrated that the inflammatory state associated with a decreased expression of lipogenic markers was more pronounced in diabetic subjects . we could not reveal any differential expression according the prediabetic or diabetic status of the subjects . this might suggest that omental overexpression of toll - like receptors could play a role in early prediabetic phases of metabolic syndrome acquisition but not in further complications . activation of tlr2 by saturated fatty acids leads to the activation of myd88-dependent signaling pathways whatever heterodimer is implied . we here demonstrate that proinflammatory products can be synthesized by 3t3-l1 cells following tlr2/tlr1 activation . we have used the well - known 3t3t - l1 murine preadipocyte cell line as an in vitro system for adipocyte generation to study the inflammatory response of both preadipocytes and adipocytes upon tlrs stimulation . the expression level of tlrs in 3t3-l1 cell line has not to be compared to what was obtained from human samples since adipose tissue is constituted of numerous different cell types ( e.g. , adipocytes , macrophages , and endothelial cells ) . our purpose was to stress out the potential responsiveness of the adipocyte or of its precursors toward stimulatory compounds of the tlr2 pathway . we had already shown that il-6 , certain chemokines ( ccl2 , ccl2 , and ccl11 ) , inducible nitric oxide synthase ( nos2 ) , and cyclooxygenase-2 ( ptgs2 ) , all important mediators of inflammation , can be induced via tlr4 activation in the 3t3-l1 cell line . tlr4 activation induced a lower secretion of il6 , ccl5 , and ccl11 in adipocytes when compared to preadipocytes , while ccl2 secretion was similar in both differentiation states . following tlr2 activation , only the secretion of il-6 was lower in differentiated adipocytes than in preadipocytes . similarly , we observed different induction factors for mrna levels of the proinflammatory enzymes nos2 and ptgs2 in this study of activation via tlr2 and in the previous study of activation via tlr4 . recently , an exhaustive study of tlr1 to tlr9 activation of adipocyte with corresponding ligands was reported demonstrating a distinct response for each receptor . as stated before , the pertinence of these observations will be strengthened when the in vivo genuine ligands will be described . we have observed that tlr2 and tlr6 expression is constant throughout differentiation of 3t3-l1 cells , whereas tlr1 and tlr4 expression is modulated in fully differentiated adipocytes . whether this could explain the weaker secretion of il-6 observed in fully differentiated adipocyte can be hypothesized . we suggest that a fine regulation of the synthesis of proinflammatory mediators could be achieved in adipose tissue via differential expression and activation of tlr family members . as for tlr4 stimulation this is in agreement with previous observation that showing tnf release in human adipose tissue is mainly due to nonfat cells . to conclude , we demonstrate here that tlr2/tlr1 activation is able to interfere with adipocyte differentiation in the 3t3-l1 cell line , as previously described for tlr4 . this could occur either directly or via secretion of adipoctye - derived proinflammatory and antiadipogenic products . this observation is in agreement with our previous study demonstrating that the expression of lipogenic factors is reduced in omental adipose tissue in correlation with inflammation increase . beside their defense function of alerting the immune system of the presence of pathogenic microorganisms , tlrs can also sense dietary lipids therefore , it is tempting to speculate that detection of abnormal level or composition of these lipids will induce a physiological response . our observations suggest that it might be the case and that , in addition to tlr4 , other tlr family members , that are functional and present in adipocytes , could play this role . unfortunately , the physiological response results in elicitation of a chronic omental adipose inflammation which contributes to metabolic syndrome .

cardiac magnetic resonance imaging ( mri ) is increasingly used as the optimum modality for cardiac imaging . an aging population and rising numbers of patients with permanent pacemakers means many such individuals may require cardiac mri scanning in the future . whilst the presence of a permanent pacemaker is historically regarded as a contra - indication to mri scanning , pacemaker systems have been developed to limit any associated risks . no reports have been published regarding the use of such devices with cardiac mri in a clinical setting . we present the safe , successful cardiac mri scan of a patient with an mri - conditional permanent pacing system .

magnetic resonance imaging ( mri ) gives superior imaging resolution and is increasingly the modality of choice for functional and anatomical imaging in cardiovascular disease . in an aging population the prevalence of patients with implanted trans - venous pacing systems means that , increasingly , such patients will require a cardiac mri study ; however , the presence of a permanent pacemaker ( ppm ) has traditionally excluded mri as a viable imaging modality . the use of mri as a viable and safe imaging modality for individuals with an implanted ppm has been an issue of debate for some time . however , there are reports of hazards both to the device and the patient , including fatalities . historically , the main concerns centered around the potential for the strong magnetic fields to move the device , cause inappropriate pacemaker stimulation , potential alterations to the device programming , and to create cardiac tissue damage through local heating with consequent alteration in lead thresholds . confidence has improved following the recent advent of mri conditional systems , approved and ce marked for conditional use with mri scanning , including cardiac mri . whilst mri scanning of anatomy remote to the heart has been increasingly reported , there remains a reluctance to use mri fields to directly image the heart and thorax . indeed , initial recommendations involved keeping the pacing device away from the isocenter of the magnet , hence precluding cardiac imaging . the concern was that the risk of damage to the device or the patient would be too high if the magnetic field was concentrated directly over the heart , as well as the increased potential for artefact attenuation of image quality . however , despite growing evidence to support the safety of these devices in mri scanning of the heart under experimental conditions , to our knowledge there are no published reports regarding their use with cardiac mri in a clinical setting . a 64-year old man presented with a history of increasing exertional breathlessness , chest tightness and intermittent fatigue shortly after undergoing left atrial ablation for paroxysmal atrial fibrillation ( af ) . following an initial diagnosis of af in 2005 , medical management with anti - arrhythmic drugs ( including amiodarone ) had been effective , but worsening symptoms necessitated left atrial ablation with pulmonary vein isolation in 2009 . subsequent to this , he reported increasing breathlessness and chest discomfort . due to persistent symptomatic sinus bradycardia given his ongoing symptoms and the uncertainty of the underlying diagnosis , the pacing system selected for implant was a medtronic enrhythm mri surescan dual chamber device and capsurefix mri leads ( figure 1 ) . this pacing system ( generator box and leads ) is the first to be designed to be compatible with mri scanning under pre - defined conditions ; in general terms , these conditions include marginal limitation of the magnetic field and ensuring that the pacemaker is well established and functioning reliably . radio - opaque labels are present on the lead ( large circle ) and device ( smaller circle ) to indicate that the device can be used with magnetic resonance imaging . chest x - ray , enlarged image of generator box . radio - opaque labels are present on the lead ( large circle ) and device ( smaller circle ) to indicate that the device can be used with magnetic resonance imaging . the patient 's past medical history included wolf - parkinson - white syndrome , with successful ablation of a right free wall accessory pathway in 1995 , moderate aortic valve regurgitation , essential hypertension , benign prostatic hyperplasia , obesity ( bmi=37 ) and mild psoriasis . physical examination revealed a regular paced rhythm , quiet aortic stenotic and regurgitant murmurs , and clear lung fields . trans - thoracic echocardiography provided non - diagnostic images as a consequence of body habitus . coronary angiography four years previously had demonstrated no significant coronary disease . in view of the wide differential diagnosis and the patient 's previous exposure to high - dose ionising radiation , mri scanning was considered the most appropriate imaging modality to gather information on coronary perfusion , left ventricular function , valvular status , pericardial constraint and pulmonary venous anatomy in a single imaging procedure , free of further ionising radiation . the scan was performed according to a local protocol designed to closely follow the conditions of use published by the manufacturers of the device . close liaison with the manufacturers ' technical representatives was maintained throughout . a 1.5 t magnetic field ( philips intera , philips healthcare , the netherlands ) was employed and specific absorption rate ( sar ) was kept to 1 watts / kg or less ; well beneath below the 2 watts / kg advised . the device pocket had healed well over the previous five months and lead thresholds were stable , comfortably below the stipulated capture thresholds of 2.0 volts at 0.4 millisecond pulse width ( table 1 ) . as the patient was not dependent on pacing , the device was programmed to the manufacturers ' advised setting of odo ( i.e. sensing only , not pacing ) during the scan , with continuous non - invasive hemodynamic monitoring . the device sensing and pacing parameters pre- and immediately post - scan did not change to any significant degree ( table 1 ) . subsequent pacing checks also proved unremarkable , with no undue changes to battery longevity . table 1characteristics of the pacing leads before and immediately after the scan.pre-scanpost-scanatriumventricleatriumventriclethreshold @ 0.4 ms ( v)0.5111sensing ( mv)3.37.83.97.6impedance ( nms)416480440528 diagnostic quality images were acquired , including first pass adenosine stress perfusion imaging , and early and late gadolinium enhanced imaging . a minor degree of artefact was reported due to dephasing ( figures 2 and 3 ) which did not compromise the quality of data interpretation . figure 2still images taken from balanced steady state free precession ( bssfp ) cine image acquisition . trans - axial view of the heart ( left hand panel ) showing lead artefact ( arrow ) in the right atrium ( ra ) and right ventricle ( rv ) . trans - axial view of the pre - pectoral pocket ( right hand panel ) showing artefact from the pacemaker generator box ( arrow ) : artefact did not compromise the quality of data interpretation . still images taken from balanced steady state free precession ( bssfp ) cine image acquisition . trans - axial view of the heart ( left hand panel ) showing lead artefact ( arrow ) in the right atrium ( ra ) and right ventricle ( rv ) . trans - axial view of the pre - pectoral pocket ( right hand panel ) showing artefact from the pacemaker generator box ( arrow ) : artefact did not compromise the quality of data interpretation . figure 3(a ) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium . ( b ) still image from a bssfp cine clip showing excellent resolution with only minimal artefact . ( c ) late gadolinium enhanced imaging showing absence of left ventricular scarring , again with minimal interference from the pacing leads . ( a ) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium . ( b ) still image from a bssfp cine clip showing excellent resolution with only minimal artefact . ( c ) late gadolinium enhanced imaging showing absence of left ventricular scarring , again with minimal interference from the pacing leads . the scan comprehensively evaluated both anatomy and function without the need for ionising radiation and without compromising the image quality . the scan was performed according to a local protocol designed to closely follow the conditions of use published by the manufacturers of the device . close liaison with the manufacturers ' technical representatives was maintained throughout . a 1.5 t magnetic field ( philips intera , philips healthcare , the netherlands ) was employed and specific absorption rate ( sar ) was kept to 1 watts / kg or less ; well beneath below the 2 watts / kg advised . the device pocket had healed well over the previous five months and lead thresholds were stable , comfortably below the stipulated capture thresholds of 2.0 volts at 0.4 millisecond pulse width ( table 1 ) . as the patient was not dependent on pacing , the device was programmed to the manufacturers ' advised setting of odo ( i.e. sensing only , not pacing ) during the scan , with continuous non - invasive hemodynamic monitoring . the device sensing and pacing parameters pre- and immediately post - scan did not change to any significant degree ( table 1 ) . subsequent pacing checks also proved unremarkable , with no undue changes to battery longevity . table 1characteristics of the pacing leads before and immediately after the scan.pre-scanpost-scanatriumventricleatriumventriclethreshold @ 0.4 ms ( v)0.5111sensing ( mv)3.37.83.97.6impedance ( nms)416480440528 diagnostic quality images were acquired , including first pass adenosine stress perfusion imaging , and early and late gadolinium enhanced imaging . a minor degree of artefact was reported due to dephasing ( figures 2 and 3 ) which did not compromise the quality of data interpretation . figure 2still images taken from balanced steady state free precession ( bssfp ) cine image acquisition . trans - axial view of the heart ( left hand panel ) showing lead artefact ( arrow ) in the right atrium ( ra ) and right ventricle ( rv ) . trans - axial view of the pre - pectoral pocket ( right hand panel ) showing artefact from the pacemaker generator box ( arrow ) : artefact did not compromise the quality of data interpretation . still images taken from balanced steady state free precession ( bssfp ) cine image acquisition . trans - axial view of the heart ( left hand panel ) showing lead artefact ( arrow ) in the right atrium ( ra ) and right ventricle ( rv ) . trans - axial view of the pre - pectoral pocket ( right hand panel ) showing artefact from the pacemaker generator box ( arrow ) : artefact did not compromise the quality of data interpretation . figure 3(a ) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium . ( b ) still image from a bssfp cine clip showing excellent resolution with only minimal artefact . ( c ) late gadolinium enhanced imaging showing absence of left ventricular scarring , again with minimal interference from the pacing leads . ( a ) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium . ( b ) still image from a bssfp cine clip showing excellent resolution with only minimal artefact . ( c ) late gadolinium enhanced imaging showing absence of left ventricular scarring , again with minimal interference from the pacing leads . the scan comprehensively evaluated both anatomy and function without the need for ionising radiation and without compromising the image quality . to our knowledge , this is the first report in a clinical setting of the safe use of cardiac mri to investigate a patient previously implanted with an mri conditional pacemaker . only minor adjustments were required to the usual scanning protocol and high quality diagnostic images were readily obtained . importantly , the patient experienced no ill effects and there was no change in pacemaker function . whilst there have been reports of mri scans undertaken on various parts of the anatomy , including the heart , of individuals with pacing devices , we demonstrate that safe high quality cardiac mri scanning with a dedicated mri conditional pacemaker device can be performed safely and successfully in a clinical setting .

purposeintra - articular knee injections are commonly performed in clinical practice for treating various knee joint disorders such as osteoarthritis and rheumatoid arthritis . when selecting the portal for injection , not only intra - articular needle accuracy but also procedural pain should be taken into consideration . the purpose of this study was to determine whether injection through anterolateral portal provokes less pain and provides better pain relief compared to superolateral portal.materials and methodsa total of 60 patients with primary osteoarthritis of the knee receiving intra - articular injections were randomized into 2 groups according to the type of portal approach ; anterolateral or superolateral . all patients received hyaluronic acid ( 20 mg ) and triamcinolone ( 40 mg ) as the first injection followed by second and third injections of hyaluronic acid on a weekly basis . underlying knee pain , procedural pain , and knee pain at 4 weeks were evaluated using visual analogue scale ( vas).resultsinjection through anterolateral portal provoked less pain ( vas , 1.51.3 ) than the superolateral portal ( vas , 1.5 vs. 2.7 ; p=0.004 ) . no differences were found in the degree of pain relief at weeks between the two groups ( p=0.517).conclusionswe recommend the use of anterolateral portal for intra - articular knee injection as it provokes less pain and comparably short - term pain relief than the superolateral portal .

intra - articular knee injection is a common , relatively simple and safe procedure done in an outpatient setting for various knee conditions . injections of corticosteroids and hyaluronic acid ( ha ) are most common for the treatment of knee osteoarthritis ( oa ) . although several portals are available for knee injection12 ) , each has its own advantages and disadvantages . accuracy of intra - articular needle placement , portal site pain and experience of the clinician are important factors to be considered before selecting a portal for knee injection . the two routinely used approaches for intra - articular knee injections are superolateral and anterolateral . the superolateral approach with the leg in extension the anterolateral approach is familiar among knee surgeons due to its routine use in arthroscopic surgery . it allows the patient to remain in a sitting position with the knee bent , and bilateral injections can be performed with ease without changing the patient 's position . it is , therefore , useful in patients whose knee can not be extended , and furthermore , it does not require manipulation of the patella4 ) . accordingly , most of the previous studies stressed upon the accuracy of intra - articular needle placement . few studies have described procedural pain and the degree of pain relief following intra - articular knee injection through various portals . hence , we sought to determine whether injection through the anterolateral portal provokes less pain and provides better pain relief than the superolateral portal . this prospective randomized controlled study was undertaken in 60 patients from 30 june 2014 to 30 october 2014 at our tertiary care center . patients were adequately educated regarding the nature of the study before the procedures . written informed consent and clearance from the local ethical committee were obtained before the initiation of the study . inclusion criteria were patients with radiological kellgren - lawrence grade ii or iii oa knee and ability to give informed consent . exclusion criteria were as follows : conditions other than primary oa , systemic diseases that may affect the results , ha and steroid injections within recent three months , allergy to ha injection , the use of warfarin or antiplatelet therapy , or the presence of any infection . patients were recruited in our outpatient department and randomly assigned to either the superolateral injection group or the anterolateral injection portal group using computer - generated permuted block randomization . there were 29 patients in the superolateral group and 31 patients in the anterolateral group . demographic features and preoperative status of the patients including the oa severity in the patellofemoral and tibiofemoral joints and mechanical tibiofemoral angle were compared between the two groups ( table 1 ) . underlying knee pain was recorded using visual analogue scale ( vas ) , where 0 cm = no pain and 10 cm = unbearable pain67 ) . all patients received weekly injections for three weeks in accordance with the assigned route on an outpatient basis under strict aseptic precautions . all procedures were carried out using 23 gauge needles blindly based on anatomic landmarks by an experienced surgeon . injection through the anterolateral portal was given with the patient in a sitting position with the knee flexed to 90 at 1 cm proximal to the joint line , lateral to the patellar tendon , the needle was directed towards the intercondylar notch ( fig . injection through the superolateral portal was performed with the patient in supine position . with the knee extended , the needle was inserted 1 cm above and 1 cm lateral to the superolateral margin of patella at a 45 angle in the cephalolateral to caudomedial direction ( fig . the first injection included ha ( 20 mg ) and triamcinolone ( 40 mg ) and subsequently , the second and third injections included only ha ( 20 mg ) . the primary outcome variable was the degree of pain measured at the portal site during the first injection , and the secondary outcome variable was the degree of pain relief , which was evaluated at 4 weeks after the last injection . procedural pain and knee pain were evaluated using a 0 - 10 vas , where 0 indicates no pain and 10 indicates the most severe pain . in order to ensure the validity and reliability of pain evaluation , a single investigator ( lee sy ) assessed pain levels for all patients . 20.0 ( ibm co. , armonk , ny , usa ) and p - values of < 0.05 were considered statistically significant . the chi - square test was used to compare categorical variables , and the student t - test or paired t - test was used to compare numerical variables . injection through the anterolateral portal provoked less pain than the superolateral portal , and no differences in the degree of pain relief at 4 weeks after last injection were found between the two groups . the mean procedural pain was lower in the anterolateral group than in the superolateral group ( 1.5 vs. 2.7 , p=0.004 ) ( table 2 ) . no intergroup differences were found in pain level ( 2.9 vs. 3.1 , p=0.517 ) or the degree of pain relief ( 2.3 vs. 2.2 , p=0.883 ) at 4 weeks after last injection . there were no immediate complications noted following injection , such as transient flushing reaction or erythema at the injection site . intra - articular injection of the knee joint is commonly performed in clinical practice and is the most common invasive procedure in sports medicine4891011 ) . although the accuracy of the intra - articular needle placement through various routes has been vastly studied , pain - related factors such as procedural pain were barely touched in the literature . the present study hypothesized that knee injection through the anterolateral portal would be less painful and provide better short - term pain relief than the superolateral portal . we found that injection through the anterolateral portal provoked less pain than the superolateral portal , and no differences in the degree of pain relief at 4 weeks after last injection were found between the two groups . findings in this study supported our primary hypothesis that injection through the anterolateral portal would provoke less pain as compared to the superolateral portal . our findings are in contrast with a previous study reporting no significant difference in procedural pain between the modified anterolateral and lateral mid parapatellar portals12 ) . on the contrary , our findings are in line with another previous study on the anterior approach for knee arthrography , where significant reduction was observed in absolute and relative degree of pain for the anterolateral route compared with the anterior paramedian route5 ) . we speculate that thinner soft tissue for the needle to transverse may be related to less pain in injection through the anterolateral portal , particularly when the knee is flexed to 90. in addition , pain detected during the superolateral approach can be explained by accidental needle collision with the bone , quadriceps tendon , and suprapatellar synovium13 ) . nonetheless , our study does not contain any data explaining why and how injections through the anterolateral portal provoke less pain than through the superolateral portal . future studies are warranted to scrutinize this issue . in our study , the degree of pain relief at 4 weeks of follow - up was comparable between the two groups . this does not support our secondary hypothesis that injections through the anterolateral portal offer better pain relief than through the superolateral portal . there are only two studies that compared clinical outcomes between different knee injection sites , and both studies found no significant difference between each other : lateral mid patellar injection vs. anterolateral injection12 ) and infrapatellar injection vs. medial knee injection14 ) . on the other hand , several studies compared clinical outcomes of ultrasound - guided versus blinded injections and reported that ultrasound - guided injections provided better short - term clinical outcomes than blinded intra - articular knee injections1516 ) . however , as the ultrasound - guided injection technique requires expensive devices and trained skills , its clinical and practical values should be evaluated according to the situation of each physician . first , the accuracy of intra - articular needle placement was not confirmed . before the initiation of this study , we considered ultrasonographic or radiographic confirmation of the accuracy of intra - articular needle placement , but it deemed impractical or unethical to expose study participants to additional expense or radiation hazards . furthermore , we noted that even though the use of needle guidance might improve the accuracy of knee injections , insufficient evidence existed to prove that increased accuracy of knee injections would lead to improved therapeutic outcome . a previous study reported that blinded knee injections were reasonably accurate in the lateral injection sites5 ) . a recent systematic review found that overall one in five blinded knee injections were inaccurate17 ) . in the systematic review , pooling data across studies suggested blinded knee injection at the superolateral portal site was most accurate ( 87% ) while injections through medial mid - patellar portal ( 64% ) and anterolateral portal ( 70% ) were less accurate . therefore , whether injection through the anterolateral portal is more accurate than injection through the superolateral portal should be elucidated in future studies . the results of this study may have been affected by the experience of the surgeon , which may limit generalization of our findings . hence , studies evaluating the degree of pain through portals of different approaches are required . finally , this study should be regarded as a preliminary study using a small sample size , which prompts future studies with larger sample sizes and sophisticated evaluation tools regarding needle placement accuracy . because of the small sample size , we could not perform subgroup analyses according to various factors that could influence the technical difficulty during injection or the degree of pain relief such as body mass index , oa , severity , and the presence or severity of patellofemoral joint . therefore , we could neither mention the effects of the confounders nor recommend individualized portal selection . it provokes less pain and provides better short - term pain relief than the superolateral portal . randomized trials to evaluate pain upon multiple routes of injections as well as accuracy of needle placement are needed .

highlightsgenitalia are a key element for male self - esteem , contributing towards mental and physical balance and influencing their social life.scrotal dartos - fascio - mio - cutaneous flap for shaft coverage offers a large amount of tissue with low donor site morbidity , avoiding unsightly scars in more visible parts of the body .

a copy of the written consent is available for review by the editor - in - chief of this journal on request . yes , the guarantor accepts full responsibility for the work and conduct of the study , had access to the data , and controlled the decision to publish .

this article discusses the severity of illness case - mix groups , and suggests a refinement to diagnosis - related groups ( drg 's ) designed to accommodate the important element of patient severity . an application of the suggested refinement is presented in a discussion of the efficient production of hospital services.the following areas are addressed . a brief summary of the goals and development of the severity of illness index , and the methodology used to collect severity of illness data on hospital inpatients.comparative analyses of the resulting case - mix groups within hospitals , and an application of severity - adjusted diagnosis - related groups case - mix definitions.the contribution of the variation in physician practice patterns to the variation in resource use per patient within a hospital.cross-hospital comparisons.some of the consequences of incorporating a patient severity refinement into the prospective payment system .

how should patient categories , or case - mix groups , be defined if they are to be used for prospective payment for inpatient care ? two criteria are important for evaluating the effectiveness of a case - mix grouping system : medical meaningfulness of the grouping system and homogeneity of resource consumption within each of the case - mix groups . in addition , if a case - mix grouping system is to be used for prospective payment , two additional criteria must be considered : independence from the medical treatment process , and administrative feasibility . with these criteria in mind , a severity of illness index was developed by a team of researchers , physicians , and nurses at the johns hopkins university . it was designed to be a medically meaningful generic classification system that could differentiate the severity of illness of hospital inpatients . because discharge abstract data contain only labels of principal and secondary diagnoses , procedures performed , age , etc . , the conventional discharge abstract data base is not rich enough to describe patient severity of illness accurately . using the patient 's total medical record , the severity of illness index assigns to each patient at discharge an overall severity score that is determined from the scores of each of seven medically meaningful dimensions chosen to reflect burden of illness . these seven dimensions , as shown in figure 1 , are : stage of the principal diagnosis the severity of illness index is described in detail in various reports ( horn , sharkey , and bertram , 1983 ; horn , 1983a ; horn and sharkey , 1983 ; horn , chachich , and clopton , 1983 ; horn , 1983b ) . to determine the severity of illness score for an individual case , a rater scores each of the seven dimensions into one of four levels of increasing severity by examining data in the patient 's medical record following discharge . definitions of each of the four levels for each dimension are provided , and raters are intensively trained to distinguish the reasons for selecting the level of each dimension . the rater then assigns an overall severity score for the patient on a four - point scale by implicitly integrating the values of the seven dimensions . the reliability and validity of the severity of illness instrument has been studied in several ways . more than 95 percent of the individual raters achieve greater than 90 percent agreement on blind re - rating of a sample of their charts after using the severity of illness index for 2 months . all the hospital data reported here had greater than 90 percent agreement rates on blind re - rating of samples of charts . the actual agreement percent , rather than agreement corrected for chance agreement , has been reported here because of the very different distributions of overall severity of illness in the samples of charts selected for blind re - rating . we did not feel that each rater 's reliability assessment should be affected because the random sample selected for that review happened to contain charts from mostly one of the severity of illness levels . however , over 90 percent of the time , the agreement percent and the agreement corrected for chance agreement statistic were within 2 percentage points of each other . thus , we found that severity of illness data can be collected in a reproducible manner . with respect to the validity of the severity of illness instrument , we used the following procedures to assure that the instrument would come as close as possible to accurately measuring patient severity of illness . content validity was assessed by presenting the criteria for classification ( definitions of severity levels ) to a panel of medical experts who systematically examined the definitions and came to a consensus . the clinical experts felt that the criteria were representative of patient severity of illness at each identified severity level . face validity was examined by asking the developers and users of the instrument to make subjective judgments as to whether or not the instrument seemed reasonable and had the ability to obtain reasonable data . empirical investigation suggests that the index is a good measure of severity , but the process of validation is constantly being examined . the severity of illness index can not be compared with another validated measure of severity because none is available at this time . the research and statistical analyses discussed below , however , present evidence that the instrument leads to an accurate measurement of illness severity . the four levels of severity are an ordinal scale from the least severe ( level 1 ) to the most severe ( level 4 ) ; the distances between successive levels have no significance and are not necessarily equal . the severity of illness index is a generic measure , referring to the patients themselves . thus , any grouping system for patient classification can be subdivided into severity of illness levels . in particular , the severity of illness index can be used within drg 's or within any other case - mix system . another feature of the severity of illness index , which it shares with other case - mix grouping systems such as drg 's and disease staging , is that it does not explicitly take into account the quality of care received by the patient . the severity of illness index asks how sick the patient is and what burden of illness the patient exhibits while in the hospital . it does not ask whether the patient 's burden of illness was naturally caused or iatrogenically caused , ( i.e. , was exacerbated by accident or infection during hospitalization ) . thus , a patient may become sicker as a result of poor quality of care . severity of illness data are being or have been collected in more than 30 hospitals in the united states and canada . these hospitals include university teaching hospitals , community teaching hospitals , and community nonteaching hospitals . the results presented here are based on those hospitals for which we had at least six months of data . these results are preliminary and will need to be studied more thoroughly when additional data become available . because of the controversies about what trim points should be used , the data have not been trimmed . this permits generalization to an entire patient population , and thus application of a prospective payment system to all patients . severity of illness data are usually collected at discharge by medical records personnel concurrent with the coding of discharge abstract data , or by utilization review personnel when a final utilization review on a patient is completed . for analysis , severity of illness data are merged with discharge abstract data and financial data on each patient . at this point , a computer algorithm takes the presence of an operating room procedure into account . the data are subdivided into three subgroups depending on whether the patient had no operating room procedure , a moderate operating room procedure , or a major operating room procedure . the designation of moderate versus major operating room procedure is based on a list developed by a surgeon panel . a major operating room procedure either requires very special skills and education or takes a long time from which to recover . the subdivision into procedure type subgroups is done for analytical purposes , but does not affect the patient 's severity rating . within each severity of illness group , we examined resource consumption as expressed by total charges , length of stay , and ancillary service charges such as laboratory , radiology , pharmacy , and routine charges . we studied severity of illness within drg 's and within major diagnostic categories ( mdc 's ) , of which the drg 's are a finer subdivision . the results are similar for all types of resource use , so we shall demonstrate them using total charges . the necessary data are being collected to take charges back to costs , but the results are not yet available . in figure 2 , we show a sample of the reduction in variance ( riv ) results obtained when patients are grouped within an mdc . the patients are placed either into drg 's ( from 5 to 48 subgroups depending on the mdc ) or into the four severity levels divided into procedure type subgroups ( up to 12 subgroups ) . is represented by the formula : the data come from a total sample of over 19,000 cases in one university teaching hospital . these results are representative of the kinds of riv results that have been seen for all the mdc 's in the hospitals in the data set . the data show that even though the severity of illness index may place patients into fewer groups ( at most 12 ) than the drg 's , the variability in resource use that is explained by the severity of illness groups is greater than the variability that is explained by drg 's . the weighted ( by sample size ) coefficient of variation statistics for the same mdc 's are shown in figure 3 . the coefficient of variation of a data set is the standard deviation divided by the mean . we see that the severity of illness groups have lower weighted coefficients of variation , and hence , by this measure , have greater homogeneity than the drg 's . analysis of variance f test statistics for the severity of illness index and for drg 's are shown in figure 4 . the analysis of variance f test indicates how different the means are between the various groups , compared with the variability within the groups . a higher f value indicates a grouping system with greater differentiation among the groups as well as better homogeneity within the groups . we see that the severity of illness groups have much higher f values than the drg 's . thus , by three common measures of homogeneity , the severity of illness groups , even though almost always fewer in number , are found to be more homogeneous with respect to resource use than drg 's . these results are representative and typical of those seen from all the hospitals collecting severity of illness data on an ongoing basis . homogeneity of resource use is a desirable feature of a case - mix grouping system for prospective payment . it implies that most cases in a group will actually have resource - use levels which are approximately that of the typical ( average , median , or whatever ) case in the group the norm used to determine the amount of prospective payment for all patients in the group . even if a particular hospital 's patient distribution is atypical , it will be reimbursed appropriately at levels that fairly reflect the resource consumption of its patients . if groups are heterogeneous , the possibility exists that a hospital 's patients in a group will cluster far from the typical a grouping system with more groups might be expected , in principle , to be able to produce groups that are more homogeneous than those produced by a system with fewer groups . for the drg 's and severity of illness case - mix systems thus , it is not just the number of groups that has a decisive influence on homogeneity , but also the fundamental conception of what is being quantified by the system . the data indicate that just the labels of principal and secondary diagnoses , age , procedures , etc . , on which drg 's are based , do not describe sufficiently well how severely ill the patients are . it was found , however , that dividing patients into severity of illness groups yields much better explanatory power . an example of these results for one hospital with more than 19,000 cases in 1 year is shown in figure 5 . patients were placed into drg 's and also classified by severity of illness level and procedure type alone , severity and procedure type within mdc 's , and severity and procedure type within drg 's . for each of these three ways to use severity of illness , the result is that the severity of illness groups are more homogeneous than the drg 's alone , as indicated by greater reductions in variance , larger f values , and lower coefficients of variation . the number of groups into which the patients were classified in this hospital for each of the case - mix grouping systems is given in parentheses in the legend . in tables 1 - 3 , examples are shown of three drg 's , each illustrating a different aspect of variability . table 1 is for drg 75 ( major chest procedures ) , which contained 47 patients whose total charges ranged from $ 1,117 to more than $ 205,000 . a patient was placed in the major operating room procedure category if any of the patient 's operations was on the major operating room procedures list . otherwise , the patient was placed into the moderate operating room procedures category . in particular , the patient with charges of $ 1,117 was classified by the drg grouper into drg 75 because of a procedure coded 33.27 ( other lung biopsy ) . our refined definitions of procedure codes do not list this procedure in the major category , nor does this patient 's resource use reflect such a designation . however , some differences are noted in resource use by severity of illness level even among those patients who have only a moderate operating room procedure . the same phenomenon is observed for patients with major operating room procedures . large differences can be found between the average resource use for patients in the same severity level who have a moderate operating room procedure versus a major operating room procedure . the coefficients of variation of each of the severity of illness groups are much smaller than the coefficient of variation for drg 75 overall . by dividing the patients into severity of illness and procedure type groups within this drg , table 2 is for drg 108 ( cardiothoracic procedures , except valve and coronary bypass , with pump ) , which contained 60 patients with total charges ranging from a little more than $ 5,000 to more than $ 289,000 . although all these patients had a major operating room procedure , some differences in resource use by severity of illness level were found . table 3 is for drg 296 ( nutritional and miscellaneous metabolic disorders , age over 70 and/or with a complicating or comorbid secondary diagnosis ) , which contained 52 patients and had a smaller range of total charges , from $ 374 to more than $ 57,000 . however , differences were still observed in resource use as severity of illness increased . in this case , however , the severity level 1 group had a rather large coefficient of variation . this is attributable to one patient who had difficulty in placement , and whose resource use was therefore much higher than would be expected for a patient in this level . one of the main reasons for adopting a prospective payment system is to encourage an efficient level of operation within the health care delivery system . within a hospital , a prospective payment system should provide specific incentives for reducing inefficiencies of hospital services and should have groups that are meaningful to the medical community . if it does , then physicians may be led to modify their behavior , if appropriate . it is also important that a case - mix system for prospective payment produce groups that are homogeneous ( by various measures ) with respect to patient resource use . because of the often great variability in resource use found within drg 's , a question arises about their effectiveness in inducing desirable modifications in physician practice patterns . severity of illness within drg 's produced the most homogeneous groups ( figure 5 ) . how much of this variability might be explained by differences in physician practice patterns , the drg and severity of illness groups were subdivided further into groups of patients treated by each individual physician . we could , by this means , compare how much of the variability of charges within drg 's could be explained by physician practice patterns , compared with how much of the variability of charges within severity - of - illness - adjusted drg 's was explained by physician practice patterns . the results of these comparisons are presented in figure 6 for several hospitals . in general , the results show that drg 's explain 30 - 40 percent of the variability in resource use . physician subgrouping within drg 's explains another 20 - 40 percent of the variability of resource use . taking severity of illness into account as well , raises the explanatory power to between 90 percent and 96 percent of the variability in charges for each of the hospitals . these results suggest that most of the variability in resource use within a hospital can be explained by : the case mix that is going to the hospital ( drg ) . the coefficients of variation for these same hospitals grouped in the same ways are shown in figure 7 . these results show that physicians do explain some of the variability in resource use . however , it is not clear if the variability results from different physicians treating the same type of patient differently ( efficiency ) or differences in patients within a drg , severity , procedure type group treated by the same physician ( classification ) . these results , as well as the contribution of poor quality of care and its effect on the definitions of severity of illness and drg 's , need further study . if a hospital 's ( or a physician 's ) resource use within a drg is higher than a typical level , it could be that the institution ( or physician ) is inefficient . it could also mean that the institution or physician is treating patients who are more severely ill . because severity - adjusted drg 's produced the most homogeneous groups in this study , patients from the whole institution were placed into the appropriate drg group and further subclassified by severity of illness level and procedure type ( drg , severity , procedure ) . as the norms of practice in each institution , we used the average resource use expressed in terms of total charges , length of stay , laboratory charges , radiology charges , routine charges , and pharmacy charges for patients in each drg , severity , procedure group . subsequently , we compared the resource use of each physician 's patients with the norms in the appropriate categories . for each physician , we then accumulated the differences between each of his patient 's resource use and the norms , controlling for drg , severity , and procedure type . the results are explained in more detail in another article ( horn , horn , and moses , 1984 ) . it was found that some physicians treated most of their patients with less resource use than the norms , and some with more . the same comparisons were also made when the patients were grouped only by drg 's , and not adjusted for severity or procedure type ( figure 8) . the two different methods of assessing an individual physician 's efficiency often led to different conclusions . these disparate results signal to a hospital administrator that the underlying causes of the differences should be investigated . only a more detailed review will show whether the differences are because of quality of care , efficiency , or treatment of more severe cases . however , the implications of these differences between drg 's and severity- and procedure - adjusted drg 's are great . where will medical practice be in the future if the wrong physicians are criticized ( or praised ) for atypical practice patterns ? the results in the previous sections have indicated that severity of illness refinements within drg 's can produce resource - use groups that are more homogeneous . however , the fact that there is a spread of severity within a drg is not necessarily a fatal problem . if all hospitals treat patient populations with the same distribution of severity of illness within each of the drg 's , one might expect variations in resource use within a drg to average out . exactly such a reliance on a law of large numbers was explicitly stated in secretary richard schweiker 's report to congress ( department of health and human services , 1982 ) in which he proposed the present prospective payment system for medicare . in figure 9 , the distribution of severity of illness within drg 's in several different types of hospitals is shown . each hospital the various shaded sub - bars represent the percent of drg 's having one severity of illness level ( homogeneous with respect to severity of illness ) , having two severity of illness levels , etc . in hospital ut1 , on the other hand , more than 60 percent of the drg 's in hospital c1 had one level of severity , and fewer than 10 percent of the drg 's had three and four levels of severity . thus , different hospitals have different numbers of drg 's with a spread of severity of illness . figure 10 shows the distribution of patients according to the number of severity levels that their respective drg 's contained . in hospital ut1 , these drg 's contained 63 percent of the hospital 's patients , compared with 35 percent or less of the patients in the other hospitals in drg 's with three or four levels of severity . only 18 percent of the drg 's in hospital ut1 were homogeneous with respect to severity , and these drg 's contained only 3 percent of the patients in the institution . in the other hospitals , no more than 31 percent of the patients were in drg 's that were homogeneous with respect to severity of illness . thus , the drg 's that are homogeneous with respect to severity contained only a minority of the patients in the study hospitals , whereas those drg 's that were heterogeneous with respect to severity encompassed the majority of patients in all five institutions . however , the distribution of those patients varied greatly by institution . the financial impact of patients in the drg 's that were heterogeneous with respect to severity of illness is shown in figure 11 . the drg 's that contain three or four levels of severity of illness in hospital ut1 ( 37 percent of all drg 's present in ut1 ) accounted for 81 percent of the total charges in hospital ut1 , compared with 52 percent or less of the charges in the other hospitals . in this respect , thus , different percentages of a hospital 's revenue are at risk in the drg 's that are heterogeneous . these findings indicate that there can be large inter - hospital differences in the distribution of severity of illness of patients treated within a drg . furthermore , although teaching hospitals frequently treat a greater proportion of more severely ill patients , the severity distributions of the patients in the study hospitals are not predicted reliably by a simple classification by hospital type ( teaching versus community ) or by number of residents available per bed . the prospective payment system based on drg 's is an average - cost - formula system . claims data in the health care financing administration medicare provider analysis and review file supplemented by discharge records from maryland and michigan were used to place patients into their designated drg 's , and cost weights per drg were constructed . these weights reflect the relative costs of treating patients in a particular drg across all hospitals . the actual drg payments are computed by multiplying the drg cost weight by standardized amounts with adjustments for region of the country , differences in wage rates , and differences in number of residents per bed . collection of severity of illness data could be accomplished simultaneously with the collection of discharge abstract data on which the drg system is based . professional review organizations , who have been designated to monitor the reliability of discharge abstract data , could also monitor the reliability of severity of illness data . implementation of a prospective payment system involves administrative issues of equity , cost and regulatory burden . an equitable distribution of payments is essential to pay hospitals fairly for the efficient production of services performed . definition of the hospital product as accurately and precisely as possible is of primary importance . unreliable discharge abstract and severity data from coder errors , incomplete records , and potential gaming of the system definitions can be audited and corrected . this problem will exist and will require expense and regulatory efforts to monitor it . to alleviate part of this problem , we are developing a computerized severity of illness information data base that is based on a 6th digit enrichment of the current coding system , the international classification of diseases , ninth revision , clinical modification . this new , richer discharge abstract data set will be the basis for obtaining severity of illness levels from computer algorithms . it will also permit researchers to define and study other case - mix grouping systems from the new discharge data . as mentioned earlier , a patient may become sicker , and hence may receive a higher severity of illness rating , either because of poor quality of care that allows the patient to develop complications , or a poor clinical choice of therapy , to which the patient does not respond promptly . the consequence of this for a prospective payment system would be a higher payment for iatrogenically caused , as well as naturally , caused , illness . this problem exists , however , in the current drg prospective payment system , with or without a severity of illness adjustment . what is needed is a good measure of quality of care to correct this problem in any prospective payment system . a per - case prospective payment system has many of the right incentives to deal with the financial crisis in the health care field . with more accurate descriptions of their products , hospitals should be able to manage themselves in a more businesslike manner , and accurate cross - hospital comparisons can be made . each individual hospital patient is different , but there are sufficient similarities among patients to allow them to be grouped into medically meaningful homogeneous resource - use groups . trade - offs between the number of groups in such a grouping system , their medical meaningfulness , their homogeneity , and the robustness of the system against manipulation by hospitals and physicians need to be investigated thoroughly to determine which of the various possible case - mix systems is most suitable for prospective payment purposes . although our results , which used the severity of illness index for case - mix purposes , are consistent within all the hospitals analyses to date , these results could be biased by the self - selection factor of the hospitals that have elected to gather severity data . more data from a greater cross - section of hospital types are needed to determine if these results will continue to hold for all hospitals .

in june 2001 , dengue transmission was detected in havana , cuba ; 12,889 cases were reported . dengue 3 , the etiologic agent of the epidemic , caused the dengue hemorrhagic fever only in adults , with 78 cases and 3 deaths . after intensive vector control efforts , no new cases have been detected .

havana is the capital city of the republic of cuba with 15 municipalities , 2,193,848 inhabitants , and a population density of 3,040/km . located in the north of the country , it covers an area of 720.84 km and has an annual average temperature of 25c . house indexes ( percentage of houses with at least one infested container ) of 0.05 to 0.91 were reported from 1997 to 2001 . in july 2001 , house indexes at the municipalities of the city varied from 0.2 to 1.5 ; however , higher figures were observed at health areas and blocks . these data demonstrate that transmission risk must be assessed in more numerous , smaller geographic areas . the entomologic surveillance and vector control activities involved 4,796 workers ; 3,278 family doctors offices ( one family doctor per 120 families and 600 inhabitants ) and 81 health areas constitute the primary health care system , and 23 hospitals comprise the second and third levels . once the santiago de cuba epidemic was detected in january 1997 ( 5,8,9 ) , specifically in havana city , the surveillance was directed at detecting dengue transmission by studying patients with undifferentiated fever and patients with suspected dengue ( patients with fever and two or more symptoms of df such as myalgia , arthralgia , headache , and rash ) . a serum sample for dengue immunoglobulin ( ig ) m detection was collected 5 days after onset of fever . igm studies were conducted first at the laboratory of the centro provincial de higiene y epidemiologia de ciudad habana ( cphe - ch ) by using the ultramicro - enzyme - linked immunosorbent assay ( elisa ) for dengue igm detection ( 10 ) . positive samples were confirmed at the national reference center , the tropical medicine institute ( ipk ) by an igm capture elisa ( 11 ) . a comprehensive study from clinical , epidemiologic , and entomologic perspectives was conducted at those health areas where case - patients were found ; a second serum sample was collected 23 weeks after illness onset to demonstrate the antibody seroconversion or a 4-fold increase in antibody titer ( 12 ) . the table shows the total number of serum samples studied from 1997 to 2002 . cphe - ch , centro provincial de higiene y epidemiologia de ciudad habana ; ipk , tropical medicine institute . through march 2002 . in june 29 , 2001 , the index case - patient was a 68-year - old white woman who lived in the 26 de julio health area of the playa municipality ; she had no history of travel outside the country . the 26 de julio health area was a residential location with a noncontinous water supply ( it received water every 2 days ) . many persons from dengue - endemic countries lived in the area , and many boarding houses also characterized this area . within 2 weeks , 20 additional df cases were serologically confirmed . a retrospective seroepidemiologic study was conducted in a radius of 1 km around the index patient to look for any patients with suspected dengue or undifferentiated fever ; 312 febrile patients , and 14 suspected df patients were found ; however , df was confirmed by serologic studies in 4 of them . all 4 case - patients had dengue igm and high titers of igg dengue antibodies . epidemiologic studies of these patients showed that the first case occurred in late may or early june . the primary case - patient was a 53-year - old white man from the same health area as the index patient . once transmission was confirmed , a proactive dengue surveillance program was established , based on information from family doctors . virologic and molecular surveillance demonstrated that dengue 3 was the etiologic agent of the epidemic . ninety - one dengue 3 isolates were obtained from samples collected at various times during the epidemic . considering the active surveillance and that specimens from all identified clinical case - patients were studied by serologic or virologic methods , the figure of confirmed cases is very close to the total number of dengue clinical cases of the epidemic . all confirmed case - patients figure 1 shows the histogram of the epidemic , and figure 2 shows the municipality distribution and the date of confirmed transmission in the city . by week 30 ( july ) new cases were detected in the arroyo naranjo municipality , and by the end of october ( week 42 ) , almost all municipalities had reported dengue transmission . dengue confirmed case notification according to onset of fever extension of the epidemic in havana city , 2001 - 2002 the wide clinical spectrum of dengue was established in the pan american health organization ( paho)/world health organization ( who ) guidelines ( 13 ) . because of the detection of dengue transmission in the city , the existence of the primary health system , and the strong dengue surveillance system that included laboratories with appropriate technology for serologic diagnosis , we decided to extend the clinical , epidemiologic , and laboratory surveillance to the study of almost all undifferentiated fever cases and those patients with a compatible classic dengue picture . a house - by - house survey for febrile cases and dengue suspected cases was performed in havana city by the family doctors . as a result , 72,162 cases ( 41,830 undifferentiated fever and 30,332 dengue suspected cases ) were epidemiologically , clinically , and serologically studied . dengue infection was confirmed in 12,889 ( 17.86% ) of the total cases . of patients with confirmed cases , 1,660 ( 12.9% ) were children and 11,229 were adults ( 87.1% ) ; 52.4% were female and 47.6% were male . dhf was diagnosed in 78 patients , all adults ( 1664 years of age ) . the main signs and symptoms detected in patients with confirmed dengue cases at the time of hospital admission were fever , 100% ; headache , 89% ; retrorbital pain , 59.2% ; arthralgia , 59.4% ; myalgia , 35.2% ; and rash , 28.1% . other symptoms such as cough , diarrhea , nausea , and vomiting were observed in 21.2% of case - patients . the peak of the epidemic occurred in october and the highest number of cases occurred on october 20 ( 241 confirmed cases ) ; 1,150 cases were confirmed by week 42 ( october 1420 ) . the onset of symptoms of the last two case - patients occurred by february 22 , 2002 . the epidemic was considered controlled 36 days later with confirmation that no possibility of transmission existed . case fatality rate was 3.8% among patients with dhf / dengue shock syndrome ( dss ) . after the first cases were detected , all patients with suspected dengue and those who were severely ill , or those classified as having dhf / dss were hospitalized , all adults at the ipk hospital and all children at the aballi and cerro pediatric hospitals . in total , 4,184 patients were hospitalized , 3,197 adults and 987 children . by the end of the epidemic in january 2002 , a broad hospitalization policy was established in areas free of vector , aedes aegpyti mosquitoes , ( all febrile and dengue suspected case - patients were hospitalized or treated at home with daily visits by the family doctor ) ( 13 ) . the vector control strategy had two phases : the first started as soon as the transmission was detected and restricted the number of cases and geographic extension of the epidemic ( the risk of expansion of the epidemic was high because of the vector indexes in havana city and other provinces).the second phase , called the intensive campaign , started at the beginning of january 2002 and interrupted transmission and , consequently , lowered the risk of dengue endemicity in approximately 70 days . the campaign was based on the principles of dengue control established by the paho guidelines ( 13 ) with the involvement of the whole community ( the head of state , governmental and political bodies at all levels , householders , community organizations , etc . ) the objectives of the intensive campaign were to control the vector and interrupt dengue transmission . massive environmental management and sanitation efforts , the elimination of breeding sites , and the elimination of adult mosquitoes were also carried out . these activities were accompanied by extensive efforts to mobilize the community , a strong program of quality control , the active media involvement , and the repositioning of tanks and different water containers . from a house index of 0.49 at the beginning of the intensive campaign , the intensive active surveillance and the hospitalization of all febrile patients and all patients thought to be infected with dengue were crucial in order to reduce the dengue transmission . at present , after 17 months since the last dengue case , strong surveillance is maintained by the six regional laboratories and the national reference center , and no additional cases have been reported . aegpyti in a regional situation in which the disease has caused unprecedented numbers of cases of df and dhf ( 1,015,420 dengue cases and 14374 dengue hemorrhagic fever , with 225 deaths have been reported to paho ) ( data provided by jorge arias , who american regional office ) . as has been stated in the paho resolution approved by the panamerican health assembly in september 2002 ( 14 ) ,

objective : to compare the functional results of carpectomy and fourcorner fusion surgical procedures for treating osteoarthrosis following carpal trauma.methods:in this prospective randomized study , 20 patients underwent proximal row carpectomy or fourcorner fusion to treat wrist arthritis and their functional results were compared . the midcarpal joint was free of lesions in all patients.results:both proximal row carpectomy and fourcorner fusion reduced the pain . all patients had a decreased range of motion after surgery . the differences between groups were not statistically significant.conclusions:functional results of the two procedures were similar as both reduced pain in patients with scapholunate advanced collapse / scaphoid nonunion advanced collapse ( slac / snac ) wrist without degenerative changes in the midcarpal joint .

posttraumatic osteoarthrosis , however , shows a predictable standardized progression , and thus treatment depends on the stage of evolution of this condition . the etiology of posttraumatic wrist osteoarthrosis is usually secondary to ligament injuries or carpal fractures.1 - 5 in posttraumatic wrist arthrosis cases , 95% are located around the scaphoid , and in 55% of the patients with arthrosis the most common pattern is called scapholunate advanced collapse ( slac ) of the wrist , which results from ligament ruptures . the evolution of this type of arthrosis is divided into the following three stages:2,6 ( 1 ) arthrosis between the styloid process of the radius and the scaphoid ; ( 2 ) arthrosis in the radial scaphoid fossa , ( 3 ) arthrosis between the capitate and the lunate . the osteoarthrosis can also result from pseudoarthrosis of the scaphoid ( scaphoid nonunion advanced collapse ( snac)).7 in this degenerative pattern , pseudoarthrosis of the scaphoid acts biomechanically in the same way as injuries to the scapholunate interosseous ligament ; as observed in cases of slac wrist , butradial scaphoid fossa was preserved when pseudoarthrosis of the scaphoid occurred.8 the snac stages are : ( 1 ) arthrosis between the styloid process of the radius and the scaphoid ; ( 2 ) arthrosis between the scaphoid and the capitate and ( 3 ) arthrosis between the capitate and the lunate . several surgical approaches for the treatment of posttraumatic osteoarthrosis of the carpal bones have been reported namely , proximal row carpectomy , fourcorner fusion , selective denervation of the wrist , partial styloidectomy of the styloid process of the radius , fusion of the scaphoid trapezium trapezoid joint , fusion of the scaphoid capitate joint , atlas fusion ( lunate capitate ) and the complete fusion of the wrist.4,5,8 - 22 this study aimed to compare the functional results of proximal row carpectomy and fourcorner fusion for the treatment of posttraumatic wrist osteoarthrosis with no effect on the midcarpal joint . twentythree patients were selected based on the inclusion and noninclusion criteria for this study ( see below ) . all patients presented wrist osteoarthrosis , with a diagnosis of slac or snac , but without involvement of the midcarpal joint . all patients were operated on by the same surgeon at the same hospital , between august 2004 and september 2007 . the inclusion criteria were the presence of wrist osteoarthrosis grade i or ii slac / snac and acceptance of the statement of free and informed consent . the exclusion criteria were presence of gross deformities in other limbs ; rheumatological conditions ; infections ; involvement of the midcarpal joints , diseased condition in the contralateral wrist and previous fractures of the distal extremity of the radius or the carpal bones . the subjective analysis was based on the disabilities of the arm , shoulder or the hand ( dash ) questionnaire and an analog pain scale.23,24 in the objective evaluation , the following were observed : wrist goniometry , grip force ( jamar ) , grip force of pulp pulp , lateral ( key ) and threefinger ( tripod ) pinches , discrimination between two points on the pulp of the second and fifth fingers , and on the dorsum of the first web , measurements of the hand and wrist volumes and the jebsen taylor functional test.25 patients underwent preoperative and postoperative assessments and the latter were done 3 , 6 and 12 months after the surgical procedure . all assessments patients who underwent proximal row carpectomy and those who had fourcorner fusion started their rehabilitation with kinesiotherapy and physical means 3 weeks and 2 months after the operation , respectively . comparison of the evolutional results within the same surgical group , between the preoperative and postoperative assessment were performed by the wilcoxon test . data on the operated and contralateral wrist were also compared using the wilcoxon test . a nonparametric mann whitney test was used to analyze the results between the two surgical groups . the significance level of 5% ( p0.05 ) comparison of the evolutional results within the same surgical group , between the preoperative and postoperative assessment were performed by the wilcoxon test . data on the operated and contralateral wrist were also compared using the wilcoxon test . a nonparametric mann whitney test was used to analyze the results between the two surgical groups . the significance level of 5% ( p0.05 ) the mean ages of the fusion and the carpectomy groups were 4210.6 and 43.410.1 years , respectively . there was no difference in the handedness of the affected limbs between the two groups . the data were divided into a direct evolutional analysis and comparative analysis within the same group and between the groups . data on direct evolutional analysis direct comparison of evolution of the parameters over time within each group are shown in table 1 . after 12 months despite the radial deviation values , all the wrist parameters decreased in both procedures . grip force was evaluated and the fusion group achieved the same values at 12 months as before surgery , this did not occur in the carpectomy group ( table 2 ) . for pinch force evaluation , the operated wrists showed volumetry values that were smaller than those of contralateral wrists by the end of the evaluations , after 12 months of surgery , in both groups . the preoperative values in the fusion and carpectomy groups were 7.6 and 8.2 , respectively . dash values corresponded to the analog pain scale values , which meant that all patients showed an improvement in their daily living and work activities . preoperative dash values in the fusion and carpectomy groups were 42.7 and 52.4 , respectively , and postoperative values were 29.9 and 37.7 , respectively . jebsentaylor test values showed that hand abilities had improved in both groups , from 57 to 40.9 seconds in the fusion group and from 74.1 to 65.2 seconds in the carpectomy group . comparative analysis is a proportional comparison of the data between the groups as follows : horizontal analysis : evaluation of the data between the operated and the contralateral wrist 12 months after the operation ( table 3 ) . vertical analysis : evaluation of the data on the operated wrist 12 months after the operation in relation to the preoperative ipsilateral data ( table 4 ) . in the horizontal analysis , the data were compared proportionally and values for the contralateral limb were used as the baseline . in the vertical analysis , the preoperative measurements were used as the baseline values in the fourcorner fusion group , 1 case of reflex sympathetic dystrophy was seen . in the proximal row carpectomy group , 3 cases of synovitis with significant wrist edema and 2 cases of reflex sympathetic dystrophy were seen . all observed complications occurred no later than the second month after the operation and were treated clinically . none of the patients presented any breakage of the synthesis material or deep infection , or any other condition that might have required further surgical intervention . data on direct evolutional analysis direct comparison of evolution of the parameters over time within each group are shown in table 1 . after 12 months despite the radial deviation values , all the wrist parameters decreased in both procedures . grip force was evaluated and the fusion group achieved the same values at 12 months as before surgery , this did not occur in the carpectomy group ( table 2 ) . for pinch force evaluation , the operated wrists showed volumetry values that were smaller than those of contralateral wrists by the end of the evaluations , after 12 months of surgery , in both groups . the preoperative values in the fusion and carpectomy groups were 7.6 and 8.2 , respectively . dash values corresponded to the analog pain scale values , which meant that all patients showed an improvement in their daily living and work activities . preoperative dash values in the fusion and carpectomy groups were 42.7 and 52.4 , respectively , and postoperative values were 29.9 and 37.7 , respectively . jebsentaylor test values showed that hand abilities had improved in both groups , from 57 to 40.9 seconds in the fusion group and from 74.1 to 65.2 seconds in the carpectomy group . comparative analysis is a proportional comparison of the data between the groups as follows : horizontal analysis : evaluation of the data between the operated and the contralateral wrist 12 months after the operation ( table 3 ) . vertical analysis : evaluation of the data on the operated wrist 12 months after the operation in relation to the preoperative ipsilateral data ( table 4 ) . in the horizontal analysis , the data were compared proportionally and values for the contralateral limb were used as the baseline . in the vertical analysis , in the fourcorner fusion group , 1 case of reflex sympathetic dystrophy was seen . in the proximal row carpectomy group , 3 cases of synovitis with significant wrist edema and 2 cases of reflex sympathetic dystrophy were seen . all observed complications occurred no later than the second month after the operation and were treated clinically . none of the patients presented any breakage of the synthesis material or deep infection , or any other condition that might have required further surgical intervention . the mean age ( 4052 years ) of patients in this study is similar to that in other studies.8,16 - 22 a period of 510 years between the trauma and the start of signs and symptoms of arthrosis implies a condition that occurs in patients who are around 35 year old.7 rigor in applying the noninclusion criteria was fundamental for obtaining a homogeneous group of patients . the use of computed tomography to evaluate the joints , particularly the midcarpal joint , contributed greatly towards homogenizing the study groups . twelve months after the operation , the overall range of wrist motion was smaller in relation to the preoperative values in both procedures ( p0.05 ) . the arc of flexion extension was , on average , 25% and 17% less in the cases of fourcorner fusion and in proximal row carpectomy , respectively . the radial ulnar deviation , on average , was , 0.3% and 10% less in fusion patients and carpectomy patients , respectively . the radial deviation in cases of fourcorner fusion was the only goniometry parameter that had improved 12 months after the operation . decreases in the overall range of motion in relation to preoperative values in proximal row carpectomy5,26 - 28 and fourcorner fusion5,29 has been reported . in our study , horizontal analysis within each group showed proportional rangeofmotion results favoring proximal row carpectomy , except for radial deviation , for which fusion was favored ( p 0.05 ) . the results presented here are consistent with other published results.8,16 - 19,22 however , others found that the arc of flexion extension in cases of fourcorner fusion was greater than the arc in cases of proximal row carpectomy.20 vertical analysis of the fourcorner fusion group showed that the grip force recovered to preoperative values ( p0.05 ) . in the carpectomy group , 95% of the preoperative grip force was recovered ( p 0.05 ) . a loss of carpal height in the proximal row carpectomy procedure owing to relative stretching of the flexor and extensor tendons has been reported.2 the improvement in grip force after the operation can be credited to pain relief.27,28 horizontal data from a series of proximal row carpectomy cases with longer followup duration have shown that between 60% and 80% of the grip force in the contralateral wrist is achieved.4,26 - 28 in contrast , only 47% of the grip force was achieved in this study . patients undergoing proximal row carpectomy may take up to 1 year to achieve complete rehabilitation.28 horizontal analysis of the grip force in patients undergoing fourcorner fusion provided the best defense of this technique , to the detriment of proximal row carpectomy , because of the preservation of grip force in relation to the nonoperated side.2,16 a 10% increase in relative grip force may occur over a 3year period between postoperative evaluations in cases of fourcorner fusion.30 grip force values of between 70% and 87% , in relation to the contralateral wrist have been reported.8,17 - 20,30 in this study , the grip force was 73% of the contralateral wrist in patients who underwent fourcorner fusion and 47% in patients undergoing proximal row carpectomy compared with the nonoperated side ( p0.05 ) . evaluation of the grip force was done by finger pinches ( key pinch , pulp pulp pinch and tripod pinch ) , together with a test to discriminate between two points . this was done to determine whether surgical procedures might have caused lesions in the peripheral nerves.23 the results obtained proved that both surgical procedures were safe . the universal analog pain scale was applied to the study patients . in the fourcorner fusion group , there was a 33% reduction in pain , compared with preoperative values ( p0.05 ) . in the proximal row carpectomy group the comparative values between groups were not statistically significant . in the vertical dash analysis , the patients who underwent fourcorner fusion or proximal row carpectomy achieved 30% and 28% evolution , respectively , compared with preoperative values ( p0.05 ) . in the vertical analysis of the jebsen taylor test,25 the patients who underwent fourcorner fusion achieved , on average , 28% evolution over the task duration , whereas the value was 12% in patients who underwent proximal row carpectomy ( p0.05 ) . in the horizontal analysis , almost all patients recovered their function compared with the contralateral side ; 98% in fourcorner fusion group and 99% in proximal row carpectomy group . both proximal row carpectomy and fourcorner fusion surgical procedures provided similar functional results for treating degenerative conditions of slac / snac without the impairment of the midcarpal joint . indication for the surgical technique should be based on several parameters , such as patient 's age , duration of immobilization , risk of pseudoarthrosis , possibility of breakage of the synthetic material , infection , duration of rehabilitation and the experience of the surgical team .

neonatal diabetes mellitus ( ndm ) , defined as persistent hyperglycemia occurring in the first months of life , is a rare cause of hyperglycemia and is often misdiagnosed as type 1 diabetes mellitus ( t1 dm ) . numerous reports have shown that the successful transition from insulin to sulfonylurea agents can be achieved in up to 90% of patients with ndm . however , most of the reports pertain to infants ; the literature is limited regarding treatment of adults with ndm . we present our experience with a patient with permanent ndm , initially misdiagnosed as t1 dm , who subsequently was successfully transitioned to oral sulfonylurea therapy after 37 years of insulin dependence .

neonatal diabetes mellitus ( ndm ) , defined as persistent hyperglycemia occurring in the first six months of life , is a rare cause of hyperglycemia with an estimated incidence of 1 in 100,000 to 1 in 260,000 live births [ 13 ] . almost all cases of ndm have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age . there are 22 known genetic causes of ndm ( mutations in 21 genes and methylation abnormalities at the 6q24 locus ) that identify different clinical subtypes of the disease . this includes transient ndm ( tndm ) , permanent ndm ( pndm ) , and complex syndromes in which ndm is often the presenting feature ( i.e. , wolcott the most common cause of ndm are mutations in the adenosine triphosphate ( atp)-sensitive potassium channel ( k - atp channel ) subunit genes abcc8 and kcnj11 which regulate the release of insulin from pancreatic cells [ 1 , 58 ] . the pancreatic -cell k - atp channels are hetero - octamers assembled by four kir6.2 subunits and four high - affinity sulfonylurea receptor 1 ( sur1 ) subunits encoded by the genes kcnj11and abcc8 , respectively [ 911 ] . kcnj11 mutations are more frequent in patients with pndm , whereas mutations in abcc8 cause tndm more frequently . the majority of infants with ndm are small for gestational age , which may be related to decreased insulin secretion in the fetus and exhibit postnatal catch - up growth with insulin therapy . the most severe defect includes marked developmental delay and early onset epilepsy , also known as dend syndrome [ 8 , 10 ] . subcutaneous insulin was routinely used in the past to treat patients with this disorder ; however , studies have shown that the successful transition from insulin to sulfonylurea ( su ) agents can be achieved in up to 90% of patients with ndm . we report a case of pndm whose treatment was successfully transitioned from insulin to oral su therapy after 37 years of insulin dependence . a 37-year - old man with history of poorly controlled diabetes with multiple microvascular complications of proliferative diabetic retinopathy , stage 3 chronic kidney disease ( ckd ) and peripheral neuropathy , presented to our institution for further evaluation . the patient reported frequent hypoglycemia and blood glucose ( bg ) excursions with bg ranging from 48330 mg / dl . review of his medical records demonstrated previous hemoglobin a1c ( hba1c ) measures as high as 12.9% . he was diagnosed with type 1 diabetes mellitus ( t1 dm ) when he was 2 weeks old and multiple daily injections of insulin ( mdi ) were initiated to control glycemia . he reported a strong family history of early - onset insulin - dependent diabetes in both of his siblings , and his mother had insulin - dependent diabetes mellitus ( unknown type ) . the patient s medical and family history warranted further investigation and consideration of other forms of diabetes . hba1c was 7.7% , c - peptide < 0.2 ng / ml ( range 0.83.2 ng / ml ) with corresponding bg of 80 mg / dl ( 65100 mg / dl ; table 1 ) . the patient s physical exam , and specifically the patient s neurological status , was otherwise normal . as genetic testing could not be afforded by the patient , a trial of su therapy was offered . glimepiride 2 mg daily ( qd ) was initiated and slowly titrated up to 8 mg twice daily ( bid ) over a 3-month period with an observed increase in c - peptide from undetectable to 0.7 ng / dl ( bg 176 mg / dl ) . glimepiride was then increased to 12 mg bid with further improvement in glycemic control . insulin detemir and insulin lispro were slowly decreased as the dose of su therapy was increased . in an attempt to increase the patient s own endogenous insulin production and achieve further insulin independence , a 7-day trial of sitagliptin 100 mg qd was initiated with a subsequent increase in c - peptide to 0.9 ng / dl ( bg 252 mg / dl ) . however , glycemic control did not improve so the sitagliptin was discontinued and a trial of dapagliflozin 5 mg qd was initiated ( despite his ckd stage 3 ) . glycemic control significantly improved , and after dapagliflozin was increased to 10 mg qd , insulin therapy was completely discontinued ( his original total daily dose of insulin was 64 units ) . the patient follows a carbohydrate - controlled diet of approximately 45 g per meal and occasionally takes 3 units of insulin lispro when planning to eat a high carbohydrate containing meal , particularly when eating out at restaurants . the addition of the sodium - glucose cotransporter-2 ( sglt2 ) inhibitor has helped tremendously with postprandial glucose control and the patient has not had any episodes of diabetic ketoacidosis . repeat hba1c after 3 months of oral anti - diabetic therapy was 6.6% , with average bg in the 70130 mg / dl range . over the last year since the initiation of su therapy , his hba1c has remained below 7% without any episodes of hypoglycemia ( table 2 ) . multiple attempts to evaluate and test his parents and siblings for ndm have been unsuccessful thus far.table 1c - peptide , glucose , and hba1c values with corresponding insulin and su regimendatec - peptide ( 0.83.2 ng / ml)glucose ( 65100 mg / dl)hba1c , % insulin regimen + dose of su7/28/14<0.2807.3det 27 units + lis 7 - 15 - 15 and glim 2 mg qd was added after labs obtained7/30/140.32917/31/14increased glim 4 mg qd8/4/140.4263det 22 units + lis 7 - 15 - 15 + glim 4 mg qd8/12/140.5306changed det to 18 units + lis 7 - 14 - 12 + increased glim to 4 mg bid8/29/140.73219/17/14increased glim to 8 mg bid , decreased lis to 3 - 6 - 59/24/140.7178det 18 units + lis 3 - 6 - 5 + glim 8 mg bid9/25/14decreased lis to 3 units with meals10/6/14stopped mealtime lis , increased glim 12 mg bid10/20/140.71757.0det 15 units + glim 12 mg bid , lis prn , add sitagliptin 100 mg qd10/28/140.9252det 15 units + glim 12 mg bid , stop sitagliptin , start dapagliflozin 5 mg qd2/23/150.62196.6det 10 units + glim 12 mg bid + dapagliflozin 10 mg qd + lis prn , 45 g of cho per meal2/25/15decreased det to 6 units3/4/15glim 12 mg bid and dapagliflozin 10 mg qd , stopped det7/15/151686.9%glim 12 mg bid and dapagliflozin 10 mg qd + lis 3 units prn for high cho meal11/16/151486.8 bid twice daily , cho carbohydrate , det insulin detemir , glim glimepiride , hba1c hemoglobin a1c , lis insulin lispro , prn pro re nata ( as needed ) , qd daily , su sulfonylureatable 2hba1c% trendyear200820092010201120142015hba1c%12.911.711.29.47.76.6 hba1c hemoglobin a1c c - peptide , glucose , and hba1c values with corresponding insulin and su regimen bid twice daily , cho carbohydrate , det insulin detemir , glim glimepiride , hba1c hemoglobin a1c , lis insulin lispro , prn pro re nata ( as needed ) , qd daily , su sulfonylurea hba1c hemoglobin a1c informed consent was obtained from the patient for publication of this case report . ndm is a monogenic form of diabetes that presents within the first six months of life . in approximately half of patients , the diabetes will be permanent , and in the remaining cases the diabetes will remit within a few weeks or months although it might relapse later in life . approximately , two - thirds of tndm cases are caused by abnormalities in an imprinted region on chromosome 6q24 [ 13 , 14 ] with activating mutations in either of the genes encoding the two subunits of the k - atp channel of the -cell membrane ( kcnj11 or abcc8 ) causing the majority of the remaining cases . in contrast , the genetic abnormality responsible for up to 30% of pndm cases remains unknown although the commonest known cause in outbred populations is mutations in the k - atp channel or ins genes . heterozygous activating mutations in the kcnj11 or abcc8 gene , which encodes the kir6.2 subunits and sur1 regulatory subunits of the k - atp channel , respectively , have been implicated and account for 30% to 58% of cases of pndm diagnosed in patients less than 6 months of age [ 6 , 11 ] . mutations in the k - atp channels lead to decreased sensitivity to atp inhibition ; consequently , the channels remain open in the presence of glucose , thereby reducing insulin secretion . sus close k - atp channels by an atp - independent route thereby causing insulin secretion studies have shown that many patients with diabetes caused by kcnj11 or abcc8 mutations can successfully switch from treatment with insulin to oral su therapy . the dose of su therapy that is required to manage glycemia in patients with ndm , particularly to achieve insulin independence , is often higher than the maximum recommended dose for the treatment of type 2 diabetes mellitus , typically needing around 0.5 mg / kg / day of glibenclamide [ 11 , 16 ] . in our case , glimepiride was chosen instead of glibenclamide ( glyburide ) because of the ever - evolving literature reporting a lower risk of hypoglycemia and lower risk of all - cause and cardiovascular - related mortality with the other readily available sus compared to glibenclamide [ 18 , 19 ] . a report by thurber et al . found that , in patients with ndm , earlier age at initiation of su treatment was associated with an improved response to su therapy . this led the authors to appropriately hypothesize that perhaps the declining sensitivity to su therapy observed with more advanced age may be due to the loss of -cell mass over time in those treated with insulin therapy , thereby highlighting the importance of early recognition and initiation of su therapy . in our patient , we were able to demonstrate a rise in c - peptide as the su dosage was increased . while it is certainly possible that the original c - peptide may have been undetectable because the patient did not follow directions ( drink orange juice prior to laboratory assessment to ensure bg is over 200 mg / dl ) the observed gradual rise in c - peptide , in the setting of hyperglycemia , as the su dose was increased , demonstrates a slow increase in endogenous insulin secretion in response to the therapy . the patient did follow the directions to raise serum glucose > 200 mg / dl at the time of subsequent c - peptide measures , as demonstrated in table 1 . while the lack of genetic testing is a significant limitation of our report , this limitation does not take away from the main point of this report , that being the importance of performing a very thorough personal and family history in patients who present with a prior diagnosis of diabetes , particularly t1 dm very early in life , as a near - miraculous transformation in management may be possible in a minority of patients , as was the case in our patient , even if genetic testing is not available / feasible . genetic testing for some conditions is available free of charge on a research basis in certain academic institutions in patients diagnosed with diabetes before 6 months of age ( e.g. , [ 2123 ] ) . to the best of our knowledge , we are the first to report the effective and safe use of sglt2 inhibitors in a patient with ndm . while the hba1c did not appear to improve significantly after the addition of dapagliflozin , the therapy did help to lower postprandial glycemic excursions , not adequately addressed by the su therapy , and helped to reduce the frequency at which the patient took a correction dose of bolus insulin , or a dose of bolus insulin to assist with glycemic control when he consumed higher carbohydrate containing meals . we report the case of a patient with long - standing poorly controlled ndm initially misdiagnosed and treated as having t1 dm . many patients with ndm treated with insulin - only therapy behave as brittle diabetics , with difficult to control glycemia leading to complications . the addition or transition to su therapy may help some of these patients achieve better glycemic control , while requiring less dependence on insulin therapy . our case highlights the importance of history taking in the management of patients with diabetes , especially the need to conduct a thorough family history , as recognition of ndm can profoundly impact the diabetes - related management of both the patient and their family members .